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LIBRARIES
MICHIGAN STATE UNIVERSITY
EAST LANSING, MICH 48824-1048

This is to certify that the
dissertation entitled

PLASMA CARNITINE, CLINICAL PARAMETERS, AND
QUALITY OF LIFE IN PATIENTS RECEIVING
HEMODIALYSIS

presented by

ALISON LEAH STEIBER

has been accepted towards fulﬁllment
of the requirements for the

PhD. degree in ,~" Nutrition

 

 

  

A"

 

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, 2.1an 05‘

Date

MSU is an Afﬁrmative Action/Equal Opportunity Institution

PLACE IN RETURN BOX to remove this checkout from your record.
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PLA

PLASMA CARNITINE, CLINICAL PARAMETERS, AND QUALITY OF LIFE IN
PATIENTS RECEIVING HEMODIALYSIS.

By

Alison Leah Steiber

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTORATE OF PHILOSOPHY
Food Science and Human Nutrition

2005

PLAS

Cami-tin:
interest II
suboptim

patients a

ABSTRACT
PLASMA CARNITINE, CLINICAL PARAMETERS, AND QUALITY OF LIFE IN
PATIENTS RECEIVING HEMODIALYSIS.
By
Alison Leah Steiber
Carnitine metabolism and the therapeutic use of camitine has been a major area of
interest in dialysis patients. The purpose of this study was to determine risk factors for
suboptimal plasma camitine concentrations and associated factors in hemodialysis
patients and to determine if a deﬁned group of chronic kidney disease (CKD), patients at
increased risk for altered camitine metabolism would respond to L-camitine with regards
to clinical parameters, perceived quality of life, and plasma acylcarnitine moieties. To
examine these issues this study was done in two phases. Phase I was a cross sectional,
observational study and phase II was a randomized, double-blind, clinical trial, both were
conduct at 3 Midwest dialysis center on patients receiving hemodialysis (HD). Phase I
resulted in subjects (n=49) who were 60:16 (meanglgSD) years of age and 48% male. 15%
had type 1 Diabetes Mellitus (DM), 29% had type 2 DM, and 25% had left ventricular
hypertrophy (LVH). The plasma free and total camitine (FC and TC), acylcarnitine (AC),
and acyl-to-free concentrations (A/F) were: 40.3:‘11.8um/L, 22.817.3um/L,
17.5:5.9pm/L, and 0.80 i027, respectively. Blood urea nitrogen (BUN), parathyroid
hormone (PTH), and ejection fraction_were positively correlated and age and left atrial
dilation were negatively correlated with TC. BUN and hematocrit were positively
correlated and age was negatively correlated with PC. Subjects who used mannitol or

were male had higher concentrations of both PC and TC, respectively. Patients in phase II

met the l]
cmemr;
duration

difadon a
ere-ups. I

period \\‘

lax'aiiabl

were fen

met the following criteria: age >18 years, on HD 3x/wk >1 year, a plasma FC
concentration of <40umol/L and presence of 2 of the following risk factors: > 65 yr, 2 yr
duration of dialysis, female, use of aspirin and/or mannitol, have type 2 DM, leﬁ atrial
dilation and/or LVH. In phase II, 50 patients were randomized into treatment and placebo
groups. Patients were blindly treated with 2 g IV camitine or placebo. The treatment
period was for 24 weeks with data collected at O, 12 and 24 weeks. Of the 50 recruited, 7
died, 7 withdrew, 2 had FC>40umol/L, and 7 did not complete the ﬁnal SF 36 tool
(available data used). Of the remaining 34 patients, the mean age was 68:14 yrs, 38%
were female, 45% had diabetes. Mean Subjective Global Assessment, Body Mass Index,
energy intake and protein intake were 6: 0.8; 28:7; 17kcals/kg and 0.7g/kg respectively.
Mean plasma TC, FC, Short Chain AC, Long Chain AC and A/F camitine ratio were
36.05+10.64, 18.9+6.5, 11.12+4.0l, 6.03+1.91 (umol/L) and 0.97+O.34. Paired t-test
analysis showed signiﬁcant improvements in the treatment group for role-functioning
(21 .2i24.7 to 53.9:72), bodily pain (53.5:35 .8 to 72.2:314), role-emotional (58.9:434
to 84.7:29.18), and the SF36 physical (36.1_+_9.6 to 39.7:85, p=0.078) composite score;
but no signiﬁcant changes in the control group. Erythropoietin dose changes were
signiﬁcantly different between treatment and placebo groups from O to 24 weeks (-
1316i2795 versus. 1464i2770 units). All acylcarnitine moieties were signiﬁcantly
increased at 12 and 24 weeks for patients in the treatment, but not control, group except
for 2-methylbutynylcamitine. In summary after 24 weeks of IV camitine therapy,
acylcarnitine moieties increased, SF36 scores improved and erythropoietin doses were

reduced.

Litre

This dissei
to David A
person I at
encourage

dietitians. i
Voluntarily
thanks to D

Organized

Another big
made the “I
and Abby It
achiei‘ed COI
ll'eatherspot

Final], and e

support f0r 3.
ha . ,
Grand

ma uh]

Cram. .\ 10m

Acknowledgements
This dissertation and all the work involved in its creation are dedicated in loving memory
to David Ainsworth and Nancy Ainsworth-Vaughn. They inspired me to become the
person I am today. This work was accomplished due to the love, support, and
encouragement of so many. Tremendous amounts of thanks must go to the physicians,
dietitians, nurses and especially the patients of the Dialysis Center of Lincoln. They
voluntarily participated in my research making the impossible, possible. Extra special
thanks to Dr. Leslie Spry for believing in this project and to Jen Strong for keeping me

organized.

Another big thank you to my lab partners, they made me laugh, laughed at me, and again
made the whole experience sane when it should have been insanity. Special thanks to Mia
and Abby for traveling with me. Our laboratory and the special environment that it
achieved could not have been possible without the leadership and caring of Dr.
Weatherspoon. She led us with respect, humor, and love and for that I am grateful. And a

huge thank you to Dr. Davis, without whom I would have not data.

Finally and eternally, I thank my family. My mother has been my best critic and best
support for as long as I can remember, this endeavor has been no exception. My children
have endured many hours in labs (especially on grant days), much time with Dad or
Grandma while I was away at meets or studying for tests and occasionally a tired and
cranky Mom. I thank them and hope they will forgive me. To my husband, I do not think

there are words to express my undying gratitude for all he has done. He is the love of my

iv

life and m

presence i

life and my soul mate. This dissertation would not have been accomplished without his

presence in my life.

 

Lntc
Lutc
Into
Acror
Chap
In

C bapt

Table of contents

List of Tables ..................................................................... I
List of ﬁgures ..................................................................... 11
List of Appendices ................................................................ III
Acronyms ......................................................................... IV
Chapter One: Introduction ..................................................... 1
Justiﬁcation and Aims ...................................................... 4
Chapter Two: Review of literature ........................................... 11
Chronic Renal Failure ............................................. 11
Hemodialysis ........................................................ 12
Carnitine ............................................................. 13
Carnitine and Diet .................................................. 14
Biosynthesis of Carnitine .......................................... 19
Carnitine Functions ................................................ 22
Carnitine and Chronic Renal Failure ............................ 23

Acyl-to-Free Carnitine Ratio, Free Carnitine, and
Acylcarnitine in Dialysis Patients ...................... 25

Potential Mechanisms for Role of Carnitine in Chronic

Kidney Disease ........................................... 27
1) Malonyl Co-A ......................................... 28
2) Free Carnitine .......................................... 3O
3) Carnitine Palmitoyl Transferase I .................. 3O
4) Acylcarnitine .......................................... 32

vi

 

Chapter

Di:
Ref

Chapter F1

Consequences of Altered Carnitine Metabolism .............. 33

Anemia ..................................................... 33

Muscle Function and Fluid Retention ................. 34
Hypertriglycerides ........................................ 36

Cardiovascular Disease .................................. 37

Quality of Life ............................................ 37

Carnitine Supplementation ....................................... 41
Chapter Three: Overall Methods ........................................... 51

Phase I Sample ......................................................... 51
Phase I Data Collection ............................................... 52
Dependent Variable Deﬁnition ...................................... 53
Independent Variable Deﬁnition .................................... 54
Phase I Data Analyses ............................................... 56
Phase II Sample Recruitment ....................................... 57
Phase 11 Data Collection ............................................. 59

Chapter Four: “Carnitine Concentrations and Clinical

Parameters in Chronic Hemodialysis Patients” .................. 68
Introduction ........................................................... 69
Methods ............................................................... 71
Results ................................................................. 74
Discussion ............................................................ 76
References ............................................................ 84

Chapter Five: “ Carnitine Treatment Improved Quality

vii

 

Chap!

Li:
F U1
Pb.

PM,

of Life Measures in a Sample of Midwestern

Hemodialysis Patients” ............................................. 93
Introduction ........................................................... 94
Methods ............................................................... 97
Results ................................................................. 1 02
Discussion ............................................................ 103
References ............................................................ 1 08

Chapter Six: “The Impact of Intravenous Carnitine

Treatment on Hemodialysis over 24 Weeks on

the Concentration and Distribution of Acylcamitines” .......... 117
Introduction ............................................................ 1 18
Methods ............................................................... 120
Results ................................................................. 123
Discussion ............................................................ 125
References ............................................................ 1 30
Chapter Seven: Summary .................................................. 141
Strengths ............................................................... 145
Limitations ............................................................ 146
Future Directions ..................................................... 147
List of Appendixes:
Phases I and H Informed Consents ................................. 153
Phases I and II Data collection Forms ............................. 159

viii

 

Lablgg
Chaptt
1.1 Sta;
1.2 PM
Chapte

3.10m

List of Tables

Meg Page Number
Chapter One

1.1 Stages of Chronic Kidney Disease ....................................... 2
1.2 Phase I and Phase II Timeline ............................................ 8
Chapter Two

2.1 Carnitine Content in Foods .............................................. 15
2.2 Carnitine Intake in Different Nutritional Intake Patterns ............ 17
2.3 Normal Carnitine Concentrations ....................................... 23
2.4 Medical Outcomes Short Form 36 Domains ........................... 40
Chapter Three

3.1 Variables Collected in Phase I ........................................... 53
3.2 Risk Factor Criteria ....................................................... 58
3.3 Data Collection Format ................................................... 61
Chapter Four

4.1 Demographic Data ........................................................ 87

4.2 Mean and Standard Deviations for Continuous

Clinical Parameters ............................................................ 88
4.3 Continuous Independent Variables Signiﬁcantly Associated

with Carnitine .................................................................. 89
4.4 Categorical Independent Variables Signiﬁcantly Associated

with Carnitine .................................................................. 9O

ix

 

4.5)

Chap
5.1C.

5.2 P1

5.4 SE

for
Chapt
6.1 Dc

6.2 Ba.

63 B &
He;
6‘4 ASS

Mo

4.5 Multivariate Linear RegreSsion Models Predicting Total, Free, and

Long Chain Acylcarnitine Values ....................................... 91
Chapter Five
5.1 Characteristics of the Study Groups at the Study .................... 111
5.2 Plasma Carnitine Concentrations by Treatment Groups ............ 113

5.3 Associations Between Descriptive Variables and
Various Baseline Carnitine Fractions ................................. 114

5.4 Short Form 36 (SF 36) scores at baseline and at Six Months

for the control and Carnitine Groups .................................. 115
Chapter Six
6.1 Descriptive Characteristics at Baseline, 12 and 24 Weeks .......... 132

6.2 Baseline Plasma Total, Free, Short, and Long Chain
Acylcarnitine Concentrations .......................................... 133
6.3 Baseline Acylcarnitine Concentrations for Total Sample and
Healthy, Fasted Controls at Baseline, 12 and 24 Weeks (umol/L). 134
6.4 Associations Between Post-treatment Plasma Acylcarnitine

Moieties and Nutritional Parameters ................................... 135

 

6.4 Tr.
Tn
6.5 m-
1111
6-6 Tn

Th:

List of Figures

ﬂgﬂg Page Number
Chapter Two

2.1 Composition of Composite Scale Scores ................................. 41

Chapter Four

4.1 Relationship of SF36p to MP in Males ................................. 92

Chapter Six

6.1 Mean Plasma Carnitine Concentrations ................................. 136

6.2 Plasma Free Carnitine at O, 12, and 24 Weeks .......................... 137

6.3 Chromatograph of One Patient at 0, 12, and 24 Weeks ............... 138

6.4 Treatment Group at O, 12, and 24 Weeks of Acetyl-

Through Hexanylcarnitine ................................................ 139
6.5 Treatment Group at O, 12, and 24 Weeks of Octanyl-

Through Tetradecanylcamitine ........................................... 140
6.6 Treatment Group at O, 12, and 24 Weeks of Hexadecanyl-

Through 9, 12-octadecadienylcarnitine ................................. 141

xi

Ac:

I.)

'JJ

Si

10. Bk

11..\r

Acronyms

1. Chronic Kidney Disease CKD

2. Kilocalorie kcal

3. Glomerular Filtration Rate GF R

4. Sodium-dependent organic cation transporter OCTN2
5. Subjective Global Assessment SGA

6. Medical Outcomes Short Form-36 SF36

7. Dialysis Center of Lincoln DCL

8. University committee on Research Involving Human

Subjects UCRIHS
9. Mid-Arm Muscle Circumference MAMC
10. Body Mass Index BMI
11. Normalized Protein Catabolic Rate nPCR
12. Standard Deviation SD
13. Diabetes Mellitus DM
14. Left Ventricular Hypertrophy LVH
15. Leﬁ Arterial Dilation LAD
16. Blood Urea Nitrogen BUN
1 7. Hemodialysis HD

18. Coenzyme-A Co-A

xii

 

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treatmcn‘
States M
The num
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Alla.

Chapter 1

Introduction
The prevalence of chronic kidney disease (CKD) has been on the rise in the United
States, at an average rate of 8% a year over the past ﬁve years. As the disease progresses
from early to advanced stages (Table 1.1), it eventually becomes necessary for dialysis
treatment to be initiated. There are currently more than 250,000 individuals in the United
States with CKD, undergoing maintenance hemodialysis or chronic peritoneal dialysis.
The number of maintenance dialysis patients will surpass a half million by the year 2010.
As prevalence of CKD increases so does the cost. In 1993 and 1999, dialysis provider
reimbursement by Medicare was 7.12 billion and 10.77 billion dollars, respectively. As
the average age in the United States increases so does the mean age of patients on
dialysis. In 2001, patients 55 to 60 years old with chronic renal failure who were
receiving dialysis had an average life expectancy after initiation of treatment of 4.5 years;

the average age for patients on dialysis was 56 years old (1).

Patients with CKD experience lower quality of life, greater morbidity, higher
hospitalization rates, and increased mortality as compared with the general population.
Characterized by uremia this disease is also associated with clinical complications such
as anemia (2), malnutrition (3), osteoporosis (4), tissue and vessel calciﬁcation (4),
hypertension (5), leﬁ ventricular hypertrophy (6), hypertriglyceridemia (7), and ﬂuid
retention (8). A number of metabolic functions are affected as kidney failure progresses

abnormal concentrations of substances such as urea begin to accumulate in the blood.

 

 

 

.- ' . ch .
l‘udncy d:
.f GFR :-

‘ Ekno:

Protein an
\

Table 1.1

Stages of Chronic Kidney Disease

 

 

 

 

 

 

 

Stage Description GF R, mL/min/1.73m2
- At increased risk 360*
1 Kidney damage with normal or T GFR” 390
2 Kidney damage with mild t GFR 60-89
3 Moderately 1 GF R 30-59
4 Severely t GFR 15-29
5 Kidney Failure <15 (or dialysis)

 

 

*with chronic kidney disease risk factors (>60 years of age, hypertension, previous
kidney damage, belonging to a ethnic or racial minority.

** GF R = glomerular ﬁltration rate

*** Bknoyan G, Latos DL, Lindberg J. 2003 (9)

Poor appetite, nausea, and restrictive dietary regimes are associated with compromised
protein and energy intakes and varying degrees of malnutrition (10). A major well noted
secondary effect of renal failure is decreased hydroxylation of vitamin D, which affects
calcium metabolism and subsequent uptake into the bones thereby causing osteoporosis.
Finally, cardiovascular complications associated with the combination of hypertension,
ﬂuid retention, left ventricular hypertrophy, and hypertriglyceridemia may act exacerbate
morbidity and mortality in the CKD population (6;7). Depending on the severity and

number of these clinical conditions, functional and mental status may be impaired to the

point that quality of life is compromised.

Dietary modiﬁcation is a cornerstone of treatment for CKD patients because of the

metabolic alterations, which occur primarily in protein and electrolyte metabolism. In the

 

 

early stages
are restricte
and receive
so1ub1e mo.
requiremeri

camitine.

RCCCTIIi'V. t
therapeuti.
ICCCIVimg
the third (
molecule

long Chai

early stages of CKD, protein and electrolytes such as potassium, phosphorus, and sodium
are restricted in an effort to preserve kidney ftmction. When patients reach stage 5 CKD
and receive dialysis, the dietary recommendations change due to protein and small water-
soluble molecules, such as camitine, being lost in the dialysis process. Protein
requirements are elevated. However, there are currently no dietary recommendations for

camitine.

Recently, camitine (3-hydroxy—4-N-trimethylammoniobutanoate) metabolism and
therapeutic administration of carnitine has been a major area of interest in patients
receiving dialysis. In mammals, acyl groups are attached to camitine via an ester bond at
the third carbon in the cytoplasm. All functions of camitine involve the esteriﬁed
molecule translocating from one cellular compartment to another. During this process
long chain acylcarnitine moieties can be removed from the cytosol or medium chain
acylcarnitine moieties from the peroxisomes (11). The movement of acylcarnitine
moieties from one cellular compartment to another may increase the likelihood of
oxidation. Long chain acylcarnitine moieties are known to have deleterious effects on
cellular membranes and metabolic functions (12). Patients with renal failure may have
increased concentrations of long chain fatty acyls or an alteration in camitine metabolism

due to their uremic status.

The primary source of camitine in humans is exogenous intake of carnitine, lysine and
methionine via food (13). Meat, poultry, and dairy products are reportedly high in total

camitine (14). Therefore, low intakes of these high protein foods may lead to decreased

 

intakes of t
as depicted
of total can
meat eating

due to decrc

Treatment v
hzu been eti‘.

patients ( 16.

Justiﬁcation

Due to alters:

and changes 1
assessment in
dialysis uh-O :
factors for am
Strategjes inc-1
Can be $31le

(ll-ill 515. This X

Wu”? Stain.

ClimCaI ,

intakes of camitine. This has been shown in individuals on lactoovovegetarian diets (15),
as depicted by lower serum camitine concentrations. Although the serum concentrations
of total camitine were not clinically abnormal, they were signiﬁcantly lower than the
meat eating controls. Dietary intake of carnitine may not be sufﬁcient in CKD patients

due to decreased intake of high protein foods.

Treatment with oral and intravenous L-carnitine (biologically active form of camitine)
has been effective in increasing plasma total and free camitine concentrations in CKD

patients (16—18). However, the results have varied in clinical endpoint beneﬁt.

Justiﬁcation and Aims

Due to alterations in dietary camitine intake, camitine losses through dialysis treatment,
and changes in camitine metabolism in CKD, it is crucial to include camitine metabolism
assessment in the management of dialysis patients. Identifying CKD patients receiving
dialysis who are at greatest risk for increased fatty acyl concentrations, or identifying risk
factors for altered camitine metabolism would facilitate determination of treatment
strategies including exogenous L-camitine. Accordingly, clinically signiﬁcant beneﬁts
can be evaluated for the overall better health and quality of life of CKD patients receiving
dialysis. This research was conducted to: 1) identify risk factors associated with altered
camitine status; 2) to determine the effect of L-carnitine administration in a sample of
hemodialysis patients selected using the previously determined risk factors on speciﬁc

clinical parameters and on plasma acylcarnitine moieties.

Aim 1:
To. determine

factors in hem

 

a H}
Clir
lun
car

Aim 2:
To determine i
camitine mew!
perceived qua}
‘1 Hr‘r
mit
hsr
par;
free

Aim 3:
10 meaSUre the
mOieiieS in CK

meiﬁlﬂlism.

& “)1
of r.

meg

Aim 1:
To determine risk factors for suboptimal plasma camitine concentrations and associated
factors in hemodialysis patients.

a. Hypothesis 1: Plasma camitine alteration varies with individual patients and
clinical variables correlated (p<0.05) with plasma total, free, short chain and
long chain acylcarnitine will identify patients at greatest risk for altered
camitine metabolism.

Aim 2:

To determine if CKD patients receiving hemodialysis at increased risk for altered
camitine metabolism will respond to L-carnitine with regard to clinical parameters and
perceived quality of life.

a. Hypothesis 2: Based on the premise that L-carnitine will increase oxidation of
mitochondrial long chain acyl camitine and increase mobilization. It was
hypothesized that a statistically signiﬁcant improvement will occur in clinical
parameters (including perceived quality of life) correlated with plasma total,
free, short, and long chain acylcarnitine.

Aim 3:

To measure the effect of treatment with intravenous L-carnitine on plasma acylcarnitine
moieties in CKD patients who are receiving hemodialysis and at risk for altered camitine
metabolism.

a. Hypothesis 3: Treatment with intravenous L-carnitine will increase oxidation
of mitochondrial long chain acylcarnitines and increase mobilization of

medium and long chain acylcarnitines out of the mitochondria leading to a

 

In order to

conduct a t
Phase 1 — a

medical re
factors for
hemodials

Ph
{1313 Were
and medi.
i“10 treatr
the Place‘
midi \\ a
“111 resp.

met-4501i

piziQma n

bé

measurable increase in medium and long chain acylcarnitine moieties in the

plasma.

In order to accomplish the aims set forth, it was determined that it would be necessary to
conduct a two phase study as follows:

Phase I — a cross sectional, observational study in which data were obtained from a
medical record review and patient examination and interview in order to determine risk
factors for suboptimal plasma camitine concentrations and associated factors in
hemodialysis patients;

Phase II - a randomized, double-blind, placebo controlled clinical trial in which
data were obtained from blood collection (plasma), patient examination and interview,
and medical record reviews. Patients selected based on deﬁned criteria, were separated
into treatment or placebo groups. The treatment group received 20mg/kg/treatment and
the placebo group received a normal saline infusion. The purpose of this section of the
study was to determine if CKD patients at increased risk for altered camitine metabolism
will respond to: 1) determine if CKD patients at increased risk for altered camitine
metabolism will respond to L-carnitine with regard to clinical parameters and perceived
quality of life and 2) measure the effect of treatment with intravenous L-carnitine on
plasma acylcarnitine moieties in CKD patients at risk for altered camitine metabolism.

The organization of this dissertation is as follows:
0 Chapter 2 —- Review of Literature, with a complete description of CKD, dialysis,
and camitine as it pertains to CKD;

0 Chapter 3 — Overview of methods used in phases I and II;

 

0 Chap

0 Chap

0 Char

0 Cha;
Chapters 4. :
correlations
camitine rat
Synthetic er
Palmityroid
intake, dun
phase “as (
tienrs Wh
dilation. 10.

more likely

0 Chapter 4 - Manuscript 1, phase I - aim 1;

0 Chapter 5 -Manuscript 2, phase II - aim 2;

0 Chapter 6 -— Manuscript 3, phase II - aim 3;

0 Chapter 7 - Summary, strengths, limitations, and future research directions.
Chapters 4, 5, and 6 focus on the study aims. Chapter 4 focuses on the baseline
correlations between plasma total, free, and acylcarnitine concentrations, acyl-to-free
camitine ratio and links to speciﬁc clinical parameters (age, weight loss, average monthly
synthetic erythropoietin and mannitol dose, triglyceride, blood urea nitrogen, and
parathyroid hormone concentrations, exercise tolerance, perceived quality of life, dietary
intake, duration of treatment and treatment time, medications and co-morbidities). This
phase was observational with no intervention occurring. Because phase I showed that
patients who were older, female, consumed less dietary protein, had more arterial
dilation, lower ejection fraction percentage, used aspirin and did not use mannitol were
more likely to have lower plasma camitine concentrations, it was determined that a
camitine intervention study would clearly establish whether or not camitine might be
beneﬁcial when these factors are present. Therefore, a randomized, double-blind, placebo
controlled clinical trial was designed for phase II, in which quality of life, synthetic
erythropoietin dosage, muscle cramping and nutritional parameters were monitored for
changes after treatment with L-carnitine. The timeline for both phases I and II is outlined

in Table 1.2.

In Chapter 5, the results of phase II, aim 2 are reported. Variables such as age, duration of

treatment, gender, medication use, and presence of disease (such as type 2 diabetes) were

 

Ph-

1 2000

 

Data
collect
ion

ffﬁe alv-

describes

regarded as risk for altered camitine metabolism. When patients from phase I were
grouped by these selected risk factors, the mean camitine concentrations for plasma total,
Table 1.2

Phase land Phase II Timeline

 

 

 

Phase I Phase II
1/2000 5/2000 7/2002 8/2002 1 1/2002 2/2002 5/2002
Data Data Baseline Intervention 12 week 24 week Data
collect analysis: data period data (ﬁnal) analysis
ion Abstract collection began collection data complete:
submission collection Abstracts

submitted

 

 

 

free and acylcarnitine decreased as the number of risk factors increased. Chapter 6
describes the effects of L-camitine treatment on plasma acylcarnitine concentrations and

on the plasma acetyl to linoleic acid ratio.

 

ta:

. Sjstem

States. 1
and Kid

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Introduction

Reference List

System URD: USRD 2001 Annual Data Report; Atlas of End Stage Renal Diseases in the United
States. Bethesda, MD, National lnstitute of Health, National Institute of Diabetes and Digestive
and Kidney Diseases, 2001.

Caruso U, Umberto, Leone L, Cravotto E, and Nava D. Effects of L-camitine on Anemia in Aged
Hemodialysis Patients Treated with Recombinant Human Erythropoietin: A pilot study. Dialysis
and Transplantation l998;27(8), 498-504.

Steiber AL. Clinical indicators associated with poor oral intake of patients with chronic renal
failure. 1 Ren Nutr. 1999;9z84-8.

Llach F, Yudd M. Pathogenic, clinical, and therapeutic aspects of secondary hyperparathyroidism
in chronic renal failure. Am J Kidney Dis.l998;32:S3-12.

Kalantar-Zadeh K, Rodriguez RA, Humphreys MH. Association between serum ferritin and
measures of inﬂammation, nutrition and iron in haemodialysis patients.
Nephrol.Dial.Transplant.2004;l9:141-9.

Erturk S, Nergizoglu G, Ates K et al. The impact of withdrawing ACE inhibitors on erythropoietin
responsiveness and left ventricular hypertrophy in haemodialysis patients.
Nephrol.Dial.Transplant. 1999;14: 1912-6.

Koniger M, Quaschning T, Wanner C, Schollmeyer P, Kramer-Guth A. Abnormalities in
lipoprotein metabolism in hemodialysis patients. Kidney Int Suppl 1999;71:8248-8250.

Zaluska W, Jaroszynski A, Bober E, Malecka T, Kozik J, Ksiazek A. [Measurement of ﬂuid
compartments using electrical bioimpedance for assessment of target weight in hemodialysis
patients]. Przegl.Lek. 2000;57:707-10.

Eknoyan G, Latos DL, Lindberg J. Practice recommendations for the use of L-Camitine in
dialysis-related camitine disorder National Kidney Foundation Carnitine Consensus Conference.
Am J Kidney Dis 2003;41:868-76.

Beto JA, Bansal VK. Medical nutrition therapy in chronic kidney failure: integrating clinical
practice guidelines. J Am Diet Assoc 2004;104:404-9.

Kemer J, Hoppel C. Fatty acid import into mitochondria. Biochim.Biophys.Acta 2000;1486: 1-17.

Hurot JM, Cucherat M, Haugh M, Fouque D. Effects of L-carnitine supplementation in
maintenance hemodialysis patients: a systematic review. .l.Am.Soc.Nephrol. 2002;13:708-14.

Rebouche CJ, Lombard KA, Chenard CA. Renal adaptation to dietary camitine in humans. Am J
Clin.Nutr. 1993;58:660-5.

Rudman D, Sewell CW, Ansley JD. Deﬁciency of carnitine in cachectic cirrhotic patients. J
Clin.lnvest 1977;60:716-23.

Lombard KA, Olson AL, Nelson SE, Rebouche CJ. Carnitine status of lactoovovegetarians and
strict vegetarian adults and children. Am J Clin.Nutr. 1989;50:301—6.

 

16. Exam :‘r
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patients.

 

l6.

l7.

l8.

Evans AM, F aull R, Fomasini G et al. Pharrnacokinetics of L-camitine in patients with end-stage
renal disease undergoing long-term hemodialysis. Clin.Pharmacol.Ther. 2000;68:238-49.

Evans A. Dialysis-related camitine disorder and levocamitine pharmacology. Am J Kidney Dis
2003;41:513-826.

Brass EP, Adler S, Sietsema KE, Hiatt WR, Orlando AM, Amato A. Intravenous L-carnitine
increases plasma camitine, reduces fatigue, and may preserve exercise capacity in hemodialysis
patients. Am.J.Kidney Dis. 2001;37:1018—28.

 

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Chapter 2

Review of Literature
Chronic Renal Failure
The kidney has many functions including a role in maintenance of water and electrolyte
(eg: potassium, sodium, chloride, calcium and phosphorus) balance, regulation of acid-
base balance, and removal of nitrogeneous waste products, primarily urea. Other
ﬁmctions of the kidney are to produce renin, erythropoietin, and 1,25-
dihydroxycholecalciferol. However, a major function of the kidney is to produce urine. It
does this by forming ultraﬁltrate from the plasma in the glomeruli and proximal tubules.
In the proximal section of the tubules a portion of sodium, chloride, and water is removed
concentrating the ultraﬁltrate. Other solutes such as potassium, magnesium, and calcium
are totally or partially reabsorbed. As the ultraﬁltrate continues through the Loop of
Henle, distal convolution, and collecting ducts, the ultraﬁltrate is reﬁned by regulation of

water, electrolytes, and hydrogen ions.

When a patient begins to lose renal mass he or she begins to adjust to this loss through an

aggregate of mechanisms known as compensatory renal hypertrophy (1). In the early

Stages of kidney failure, the kidney begins to increase in weight and cross-sectional
surface area. This increase in surface area is due to enlargement of glomeruli and
proximal convoluted tubules. This enlargement takes place for the most part in the cortex
and minimally in the medulla. As the patient continues to lose renal mass, each nephron

that remains must excrete more water and solutes in order for homeostasis to continue.

ll

However, nephrons have limits and eventually this loss of renal mass results in

irreparable kidney damage (1 ).

Glomerular ﬁltration rate (GFR) is one method to measure the progressive loss of renal
mass and kidney function. In the clinical setting, the most common method for
determining GFR is by comparing the amount of creatinine in urine to the amount
measured in the plasma. Normal GFR for an adult human is 125mL/minute. However, as
renal function declines, the amount of water and solutes ﬁltered begins to decline until it
reaches approximately 10 to 15 mL/minute (2). At this level of renal ﬁrnction, it is
common for some kind of renal replacement therapy to begin in the form of dialysis.
Previously referred to as end stage renal disease; however, the term commonly used now
is CKD Stage 5 (3). The three most prevalent diseases leading to dialysis treatment in
2002 were diabetes mellitus, 45%; hypertension, 27%; and glomerulonephritis, 9% (4;5).
There are two basic types of dialysis. The ﬁrst, hemodialysis, is done three to four times
per week and requires an internal or external vascular access. The second type of dialysis
is peritoneal dialysis, which is conducted daily, within the peritoneal cavity of the patient.
This study focuses on patients receiving hemodialysis. Hence, a more in-depth

description of this type of dialysis is provided.

Hemodialysis
Hemodialysis is deﬁned as "the removal of certain elements from the blood by virtue of
the difference in the rates of their diffusion through a semi—permeable membrane, for

example, by means of a hemodialysis machine or ﬁlter" (3). The process begins by

12

placing an access into the blood stream via a central line (e.g. subclavian or internal
jugular) or an arterio-venous ﬁstula. Blood is routed out of the body through a pump,
which can be set at different speeds depending on the capability of the access, into a
ﬁlter. The ﬁlter is in a "bath" of dialysate containing a mixture of ions such as calcium,
potassium, and magnesium. Due to hydrostatic and oncotic pressure exerted by the
dialysate and ﬁlter urea, sodium, potassium, phosphorus, excess ﬂuid and some other
water-soluble molecules such as albumin and camitine are removed from the blood. The
blood is returned to the body via the access. Hemodialysis treatment is typically done
three times a week. However, this may vary for very small or very large individuals from
as little as one time a week to as much as daily. Approximately 70% of plasma free and
acetyl camitine is lost through the hemodialysis membrane every time the patient is

dialde (6).

Carnitine

Carnitine is a compound synthesized from the amino acids lysine and methionine in the

kidney, liver, and brain. Approximately 75% of total body camitine needs originate from

food sources of carnitine, lysine, and methionine. The other 25% is generated from

metabolic conversion. Unlike other tightly regulated nutrients (i.e. calcium, iron, sodium,
potassium) in healthy adults, there is an association between dietary intake of carnitine
and plasma camitine concentrations (r = 0.64, p<0.05) (7). The status of carnitine in
humans varies by body composition, gender, and overall diet. In lean adults when
camitine intake is restricted, plasma total and free camitine concentrations and urine free

carni tine excretion decrease. However, when obese individuals are placed on a camitine-

restricted diet, the decrease tends to be blunted (8). When comparing total camitine in
female and male adults, females tend to have lower plasma concentrations and lower

urinary excretion than males (9).

Carnitine and Diet

Dietary camitine is found primarily in foods from animal sources. However, lower
amounts are found in grains, fruits, and vegetables. In Table 2.1, a list of foods with
corresponding camitine content generated from the literature is provided. The
methodology used to analyze camitine content of these food items was either via
radioenzymatic assay, which has been well validated, or via the spectrophotometric
method, which when used for biological samples may be inaccurate. Furthermore, the
camitine amounts listed are ambiguous since the state of the food items and speciﬁc
details about the food such as raw versus cooked and if cooked, which cooking method

are not provided.

Nutritional intake patterns, including diet quality, probably have a greater impact on total

body camitine content than camitine content in single foods. Individuals with minimal to

no animal protein intake, such as vegans (9-11) or people with predominately cereal
based diets (12) have lower plasma camitine concentrations compared to people
consuming diets including animal proteins. Lombard et a1. (9) reported that adult and
children who were lactoovovegetarians and vegans, have lower concentrations of plasma

free and

Table 2.1

Carnitine Content in Foods

 

 

 

 

 

 

 

 

 

Food Item gmol Tota_l__Carnitine/ 100g Reference
food

Animal Products:

Steak, prepared 525 (l 3)

Beef (tenderloin, shoulder, 3691.4 — 4160.5 (14)

rump), raw

Ground Beef, prepared 300 (1 3)

Beef liver, raw 160.4 (14)

Chicken, prepared 60 (13)

Egg, chicken, prepared 5 (13)

 

Grain products (dry, unless
otherwise speciﬁed):

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Comﬂakes 59 (13)
Grits 5 1 (1 3)
Rice 44 (13)
Bread 10.9 (13)
Graham Crackers 5 (13)
Oat seedling (80-90 hour) 8.64 (15)
Wheat germ 7.41 (15)
Wheat seed 2.47 (15)
Oat seed 0.62 (15)
Barley seed 0 (15)
Fruit: (15)
ﬁrplesauce 19.5 (1 3)
Pears 17 (13)
Orange juice 11 (l 3)
Peaches 10 (13)
A plejuice 8 (13)
Pineapple 6.5 (13)
LVegetables: (13)
[l‘omatoes 18 (13)
LCauliﬂower 8.64 (151
l ASparagus 8 (13)
Peas 7.2 (13)
LGreen beans 5 (13)
LAvocado 4.94 (15)
! Spinach leaf, cabbage head 0 (13:13:15)
leaf, carrot, potato
Legumes:
Peanut seed (minus seed 0.62 (15)

 

coat)

 

 

15

cow V] 16.4 (13)

cow 100ml 259.8“ (16)
human 50.0“ 16

* analyzed by ﬂow-injection analyses with an immobilized camitine acetyltransferase
bioreactor. Method based on spectrophotometric determination (l7)

 

total carnitine and have lower urinary excretion of total camitine compared to similar age
groups with animal proteins in their diet. The small difference in plasma camitine,
although statistically signiﬁcant is probably not biochemically or clinically signiﬁcant
due to the lack of evidence of pathophysiological consequences. However, children with
kwashiorkor (due to a low protein intake) have been reported to have decreased plasma
camitine concentrations when compared with healthy controls. The plasma camitine was

positively correlated with long chain fatty acid oxidation (1 8).

Fat content of the diet has been reported to alter camitine status as well. For example,
healthy adult subjects fed a high fat, low carbohydrate diet have signiﬁcantly higher
concentrations of plasma total and free camitine and higher urinary excretion of total,

free and acyl camitine by day 6 of the diet compared to subjects fed a low fat, high
carbohydrate diet (10;1 l). The mechanism for this extremely interesting phenomenon is
yet unknown. Table 2.2 outlines total camitine content from a variety of nutritional intake

patterns as reported in the literature for healthy and unhealthy individuals.

Carnitine Intake in a Variety of Nutritional Intake Patterns

Table 2.2

 

 

 

 

 

 

 

 

Nutritional Intake TotaLCgarnitine Analysis/Information Reference
Patte_rg gmol/day $22829

Far East Indian: Calculated based on values (12)
Middle income 92.5: 12.3 from Rudman et a1, 1977

Low income 55.5:123

L-Carnitine restricted 6414:1687 Radioenzymatic (19)
diet

Cirrhotic patients 29* Spectrophotometric (13)
Normal controls 410*

Wisconsin adults Calculated based on values (7)
(normal omnivores): obtained from personal

Females 1827:3296 communications with P.

Males 2901:5185 Borum

 

 

 

"‘ Standard deviation not provided by reference

Carnitine can also be synthesized from the amino acids lysine and methionine in the
body. There are two sources of these amino acids, dietary and endogenous protein
degradation. The amount of lysine and methionine in the diet can therefore affect
camitine biosynthesis, especially if the intake of carnitine is negligible, such as in vegans
(20;2 1). In 75 individuals, Krajcovicova-Kudlackova et al. (20), demonstrated signiﬁcant

differences in plasma free camitine, dietary methionine, and dietary lysine in

17

vegans/lactoovovegetarians and omnivores. Additionally, they found signiﬁcant positive
correlations between plasma free camitine and dietary lysine, and plasma free carnitine
and dietary methionine in vegans and lactoovovegetarians, but not in omnivores. Dietary
lysine and methionine, although essential are distantly removed from the camitine
biosynthesis process as outlined below. It is surprising that these amino acids are

correlated with plasma carnitine and warrants further investigation.

Micronutrients such as vitamin C, iron, pyridoxine and niacin are necessary for carrritine
biosynthesis. If intake of these is insuﬁcient, then camitine status may be compromised
(22-24). Plasma camitine concentrations were examined in guinea pigs deﬁcient in
vitamin C. The vitamin C deﬁcient state decreases butyrobetaine hydroxylation (25;26).
However, the extent to which overall camitine biosynthesis is affected is unknown. In
iron deﬁcient individuals, camitine status was positively correlated with serum ferritin
concentrations (23;27). As serum ferritin concentration increased to within normal
values, the concentration of plasma camitine also returned to normal. Pyridoxine intake
does not have a large impact on camitine biosynthesis. However plasma short-chain
acylcarnitine decreased without a signiﬁcant change in plasma total camitine when
human subjects were fed a pyridoxine restricted diet (28). While these studies give
insight to the role of micronutrients in camitine homeostasis, the key issue in body

camitine status appears to be the overall quality of the diet.

Absorption of dietary camitine through the gastrointestinal tract depends on the oral load

consumed (19;29;30). For instance, in adults a high load diet (>6g camitine per day)

18

approximately 5 to 15% of the oral carnitine is absorbed. Conversely, in a low load diet
(<1 g camitine/day) greater than 75% of the camitine is absorbed. The unabsorbed portion

is degraded by bacteria in the colon into trimethyl amine and y-butrobetaine (19;29;30).

To date, there are no known dietary components which impair absorption of camitine nor
is there a known toxicity associated with the commonly ingested amounts dietary
amounts of carnitine. Although camitine is found in high protein foods and hemodialysis
patients have elevated protein needs (0.8g protein/kg for healthy vs 1.2g protein/kg for
hemodialysis, respectively), patients are often unable to consume these high protein foods

due to poor appetites or aversions to meats associated with uremia (31).

Biosynthesis of Carnitine
In humans, the rate of carnitine biosynthesis is determined by the availability of
trimethlyllysine and its ability to cross the mitochondrial membrane in order to reach the
intrarnitochondrial site of trimethyllysine hydroxylase activity (32). In humans,
trimethyllysine hydroxylase plays a role in camitine synthesis regulation. The regulatory
effect of trimethyllysine was demonstrated by Rebouche et al (21), when supplemental
trimethyllysine was given to humans, and an 8-fold increase in camitine biosynthesis
occurred. Humans tend to synthesize approximately 1-2umol/kg/day (19). Carnitine is
found in all biological tissues, although ninety-seven percent is located in skeletal

muscle (33).

19

In humans, 30 to 50% of exogenously and endogenously produced trimethyllysine is
converted to camitine; the remainder is excreted in the urine (21). Within the kidney, 98-
99% of tubular resorption of free camitine occurs, unless the transporters become

saturated (19). Healthy subjects excrete camitine at a rate of 5 umol/kg/day, which, for an

80 kg person is 2800 umol/week.

Carnitine synthesis is a multi-step process, which begins with the addition of a hydroxyl
group to the third carbon of lysine by the enzyme trimethyllysine dioxygenase
(hydroxylase) in the mitochondria of the kidney, liver, heart, muscle, and brain. This step
requires 2-oxoglutarate, Fey, and molecular oxygen as cofactors as well as vitamin C to
maintain iron in the ferrous state (34-36). The product of this reaction is 3-hydroxy-N6-
trimethyllysine, and it appears to be the only step in which the enzyme is located in the

mitochondria.

Conversion of 3-Hydroxy-N6-trimethyllysine to 4-N-trimethylammoniobutanal and
glycine is catalyzed by 3-hydroxy-N6-trimethyllysine aldolase. This enzyme is located in
the cytosol, with the highest activity found in liver (34). Pyridoxine is required for
aldolase activity because it uses pyridoxal 5’-phosphate as a cofactor. 4—N-
trimethylammoniobutanal dehydrogenase catalyzes the formation of 4-N-
trimethylammoniobutanoate, also known as butyrobetaine, from 4-N-

trimethylarnmoniobutanal using niacin in the form of NAD as a hydrogen acceptor.

20

4-N-trimethylammoniobutanoate enters the circulation and then enters the kidney and the
liver through an active transport mechanism. In humans, the activity of butyrobetaine
dioxygenase is signiﬁcantly higher in the kidney (3-16 fold) than in the liver, while the
activity in brain is 50% that of liver. In the kidney or liver cytosol, 4-N-
trirnethylammoniobutanoate is hydroxylated on the third carbon to form L-carnitine. This

reaction requires Fez: molecular oxygen and vitamin C for activity (34).

After L-carnitine is synthesized, it is transported through the circulation and taken up by
other tissues through active sodium-dependent transport. This sodium-dependent organic
cation transporter (OCTN2) (37) has been cloned and characterized. The OCTN2
transporter is probably responsible for camitine transport into the tissues and is involved
in tubular reabsorption of carnitine in the kidney. Recently, other transporters, OCTNI

(37;38); , OCTN3 (39), CT2 (40) and ATB°’+ (41), have been identiﬁed to carry camitine.

Carnitine is located in separate compartments of the body with differing rates of turnover
(42) and the muscle tissue to plasma ratio in humans is high at 100:1 (3 7). Liver contains
approximately 500 to 1000 nmol total camitine per gram wet weight. Liver camitine
rapidly interacts with plasma carnitine and has a half-life of one to two hours.
Alternatively, skeletal muscle which contains 3000 to 5000 nmol total camitine per gram
wet weight, does not readily communicate with plasma, and has a half-life of several days
(42). Therefore, changes in camitine content in liver rapidly appear in plasma, whereas

changes in skeletal muscle content may not be as readily apparent in plasma. The two

21

primary forms of plasma camitine are free and acyl.These forms can readily be converted

from one to the other.

Carnitine Functions

Carnitine has many potential functions. The ﬁrst has been well established as a transport
molecule for long chain fatty acyls crossing the inner mitochondrial membrane. Carnitine
acyl transferase I catalyzes the formation of acylcarnitine from fatty acyl—CoA. This
process is regulated by malonyl-CoA. As malonyl-CoA concentrations increase, camitine
palrnitoyl transferase-I becomes inhibited (43). Camitine/acylcarnitine translocase
enzyme then catalyzes the transportation of the acylcarnitine into the mitochondrial
matrix (44) where camitine is separated from the fatty acyl, so the fatty acyl can enter the
B-oxidation pathway to be broken down to form ATP. Second, camitine functions in
exporting acetyl- and chain-shortened acyl products from the peroxisomes preserving the
cellular CoA homeostasis (44). Finally, camitine releases mitochondrial CoA from acyl-
CoA, when the free CoA supply becomes limited, due to activation and subsequent
accumulation of metabolites in the mitochondrion, allowing the CoA to be used for other
functions (43). Normal plasma concentrations of total, ﬁee, short and long chain
acylcarnitine established by Hoppel (45;46) and for total camitine concentrations for men

and women established by Borum (47) are depicted in Table 2.3.

22

Table 2.3

Normal Plasma Carnitine Concentrations

 

Me of Carnitine

 

Total 46.1 :1; 10um/L
males = 51.7:10.8um/L

females = 43.8:11um/L

 

 

 

Free 36.7: 7.6um/L
Short chain 5.7: 3.5 urn/L
acylcarnitine

Long chain 3.7 : 1.5um/L
acylcarnitine

 

 

 

When compared with healthy subjects, patients with CKD are reported to have higher
acylcarnitine concentrations and lower plasma free camitine concentrations; whereas, the

total camitine concentrations remain within the normal range (48).

In healthy subjects, the most prevalent form of plasma acylcarnitine is acetylcamitine.
Following camitine removal, coenzyme-A (Co-A) is attached to acetate and acetyl Co-A
is formed. Acetyl Co-A is a substrate for many metabolic pathways and in a healthy

population is the primary metabolite resulting from B-oxidation. However, in plasma of

23

CKD patients, increased amounts of medium and long chain acylcarnitine concentrations
are reported in the literature (48-50). The increase in medium and long chain
acylcarnitine moieties may be due to an alteration in B-oxidation and/or a decrease in
excretion of these metabolites. Long chain fatty acyls have deleterious effects on cellular
membranes and metabolic ﬁmctions. Hypothesized mechanisms for the negative effects
are 1. interference with sodium-potassium transporters in cell membranes (51) and 2.

detergent effects on cellular phospholipid membranes (52).

Carnitine and Chronic Renal Failure

The exact role of carnitine in hemodialysis patient metabolism is unclear. Patients
receiving hemodialysis are thought to have secondary versus primary camitine deﬁciency
(53). Secondary camitine deﬁciency is associated with a less obvious alteration in
biochemical markers (plasma free-, acylcarnitine and acyl-to-free camitine ratio) than
primary camitine deﬁciency, and is typically caused by an underlying genetically
determined inborn metabolic error, an acquired disease (e. g. chronic renal failure,
Fanconi Syndrom) or an iatrogenic factor (53). On September 8-10, 2002, The National
Kidney Foundation held a consensus conference on camitine, which established the term
dialysis-related camitine disorder (DCD) to describe the symptoms associated with
altered camitine metabolism (54). The four main types of symptoms that have been
reported in the literature as accompanying altered camitine metabolism are: anemia
hyporesponsive to synthetic erythropoietin, intradialytic hypotension, cardiomyopathy

and skeletal muscle weakness (55). However, even though the term DCD and its

24

associated conditions were established, it is not completely accurate because it implies
that dialysis is the cause of the altered metabolic state of camitine. However, in reality

the actual mechanism of the alteration is unclear.

Prior to patients requiring dialysis, they gradually lose the ability to produce and ﬁlter
urine and eventually the patient produces no urine at all. As the glomerular ﬁltration rate
drops, the concentrations of plasma acylcarnitine begin to rise. Normally these
acylcarnitine molecules are lost in the urine, but as urine production decreases, plasma
acylcarnitine concentrations increase. Controversy exists on the cause of the altered
plasma camitine concentration. The question is whether an actual decrease in plasma
camitine concentrations occurs thus causing metabolic consequences in hemodialysis
patients or the metabolic consequences are caused by a defect in one of the B-oxidation

enzymes of the mitochondrial matrix.

In pre-stage 5 CKD patients, Rodriguez-Segade et al (56) showed a plasma acyl-to-free
camitine ratio that was signiﬁcantly higher than healthy controls. This higher ratio
indicates that not only are the concentrations of acylcarnitine higher with a subsequent
ratio increase, but possibly the actual metabolism of carnitine may be altered in patients
with uremia. In 1987, Ricanati et al (50) argued that the concentrations of muscle, liver
and acylcarnitine derivatives for hemodialysis patients were within normal ranges. The
patients in this study were also pre-stage 5 chronic kidney disease at the time of data
collection, were therefore a comparable p0pulation. Later, Hoppel et al (49) showed that

although the amount of plasma total and acylcarnitine was not different in hemodialysis

25

patients, the types of acylcarnitine did differ signiﬁcantly. Data from these studies
suggest that in the case of pre-dialysis patients, the plasma concentration of camitine may
not be as indicative of altered B-oxidation metabolism, as the type of plasma

acylcarnitine.

Acyl-to—Free Carnitine Ratio, Free Carnitine, and Acylcarnitine in Dialysis Patients:
In subjects without chronic renal failure, camitine is excreted through the kidneys. Both
plasma free and acylcarnitine pass into the urinary ultraﬁltrate, but 99% of the free
camitine is preferentially reabsorbed in the tubules. Plasma acetylcamitine, a 2-carbon
acylcarnitine moiety is excreted at a rate four times greater than that of free camitine
(57). This increased excretion of acetylcamitine may keep the acyl-to-free camitine ratio
low. Data on healthy adults from Hopple and Brass (45) suggest that a normal acyl-to-
free ratio is 0.26. In pediatric patients, a plasma acyl-to-free camitine ratio of 2 0.4 can
deﬁne a camitine deﬁciency (53). In dialysis patients the ratio is typically between 0.6

and 0.9 (58), which may be elevated (58).

When patients reach stage 5 CKD and begin dialysis, plasma ﬁee camitine is lost. As a
small, water-soluble molecule, camitine passes through the dialysis membrane and
accumulates in the dialysis ﬂuid (33;44;58;59). The plasma free camitine is lost through
the dialysis membrane at a rate similar to acylcarnitine (about 70%), affecting the ratio of

acyl-to—free camitine in hemodialysis patients (6).

26

Plasma from patients with chronic renal failure contains elevated concentrations of
acylcarnitine formed from accumulating camitine metabolism intermediates at the
expense of free camitine (42). This occurs for example, when acetate is used as an
alkalizing anion in the dialysate ﬂuid. Acetate is activated for metabolism by coupling to
free CoA-SH reducing the intramitochondrial concentration of the essential cofactor (47).
CoA-SH is also required for the oxidation of 2-oxog1utarate in the citric acid cycle.
Inadequate CoA-SH concentrations can slow the metabolism of acetate. In the presence
of adequate camitine, the transfer of fatty acids from acyl-CoA to camitine, followed by
an export of acylcarnitine to the cytosol can regenerate CoA-SH. If acetate overload has
depleted the concentrations of free camitine, then both CoA-SH depletion and acyl-CoA

buildup will limit acetate metabolism, and the anion will increase evan higher.

Therefore, even when free plasma camitine concentrations appear adequate, they may not
be sufficient to handle the high acetate loads potentially experienced during
hemodialysis. Currently, dialysate in most centers consists of predominately bicarbonate
and minimal amounts of acetate. However, based on ﬁndings of Borum (47), a problem
may still exist even with low acetate concentrations. Additionally, the total camitine
concentrations in tissues are reduced in some people receiving hemodialysis (42). When
measured, free plasma camitine concentrations, in hemodialysis patients appear to drop

during dialysis the return to normal requires several hours (6;59).

Studies conducted with hemodialysis patients indicate that this population has the

potential for low plasma concentrations of free-camitine (52;60). Giovenoli had 26

27

patients, who had been receiving hemodialysis for a minimum of one year. When the
patients were divided into separate groups for three different methods (oral,
interdialysate, or intravenous) of L-camitine administration, the baseline measures of
muscle ﬁee camitine were below normal at 24.2 uM/liter, 19.2 uM/liter, and 28.0
uM/liter, respectively. Normal concentrations of free camitine in the muscle. were set at
36.7 i 7.6 uM/liter for men and women by Brass and Hoppel (45). Matsurnura et a1 (52)
found that 26 patients, who had been receiving hemodialysis for a minimum of one year,
had an average plasma free camitine concentration of 21 .8 uM/liter at baseline. These
sub-optimal concentrations may be contributing to defects in metabolism of long chain
fatty acids in hemodialysis patients and hence symptoms, such as: fatigue, muscle

cramps, poor muscle strength, poor appetite and decreased quality of life.

Recommendations and Implication for Carnitine in Chronic Kidney Disease
In the year 2002, L-carnitine was approved by the Centers for Medicare & Medicaid ,
Services (CMS) for national reimbursement in hemodialysis patients with either

erythropoietin resistance or chronic hypotensive episodes during dialysis treatment.

There are at least four possible key metabolic areas, for which camitine treatment may
have important clinical outcomes for hemodialysis patients. These are: l) malonyl-CoA
concentrations — due to its role in regulating camitine palmitoyl transferase-I and thus its
role in hepatic mitochondrial fatty acyl oxidation, 2) plasma free camitine concentrations
— the availability of this substrate could affect the rate of the camitine palmitoyl

transferase-I enzyme, 3) camitine palmitoyl transferase-I — due to its role in forming

28

acylcarnitine compounds, and 4) plasma acylcarnitine concentration— due to the reported

variety of acyl forms unlike normal controls in CKF patients (49).

l) Malonyl-CoA

Malonyl-CoA has regulatory affects on camitine palmitoyl transferase-I (43). Therefore
nutrients or hormones which affect malonyl-CoA may ultimately impact long chain fatty
acyl oxidation or camitine metabolism. Kilocalorie and protein intake impact malonyl-
CoA concentrations. As kilocalorie intake decreases so do malonyl-CoA concentrations,
which in turn causes the inhibitory effect of malonyl-CoA to be removed. The kilocalorie
and protein requirements for hemodialysis patients are estimated to be between 30 and 35
kilocalories per kilogram and 1.2 to 1.4 grams protein per kilogram for those patients
with a normal body weight (3).

I. Kilocalorie, Protein, Normalized Protein Catabolic Rate and Blood Urea Nitrogen
Kilocalorie (kcal) and protein consumption are important to assess associations with
plasma total, free, acylcarnitine and acyl—to-free camitine ratio. Kilocalorie intake can
impact the insulin to glucagon ratio, which can affect the inhibitory action of malonyl-
CoA on camitine palmitoyl transferase, the enzyme responsible for combining the long
chain fatty acyl and camitine (61). Protein intake will be examined through the
calculation of the normalized protein catabolic rate. When the normalized protein
catabolic rate is calculated interdialytic rise in blood urea nitrogen, time between dialysis
treatments, and urine urea nitrogen are used to determine the rate of protein turnover in
the body (3). This measure can be an indicator for adequacy of protein consumption.

Blood urea nitrogen is a laboratory measure used in renal failure patients to monitor the

29

serum uremic status. The accepted normal ranges for a patient with renal failure are 60 to
100 mg/dl (3). A high blood urea nitrogen value might indicate a number of situations,
including a high intake of total or low biological value protein, a gastrointestinal bleed or
inadequate dialysis. High protein foods are the primary sources of carnitine. Therefore, a
diet with _>_ 1g protein/kg body weight in total protein could potentially have more

camitine than a diet with S 1 g protein/kg body weight.

II. Nutritional Status

Patients with CKD have an increased risk for protein-energy malnutrition. An
inexpensive, noninvasive method of determining nutritional status is subjective global
assessment. The subjective global assessment is a series of questions about appetite,
bowel functions (e. g. constipation, diarrhea), daily activities, and weight changes
combined with a brief physical examination. Each question and the physical examination
are answered on a seven-point scale. The reliability and validity of this test have been
shown in studies with a small sample size (62-64). Speciﬁcally, in hemodialysis patients
subjective global assessment has been positively correlated with albumin, pre-albumin,
body mass index and other well established indicators of morbidity and mortality (62;64-

66).

2) Free Carnitine
Between ten and seventy percent of hemodialysis patients have been reported to have
some type of malnutrition which might be accompanied by muscle mass loss given that

the primary site for camitine storage in the body is skeletal muscle (3). The diet is the

30

 

primary source of camitine, especially high protein foods, hemodialysis patients, who are
not consuming sufﬁcient kilocalories and protein to maintain skeletal muscle, may not be
consuming sufﬁcient free camitine to replace the reported camitine lost in the dialysate.
Hence, sub-optimal plasma concentrations of carnitine might be a serious consequence of
the combination of these two factors. Borah eta l (67) demonstrated that hemodialysis
patients who did not consume _>_ 1.0 grams of protein per kilogram could not maintain

positive nitrogen balance.

Free camitine is lost at a similar rate to acylcarnitine into the dialysate, compared to
healthy patients. Acylcarnitine is lost at a greater rate (54). Therefore, patients who
receive longer (greater than three hours) dialysis for each treatment and have received it
for a longer duration (in months or years) may have a greater potential for free camitine

loss.

3) Carnitine Palmitoyl Transferase—I

Carnitine palmitoyl transferase-I may be affected, as previously mentioned, by malonyl-
CoA and free camitine concentrations. Additionally, long chain fatty acyl-CoA
availability and/or free fatty acids may affect camitine palmitoyl transferase-I by
stimulating and inhibiting the enzyme (43). There has been some evidence to suggest
high serum calcium ions due to increased concentrations of parathyroid hormone may

interfere with camitine palmitoyl transferase-I (68) as well.

31

 

 

 

I. Para!
Chronic
(active \
h}'dFOX}'(
dihydrox
including
demonstr:
chain fart)
an effect 0
the long cl
“”35 hyped
failure exis
resulted in 1'
Calcium in t
Micrase 1
Calcium Chm

When Calcium

4') 4051mm“
Die acl’lcarnjl

Hoppel er a] (4

”Sand“)

b" r .

I. Parathyroid Hormone

Chronic Renal Failure patients have low concentrations of l,25—dihydroxycholecalciferol
(active vitamin D) because the ﬁnal step of activation (hydroxylation of 25-
hydroxycholecalciferol) occurs in the kidney. Low concentrations of 1,25-
dihydroxycholecalciferol can trigger a number of events leading to hyperparathyroidism,
including low serum calcium concentrations. In 1987, Smogorzewski et al. (69)
demonstrated that chronic renal failure is associated with impaired oxidation of long
chain fatty acids by skeletal muscle possibly due the lack of availability of carnitine or to
an effect on the enzyme, camitine palmitoyl transferase, responsible for the formation of
the long chain fatty acylcarnitine complex. In a later study by the same scientists (68), it
was hypothesized that a connection between parathyroid hormone and chronic renal
failure existed. This hypothesis posits that elevated parathyroid hormone concentrations
resulted in increased calcium ions in the skeletal muscle. These high concentrations of
calcium in the molecule may interfere with the action of the camitine palmitoyl
transferase l and thus affect long chain fatty acyl oxidation. Pema et al. (68) used a
calcium channel blocker and found a positive effect on camitine palmitoyl transferase,

when calcium concentrations in the skeletal muscle were controlled.

4) Acylcarnitine

The acylcarnitine concentrations may be elevated or altered in hemodialysis patients.
Hoppel et a1 (49) found an increase in the various types (e. g. acylcarnitine with 14
carbons and 10 carbons) of acylcarnitines that was not found in healthy controls. In

healthy controls ninety-ﬁve percent of the acylcarnitines were acetylcamitine compared

32

 

 

to Chron
and thes

COA del

Conseqt
Clinical 1
anemia, 1

cardiac dr

to chronic renal failure patients, who had a wide range of medium chain acylcarnitines
and these differences have been hypothesized by Hoppel et al to be caused by an acyl-

CoA dehydrogenase defect rather than by a camitine deﬁciency (49).

Consequences of Altered Carnitine Metabolism
Clinical manifestations of camitine metabolism may include but are not limited to:
anemia, ﬂuid retention and muscle cramping or muscle weakness, hypertriglyceridemia,

cardiac defects and poor perceived quality of life measurements.

Anemia

Anemia in the hemodialysis patient is usually monitored by measuring hematocrit and
hemoglobin concentrations. It is of special concern to the hemodialysis patient because as
the kidneys fail they produce less erythropoietin. Erythropoietin is a glycoprotein whose
function is to stimulate the division and differentiation of committed erythroid
progenitors in the bone marrow. Therefore, a large percentage of hemodialysis patients
receive synthetic erythropoietin to replace what is not being made. Epogen® is the

Amgen Inc. trademark for epotin alpha, which is the proper name for recombinant human

erythropoietin.

Long chain fatty acyls are hypothesized to inhibit Na/K ATPase activity of the red blood
cell membrane. Labonia et a1 (51) showed that treating 8 hemodialysis patients with oral
(l gram/day) and intravenous (1 gram 3 times per week post-dialysis treatment) L-

camitine increased the Na/K ATPase activity (p<0.05). Matsumura et al (52) showed low

33

 

plasma
associa
patients
microci
receiver
(511. Tr
camitine
enthroc
patients
[700111115 1

When [1".

Muscle

Milnnito
lnhibitin
osmo 1 a1 1
[Bed dur
imSTdia}
that Don

’ .1 .
SQUUQTI

plasma total (r = -0.56, p<0.01) and acylcarnitine (r = -0.58, p<0.01) concentrations were
associated with accelerated erythrocyte osmotic fragility in twenty-six hemodialysis
patients. Nikolaos et a1 (70) reported a signiﬁcant reduction in deformability (inﬂuences
microcirculation, tissue oxygen delivery and life span) of 15 hemodialysis patients who
received 30 mg intravenous L-carnitine/kg/dialysis treatment for 3 months. Labonia et a1.
(51), Trovato et al. (71) and Nikolaos et al (70) showed increases in hematocrit with
camitine treatment. The mechanism for this is thought to be the stabilization of the
erythrocyte membrane by facilitation of lipid uptake. Treatment of 31 adult hemodialysis
patients with 1 gram L-carnitine intravenously at the end of each dialysis treatment for 6

months resulted in decreases in Epogen® doses (p<0.05) during the treatment period.

When the L-camitine treatment stopped the Epogen® doses increased (72).

Muscle Function and Fluid Retention

Mannitol is a diuretic which increases the osmotic pressure of the glomerular ﬁltrate,
inhibiting tubular reabsorption of water and electrolytes, and elevates blood plasma
osmolality, resulting in enhanced water ﬂow into the extracellular ﬂuid (73). Mannitol is
used during some hemodialysis treatments when a hemodialysis patient has a large
interdialytic weight gain to aid in removing excess ﬂuid without causing the cramping
that normally occurs when large amounts of ﬂuid are removed(73). Hypertonic saline
solution can also be used for similar effect in hemodialysis patients. L-camitine treatment
has been postulated to aid in decreasing muscle cramping from ﬂuid removal.
Bellinghieri et al (59) was able to report a decrease in the number of patient reported

cramps during dialysis (p<0.05) when 2 grams/day of oral camitine treatment was used in

34

 

l-l hemr
nor deta

L—carnit

A study .
patients.

atrophy (
different

dial} sis s
reeeived '
Week [Tea
ﬁbers, su;
“We test:
GUI of 5, z
ShOXKed if
camitine (

IaCk of in:

In 1998. S
Realm—635‘
treatment.

R'mpltvtmi

14 hemodialysis patients for 60 days. This data, although interesting is not quantitative
nor detailed. Therefore it is difﬁcult to determine if the observed effect is attributable to

L-camitine.

A study on the effect of L-carnitine on type I and Ila skeletal muscles in hemodialysis
patients, demonstrated that at baseline, all of the patients had some type of muscle
atrophy (60). The patients were separated into three groups and each group received a
different treatment regimen. The ﬁrst group had 0.0725 mM/L of carnitine added to the
dialysis solution, the second group received two grams L-carnitine orally and the third
received two grams intravenously at the end of each dialysis session. After a twenty-four
week treatment period there was a seven percent increase in the diameter of type I and Ila
ﬁbers, suggesting an increase in muscle strength. When muscle strength, pain and cramps
were tested, muscle strength improved in 7 out of 16 patients, muscle pain regressed in 2
out of 5, and muscle cramps decreased in 6 out of 10 patients (60). All three groups
showed improvements in muscle function. It was hypothesized that patients with higher
camitine concentrations would have a greater tolerance for exercise. A limitation was the

lack of information regarding the patients exercise patterns before and during the study.

In 1998, Sakurauchi et al. (74) treated thirty hemodialysis patients with muscular
weakness, fatigue or cramps/aches with intravenous L-carnitine. After twelve weeks of
treatment, sixty-six percent of the patients had at least some improvement in muscular

symptoms. This data was patient reported and no detailed information on in cramp

35

 

intenslt)

Hipertr
Long Chg
acids Can
fall) acid
accumul'd
hemodialj
a cross-m
one hundr
concentral
triglycerid
concentrat

('75). In [ht

forty mg'l'

A 1998 stu
“Eight intr;
concentratir
hgher 3.1710
Fl’ﬂlﬁﬁnor.

int -
he EllSal

intensity nor number of cramps per treatment was given. Therefore, the study ﬁndings

need to be interpreted cautiously.

Hypertriglyceridemia

Long chain fatty acids have two main pathways for metabolism. The long chain fatty
acids can use camitine to enter the mitochondria and begin B-oxidation or the long chain
fatty acids can be formed into triglycerides. If camitine metabolism is abnormal, an
accumulation of triglycerides may occur. Vacha et al. (75) treated twenty-nine chronic
hemodialysis patients with twenty milligrams per kilogram of intravenous L—carnitine in
a cross-over design study. Each group of patients received both placebo and treatment for
one hundred and twenty days. Patients with high-density lipoprotein cholesterol
concentrations below forty milligrams per one hundred milliliters had signiﬁcant
triglyceride lowering effects during the treatment period. Mean triglyceride
concentrations werereduced from 365: 13 mg/ 100 ml to 181:6 mg/ 100 ml (p<0.001)
(75). In those patients with the high-density lipoprotein cholesterol values greater than

forty mg/100 ml no signiﬁcant change was seen with L-camitine treatment.

A 1998 study by Elisaf et al (76), on 28 dialysis patients treated with 5 mg/kg body
weight intravenous L-camitine over 6 months, reported signiﬁcantly lower triglyceride
concentrations (r = 0.34, p<0.05). The results from this study might have been stronger if
higher amounts of L—camitine were used (i.e. 20 mg/kg body weight intravenously).
Furthermore, high density lipoprotein cholesterol concentrations were not controlled for

in the Elisaf et al (76) study which may be a limitation. Stefanutti et al (77) examined L-

36.

 

 

carnitir
values

signiﬁc
l g 3 tir
270:36

normal 1

C ardios
Dialysis
Cause of
cOrltr'ibut
Pmbabl}
hl‘Perten:
The hem
“W car
metal50115

Carib-0m V

camitine treatment effects on a population of hemodialysis patients with triglyceride
values between 170 and 260 mg/dl and between 220-260 mg/dl. These patients showed
signiﬁcant decreases in serum triglyceride concentrations after 30 days of treatment with
l g 3 times a day of oral L-carnitine (p50.02). The mean triglyceride values dropped from
270:36.1 mg/dl to 249:52.1 mg/dl (77), which is clinically signiﬁcant, but not to within

normal concentrations ($220 mg/dl).

Cardiovascular Disease

Dialysis treatment has improved to a point where the treatment itself is not the main
cause of mortality. Rather, secondary causes such as cardiac disease are major
contributors to mortality in this population. Cardiac disease in the renal patient is
probably multifactoral. Hemodialysis patients have symptoms such as hyperlipidemia,
hypertension and left ventricular hypertrophy, which all contribute to cardiac disease.
The heart muscle uses non-esteriﬁed free fatty acyls as an energy source and therefore
needs camitine to transport fatty acyls beyond hexoate (78). Defects in camitine
metabolism have been linked to cardiac disorders such as ischemic injury and

cardiomyopathy.

Quality of Life

As treatment methods improve and the length of life on dialysis increases quantiﬁable
measures of quality of life have become increasingly important in determining risk of
mortality. Measurement of patients’ perceived quality of life is therefore an important

outcome assessment in CKDl. Many assessment tools are currently being used for CKD

37

 

 

patients. 1
Powers Q1
patients. 11
on the pati

(biochemic

One of the
been Show
plasma con
(4172;83-8
Well being c
months and
Disease QUt
POSitit'e imp
measured m
being 0n 1y 0'

of the St UdV '

patients. Heacock et al (79) studied perceived quality of life using The F errans and
Powers Quality of Life Index for associations with nutritional status in hemodialysis
patients. In this study the researchers began to investigate the effect of nutritional status
on the patient’s overall quality of life (79). It showed that the more nutritionally sound

(biochemical and anthropometric), the better the quality of life.

One of the better known tools, the Medical Outcomes Study Short-Form-36 (SF 36), has
been shown by Kutner and others to have good reliability and validity (80-82). Higher
plasma concentrations of camitine have been associated with better quality of life
(42;72;83-85). Sloan et al (86) demonstrated that an increase in the physical and general
well being components of the SF36 after oral treatment with 1 g L-carnitine for 1 ‘/2
months and Brass (83) reported an improvement in the fatigue portion of the Kidney
Disease Questionnaire. Both of these ﬁndings suggest that L-carnitine might have a
positive impact on perceived quality of life by the patient. However, Sloan et al (86)
measured the SF36 every month and the improvement in the physical and general well
being only occurred after the ﬁrst month of treatment but disappeared for the remainder
of the study. The lack of subsequent signiﬁcant ﬁndings may be attributed to the
methodology used in the study. The L-carnitine was administered orally. When L-
camitine is administered in this manner the gastrointestinal tract breaks the undigested
portions down by bacterial degradation into y-butrobetaine or trimethylamine, which are
typically excreted in the urine (19;29;30). Patients with stage 5 CKD do not produce

signiﬁcant quantities of urine and therefore cannot excrete these by-products. Therefore,

38

 

the serur

any bene

The SF3<
health-rel
The dome
activity (;
componer
composite
0Plimal ir.

interx'emit

the serum concentrations of the products may increase to a detrimental level, and negate

any beneﬁcial effects the initial treatment of L-carnitine may have had.

The SF 36 has 8 different sub-scales, known as domains, related to self-perception of
health-related quality of life (Table 2.4). The range for each of the 8 domains is 0 to 100.
The domains may be examined individually, more speciﬁcally those related to physical
activity (physical function, role-functioning, general health and vitality) and the physical
component score (Figure 2.1). It is beneﬁcial to assess the individual scores and the
composite scores to more speciﬁcally determine which areas the patient feels are sub-
optimal in their life. Be determining the speciﬁc are which is low, subsequent

interventions can be determined.

39

Table 2.4

Medical Outcomes Short Form 36 Domains

 

Scale

Low Score Indicators

 

Physical functioning

Limitations in physical activity, inc. bathing &

dressing

 

Role-Physical

Limited work due to health

 

ole Emotional

Emotions, limited daily function, and work

 

 

Social Function

Physical & emotional signs/symptoms limit

social activities

 

 

 

 

 

Bodily Pain Severe limiting pain

Mental Health Feels nervous & depressed all the time
Vitality Feels tired & worn out all the time
General health Health much worse than previous year

 

 

40

 

Ph}'Slr
Role-l

Bodil}

Genera
Vitalit;
Social

Role-E;

Mental

Figure 2.2

Carnitine
L‘Cfll’l'titin
Catheter 31‘
Camitirie h
inexpensh
becn Show
may effect.

flltrctions a

Composition of Composite Scale Scores

Physical Functioning

 

Role-Physical Physical Health
Bodily Pain
General health
Vitality
Social functioning * Mental Health

 

 

 

 

Role-Emotional

Mental Health

Figure 2.]. Domains included in the physical and mental composite scores

Carnitine Supplementation

L-camitine can be given orally on a three times per day basis or intravenously via a
catheter after dialysis treatment 3 times a week. The two methods of administering L-
camitine have potential positive and negative effects. First, oral administration can be
inexpensive and effective. Daily, oral doses of L—carnitine of between 2 and 6 grams have
been shown to have a systematic bioavailability on average of 18% (19). Oral L—carnitine
may effectively carry camitine to the liver during a “ﬁrst-pass effect”. Because camitine
functions as a carrier protein for long chain fatty acids, into the mitochondria for B-
oxidation, the delivery of carnitine to the liver may facilitate the efﬁciency of oral L-
carnitine in the reduction of serum triglycerides, by increasing the number of long chain

fatty acyls, which cross the mitochondrial membrane and enter B-oxidation (42).

41

 

 

L'nfortu
interfere
include 1
gastroint
C omplia
the numt
are on m;
hemodial
or profou
diabetes;

(.87),

Comer-Se
treating“t
Carnitine

admmlSle
(42:70-7:
tram is co
by [he Pat
times Der
researcher
TDCreaSe l
11”:

e acﬁ'il‘lt

Unfortunately, the following potentially negative attributes of oral L-carnitine could
interfere with consistent delivery of L-carnitine to the hemodialysis patients. These could
include patient compliance, and compromised absorption through a malfunctioning
gastrointestinal tract. The researcher or clinician cannot control patient compliance.
Compliance issues may include the number of tablets the patient chooses to consume and
the number of times per day they choose to take the tablets. Many hemodialysis patients
are on multiple oral drugs, and they could become overwhelmed. Furthermore, 45% of
hemodialysis patients have diabetes (5) and 20-3 0% of these patients may have slightly
or profoundly impaired gastrointestinal tracts due to gastroparesis (a complication of
diabetes) so that the rate or amount of drug absorbed could vary from patient to patient

(87).

Conversely, a dialysis nurse gives intravenous L-Camitine at the end of the dialysis
treatment, and therefore compliance is no longer an issue. Although the intravenous
camitine is only administered 3 times per week versus daily, intravenous L-carnitine
administered in this way has been shown to have positive metabolic effects in studies
(42;70-72;76;88). Because it is given directly into the blood stream, the gastrointestinal
tract is completely bypassed, and consequently the exact amount of L-carnitine received
by the patient is known. In 1998, Caruso et a1 (72) used 1 gm of intravenous L—carnitine 3
times per week in 31 hemodialysis patients with various etiologies for 6 months. These
researchers were able to show a decrease in Epogen® doses (to improve anemia) and an
increase in total and free plasma camitine concentrations. Caruso et a1 (72) did not report
the acyl-to—free plasma camitine ratio, nor did they report whether the acyl-to-free plasma

Carnitine ratio decreased after supplementation with L-carnitine. Elisaf et al., (76) used 5

42

 

 

mg per 1:1
hemodialy
triglycerid
showed an

camitine. l

The Food 2
for the tree
has been ap

effects repo

mg per kg of body weight of intravenous L-carnitine 3 times per week, on 28
hemodialysis patients, for 6 months and induced a signiﬁcant decrease in serum
triglycerides of those patients who initially had above normal triglycerides. They also
showed an elevation in plasma free and total camitine after supplementation with L-

carnitine. Elisaf et a1 (76) did not report any acyl-to-free camitine ratios.

The Food and Drug Administration has approved both oral and intravenous L-camitine
for the treatment of both camitine abberations. However, only intravenous L-carnitine
has been approved for use in hemodialysis patients. It is well tolerated with no major side

effects reported (13;89-91).

43

 

10.

ll.

12.

13.

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Ertle .1
and lie

 

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free L-t‘
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Pharms

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2000;491:

l3.

 

 

 

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1996:1515.

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the rllltritit:
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mmelhjvlam

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45

 

27. E

 

RE

KC

5‘.
a)

[HUB

1%..

..).

S. DC Sh.

.3. 6.
a). s D

27.

28.

29.

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46

 

40.

41.

48.

(It
I-J

(l!
O)

C2
C2

Chi

40.

41.

42.

43.

44.

45.

46.

47.

48.

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47

 

 

 

 

U!
I _

55
56.
57.
58. r
r
l-
59. E
St
he
60. G;
ca.
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mir
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001111
2000
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“chm,

54.

55.

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48

 

 

 

67

68.

69.

78.

79.

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16\
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on .
Ster
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the 1]
10¢}

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67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

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Anemia in Aged Hemodialysis Patients Treated with Recombinant Human
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2.

Sakurauchi Y, Matsumoto Y, Shinzato T et al. Effects of L-carnitine
supplementation on muscular symptoms in hemodialyzed patients. Am.J.Kidney
Dis. 1998;32:258-64.

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treatment on hypertriglyceridemia in hemodialysis patients: decisive role of low
levels of high-density lipoprotein-cholesterol. Am J Clin.Nutr. 1983;38:532-40.

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on lipid parameters in hemodialysis patients. Am J Nephrol. 1998;18:416-21.

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carnitine on plasma lipoprotein fatty acids pattern in patients with primary
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Relationship Between Nutritional Status and Quality of Life in Chronic
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49

 

 

 

80.

81.

82.

84.

85.

86.

87.

88.

89.

 

 

 

L-cami'
capacit;

Zamba .
haemod
199495

 

Goral 8,
disease. .

Sloan R5
hemodia
1998:3121

De SCllUt
bCNEQn

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Labonia
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' Bartel L1

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' Konig 8.

Urinary C.‘

80.

81.

82.

83.

84.

85.

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89.

90.

91.

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L-carnitine increases plasma camitine, reduces fatigue, and may preserve exercise
capacity in hemodialysis patients. Am.J.Kidney Dis. 2001;37:1018-28.

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haemodialysis patients during ultraﬁltration. Nephrol.Dia1.Transplant.
1994;9z587-8.

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disease. J .Ren Nutr. 1998;8z1 18-21.

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between gastric emptying and clinical and biochemical factors in chronic
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Labonia WD. L-carnitine effects on anemia in hemodialyzed patients treated with
erythropoietin. Am.J.Kidney Dis. 1995;26:757-64.

Bohmer T, Bergrem H, Eiklid K. Carnitine deﬁciency induced during intermittent
haemodialysis for renal failure. Lancet 1978;12126-8.

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Konig B, McKaigney E, Conteh S, Ross B. Effect of a lipid load on blood and
urinary carnitine in man. Clin.Chim.Acta 1978;88:121-5.

50

 

 

This stud)
study and
Phases 1 a
(DC L1. in
Columbus
following

1. Li;

M

f»)

Cr
3. T1
4. 7}

Patients“

rtiadllthe

Sign the c.

Chapter 3
Overall Metl_|_o_(_l_s_
This study was conducted in two phases; phase I was a cross sectional, observational
study and phase II was a randomized, double-blind, placebo controlled, clinical trial.

Phases I and II were conducted at facilities owned by the Dialysis Center of Lincoln

(DCL), in Nebraska. These sites were Lincoln, NE (2 sites), Beatrice, NE (1 site) and

 

Columbus, NE (1 site). Approvals to conduct the studies were obtained from the
following (Appendix A):

1. University Committee on Research Involving Human Subjects (U CRIHS) at

Michigan State University, East Lansing, MI

2. Community Institutional Review Board in Lincoln, NE

3. The Administrator at DCL

4. The Medical Director of DCL
Patients who participated in either phase had an informed consent read to them and/or
read it themselves, were given the opportunity to ask questions, and were then asked to

sign the consent (Appendix B) if they agreed to participate in the study.

Phase 1 sample
The following study inclusion criteria were used:
0 patients _>_ 18 years of age
0 receiving a minimum of four hours of bicarbonate hemodialysis treatments, at
least three times per week

0 have received dialysis treatment for a minimum of one year.

51

 

Patients “er"

 

0 Cum“

0 had 3 ~‘
0 had a d
pregna'
lhese exclusit‘"
alter camitine r

Therefore. path

 

increased doses
Patients who ha
disease may cor.
were investigate

have altered ear

Phase I Data C
Data for phase 1
renal unit at DC

Table 3.1 outlin

Patients were excluded if they met the following criteria:

0 currently or previously treated (within the last two months) with L-carnitine,

0 had a severe blood loss within the past week prior to commencement of the study,

0 had a disease affecting skeletal muscle function, severe liver disease and/or were

pregnant.

These exclusion criteria were selected to minimize any confounding issues, which would
alter camitine metabolism beyond the effect of dialysis treatment or dietary intake.
Therefore, patients with severe blood loss were excluded because they are typically given
increased doses of erythropoietin and iron supplements to help increase blood volume.
Patients who have diseases affecting skeletal muscle function were excluded because the
disease may confound the correlation between camitine and the exercise parameters that
were investigated. Additionally, patients with severe liver disease and/or pregnancy may

have altered camitine metabolism for reasons other than uremia.

Phase I Data Collection
Data for phase I were collected by the researcher and a dietitian (MS, RD) working in the
renal unit at DCL. The data were collected during the ﬁrst week of January in 2000.

Table 3.] outlines all the data that were collected.

52

 

Table 3.]

Variables Collected in Phase I

 

 

 

 

 

treatment

treatment time

Variable Format Collected Units
Total, free, short and long Plasma, pre-dialysis pmol/L
chain acylcarnitine

Triglycerides Serum, pre-dialysis (all) mg/dl
Blood urea nitrogen mg/dl
Parathyroid hormone pg/ml
Hematocrit %
Weight status Digital Scale, post—dialysis Kg
Treatment time/dialysis Medical record, actual Minutes

 

 

 

 

 

 

Erythropoietin dose Medical record Mean unit/month
Mannitol dose Medical record Mean dose/month
Medication use: aspirin, Medical record Currently taking: yes/no
lipid lowering

Time from initial dialysis Medical record Years

treatment

Age Medical record Years

Exerciser Medical record Yes/no

Perceived dyspnea Scale l-20

Perceived exertion Scale 1-20

 

Medical Outcome Short
Fonn-36

Perceived Quality of Life:

SF 36 Questionnaire

l-100, 8 domains

 

 

 

 

 

Diabetes Mellitus Medical Record Presence thereof indicated

Cardiovascular disease by yes/no for each co-

Left ventricular morbidity

hypertrophy

Left arterial dilation

Dietary protein and 24 hour and typical day Mean g/day and kcal/day,

kilocalorie intake recall reported as total and per kg
body weight

 

Dependent Variable Definition

The dependent variable for this study was plasma camitine (total, free, short and long

chain acylcarnitine). A Registered Nurse (RN) or the phlebotomist at DCL drew the

participating patient’s blood on a Wednesday or Thursday of the ﬁrst week of the month

53

 

 

in accordance with the patient’s routine monthly blood draw. No patient had been without
dialysis prior to the blood draw for longer than 48 hours. A 1 ml aliquot of plasma from
the blood sample was collected in a separator tube and frozen at ~80° C until it could be
analyzed. The plasma was analyzed for free, short and long chain acylcarnitine using the
radioenzymatic assay as outlined by Brass and Hoppel (1 ). Total camitine was calculated

as the sum of the free, short, and long chain acylcarnitine values.

Independent Variable Definition

Weight and height, all medication use and dose, age, exercise patterns and tolerance,
treatment information, and presence of co-morbidities data were collected from patients’
medical records (some paper and some computerized). Weight and height were used to
calculate body mass index and body weight was used to express kilocalorie (kcal) and

protein intake per kilogram (kg).

Information on medication use was collected speciﬁcally related to lipid lowering
medications, due to their effect on serum triglycerides and their potential to confound
plasma carnitine and serum triglycerides concentrations. Additionally, consideration was
given to medications that might affect cellular acetyl Co-A concentrations such as
aspirin. Synthetic erythropoietin dosage was collected based on previous literature, which
suggested an association between the prescribed dose and plasma camitine

concentrations (2).

54

 

Data on co-morbidities such as diabetes, cardiovascular disease, and hypertension were
collected not only to describe the sample analyzed, but also to provide information on
what disease states might or might not affect camitine status in the sample. The data on
leﬁ ventricular hypertrophy (LVH) and left arterial dilation (LAD) were obtained from
the medical chart based on ﬁndings from echocardiography conducted no more than 6
months previously. Not all patients had this information. Therefore, data from only a

subset of patients were analyzed for LVH and LAD.

Laboratory data were extracted from routine monthly laboratory proﬁles of each patient.
These serum markers were obtained at the same time the camitine samples were drawn.
The serum was analyzed by Spectra Laboratories (Fremont, California). Biomarkers such
as serum blood urea nitrogen and serum albumin were determined to assess nutritional
status, as well as serum triglycerides (lipid metabolism), and hematocrit (iron status).
Parathyroid hormone concentrations were assessed because previous literature (3-5)

indicated associations between these variables and plasma camitine concentrations.

Patients who are dialyzed at DCL are given a “prescription” to exercise when they
initially begin their treatment at the facility. This prescription is a doctor’s order for an
exercise physiologist to assess a patient’s exercise capacity and make an exercise
recommendation. For the majority of patients, this entails riding a stationary bike while
receiving dialysis treatment. The stationary bike is attached by chains to the lower part of
the dialysis chair. On the day of data collection, patients who biked during their dialysis

treatment were considered “exercisers”. The rate of perceived exertion and perceived

55

 

dyspnea were used to deﬁne exercise tolerance (6). Both of these scales use a 1 to 20
rating with 20 as the most exertion or dyspnea and 1 the least. Patients who bike do so
almost every dialysis session. Each time a patient bikes, the rate of perceived exertion

and perceived dyspnea as reported by the patient are recorded into the computerized

record.

Measurement of patients’ perceived quality of life is an important outcome assessment
tool. The SF36 has eight different scales related to self-perception of health-related
quality of life. The range for each of the eight scales is 0 to 100. From the 8 domains,
weighted means are calculated to form 2 composite scores: physical and mental. The
SF36 was administered and scored by trained social workers from DCL, and the most
recent results were used (Appendix C). If an SF 36 had not been done in the past six
months, it was done on the day of blood draw by one of the social workers. Patients at

DCL are given the SF36 biannually, and are therefore familiar with the tool.

During the week of the blood draw, two dietary recalls (24 hour and usual day) were

conducted on each patient participating in this study by either the dietitian at DCL or the
investigator. The completed recalls were analyzed for average kcal and protein content
and presented as both a total value and as the total divided by the patients’ weight in kg.
Patients of differing weight have differing kcal and protein requirements. Therefore, the
total intakes were divided by weight to standardize the intake between all patients.

Analysis of the dietary recalls was done using the software program Nutritionist V

produced by N2 Computing, 1999.

56

Phase I Data Analyses

Descriptive statistics were done on both the independent and the dependent variables.
The continuous independent variables were assessed for associations with the dependent
variables: total, free and acyl (short chain acyl + long chain acyl) plasma camitine
concentrations using the Pearson’s Correlation Coefﬁcient unless the variables were

skewed, then Spearman’s Correlation Coefﬁcient was used. Each variable was plotted to

 

determine whether it was normally distributed or not. Patients were separated based on
gender, mannitol use, presence of diabetes (type 1 or type2) for analysis. Independent t-
test analyses were done for the binary variables to determine associations with the
dependent variables and the continuous independent variables. Independent t-test
analyses were also used to compare mean protein and energy intake between patients
treated or not treated with mannitol. To determine whether any of the independent
variables were predictors (when controlling for potential confounders) of the dependent
variables, multiple linear regression was used. Since the purpose of this study was to
identify important variables for the next phase of the project. correlations between
outcome variables typically used in dialysis care and camitine concentrations were

deemed signiﬁcant using a p value of less than 0.05.

Phase H Sample Recruitment
This was a prospective, randomized, double blind, clinical research study. The inclusion
criteria for phase II were:

patients 2 18 years of age,

57

0 receiving a minimum of three hours of bicarbonate, low acetate hemodialysis

treatments, three times per week, for at least one year,
Based on phase I study ﬁndings, patients admitted into the study needed to have two or
more risk factors for a compromised plasma camitine status (Table 3.2).

Table 3.2

Risk Factor Criteria

 

1. 65 years old

 

2. 1 year duration of treatment

 

3. female

 

4. use of aspirin and/or mannitol

 

5. presence of type 2 diabetes mellitus, left atrial dilation and/or

left ventricular hypertrophy

 

 

 

The exclusion criteria below were established on the same basis as in phase I, to

minimize confounders:

0 current or previous treatment (within the last two months) with L-carnitine,

a severe blood loss within the past week prior to commencement of the study,

0 disease affecting skeletal muscle ﬁinction,

a severe liver disease,

0 pregnancy
The Federal Drug Administration has approved intravenous L—carnitine for use in the

hemodialysis population if plasma free camitine concentrations are less than 40 nmol/L.

Therefore, when a patient’s plasma free camitine concentration was greater than 40

58

umol/L the patient was excluded from the study. Patients included into this study signed

a consent form and were randomized into control and treatment groups.

Phase 11 Data Collection

The variables and data collection points are outlined in Table 3.3. The researcher and a
senior level nutrition student from Michigan State University collected data At each data

collection time, they ﬂew to Lincoln, NE and conducted patient recruitment, patient

 

interviews and physical examinations, medical record reviews and arranged for blood
collection and transport to the laboratory of Dr. Alan Davis in Grand Rapids, MI. For any

patients who were not available at the time of their visit, the renal dietitians who worked

at DCL facilities collected the missing data.

Variable Definition

Patients had their blood drawn for plasma camitine analysis during the study on the ﬁrst
week of the baseline month, the 12‘h week and the 24th week in accordance with routine
monthly blood draws. These periods for data collection were selected after an extensive
review of the literature revealed these to be the most common data collection points. A
renal registered nurse or dialysis technician collected a 1 ml aliquot of plasma from the
blood sample in a heparinized tube. The blood sample was spun, the plasma was
transferred via a pipette into a new test tube, and then the plasma was frozen to -80
degrees. The plasma was sent packed in a sealed polystyrene container on dry ice to the
Spectrum Laboratory in Grand Rapids, M1, for determination of total and free camitine

using the radioenzymatic assay outlined by Brass and Hoppel (1 ;7). In this laboratory,

59

when the samples of plasma were thawed for analysis. A portion was separated, refroze,
and sent to Dr. Charles Hoppel’s laboratory at the Louis Stokes Veterans Administration
Medical Center in Cleveland, OH for analysis of both acid soluble and insoluble
acylcarnitines using high performance liquid chromatography with mass

spectrometry (8).

60

Table 3.3

Data Collection Format

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Baseline Montth 12 weeks 24 weeks
Carnitine:
'l'olttl X X X
l’rec X X X
Acyl (FA Lengths) X X X
A/F ratio X X X
Real/protein intake/nPCR X X X
Subjective (ilobal X X X
Assessment. MAMC
Short-Form-36 X X
BUN, Hct, Hgb, TG, Alb, X X
PTH“. HDI.‘
Mean Epogen® dose X
IDWG, X X X
mannitol/hypertonic
Dialyzer type, Aspirin use X X

 

 

 

 

 

Bold variables = primary clinic outcomes
Abbreviations: A/F ratio=acyl to free plasma camitine ratio, Kcal=kilocalories,
nPCR=normalized protein catabolic rate, MAMC=mid~arm muscle circumference,
BUN==serum blood urea nitrogen, Hct=hematocrit, Hgb=serum hemoglobin, TG=serum
triglycerides, Alb=serum albumin, PTH=serum parathyroid hormone, HDL=high density
lipoproteins, IDWG=interdialytic weight gain, ASA=aspirin use

61

Two dietary recalls were collected using the multiple pass method (9), a 24 hour recall

and a typical day 24 hour recall. The Multiple Pass Method of conducting a 24 hour

 

recall was developed by the United States Department of Agriculture for the National
Health and Nutrition Examination studies. This technique was used to gather recall
assessment data in phase 11 because of its increased accuracy (9). The recalls were
analyzed using Nutritionist Pro., produced by First Databank, 2002. The total kcal and
protein values ﬁ'om the recalls were averaged in order to account for individual

variations and presented as total kcals, total protein (g) or kcal/kg and protein g/kg

Normalized PCR is a calculated valued used to assess total body protein turnover and
dietary protein intake in hemodialysis patients. The following equation is used to
calculate nPCR. In this study, the association of nPCR and plasma camitine is examined

in conjunction with actual dietary protein intake.

PCR = 0.22 + 0.036 X interdialytic rise in BUN X 24
interdialytic interval in hours

To normalize the PCR: nPCR = PCR/weight (kg)

As discussed in the review of literature (Chapter 2), SGA is a nutritional assessment tool

recommended by the National Kidney Foundation’s Guidelines for practitioners in
dialysis (Kidney/Dialysis Outcome Quality Initiative). In phase II, the format for SGA
was changed. Instead of using the A, B, C method originally presented by Detsky et al
(10), as was done in phase I, the seven point SGA form designed by Linda McCann (11)
was used (Appendix C). This form allows for greater ﬂexibility in scoring. In small
validation studies, it has been shown to have higher correlations with nutritional markers
such as albumin and mid-arm muscle circumference (MAMC) (12:13). The SGA is a

subjective measure. Therefore, in order to obtain more objective data on body

62

composition, the MAMC (14) was used to assess lean body mass. Mid-arm muscle
circumference is calculated from the mid-arm-circumference and tricep skin fold
measurements. While tricep skin fold is an indicator of body fat, the MAMC is used as an
indicator of lean body mass by comparing the results to the ﬁndings reported by

NHANES. The MAMC is inexpensive and relatively simple to conduct.

The SF36 is a perceived quality of life assessment, which has eight domains used to

comprise either a mental or a physical composite score. Each domain was scored on a

 

scale of 0 to 100, with 100 being the highest perception of functioning. The domains are
physical functioning, role-physical, bodily pain, general health, mental health, role-
emotional, social functioning, and vitality. The SF-36 surveys were administered and
scored by the social workers at DCL at baseline and at week 24 of the study, (Appendix

C).

Data for laboratory parameters (blood urea nitrogen, serum triglycerides, serum
albumin, hematocrit and hemoglobin, high density lipoproteins and parathyroid

hormone) were taken from the monthly reports routinely collected by the facilities.

Erythropoietin doses, muscle cramping data, hypertonic saline or mannitol doses, and
pre- and post-dialysis weights were collected from the Nursing Flow Sheet recorded by
the dialysis technicians or nurses at each dialysis session (Appendix C). Disease

etiologies and co-morbidities, initial dialysis treatment data, treatment duration and

63

procedures, medications, height, age, gender, were collected from computerized medical

records at DCL.

In both phases, random data audits were conducted to ensure consistency and accuracy of
collection and database entry. For example, in every 5th or 6th patient a second SGA was
performed by one of the other data collectors. Results from the two assessments were
compared. If signiﬁcant variation occurred, a third assessment was conducted. This same
procedure was followed for MAMC and 24 hour recall data. Additionally, when data
were entered into a Microsoft Excel spreadsheet and SPSS software, random checks with

the original data were conducted for accuracy.

Phase II Intervention Procedures
Patients eligible to participate in the study were randomized into either the treatment or
the control group by a co-investigator who did not participate in the data collection
process. This investigator and the pharmacist at DCL were the only persons not blinded.
Those patients who were in the treatment group received 20mg Carnitor® per kg actual
body weight, post hemodialysis treatment, three times per week intravenously. The
camitine was administered to the patients in an infusion of 100ml of normal saline over
10 minutes by the dialysis center nursing staff. The patients in the control group received
an intravenous placebo of 100 ml normal saline over 10 minutes. The amount of carnitine
needed was calculated using the patients’ dry body weight determined at baseline of the
study. By diluting the camitine in normal saline and giving it in an inﬁrsion, the potential

odor of the drug was masked and blindness maintained. The treatment period was for 24

weeks. In accordance with UCRIHS at Michigan State University and Sigma Tau
Pharmaceuticals, Inc., the providers of L-carnitine, adverse events were monitored by the
nursing staff and reported by the researcher to the University Committee for Research in

Human Subjects and Sigma Tau Pharmaceuticals.

Phase 11 Data Analysis

These data were analyzed using SPSS version, 11.0, 2001. The change in clinical
outcomes from baseline to post-intervention for each group of patients was examined by
dependent t-tests and repeated measure analysis. Dependent t—tests can measure
differences in mean values between groups that are connected in some manner such as
one variable collected at two different time periods on the same person. Repeat measure
analysis allows all the data to be placed into the analysis and analyzed for time and group
effect. Associations between dependent and independent variables were determined by
Pearson’s correlations or Spearman’s correlations depending on the nature of the
variable. Some of the variables were analyzed for multivariate relationships with multiple
linear regressions to determine predictability of the independent variables for the

dependent variable.

65

 

10.

11.

12.

13.

Reference List

Brass EP, Hoppel CL. Carnitine metabolism in the fasting rat. J Biol.Chem.
1978;253:2688-93.

Caruso U, Umberto, Leone L, Cravotto E, and Nava D. Effects of L-carnitine on
Anemia in Aged Hemodialysis Patients Treated with Recombinant Human
Erythropoietin: A pilot study. Dialysis and Transplantation l998;27(8), 498-504.

Pema AF, Smogorzewski M, Massry SG. Verapamil reverses PTH- or CRF-
induced abnormal fatty acid oxidation in muscle. Kidney Int 1988;34:774-8.

Smogorzewski M, Piskorska G, Borum PR, Massry SG. Chronic renal failure,
parathyroid hormone and fatty acids oxidation in skeletal muscle. Kidney Int
1988;33:555-60.

Stefanutti C, Vivenzio A, Lucani G, Di Giacomo S, Lucani E. Effect of L—
camitine on plasma lipoprotein fatty acids pattern in patients with primary
hyperlipoproteinemia. Clin.Ter. 1998;149: 1 15—9.

O'connor PJ, Raglin J S, Morgan WP. Psychometric correlates of perception
during arm ergometry in males and females. Int J Sports Med. 1996;17:462-6.

Hoppel C. Determination of Carnitine. Techniques in Diagnostic Human
Biochemical Genetics: A Laboratory Mannual. Wiley-Liss, Inc. 1991:309-26.

Minkler, P., Ingalls, S, and Hoppel, C. Strategy for the isolation, derivatization,
chromatographic separation and detection of camitine and acylcarnitines.
publication in process.

Moshfegh AJ, Borrud L, Perloff B, et al. Improved Method for the 24-hour
Dietary Recall for Use in National Surveys. FASEB Journal 1999;13:A603
(abstr).

Detsky AS, McLaughlin JR, Baker JP et al. What is subjective global assessment
of nutritional status?. J PEN J Parenter.Enteral Nutr. 1987;11:8-13.

McCann L. Using subjective global assessment to identify malnutrition in the
ESRD patient. Nephrol.News Issues 1999;13:1 8-9.

Jones CH, Wolfenden RC, Wells LM. Is subjective global assessment a reliable
measure of nutritional status in hemodialysis? J Ren Nutr. 2004;14:26-30.

Visser R, Dekker FW, Boeschoten EW, Stevens P, Krediet RT. Reliability of the
7-point subjective global assessment scale in assessing nutritional status of
dialysis patients. Adv.Perit.Dial. 1999;15:222-5.

66

14. Clinical practice guidelines for nutrition in chronic renal failure. K/DOQI,
National Kidney Foundation. Am J Kidney Dis 2000;35:81-140.

67

Chapter 4
Phase I, Aim 1
Carnitine Concentrations and Clinical Outcome Parameters
In Chronic Hemodialysis Patients
Steiber AL, Weatherspoon LJ, Spry L, Davis AT. Clinical Nutrition (2004) 23, 27—34
Abstract

Carnitine metabolism and the therapeutic use of camitine has been a major area of
interest in dialysis patients. The purpose of this study was to determine whether any
correlations exist between camitine status and selected clinical parameters in
hemodialysis (HD) patients. This study was an observational study of data from patients
receiving HD at a Midwest dialysis center. The subjects (n=49) were 60: 16 (mean:SD)
years of age and 48% male. Fifteen percent of the subjects had type 1 diabetes mellitus
(DM), 29% had type2 DM, and 25% had left ventricular hypertrophy (LVH). The serum
free and total carnitine, and acylcarnitine concentrations were: 40.335118 nmol/L,
22.8:73 umol/L, and l7.5:5.9 nmol/L, respectively. The serum acylcarnitine to free
camitine ration (A/F) was 0.80:0.27. Serum blood urea nitrogen (BUN), parathyroid
hormone and ejection fraction were positively correlated and age and left atrial dilation
(cm) were negatively correlated with serum total camitine (p<0.05). BUN and hematocrit
were positively correlated (p<0.05) and age was negatively correlated with plasma free
camitine. Subjects who used mannitol or were male had signiﬁcantly higher
concentrations of both plasma free and total camitine, respectively (p<0.05). Subjects
using aspirin had lower concentrations of serum total carnitine (p<0.10). These results

suggest certain subgroups of patients may need to be targeted for further studies with

68

camitine replacement therapy, i.e. long-term patients, older patients, patients with leﬁ
ventricular hypertrophy and left atrial enlargement, females and patients on aspirin
therapy.

Introduction
Carnitine has been shown to be beneﬁcial in the treatment of uremic manifestations of
dialysis patients such as anemia, ﬂuid retention, muscle weakness, and poor quality of
life scores (1-9). Seventy-ﬁve percent of carnitine in the body is ingested and the other
25% is synthesized in the kidney, brain, and liver. In adults, the source of camitine is
primarily red meats and in infants, human milk (10). Although camitine is found in high
protein foods and hemodialysis patients have elevated protein needs (1 l), the patients are
often not able to consume sufﬁcient amounts of these high protein foods to meet their

needs due to poor appetites or aversions to meats caused by uremia.

The primary function of carnitine is to transport long-chain fatty acyl-CoA into the
mitochondrial matrix. Other functions of carnitine are exporting products of B—oxidation
from peroxisomes and releasing mitochondrial CoA from acyl—CoA when free CoA

supply is limited.

Carnitine acyl transferase I catalyzes the formation of acylcarnitine. The
camitine/acylcarnitine translocase enzyme catalyzes the transportation of acylcarnitines
into the mitochondrial matrix (12) where they can be transacylated to acyl-CoA. These
acyl-CoAs can then enter the B-oxidation pathway to be broken down to form ATP.
Studies that have been conducted with hemodialysis (HD) patients suggest that this

population has the potential for low concentrations of plasma and tissue free-camitine

69

 

and/or altered camitine metabolism (2,13). These low concentrations may lead to defects
in the way patients on HD metabolize long chain fatty acids, and possibly are associated
with some of the previously mentioned symptoms, such as fatigue, muscle cramps, poor
muscle strength, poor appetite, and decreased quality of life. Supplementation of patients

on hemodialysis has been shown to increase plasma camitine concentrations.

It may be that low plasma camitine, especially acylcarnitine, concentrations are
associated with poor clinical outcomes in certain patients on hemodialysis. Clinical
parameters that are associated with anemia, muscle cramping, quality of life, and
nutrition were targeted. By establishing these associations with camitine, a sub-group of
patients, we hypothesized had increased risk for low camitine values and as well as poor
clinical outcomes can be targeted for intervention. The purpose of this study was to
determine if correlations exist between various parameters of plasma camitine and
speciﬁcally deﬁned clinical and demographic data. In particular, the following questions
were addressed: 1) Are there associations between low plasma camitine concentrations
and the following variables: average erythropoietin dose/month, average mannitol
dose/month, hematocrit, triglyceride, parathyroid hormone (PTH) and blood urea
nitrogen (BUN) concentrations, exercise tolerance, SF 36 score, dietary protein intake and
kilocalorie, years on HD, treatment time (minutes/hours), co-morbidities (presence of
diabetes, left arterial dilation and left ventricular hypertension), medications, age and
weight status? 2) What is the mean acyl-to-free plasma camitine ratio in this
hemodialysis patient population? 3) Is there a correlation between acyl-to-free plasma

camitine ratio and the clinical parameters noted above?

70

Methods
This study was conducted at the Dialysis Center of Lincoln (DCL) in Lincoln, Nebraska.
Required approvals for conducting this study were obtained from the Medical Director
and the Administrator at DCL and the University Committee on Research Involving

Human Subjects (UCRIHS) at Michigan State University.

Sample

The population from which the sample was taken included patients _>_ 18 years of age who
had been receiving a minimum of four hours of bicarbonate hemodialysis treatments,
three times per week for at least one year. Exclusion criteria were: current or previous
treatment (within the last two months) with L-camitine, a severe blood loss, disease
affecting skeletal muscle function, severe liver disease and pregnancy. Patients with
severe blood loss were excluded because they are typically given increased doses of
erythropoietin and iron supplements to help increase blood volume. Patients who have
diseases affecting skeletal muscle function were excluded because the disease may
confound the impact of carnitine on the skeletal muscle ﬁbers. Additionally, patients with
severe liver disease and/or pregnancy may have altered camitine metabolism for reasons

other than uremia Patients included in this study signed a consent form.

Procedures
Each patient, who signed a consent form, had their blood drawn by a Registered Nurse
(RN) or the phlebotomist at DCL on a Wednesday or Thursday of the ﬁrst week of the

month in accordance with the patient’s routine monthly blood draw. No patient had been

71

without dialysis prior to the blood draw longer than 48 hours. A 1 ml aliquot of plasma
from the blood sample was collected in a separator tube and frozen at —-80° C until it
could be analyzed. The plasma was analyzed for free, short and long chain acylcarnitine
using the radioenzymatic assay as outlined by Brass and Hoppel (14). Total camitine was

calculated as the sum of the free, short, and long chain acylcarnitine values.

The remainder of the laboratory data were extracted from the routine monthly laboratory
proﬁle. These variables were analyzed by Spectra laboratories (Fremont, California).
Chronological age at the time of blood draw, the average erythropoietin dose/month and
the average mannitol dose/month, a current medication list, the patient’s most recent
history and physical, the rate of perceived exertion and the perceived dyspnea, and the
patient’s most recent dialysis prescription were also obtained. The data on left ventricular
hypertrophy (LVH) and left arterial dilation (LAD) were obtained from the medical chart
based on ﬁndings from previous echocardiography. Not all patients had this information.

Therefore, data from only a subset of patients were analyzed for LVH and LAD.

On the day of data collection patients who biked during their dialysis treatment were
considered “exercisers”. The rate of perceived exertion and perceived dyspnea were used
to deﬁne exercise tolerance (15). Both of these scales use a 1 to 20 rating with 20 as the
most exertion or dyspnea and 1 the least. The patients who bike, do so almost every
dialysis session. Each time a patient rides the bicycle, the patient’s rate of perceived
exertion and perceived dyspnea ﬁve minutes into the biking session were recorded into

the computer system.

72

Measurement of patients’ perceived quality of life is an important outcome assessment
tool. One of the better known tools, the Medical Outcomes Study Short-Form—36 (SF 36),
has been shown by Kutner and others to have good reliability and validity (16; l 7). Sloan
et al (18) demonstrated an increase in the physical and general well being components of
the SF 36 after oral supplementation with 1g L-carnitine for six months. The SF 36 has
eight different scales related to self-perception of health-related quality of life. The range

for each of the eight scales is 0 to 100.

The two components examined during our study were the physical and mental
components. The quality of life assessment tool, SF36, was administered to the patients at
DCL every six months by social workers. The forms were scored by the social workers
and the most recent results of the SF 36 for the patients who were participating in this
study were used. If an SF36 had not been done in the past six months, it was done on the
day of blood draw by one of the social workers at DCL. During the week of the blood
draw, the dietitians at DCL and the investigator conducted two dietary recalls (24 hour
and usual day) on each patient participating in this study. The completed recalls were
analyzed for average kcal and protein content. The analysis of the dietary recalls was

done using the software program Nutritionist V produced by N2 Computing, 1999.
Analysis of Data

Descriptive statistics were done on both the independent and the dependent variables.

The continuous independent variables were assessed for associations with the dependent

73

variables: total, free and acyl (short chain acyl + long chain acyl) plasma camitine
concentrations using the Pearson’s Correlation Coefﬁcient. However, when the variables
were skewed, Spearman’s Correlation Coefficient was used. To better describe, the
patients they were separated based on gender, mannitol use, presence of diabetes (type 1
and 2) and the presence of LVH. T-test analyses were done for the binary variables to
determine associations with the dependent variables and the continuous independent
variables. T-test analyses were also used to compare mean protein and energy intake
between patients on and not on mannitol. To determine whether any of the independent
variables were predictors (when controlling for potential confounders) for the dependent
variables, multiple linear regression was used. Since the purpose of this study was to
identify important variables for the next phase of the project, a prospective, randomized,
controlled camitine supplementation study, correlations between outcome variables
typically used in dialysis care and camitine concentrations were deemed signiﬁcant using

a p value of 0.05, and Bonferroni adjustments were not done.

Results

A total of 49 patients met the inclusion criteria. Patient data are described in table 4.1.
The mean plasma total, free and acylcarnitine concentrations were 40.3 _t 11.8 uM/L
(SD), 22.8: 7.3 uM/L and 17.5 : 5.9pM/L respectively. The acyl group is comprised of
the short chain and long chain acylcarnitines, the means of which were 10.27 :1; 4.8uM/L
and 7.07:2.7uM/L, respectively. The mean ratio of acyl—to-free plasma camitine was

0.80 1: 0.27. The mean and standard deviation for the continuous variables are found in

74

table 4.2. Signiﬁcant correlations between camitine and the independent variables are
shown in tables 4.3 and 4.4. Secondary disease states such as: type 1 diabetes and left
ventricular hypertrophy (LVH) were trending toward statistical signiﬁcance with free
camitine. Kilocalorie intake was not signiﬁcantly correlated with plasma total, free, short

chain, or long chain acylcarnitine.

To determine which sub-groups of patients were most at risk for low plasma camitine
concentrations, the patients were separated by gender, type of diabetes, and presence of
LVH. Males, but not females, had a positive correlation between the plasma A/F ratio
and SF 36 physical component (SF 36p) as seen in ﬁgure 4.1 (r= 0.54). When the subjects
were separated into those with type 1 diabetes (n=6) and those without it, there was a
negative correlation between age and the A/F ratio (r= —0.83) in subjects with type 1
diabetes and strong negative correlations between age and free camitine and total

camitine in patients with LVH.

To examine the relationships between plasma camitine, mannitol use, kcal/protein intake
and intradialytic weight gain the following analyses were done. First, a one-way anova
was used to compare mean protein and kcal intake, mean normalized protein catabolic
rate and mean intradialytic weight gain at the time of blood draw between patients on
mannitol and those who were not on mannitol. No signiﬁcant difference was found
between the means for any of the variables. The second analysis completed was to select
those patients receiving mannitol and conduct Pearson’s correlation coefﬁcient analyses

between kcal and protein intake and the camitine moieties. Signiﬁcant correlations were

75

found between protein intake and both plasma long chain acylcarnitine (r=0.57, p<0.05)
and free camitine (r=0.48, p<0.05) concentrations. There were also trending correlations
between kcal intake (r=0.39, p<0.05) and long chain acylcarnitine and signiﬁcant

correlations between kcal intake and free camitine (r=0.51, p<0.05).

When Multiple Linear Regression Models were applied to the independent variables, the
models found in Table 4.5 signiﬁcantly predicted Total, Free and Long Chain

Acylcarnitine but not Short Chain Acylcarnitine.

Discussion
The hemodialysis population is getting older and is tending to have more co-morbidities;
by establishing these associations with camitine, a sub-group of patients who is at
increased risk for low camitine values and thereby poor clinical outcomes can be targeted
for intervention. Carnitine intervention in these subgroups may improve the patients’

quality of life and overall mortality.

In healthy adult subjects the mean acyl-to-free plasma camitine ratio is 0.26. (13). In
pediatric patients, the only population for which established abnormal cut points have
been speciﬁed, an acyl-to-free plasma camitine ratio 3 0.4 is used to deﬁne camitine
deﬁciency (19). The mean acyl— to-free plasma camitine ratio in this study was 0.8 i

0.27.

All of the patients had acyl-to-free camitine ratios above the deﬁned pediatric level of

carnitine deﬁciency.

76

In our study, the acyl-to—free plasma camitine ratio was associated with duration of
treatment, triglyceride and serum parathyroid hormone. When just males were examined,
the ratio was positively correlated with the physical component of the quality of life
indicator, short-form-36 and in patients with type 1 diabetes the ratio was positively
correlated to age. It is unclear why males and not females have this association.
However, it may be connected to the fact that males may perceive quality of life to being

more closely tied with physical ability than females.

Currently acyl-to-free plasma camitine ratios have not been examined in many studies.
However, in 1992 Golper et al.(20) reported that HD dialysis patients had acyl-to-free
plasma camitine ratios between 0.6 and 0.9. Debska-Slizien et al.(21) in an observational
study involving 37 hemodialysis patients and 29 controls found increased acyl-to-free
plasma ratios when compared to the control group, and in the hemodialysis group a
positive correlation was seen between acyl-to-free plasma camitine ratio and high-density
lipoproteins. Alhomida (22) studied the effect of hemodialysis on peripheral serum
lymphocyte concentrations and plasma camitine. When he compared healthy controls to
27 HD patients he found no difference in the lymphocyte concentrations between the two
groups, but they did ﬁnd the acyl-to-free plasma camitine to be higher in hemodialysis
patients when compared to the controls (p<0.01). When Kopple et a] (8) conducted a
study with 8 hemodialysis patients, they looked at the camitine content in the muscle and

found muscle acyl-to-free camitine concentrations were similar to healthy controls.

77

Neither Kopple et al (8), Golper and Ahmad (23), nor Alhomida (24) reported

correlations between any clinical variables and the acyl-to-free plasma camitine ratio.

Labonia et al (25), Nikolas et al (26), Caruso et a1 (1), and Trovato et al (27) have all
addressed the relationship between plasma camitine concentrations and red blood cells.
Labonia (28) studied the effect L—carnitine had on the red blood cell sodium transport
mechanism. These researchers found a decrease in the sodium-potassium pump activity
in uremic, end-stage renal disease patients, (n=8) compared to controls (p<0.05). After
supplementation with L-carnitine the sodium-potassium pump activity increased. The
authors of this study stated that in uremic patients, there existed sodium—potassium pump
inhibitors, possibly long chain fatty acids. The L-camitine supplementation could
increase fatty acid transport and oxidation thereby decreasing the concentration of the
sodium-potassium pump inhibitors. The improved pump activity could stabilize the red
blood cell membrane and increase the half-life of the cell. Labonia et al (29) as well as
Nikoloas et al, (30) Caruso et al (1) and Trovato et al (31) found increases in either
hematocrit or hemoglobin with the supplementation of L—carnitine. In our study, we also
found a positive correlation between hematocrit and free camitine. However, when this
data was subjected to the multi-variate analysis no correlations were found. Additionally,
we did not ﬁnd any correlations between erythropoietin amounts and camitine

concentrations.

Serum parathyroid hormone (PTH) positively correlated with total, free and acyl-to—free

plasma camitine concentrations. In 1987, Smogorzewski et al. (32) demonstrated that

78

chronic renal failure is associated with impaired oxidation of long chain fatty acids by
skeletal muscle possibly due the availability of carnitine or to an effect on the enzyme,
camitine palmitoyl transferase, responsible for the formation of the long chain fatty acid-
camitine complex. In a later study, by the same laboratory (33) the researchers felt a
connection between PTH and chronic renal failure existed and was due to elevated PTH
concentrations causing increased calcium ions in the skeletal muscle. These high
concentrations of calcium were postulated to be interfering with the action of the
carnitine palmitoyl transferase and thus affecting long chain fatty acid oxidation. In our
study the correlation between serum PTH and the acyl-to-carnitine ratio was positive. As
PTH concentrations increased, so did the acyl-to-free camitine ratios, possibly indicating
that higher PTH concentrations negatively affect the metabolism of acylcarnitine.
Bellingheri et al. (34) showed decreases in muscle cramping and interdialytic ﬂuid gains
in 14 dialysis patients after supplementation with oral camitine. Mannitol, (a drug used to
decrease muscle cramping while on dialysis) was used as a clinical outcome to represent
the muscle cramping. When we examined patients on mannitol the drug used for muscle
cramping due to rapid ﬂuid removal on hemodialysis, we found higher plasma total, free
and acylcarnitine concentrations in patients using mannitol (p<0.05). We then examined
the relationship with carnitine and protein and found a positive correlation between
protein intake and plasma long chain acylcarnitine concentrations. This relationship is
logical because camitine is found in high protein foods. Blood urea nitrogen
concentrations were also positively correlated with free and total camitine concentrations.
Nitrogen and camitine are both found in high protein foods and this may account in part

for the concurrent increase in blood urea nitrogen and camitine concentrations. It is

79

possible that those patients who have higher consumption of food (including high
protein/camitine foods) are also consuming more ﬂuids and in turn must have more ﬂuid

removed.

Recently, Elisaf et al. (5) showed a decrease in the mean triglyceride concentration of 28
HD patients who had received intravenous camitine supplementation (p<0.05), again
possibly decreasing their risk for cardiovascular accidents. We found a positive
correlation between serum triglyceride concentrations and the acyl-to-free plasma
camitine ratio (p<0.05), but not with free or total camitine. Correlations between left
ventricular hypertrophy (suggestive of hypertension) and left arterial dilation (suggestive
of dilated cardiomyopathy) were found in our study. These results are not clear since the
pathophysiology of these two disease states are basically opposed. Furthermore, it is
difficult to ascertain correlations such as these with retrospective left ventricle
hypertrophy data obtained via chart reviews because of the inconsistent nature of this
data. These data can only direct future research to an area that needs further clariﬁcation
from research involving more precise methods of determining left ventricular

hypertrophy and its association with camitine.

One of the secondary roles of carnitine is the release of mitochondrial coenzyme A (CoA)
from acyl-CoA when the free CoA supply becomes limited due to activation and
accumulation of metabolites in the mitochondrion (35). Medications such as aspirin can
affect the free CoA supply and therefore result in the release of mitochondrial CoA by

camitine. Patients on hemodialysis take aspirin because of its anti-clotting properties

80

during the dialysis process and for underlying heart disease. It was hypothesized that
patients taking medications that ultimately affect CoA concentrations could have an
impact on plasma camitine concentrations due to the increased need for CoA to
metabolize these medications. Subsequently, we did ﬁnd lower concentrations of total

camitine in patients taking aspirin (p<0.05).

Similar to the ﬁndings by Caruso et al (1) we found a positive correlation between age
and total and free plasma camitine concentrations. As patients’ age they lose muscle mass
and 97% of the camitine in the body is stored in the skeletal muscle. Therefore, as they

lose muscle, they may also lose camitine stores.

In summary, this study has shown that HD patients have elevated acyl-to-free ratios and
these ratios correlate with the physical component of the SF 36 in males and age in type 1
diabetic patients. F urtherrnore, certain patients, such as older, females, and those on
aspirin therapy or those with low dietary protein intake may be at greater risk for lower
concentrations in plasma camitine status. Finally, plasma free and total camitine status
appears to be further compromised as age increases; and with increases in BUN, Hct and
PTH. These results suggest certain subgroups of patients may need to be targeted for
further studies with camitine replacement therapy, i.e. long-term patients, older patients,
patients with LVH and left arterial enlargement, females, and patients on aspirin therapy.
Therapy could be targeted to these patients and outcomes monitored. In addition, the

SF36 assessment may be a useful outcome monitor in males and type 1 diabetics. The

81

data from this study warrant more in-depth investigation of the role of camitine in this

vulnerable patient group.

82

Acknowledgement:
A special thank you to Sigma Tau Pharmaceuticals for their unrestricted grant; and
Marlys Burgett, Jen Strong and Mary Lou Buss at the Dialysis Center of Lincoln; Alan T.
Davis was supported by a grant from the Blodgett Butterworth Health Care Foundation

83

10.

11.

12.

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Anemia in Aged Hemodialysis Patients Treated with Recombinant Human
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Matsumura M, Hatakeyama S, Koni I, Mabuchi H, Muramoto H. Correlation
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. Zaruba J, Probst W, Binswanger U. L-camitine improves vascular reﬁlling in

haemodialysis patients during ultraﬁltration. Nephrol.Dia1.Transplant.
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Vacha GM, Giorcelli G, Siliprandi N, Corsi M. Favorable effects of L-carnitine
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Elisaf M, Bairaktari E, Katopodis K et al. Effect of L.carnitine supplementation
on lipid parameters in hemodialysis patients. Am J Nephrol. 1998;18:416-21.

Stefanutti C, Vivenzio A, Lucani G, Di Giacomo S, Lucani E. Effect of L-
carnitine on plasma lipoprotein fatty acids pattern in patients with primary
hyperlipoproteinemia. Clin.Ter. 1998;149:115-9.

Bellinghieri G, Savica V, Mallamace A et al. Correlation between increased
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Kopple JD, Hoppel C, Casaburi R, Storer T, Qing D. Serum and Skeletal Muscle
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Ferreira IM. Quantiﬁcation of non-protein nitrogen components of infant
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Beto JA, Bansal VK. Medical nutrition therapy in chronic kidney failure:
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Iacobazzi V, Naglieri MA, Stanley CA, Wanders RJ, Palmieri F. The structure
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Brass EP, Hoppel CL. Carnitine metabolism in the fasting rat. J Biol.Chem.
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O'connor PJ, Raglin J S, Morgan WP. Psychometric correlates of perception
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Kutner NG. Assessing end-stage renal disease patients' functioning and well-
being: measurement approaches and implications for clinical practice. Am J
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Curtin RB, Lowrie EG, DeOreo PB. Self-reported functional status: an important
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Sloan RS, Kastan B, Rice SI et al. Quality of life during and between
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De Vivo DC, Bohan TP, Coulter DL et al. L-camitine supplementation in
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Golper TA, Wolfson M, Ahmad S et al. Multicenter trial of L-camitine in
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Debska S, Kawecka A, Wojnarowski K et al. Correlation between plasma
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Alhomida AS, Sobki SH, al Sulaiman MH, al Khader AA. Inﬂuence of sex and
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Golper TA, Wolfson M, Ahmad S et al. Multicenter trial of L-carnitine in
maintenance hemodialysis patients. I. Carnitine concentrations and lipid effects.
Kidney Int. 1990;38:904-1 1.

Alhomida AS. Effect of haemodialysis on peripheral lymphocyte camitine levels
in patients with chronic pyelonephritis. Br.J.Biomed.Sci. 1999;56:194-8.

Labonia WD, Morelli OH, Jr., Gimenez MI, Freuler PV, Morelli OH. Effects of
L-carnitine on sodium transport in erythrocytes from dialyzed uremic patients.
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Trovato GM, Iannetti E, Murgo AM, Carpinteri G, Catalano D. Body composition
and long-term levo—carnitine supplementation. Clin.Ter. 1998;149:209-14.

Labonia WD, Morelli OH, Jr., Gimenez MI, Freuler PV, Morelli OH. Effects of
L-carnitine on sodium transport in erythrocytes from dialyzed uremic patients.
Kidney Int. 1987;32:754-9.

Labonia WD, Morelli OH, Jr., Gimenez MI, Freuler PV, Morelli OH. Effects of
L—carnitine on sodium transport in erythrocytes from dialyzed uremic patients.
Kidney Int. 1987;32:754-9.

Nikolaos S, George A, Telemachos T, Maria S, Yannis M, Konstantinos M.
Effect of L-carnitine supplementation on red blood cells deformability in
hemodialysis patients. Ren Fail. 2000;22:73—80.

Trovato GM, Iannetti E, Murgo AM, Carpinteri G, Catalano D. Body composition
and long-term levo-carnitine supplementation. Clin.Ter. 1998;149:209-14.

Smogorzewski M, Piskorska G, Borum PR, Massry SG. Chronic renal failure,
parathyroid hormone and fatty acids oxidation in skeletal muscle. Kidney Int
1988;33:555-60.

Pema AF, Smogorzewski M, Massry SG. Verapamil reverses PTH- or CRF-
induced abnormal fatty acid oxidation in muscle. Kidney Int 1988;34:774-8.

Bellinghieri G, Savica V, Mallamace A et al. Correlation between increased
serum and tissue L-camitine levels and improved muscle symptoms in
hemodialyzed patients. Am J Clin.Nutr. 1983;38:523—31.

Rebouche CJ, Chenard CA. Metabolic fate of dietary camitine in human adults:

identiﬁcation and quantiﬁcation of urinary and fecal metabolites. J Nutr.
1991 ;121 :539-46.

86

Table 4.1

Demographic Data

 

 

 

 

 

 

 

 

 

 

 

Age (meaniSD) 60il6
Males 48%
Mannitol use 46%
Erythropoietin use 88%
Patients who biked during dialysis 46%
Patient on lipid lowering medications 25%
Aspirin use 35%
"Type 1 Diabetes Mellitus 15%
"Types 2 Diabetes Mellitus 29%
"Left Ventricular Hypertrophy 25%
MCongestive Heart Failure 15%

 

 

 

*from a subset of patients (n=43) with this data available
"per a retrospective chart review

87

Table 4.2

Mean and Standard Deviations for Continuous Clinical Parameters

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Continuous Variable MeaniSD“
Serum Blood Urea Nitrogen 51 :20 mg/dl
Serum Parathyroid Hormone 238 : 188 pg/ml

Serum Hematocrit 36:4
Left Arterial Dilation (n=12) 4.2:0.6
Ejection Fraction % (n=l4) 51:15.2
Monthly ave Epogen dose 1100 : 3631 units
Monthly ave Mannitol dose 15.2:5.3
Serum Triglyceride 169 : 107 mg/dl
Respiratory Perceived Exertion (n=1 7) 10:3
Perceived Dyspnea (n=1 7) 0.8: 1.3
Kilocalorie 1516 : 563
Protein 72 : 30 g
Duration on Dialysis 45 : 34 m
Treatment Time 3.95 : 0.73 hr

 

 

 

* Data are presented as mean : SD for n=49, unless otherwise reported

88

Table 4.3

Continuous Independent Variables Signiﬁcantly Associated with Carnitine“

 

 

 

 

 

 

 

Independent Variable Total Carnitine Free Carnitine Long Chain
Acyl Carnitine
Age r = -0.32 r = -0.41 r =—0.41
Dietary Protein r=0.33
Intake
Serum Blood Urea r = 0.29 r = 0.41 r=0.50
Nitrogen
Serum Parathyroid *r = 0.33 *r = 0.36 **r=0.34
Hormone
Serum Hematocrit r = 0.30
Left Arterial r = -0.67 r = -0.62 r=-0.80
Dilation
Ejection Fraction % r = 0.55 r=0.52

 

 

 

"‘ Pearson’s Correlation Coefﬁcient, p < 0.05
** Spearman’s Correlation Coefﬁcient, p< 0.05

89

Table 4.4

Categorical Independent Variable Signiﬁcantly Associated with Carnitine

 

 

 

Independent Variable Total Carnitine Free Carnitine
Gender
Female 37.9:2.08 21 .0:1 .29“
Male 43.1:2.73 25.0:1.60
Aspirin
37.4:2.6l** 21.2:1.59
Yes
42.5:2.20 24.0:1.35
No
Mannitol
Yes 45.3:2.56* 25.3:1.54*
No 36.1:2.00 20.6: 1 .28

 

 

Data are presented as mean : SE for n=49
* = p<0.05, ** = p=0.08

90

 

Table 4.5
Multivariate Linear Regression Models Predicting Total, Free, and Long Chain

Acylcarnitine Values

 

Model 1* Model 2** Model 3***

 

Predictive Variables (IS—Coefﬁcients) (Ii-Coefﬁcients) (ii-Coefﬁcients)

 

 

 

 

 

 

 

Blood Urea Nitrogen 0.412 0.437 0.469
(mg/d1)

Age (years) - 0.185 - 0.249 - 0.330
Weight (kg) 0.176 0.204 - 0.116
Parathyroid Hormone 0.253 0.203 0.034
(Pg/mL)

Gender - 0.161 - 0.157 - 0.276
Protein Intake (g) - 0.008 0.061 0.097
Pre-Albumin (mg/dL) - 0.105 - 0.039 - 0.284

 

 

 

*RI= 0.44, p<0.05 predicting total camitine
** R2= 0.55, p<0.05 predicting free camitine
** R2= 0.62, p<0.05 predicting long-chain acylcarnitine

91

Figure 4.1

Relationship of Short F orm-36 Physical Composite Score to Plasma Acyl-to-Free

Carnitine Ratio in Male Hemodialysis Patients

 

Relationship of SF-36p to AIF in Males

 

 

 

 

 

o Males

 

 

A—S—L—t
sunbeam

 

P
on

- females

 

 

 

j -— Linear
’ (Males)

 

 

 

 

acyl-to-froe camitine ratio

 

 

 

9.09
ON-ha

 

O

 

100
short form-36 physical

 

*r=0.54, p<0.05

92

 

Chapter 5
Phase II, Aim 2
Carnitine Treatment Improved Quality of Life Measures in a
Sample of Midwestern Hemodialysis Patients
Alison L. Steiber, Alan T. Davis, Leslie Spry, Jennifer Strong, Gina Miller,
Mary Lou Buss, Michelle M. Ratkiewicz, Lorraine J. Weatherspoon
Abstract

Previously we demonstrated that selected groups of hemodialysis (HD) patients might be
more likely to have abnormalities of camitine metabolism. The purpose of the present
study was to examine the effects of carnitine therapy in these selected groups of HD
patients on quality of life measures and erythropoietin dose. This was a double-blind,
randomized controlled trial, in which 50 HD patients were treated with either 2 g IV
camitine or placebo. The treatment period was for 24 weeks. Thirty-four patients (15 in
the treatment group) completed the study. The mean age was 69:15 yrs, 35% were
female and 44% had diabetes. Mean initial plasma total, free, short chain acyl and long
chain acyl camitine concentrations (nmol/L; meaniSEM) were 35.9: 1 .8, 18.2:tl.1,
l 1.6:0.6 and 60:03, while the plasma acyl-to—free camitine ratio was 1.02:0.05. With
respect to the Medical Outcomes Short F orm-36 (SF36), improvements from baseline
were noted in the treatment group (n = 13) for role-physical (33.9: 1 .9 to 43.2:3.0,
p<0.05) and the SF36 physical component summary score (36.1:2.7 to 39.7:2.3,
p=0.09), relative to changes in the control group (n = 14). The erythropoietin dose over
the 24 week period was reduced from baseline in the treatment group, relative to the

placebo group (-1.62i0.91 versus 1.33i0.79 units erythropoietin dry weight'l

93

hemoglobin concentration", respectively, p<0.05). After 24 weeks of IV camitine
therapy, SF 36 scores were improved and erythropoietin doses were reduced in HD
patients, relative to the control group.

Introduction
L-carnitine is a compound found in high protein foods and is produced by the liver,
kidney, and brain. The metabolic ﬁmctions of carnitine are to transport fatty acyl-CoA
into the mitochondrial matrix, export products of B-oxidation from peroxisomes, and
release mitochondrial CoA from acyl-CoA when free CoA supplies are limited. Research
has demonstrated altered free and acylcarnitine concentrations in patients receiving

hemodialysis treatment (1-3).

Recently, L-carnitine treatment was approved by the Centers for Medicare & Medicaid
Services (CMS) for national reimbursement in hemodialysis patients with either
erythropoietin resistance or chronic hypotensive episodes during dialysis treatment.
Following the establishment of the CMS reimbursement policy, a camitine consensus
group performed a thorough review of the existing literature, and determined that there
were four main types of symptoms that may accompany altered camitine metabolism in
dialysis patients: anemia hyporesponsive to synthetic erythropoietin, intradialytic
hypotension, cardiomyopathy and skeletal muscle weakness (4). The current title for this

set of symptoms is “dialysis-related camitine disorder” (DCD).

The mechanism for DCD has yet to be determined and in fact is probably multi-factorial.

Research has been done to suggest that the alteration of camitine may occur prior to

94

dialysis being initiated (5;6) and therefore the alteration may be due, in part, to dietary
intake or uremia itself. High protein foods, containing higher amounts of L-carnitine, are
often restricted in the diets of patients with renal insufﬁciency to preserve kidney
function and decrease uremic side effects. Uremia itself has been shown to alter fatty acid
(7) and amino acid (8) composition in patients. Therefore, uremia may alter the type of
carnitine (free or acylated), the utilization, or the function of carnitine as well.

Furthermore, as the kidney fails, the synthesis of carnitine may decrease.

Previous work in this lab has shown that a subset of hemodialysis (HD) patients may be
at greater risk for plasma camitine alterations (1 ). This group of hi gh-risk hemodialysis
patients has lower plasma free camitine concentrations, but normal total camitine
concentrations. Administration of L-carnitine as a therapeutic agent in this group of
patients increases plasma free camitine concentrations and by mass action leads to
removal of long and medium chain acylcarnitine moieties. The acylcarnitines will leave

the cell and be removed from the plasma by dialysis.

In an effort to show that L-carnitine treatment can beneﬁt all dialysis patients, the general
hemodialysis population has been the target for the majority of the previously reported
camitine research. However, Elisaf et al (9) found there were certain patients who
responded to levocamitine and other patients who did not. In contrast to theories from
previous studies, the purpose of this study was to select a particular section of the
hemodialysis population, determined to be at increased risk for altered camitine

metabolism by the criteria established previously (1 ), and monitor these patients for

95

beneﬁts from L-carnitine treatment, including perceived quality of life and selected

clinical outcomes.

96

—'——————— .._... .. ._--._- Mn- .. .

 

Subjects and Methods
Subjects
This study was conducted at a Midwestern dialysis center. Required approvals for
conducting this study were obtained from the Medical Director and the Administrator at
the Dialysis Center of Lincoln, the Community Institutional Review Board in Lincoln,
NE, and the University Committee on Research Involving Hmnan Subjects at Michigan
State University. All procedures followed were in accordance with the ethical standards

of Michigan State University’ Committee on Research Involving Human Subjects.

The population from which the sample was taken included patients _>_ 18 years of age,
who had been receiving a minimum of three hours of bicarbonate, low acetate
hemodialysis treatments, three times per week and who had been receiving dialysis for at
least one year. In addition, those patients admitted into the study met two or more risk
factors for a compromised serum camitine as established in our previous study (1).
These risk factors were 65 years of age or older, at least one year duration of treatment,
female, use of aspirin and/or mannitol, and the presence of type II diabetes mellitus, left
atrial dilation and/or left ventricular hypertrophy. Exclusion criteria were current or
previous treatment (within the last two months) with L—carnitine, a severe blood loss,
disease affecting skeletal muscle function, severe liver disease, pregnancy and/or free

camitine > 40umol/L.

Patients with severe blood loss were excluded because they are typically given increased

doses of Epogen® and iron supplements to help increase blood volume. Patients who

97

have diseases affecting skeletal muscle function were excluded because the disease may
confound the impact of carnitine on the skeletal muscle ﬁbers. Patients with severe liver
disease and pregnancy may have altered camitine metabolism for reasons other than
uremia. Additionally, in order to comply with the Federal Drug Administration’s criteria
for supplementation with intravenous levocamitine in the hemodialysis population, all
patients with a plasma free camitine concentration greater than 40 nmol/L were excluded.
Patients included into this study signed a consent form and were randomized into control

and treatment groups.

Procedures

This was a prospective, randomized, double blind, clinical research study performed
between September, 2001 and March, 2002. The variables and their times of collection
are outlined in table II. The patients had their blood drawn for camitine analysis during
the study on the ﬁrst week of the baseline month and at 12 and 24 weeks, in accordance
with routine monthly blood draws. The blood sample was collected in a heparin tube,
from which the plasma was obtained and stored at ~80° C until analysis. Plasma camitine

concentrations were analyzed as described previously (10;11).

The mean erythropoietin dose was recorded monthly during the course of the study. The
erythropoietin administration data were analyzed as the change ﬁ'om baseline to post-
treatrnent. Erythropoietin usage was analyzed using an erythropoietin resistance index,
created by dividing the patients’ pre- and post-treatment erythropoietin doses by the

patients’ dry weight and their hemoglobin concentration.

98

To determine changes in Kcal and protein consumption, dietary recalls were collected
using the multiple pass method (12), a 24 hour recall and a typical day recall. These
values were averaged in order to account for individual variations. Nutritionist V
produced by N2 Computing (First Databank, Inc., San Bruno, CA) was used for nutrient
analysis. Normalized Protein Catabolic Rate (nPCR) was calculated using the following
formula:

PCR = 0.22 + 0.036 X interdialytic rise in BUN * 24/Interdialytic interval in hours

To normalize the PCR: nPCR = PCR/weight (kg)

The seven point SGA form (13) and mid-arm muscle circumference (MAMC) (14) were
used to assess nutrition status at baseline, 12 weeks, and 24 weeks. SGA is a subjective
tool used to assess nutritional status and does not require laboratory values. The tool
combines a series of dietary, gastrointestinal, clinical, and functional status questions in
combination with a brief physical exam to assess nutritional status. MAMC was used as a

surrogate marker of lean body mass.

The Medical Outcomes Short F orm-36 (SF 3 6) surveys were administered and scored by
the social worker at the dialysis centers (15) at baseline and at 24 weeks. The SF36 is a
perceived quality of life assessment, which has eight domains used to comprise either a
mental (MCS) or a physical (PCS) composite score. Each domain was scored on a scale
of 0 to 100, with 100 being the highest perception of functioning. The domains are

physical functioning, role-physical, bodily pain, general health, mental health, role-

99

emotional, social functioning, and vitality. The MCS and PCS were normalized as
described by Ware and Kosinski (16). Further data were collected from medical records

and random data audits were conducted to ensure consistency and accuracy of collection.

Treatment

Those patients who signed an informed consent and met basic inclusion criteria were
screened for selected “high camitine risk” criteria (Table I). The purpose for the high
camitine risk criteria was to get a sample of hemodialysis patients most likely to have
altered camitine metabolism. The criteria used has was taken from previous studies
conducted with camitine (l ). The patients must have met a minimum of two of the
criteria in table I to participate in the study. Those patients eligible to participate in the
study were randomized into either the treatment or the control group by a co-investigator
who did not participate in the data collection process. This investigator and the
pharmacist were the only persons not blinded. Those patients who were in the treatment
group received 20mg/kg Carnitor® (Sigma-Tau Pharmaceuticals, 800 South Frederick,
Gaithersburg, MD 20877) actual body weight intravenously, post hemodialysis treatment,
three times per week intravenously. The camitine was administered to the patients in an
infusion of 100 ml of normal saline over 10 minutes by the dialysis center nursing staff.
The patients in the control group received an intravenous placebo of 100 ml of normal
saline over 10 minutes. The amount of carnitine needed was calculated using the patients’
dry weight from the baseline of the study. By diluting the camitine in normal saline and

giving it in an infusion, the potential odor of the drug was masked and blindness

maintained. The treatment period was for 24 weeks.

100

Analysis of Data

Data were analyzed using SPSS version 11.0 (SPSS Inc., Chicago, IL). For all of the
quantitative variables except camitine, the difference in the amount of change between
the control and treatment groups was determined using the analysis of covariance, using
the baseline measurement as the covariate. The camitine data for the baseline, three
month, and six month periods were analyzed using a repeated measures analysis of
variance. Differences in descriptive variables were determined using either the t-test for
quantitative variables, or the xz test for nominal variables. Associations were analyzed
using Pearson’s correlation coefﬁcient. The variables were analyzed for multivariate

relationships using multiple regression. Signiﬁcance was assessed at p < 0.05.

101

w
Of the 50 study patients, two were removed due to plasma free camitine concentrations >
40umol/L, seven voluntarily withdrew (one prior to baseline collection), and seven died
from causes unrelated to camitine. Of the 34 remaining patients (15 treatment, 19
placebo), seven declined to complete the ﬁnal SF36 survey. However, data for the other
variables were collected on these seven patients. For the variable SF 36, there were 13 in

the treatment group and 14 in the placebo group.

Table 5.1 outlines the baseline descriptive data for the study patients. The primary
etiologies for the patients on hemodialysis were diabetes and hypertension. At baseline
there were signiﬁcant differences between treatment and placebo groups for the variable
nPCR, while the remaining baseline descriptive and primary outcome variables were not
signiﬁcantly different between the two groups. There were no statistically signiﬁcant
differences at baseline for any of the camitine variables between the two treatment
groups (Table 5.2). There were numerous correlations between the camitine ﬁactions at
baseline and some of the descriptive parameters (Table 5.3). When the independent
predictors were analyzed using multiple regression to determine predictors for the three
month MAMC measurements, baseline plasma free camitine and baseline BMI were

signiﬁcant (r = 0.83).

The primary outcome variable for this study was perceived quality of life questionnaire,

SF3 6. There were a statistically signiﬁcant improvement in the camitine group for role

Physical (Table 5.4), with a trend towards signiﬁcance noted for the PCS (p = 0.09),

102

 

relative to the control group. In addition, there were no statistically signiﬁcant differences
for the baseline SF-36 values between the treatment and control groups. The signiﬁcance
level for the baseline difference in role physical score was 0.053. However, when the
baseline measurements of the role physical score were used as the covariate in the

ANCOVA, they did not achieve statistical signiﬁcance.

There was a signiﬁcant difference in the change in utilization of erythropoietin in the
camitine treated group, relative to the controls. Relative to baseline, at six months the
camitine group showed a decrease of 1.62i0.91 units erythropoietin dry weight'1 [Hb]",
relative to an increase of 1.33d:0.79 units erythropoietin dry weight'1 [Hb]'1 in the control
group. Of the other variables analyzed for changes between pre- and post-treatment with
camitine (dietary intake, nPCR, SGA, MAMC, BUN, triacylglycerols, hemoglobin,
hematocrit), none demonstrated statistically signiﬁcant changes between the treatment
and the control group.

Discussion
The main endpoints for this study were the Medical Outcomes Short Form-36 domains
and the physical composite score. Within the treatment group, a signiﬁcant increase was
seen in the SF36 domain role physical, relative to controls. Furthermore, the physical
composite score was trending strongly toward signiﬁcance. This was particularly striking

given the small sample size and the high attrition rate experienced in this study.

The SF 36 is a self-reported assessment tool designed to measure the patient’s health

related quality of life. It has been well validated in the hemodialysis population by Kutner

103

(17;18) and others (19;20). As a group, the baseline mean composite scores (prior to
normalization) found in our study were similar to the ones documented by Curtin, Lowrie
and DeOreo (20) for the physical composite score but slightly higher than the reported
mental composite score.

Other interventions, such as exercise, have been reported to increase the SF 36 scores of
end stage renal disease patients (21). However, previous studies with levocamitine
treatment have been unable to demonstrate increases in the physical and mental
composite score of the SF36. Sloan et al (3) reported ﬁndings from a randomized clinical
trial where the levocamitine was given orally for six months. In the Sloan study, the SF36
was given every one-and-a-half months, after the ﬁrst one-and-a-half month period, an
improvement could be seen in the physical functioning and general health domains.
However, this effect seemed to disappear by the sixth month. Baseline descriptive data
were not reported for the Sloan study, therefore it is difﬁcult to compare the two groups.
However, it may have been the route of administration of the levocamitine that led to the
discrepancy between the two studies. Oral levocamitine is only partially absorbed and a
bacteria-mediated process degrades the unabsorbed portion (22;23). The by-products of
this degradation process, trimethylamine and y—butrobetaine, may be toxic and are
excreted by the kidney. Patients with end stage renal disease are not able to excrete these
by-products. Therefore, it is possible the by-products increase in concentration toxic

level (24).

The Chief trial (2) was recently conducted using quality of life as one of its main end-

points. In this trial, patients were given intravenous levocamitine for six months and the

104

Kidney Disease Questionnaire (KDQ) was the assessment tool used to measure perceived
quality of life. The KDQ is validated for measuring quality of life in patients with end
stage renal disease, and it was assessed at baseline, midpoint and at the end of the trial
(2). The subjects in this trial were younger than in this trial (mean age 42-48 verses 67
years), the Chief trial treatment groups had fewer patients with diabetes (1 1-25% verses
45%), and their mean nPCR was higher (1.02-1.17 verses 0.9). At the end of the Chief
trial, the researchers did not ﬁnd an improvement in the total score of the KDQ.

However, they did ﬁnd a signiﬁcant improvement in the fatigue portion of the tool.

The results found in this study may have been in part to the way the patients were
selected or the elderly, co-morbid status of the patients. Other factors that may have

played a role are the patient’s familiarity with the SF36 and the people administering it.

The difference in the change of the erythropoietin doses between the treatment and
placebo groups is not unexpected. Other researchers such as Caruso et a1 (25) and
Kletzrnayr et al (26) have reported improvements in erythropoietin dosing with
levocamitine treatment. Caruso et al. speciﬁcally reported this ﬁnding in hemodialysis
patients greater than 65 years of age (25), whereas Kletzrnayr et al found anemia and
erythropoietin resistance index responders and nonresponders. In the Kletzrnayr et a1
study those patients who responded had signiﬁcantly reduced erythropoietin resistance
indexes (26). About 50% of the treatment group in this study were responders to

levocamitine treatment.

105

Although nutritional status was not affected by levocamitine treatment, it did seem to be
associated with baseline plasma camitine status. The independent variables that were
correlated with baseline camitine were for the most part nutritional assessment indicators
(nPCR, serum BUN, albumin, dietary protein intake, and SGA). All of the correlations
were positive, indicating the better the nutritional status, the higher the baseline plasma

camitine concentrations.

Furthermore, baseline plasma camitine was associated with 12-week BMI and MAMC
values and as can be seen by the multiple linear regression model, plasma free camitine is
predictive, along with BMI, for the 12-week MAMC. Plasma free camitine may be an
early indicator of lean body mass atrophy. Plasma free and acyl carnitine are lost in the
dialysis process. The body stores (97% of which are in the muscle) may be partly
responsible for restoring plasma camitine concentrations, especially when the diet is low

in levocamitine sources (red meats, poultry, and dairy).

The limitations of this study were low subject numbers and high attrition due to death,
voluntary withdrawal, and refusal to participate. The study was done solely by the
volunteer time and effort of the patients, nurses, social workers and dietitians at the
dialysis unit. Therefore, missing data did occur and patients were less likely to complete
the quality of life questionnaire. Strengths of the study included the knowledge of the
social workers with the SF 36, patient randomization with a placebo group and double

blinding the participants throughout the study period.

106

In conclusion, this study was unique due to the speciﬁc selection criteria of the
participants and the dietary data collected. Quality of life was signiﬁcantly improved in
the treatment group of this hemodialysis patient sample in the area of the domain role-
physical, with a trend towards improvement in the physical composite score.
Furthermore, mean erythropoietin dose changes between the groups were signiﬁcantly
different, with the treatment group dose decreasing and the placebo group increasing.
Signiﬁcant associations were found between the dependent variables (total, free, short
chain acyl and long chain acylcarnitine) and many nutrition related independent
variables. Future studies could focus on ﬁrrther narrowing the selection criteria and more
detailed investigation of the relationship between quality of life and physical functioning

with levocamitine treatment.

107

10.

Reference List

Steiber AL, Weatherspoon LJ, Spry L, Davis AT. Serum camitine concentrations
correlated to clinical outcome parameters in chronic hemodialysis patients.
Clin.Nutr. 2004;23:27-34.

Brass EP, Adler S, Sietsema KE, Hiatt WR, Orlando AM, Amato A. Intravenous
L-carnitine increases plasma camitine, reduces fatigue, and may preserve exercise
capacity in hemodialysis patients. Am.J.Kidney Dis. 2001;37:1018-28.

Sloan RS, Kastan B, Rice SI et al. Quality of life during and between
hemodialysis treatments: role of L-carnitine supplementation. Am.J.Kidney Dis.
1998;32:265-72.

Eknoyan G, Latos DL, Lindberg J. Practice recommendations for the use of L-
Carnitine in dialysis-related camitine disorder National Kidney Foundation
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Ricanati ES, Tsemg KY, Hoppel CL. Abnormal fatty acid utilization during
prolonged fasting in chronic uremia. Kidney Int Suppl 1987;22:8145-8148.

Rodriguez-Segade S, Alonso le, Paz M et al. Carnitine concentrations in
dialysed and undialysed patients with chronic renal insufﬁciency.
Ann.Clin.Biochem. 1986;23 ( Pt 6):671-5.

Ando M, Sanaka T, Nihei H. Eicosapentanoic acid reduces plasma levels of
remnant lipoproteins and prevents in vivo peroxidation of LDL in dialysis
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Nohara Y, Suzuki J, Kinoshita T, Watanabe M. Creatinine inhibits D-amino acid
oxidase. Nephron 2002;91:281-5.

Elisaf M, Bairaktari E, Katopodis K et al. Effect of L-carnitine supplementation
on lipid parameters in hemodialysis patients. Am J Nephrol. 1998;18:416-21.

Brass EP, Hoppel CL. Carnitine metabolism in the fasting rat. J Biol.Chem.
1978;253:2688-93.

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Hoppel C. Determination of Carnitine. Techniques in Diagnostic Human
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Moshfegh AJ, Borrud L, Perloff B, et al. Improved Method for the 24-hour
Dietary Recall for Use in National Surveys. FASEB Journal 1999;13zA603
(abstr).

McCann L. Using subjective global assessment to identify malnutrition in the
ESRD patient. Nephrol.News Issues 1999;13:18-9.

Clinical practice guidelines for nutrition in chronic renal failure. K/DOQI,
National Kidney Foundation. Am J Kidney Dis 2000;35:81-140.

Ware JE, Kosinski M, Gandek B. SF-36 Health Survey. Lincoln, RI: Quality
Metric Incorporated, 2002.

Ware JE, Kosinskia M. SF-36 Physical and Mental Health Summary Scales: A
Manual for users of version 1. Second Edition. Lincoln, RI: Quality Metric
Incorporated, 2002.

Kutner NG, Brogan D, Fielding B. Physical and psychosocial resource variables
related to long-term survival in older dialysis patients. Geriatr.Nephrol.Urol.
1997;7z23-8.

Kutner NG. Assessing end-stage renal disease patients' functioning and well-
being: measurement approaches and implications for clinical practice. Am J
Kidney Dis 1994;24:321-33.

Rettig RA, Sadler J H, Meyer KB et al. Assessing health and quality of life
outcomes in dialysis: a report on an Institute of Medicine workshop. Am J Kidney
Dis 1997;30:140-55.

Curtin RB, Lowrie EG, DeOreo PB. Self-reported functional status: an important
predictor of health outcomes among end—stage renal disease patients. Adv.Ren
Replace.Ther. 1999;6:133-40.

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Painter P, Carlson L, Carey S, Paul SM, Myll J. Physical functioning and health-
related quality-of—life changes with exercise training in hemodialysis patients. Am
J Kidney Dis 2000;35:482-92.

Rebouche CJ, Chenard CA. Metabolic fate of dietary camitine in human adults:
identiﬁcation and quantiﬁcation of urinary and fecal metabolites. J Nutr.
1991;121:539-46.

Sahajwalla CG, Helton ED, Purich ED, Hoppel CL, Cabana BE. Multiple-dose
pharmacokinetics and bioequivalence of L-carnitine 330-mg tablet versus l-g
chewable tablet versus enteral solution in healthy adult male volunteers. J
Pharm.Sci. 1995;84:627-33.

Evans A. Dialysis-related camitine disorder and levocamitine pharmacology. Am
J Kidney Dis 2003;41:813-826.

Caruso U, Umberto, Leone L, Cravotto E, and Nava D. Effects of L-carnitine on
Anemia in Aged Hemodialysis Patients Treated with Recombinant Human
Erythropoietin: A pilot study. Dialysis and Transplantation 27(8), 498-504. 1998.

Kletzrnayr J, Mayer G, Legenstein E et al. Anemia and camitine supplementation
in hemodialyzed patients. Kidney Int. Suppl 1999;69:893-106.

110

Table 5.1

Characteristics of the study groups at the start of the study]

 

Placebo Group (n = 19)

Carnitine Group (n == 15)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Age (y) 69.4i3.4 67.6:t3.9
Women (%) 21.1% 53.3%
Etiology of Disease

Diabetes (%) 47.3% 40.0%

Hypertension (%) 21.1% 26.7%

Other (%) 31.6% 33.3%
Duration of treatment (y) 4.0:1:0.5 3.8:t0.6
BMI (kg/m2) 28.6il.4 30.4:13
MAMC (%) 22521:] .0 22.5:1:2.0
Dietary Intake Energy 1194196 1374i99
(kcal/d)
Dietary Intake Protein (g/d) 47.5:t4.6 54.8d:5.5
nPCR2 0.78:0.03 0.92:0.05
BUN 39912.3 45.1:1:3.7
Albumin (g/dl) 3.8:t0.1 3.7i0.1
Triacyl glycerol (mg/d1) 173 43:26.4 180.4i19.0
HDL 41.6i2.7 38.1:t4.l
Hemoglobin (g/dl) l 15:03 11.3:t0.4
Hematocrit (%) 36.3i0.9 36.9:t1.2

 

Ill

 

1 All quantitative data are expressed as mean-tSEM. BMI, Body mass index; MAMC,
Midarm muscle circumference; nPCR, normalized protein catabolic rate; BUN, blood
urea nitrogen; HDL, high density lipoprotein.

2 Signiﬁcantly different at p < 0.05, using the unpaired t-test.

112

Table 5.2

Plasma camitine concentrations by treatment groupl

 

Control Carnitine

Group Group

 

Baseline 12 weeks 24 weeks Baseline 12 weeks 24 weeks

(n = 24) (n = 22) (n = 18) (n = 25) (n = 20) (n = 14)

 

 

 

 

 

Total
334.8141.
Carnitine 35.7120 34.4131 34213.2 39.1129 184.1127.2
1
(nmol/L)
Free
177.9117.
Carnitine 19011.4 18711.8 15111.4 20611.8 12571182
8
(nmol/L)
SCAC
10.8106 ll.311.3 12611.8 11.7110 54.5197 90717.9
(nmol/L)
LCAC
5810.3 4.4104 65011.15 6.7105 27315.0 66.21226
(nmol/L)
Acyl/Free

Carnitine 09610.08 0.9010.07 1.5010.26 0.9410.06 0.7010.06 0.8910.10

Ratio

 

 

 

‘ All data are expressed as mean1SEM. SCAC, short chain acylcarnitine. LCAC, long
chain acylcarnitine. All variables had signiﬁcant effects for treatment, duration of
treatment, and the interaction between treatment and duration of treatment, using a
repeated measures analysis of variance and signiﬁcance assessed at p < 0.05.

113

Table 5.3

Associations between descriptive variables and various baseline camitine fractions.l

 

 

 

 

 

 

 

 

 

 

 

 

Free Carnitine SCAC LCAC Total
Carnitine
Baseline
nPCR 0.39 0.35 0.38
Serum BUN 0.60 0.37 0.47 0.59
Sertun [Albumin] 0.41
Protein Intake 0.38 0.32
Serum 0.35
Triacylglycerol
Serum HDL 0.40
3 Months
MAMC 0.50 0.39 0.49
BMI 0.31

 

I All values represent Pearson’s r, and are signiﬁcant at p < 0.05. SCAC, short chain
acylcarnitine; LCAC, long chain acylcarnitine; nPCR, normalized protein catabolic rate;
BUN, blood urea nitrogen; HDL, high density lipoprotein; MAMC, Midarm muscle

circumference; BMI, Body mass index.

114

 

Table 5.4
Short Form 36 (SF 36) scores at baseline and at six months for the control and camitine

groups.l

 

Control (n = 14) Carnitine (n = 13)

 

 

 

 

 

 

 

 

 

 

 

 

Baseline 6 Months Baseline 6 Months
Physical 30313.0 29.2133 37.0123 35.9125
Function
Role Physical2 40.6126 40113.0 33911.9 43.2130
Bodily Pain 50.2124 47913.9 42914.3 50913.7
General Health 40313.5 39213.4 42911.9 43.2123
Vitality 46314.0 48.0133 44.1126 46.1124
Social Function 41.8133 46413.2 44.7135 50512.7
Role Emotion 43312.9 47.0128 42413.8 50.5126
Mental Health 49513.7 46214.4 50213.4 51.4127
PCS3 37.3127 35.7132 36.1127 39.7123
MCS 49.6135 51.8128 49.7137 54.2122

 

 

All data are expressed as meanisEM. PCS, physical component summary measure;
MCS, mental component summary measure. All scores were normalized to the 1998
general United States population, as described (16).

2 The change from baseline in the camitine group was signiﬁcantly greater than the

change from baseline in the control group, using ANCOVA, at p < 0.05.

115

3 The change from baseline in the camitine group showed a trend for a greater increase
from baseline than the change from baseline in the control group, using ANCOVA, at p =

0.09.

116

Chapteré
PhaseII,Aim3

The impact of intravenous camitine treatment over 24 weeks on the concentration and

distribution of acylcarnitines in hemodialysis patients

Steiber AL, Weatherspoon LJ, Hoppel CL, Minkler, P, Spry L, Davis AT

Alum

The purpose of this study is to examine the effect of camitine supplementation, on

acylcarnitine (AC) detected in plasma in a hemodialysis (HD) population at risk for

compromised camitine status. The study was a double blind, randomized trial conducted

over 24 weeks. Fifty patients met the previously deﬁned inclusion and exclusion criteria

and were randomized into either a control (IV normal saline) or a treatment (IV L-

camitine at 20 mg/kg) group. Blood collection occurred at randomization, and weeks 12,

and 24. Total camitine (TC) and free camitine (F C) were analyzed using radioenzymatic

assay; and AC moieties were analyzed using HPLC/mass spectrometry. At 24 weeks, 32

completed the study. Mean age was 68:14 yrs, 38% were female, 45% had diabetes.

 

 

All p<0.05

Treatment

 

Total

Carnitine

334:154

34:13

 

Free

Carnitine

l 78:67

15:6

 

Short
Chain
Acylcamit

ine

90:30

13:8

 

Long
Chain
Acylcamit

DC sum

0.7:0.3

0.3:0.2

 

Ayclcarni
tine to
Free
Carnitine
ratio
0.9104

l.5:l.1

 

acetylcam
itine/other
Acylcarnit

ine

85:15

49:15

 

117

 

With camitine treatment F C and AC increased. In the treatment group the magnitude of
the change in AC depended on the acyl moiety. The largest increased was in C2 with 700
fold. Medium acyls increased intermediately the greatest amount C 10/3x, C8/5x, C6/2x
and C4/6x. These data indicate an increased metabolism of LCACs in hemodialysis
patients.

1.1-1mm”.
In chronic kidney disease patients receiving hemodialysis (CKDH), camitine metabolism
has been of interest because of its potential effects on muscle fatigue and quality of life.
Patients with CKDH frequently are affected by secondary conditions such as anemia,
myopathy, hyperparathyroidism, left ventricular hypertrophy, hypertension and
osteoporosis. Many CKDH patients have altered plasma camitine concentrations and
altered acyl-to-ﬁ'ee ratios (1-3). When compared with healthy subjects, patients with
CKDH are reported to have higher acylcarnitine concentrations and lower plasma free
camitine concentrations; whereas, the total camitine concentrations remain within the

normal range (1).

In healthy subjects, the most prevalent form of plasma acylcarnitine is acetylcamitine.
Following camitine removal, coenzyme-A (Co-A) is attached to acetate and acetyl Co-A
is formed. Acetyl Co-A is a substrate for many metabolic pathways and in a healthy
population is the primary metabolite resulting from B—oxidation. However, in plasma of
CKDH patients, increased amounts of medium and long chain acylcarnitine
concentrations are reported in the literature (1;4;5). The increase in medium and long

chain acylcarnitine moieties may be due to an alteration in B-oxidation and/or a decrease

118

in excretion of these metabolites. Long chain fatty acyls have deleterious effects on
cellular membranes and metabolic functions. Hypothesized mechanisms for the negative
effects are 1. interference with sodium-potassium transporters in cell membranes (6) and

2. detergent effects on cellular phospholipid membranes (7).

It is possible that elevation of an individual acylcarnitine or a group of acylcarnitine
moieties can be identiﬁed as a marker for altered camitine metabolism. Additionally,
increased concentrations of acylcarnitine moieties may have negative eﬂ‘ects on clinical
parameters associated with camitine metabolism. Clinical parameters associated with
camitine concentrations are dietary protein, serum albumin, serum blood urea nitrogen,

serum hemoglobin and hematocrit, presence of diabetes mellitus, or left ventricular

hypertrophy (1;8;9).

Treatment with L-camitine (biologically active form) may effectively drive long chain
acylcarnitine moieties through B—oxidation by mass action, thus decreasing mitochondrial
acylcarnitine concentrations and increasing plasma acylcarnitine concentrations. As with
many dysfunctions, not all patients have the same degree of alteration. Therefore, to be
able to detect a true treatment effect, a sample of patients with signiﬁcant camitine
metabolism alteration, would be necessary. We previously demonstrated that patients
with some deﬁned camitine risk facotrs had serum camitine abnormalities (1). Therefore,
the purpose of this study was to: 1) identify acyl camitine moieties present in the plasma
of a group of hemodialysis patients at risk for abnormal camitine status and 2) determine

if treatment with L-carnitine alters acylcarnitine moieties in the patients.

119

Maths!!!
This study is one portion of a larger randomized, double-blind, placebo controlled clinical
trial. The study was conducted at a midwestem dialysis center. Required approvals for
conducting this study were obtained ﬁ'om the Medical Director and the Administrator at
the Dialysis Center, the Community Institutional Review Board, and the University
Committee on Research Involving Human Subjects at Michigan State University. All
procedures followed were in accordance with the ethical standards of the Committee on

Research Involving Human Subjects and all patients signed an informed consent prior to

participating the in the study.

Subjects

This data was obtained from a randomized, double-blind, clinical trial. The patients were
enrolled in the clinical trial based on the following inclusion criteria: _>_ 18 years of age,
receiving a minimum of three hours of bicarbonate, low acetate hemodialysis treatments,
three times per week, for at least one year. Additionally, patients admitted into the study
had two or more risk factors for a compromised camitine status: _>_ 65 years old, 1 year
duration of treatment, female, use of aspirin and/or mannitol, or presence of type 2

diabetes mellitus, left atrial dilation and/or left ventricular hypertrophy.

Exclusion criteria were: current or previous treatment (within the last two months) with
L-carnitine, a severe blood loss, disease affecting skeletal muscle function, severe liver
disease, pregnant, and free camitine > 40umollL. Patients with severe blood loss were

excluded because they are typically given increased doses of synthetic erythropoietin and

120

iron supplements to help increase blood volume. Patients who have diseases affecting
skeletal muscle ﬁrnction were excluded because the disease may confound the impact of
camitine on the skeletal muscle ﬁbers. Patients with severe liver disease and pregnancy
may have altered camitine metabolism for reasons other than uremia. Additionally, in
order to comply with the Federal Drug Administration’s criteria for supplementing with
intravenous levocamitine in the hemodialysis population, all patients with a plasma free

camitine concentration > 40 were excluded.

Procedures

Patients had their blood drawn for camitine analysis on O, 12 and 24 weeks, in
accordance with routine monthly blood collection. Plasma from the blood sample was
spun and collected in a heparin tube, frozen to -80°C and shipped with dry ice to
Spectrum Laboratory in Grand Rapids, MI. Radioenzymatic assay was used to determine
concentrations of total camitine, free camitine, short chain acylcarnitine, long chain
acylcarnitine, and the plasma acyl-to-free camitine ratio (10). In Grand Rapids, when the
samples were thawed for analysis a portion was separated and re-ﬁ'ozen for shipment to
Louis Stokes Veterans Administration Medical Center in Cleveland, OH where analysis
of acylcarnitines using high performance liquid chromatography with mass spectrometry
(HPLC/MS/MS) was conducted (1 1).

In addition to the plasma samples, descriptive and nutritional parameters were collected

from routine laboratory reports, medical records, and patient interviews. During patient

interviews, the 7-point version of Subjective Global Assessment (SGA) (12;13), two

121

multiple pass method 24 hour recalls(l4), and a Medical Outcomes Short F orm-36

(SF36) (15,16) were conducted.

The SGA tool was used to rank the presence of malnutrition using diet, gastrointestinal,
clinical, and ﬁrnctional status questions in combination with a brief physical exam to
assess nutritional status. Dietary intake of kilocalories and protein were measured with
two 24 hour recalls preformed for a typical dialysis and non-dialysis day with the
multiple pass method (14). The two day intake data were averaged (kcal/day or protein
g/daY), then calculated to kcal/kg/day and protein g/kg/day. The SF36 is a tool used to
measure perceived quality of life. It has eight domains, which were scored from 0-100,
from these eight domains; two composite scores (physical and mental) are calculated by a
weighted mean (17).

Treatment with Irv-Carnitine

During the clinical triaL patients randomized to the treatment group, received 20mg/kg
Carnitor® actual body weight, post hemodialysis treatment, three times per week
intravenously. L—carnitine was administered to the patients in an infusion of 100ml of
normal saline over 10 minutes by the dialysis center nursing staff. Patients randomized to
the placebo group received an intravenous placebo of 100 ml normal saline over 10
minutes. The amount of carnitine administered was calculated using the patients’ dry
weight from baseline. By diluting the camitine in normal saline and giving it in an
infusion, the potential odor of the drug was masked and blindness maintained. The

treatment period was for 24 weeks.

122

Analysis of Data
Data were analyzed using SPSS version, 11.0, 2001. Descriptive, correlation analysis,

and one-way AN OVA analyses were performed to detect associations, mean differences,

and changes from pre- and post-intervention periods. Signiﬁcance was deﬁned at p<0.05.

M

Fiﬁy patients were recruited for the study. Two were removed from the analyses due to
high plasma free camitine (>40 umol/L), seven voluntarily withdrew ﬁ'om the study (one
prior to baseline data collection), and seven died during the study. When baseline
characteristics where compared between patients who completed the 24 week trial and
those who did not, there were no perceivable differences. Descriptive characteristics at 0,
12 and 24 weeks are reported in Table 6.1. The patients had the following primary
etiologies 36% diabetes mellitus type 2, 28% hypertension, 9 % diabetes mellitus type 1,
6% obstructive uropathy and 21% other etiologies. Additional descriptive can be found

in Chapter 5 (Steiber et al. submitted for publication).

Baseline plasma total, ﬁ'ee, short and long chain acylcarnitine concentrations for all the
patients (n=47) and for healthy fasted controls (n=30) are shown in table 6.2. Baseline
plasma acylcarnitine concentrations for healthy, fasted controls (n=29) and all study
participants with a baseline free camitine < 40 nmol/L are found in table 6.3. After
recruitment, patients were randomized into treatment (n= 23) and placebo (n=24) groups.

There were no mean differences in acylcarnitine moiety concentrations between the

123

placebo, the healthy control or the treatment groups at baseline. The mean plasma total,
ﬁ'ee, short, and long chain acylcarnitine concentrations ﬁ'om each group are found in
ﬁgure 6.1. Figure 6.2 shows the linear increase of plasma free camitine throughout the

24 week study.

During the 24 week treatment period, the acylcarnitine moieties within the placebo group
remained stable. The treatment group has signiﬁcantly higher values in all acyl moieties
at 12 and 24 weeks when compared with the placebo group, except isovaleryl, which
never reached statistical signiﬁcance. In addition, 2-methylbutynyl reached a statistical
signiﬁcance only at week 24. Figure 6.3 is a chromatogram of a treatment group patient’s
acylcarnitine moieties ﬁom baseline to 12 and 24 weeks. Figures 6.4a-d depict mean
acylcarnitine changes which occurred within the treatment group from baseline to 12 and
24 weeks. Measuring the change in the ratio of plasma acetylcamitine to measurable sum
of propionyl to 9,12-octadecadienyl is also an indicator of change due to L-carnitine

treatment and this can be found in Figure 6.5.

Baseline statistical analyses with acylcarnitine concentrations reveal correlations with
laboratory values, anthropometric measures, and dietary factors table 6.4. Additionally,
the overall nutritional status assessment tool, subjective global assessment, was positively
correlated with decanylcarnitine (r=0.30, p<0.05), laurylcarnitine (r=0.32, p<0.05), and

palmitylcamitine (F045, p<0.01).

124

Within the treatment group, the magnitude of change in acylcarnitine depended on the
acyl length. The largest increase was a 700 fold increase in acetylcamitine. Medium
acylcarnitines increased intermediately, with the largest increases in butynylcarnitine (6

fold), octanylcamitine (5 fold), decanylcarnitine (3 fold), and hexanylcarnitine (2 fold).

2 . .
The data presented in this paper are the ﬁrst to be reported in the medical literature
regarding plasma acylcarnitine moieties in a deﬁned sample of pre- and post-treatment
hemodialysis patients. At baseline the mean free, short, and long-chain acyl lengths were
outside normal parameters (10) and parameters established by Borum (18). Plasma free
camitine was on average 50% less than normal values; whereas plasma short and long

chain acylcarnitine concentrations were approximately double normal values.

At baseline the mean concentration of acetylcamitine for the whole sample was not
signiﬁcantly different than the healthy, fasted controls. Acetylcamitine is the primary
substrate for many metabolic pathways. However when camitine metabolism is altered
theoretically, acetylcamitine concentrations would decrease. Recently, Hoppel et al
(submitted for publication) reported signiﬁcantly lower concentrations of acetylcamitine
within their dialysis sample. These patients diﬂ‘ered from the ones in our study by many
factors. Primarily they were not selected for risk of altered camitine metabolism which
was the case with our patients. Additionally, the patients in the study by Hoppel (2005)

were and they were younger, had greater ethnic diversity, and a lower frequency of

125

diabetes (2). It is unclear why this younger, potentially healthier sample had lower

acetylcamitine concentrations than our older sample.

When examining the results for acylcarnitine concentrations greater than acetylcamitine,
between the dialysis sample and the healthy, fasted, controls, the data may appear to be
conﬂicting. In table 6.2 dialysis patients’ values are reported as signiﬁcantly different
than the healthy, fasted control values. This ﬁnding contradicts table 6.3 which reports
dialysis acylcarnitine moieties are not signiﬁcantly different than the healthy, fasted
controls. The difference occurs because HPLC/MS/MS in this analysis measured 19
acylcarnitine moieties. However, over 39 acylcarnitine moieties exist. Therefore, when
the radioenzymatic assay measured short and long chain acylcarnitine, it captured all the
acyl moieties, not just those detected in the HPLC/MS/MS analysis. The acylcarnitine
moieties not captured by HPLC/MS/MS are the ones elevated, hence the discrepancy
between high concentrations found by radioenzymatic assay and normal concentrations

by HPLC/MS/MS.

As anticipated, following treatment with L-carnitine, acylcarnitine moieties, except for
isovaleryl- and 2-methylbutyrylcarnitine, were signiﬁcantly higher in the treatment group
than in the placebo group. Additionally, 24 week acylcarnitine concentrations were
higher than baseline concentrations within the treatment group. However, within the
treatment group, when 12 week acylcarnitine concentrations where compared to 24 week
acylcarnitine concentrations, a signiﬁcant difference was not found. Pharmacologically,

homeostasis is found when the treatment drug reaches a stable concentration in the

126

homeostasis was not reached at 12 weeks but may have been reached by 24 weeks.
Conversely, Evans et al (19), suggested that steady-state was reached by 9 weeks in a
pharmacokinetic trial conducted on 12 hemodialysis patients when free and
acetylcamitine were analyzed. In our study, although the free camitine continued to rise
throughout the 24 weeks of treatment by week 12 the acylcarnitine concentrations did

stabilize for some patients.

The variables associated with acylcarnitine concentrations were all surrogates for
nutrition status and nutritional intake (dietary protein intake, weight, MAMC, SGA,
nPCR, BUN, serum albumin, hematocrit and SF 36, general health domain). This is not
surprising, in previous work we and others have found correlations with total, free and
acylcarnitine and nutrition parameters (1 ;8;20). Additionally, serum albumin is bound to
long chain fatty acyls for transport, therefore it is not surprising that the concentrations of
long chain fatty acids correlate positively with serum albumin. It is possible that classes
of acylcarnitine moieties may be used as indicators for clinical or metabolic alterations

that could beneﬁt from camitine treatment.

The body’s predominate source of camitine is dietary camitine, lysine and methionine
and the predominate storage site is muscle. Currently, national food and nutrient
databases, such as the United States Department of Agriculture’s Handbook 8, do not
contain information on camitine content within foods; however, meat and dairy sources
are believed to have the highest concentrations (21,22). Dietary protein intake was

positively correlated with total and free camitine, but short and long chain acylcarnitine

127

did not (Steiber et al., manuscript submitted for publication). It is interesting that 2-
methylbutyrylcarnitine, but no others, was signiﬁcantly associated to the actual intake of
protein. Whereas, indicators of visceral and somatic protein status such as serum
albumin, nPCR and serum BUN are correlated with medium chain acylcarnitine moieties
such as: tetradecanyl, dodecanyl, octadecanyl, hexadecanyl (serum albumin); short chain
acylcarnitine moieties such as: butynyl (nPCR) and acetyl, propionyl, butynyl, and
isobutynyl (serum BUN). The differences in associations may derive from the multiple
aspects that affect visceral and protein status which are not due to protein intake. Two of
these aspects many be metabolic stress (inﬂammation, wound healing, fever, uremia) and
treatment effect (dialysis, medications). Further investigation with advanced biochemical
and anthropometric techniques such as C-reactive protein, lipid peroxidation parameters,

and DEXA are warranted to examine these relationships.

In summary, plasma acylcarnitine concentrations in CKD patients receiving hemodialysis
are altered. Treatment with L-carnitine does increase total and ﬁee camitine as well as
the acylcarnitine moieties. Steady-state of free camitine does not occur prior to 12 weeks
but may occur near 24 weeks. Finally, classes of acylcarnitine concentrations are directly
correlated to visceral and somatic nutrition indicators. Future, research will be needed
analyze all the possible camitine moieties and to establish associations between

nutritional status and acylcarnitine moieties.

128

metabolisms. Lipid peroxidation may affect lipid metabolism and thereby acylcarnitine
moiety concentrations and ﬁnally, DEXA measures lean body store which is the primary

location for camitine storage.

In summary, plasma acylcarnitine concentrations in CKD patients receiving hemodialysis
are altered. Treatment with L-carnitine increases total and free camitine as well as the
acylcarnitine moieties. Steady-state of free camitine does not occur prior to 12 weeks but
may occur near 24 weeks. Therefore, outcome measures established to measure the
effectiveness of carnitine treatment should be done at a minimum, up through 24 weeks.
Finally, classes of acylcarnitine concentrations are directly correlated to visceral and
somatic nutrition indicators. Future, research will be needed analyze all the possible
camitine moieties and to establish associations between nutritional status, factors

impacting nutrition status, and acylcarnitine moieties.

129

10.

ll.

12.

13.

Reference List

. Steiber AL, Weatherspoon LJ, Spry L, Davis AT. Serum camitine concentrations

correlated to clinical outcome parameters in chronic hemodialysis patients.
Clin.Nutr. 2004;23:27-34.

Brass EP, Adler S, Sietsema KE, Hiatt WR, Orlando AM, Amato A Intravenous
L-carnitine increases plasma camitine, reduces fatigue, and may preserve exercise
capacity in hemodialysis patients. Am.J.Kidney Dis. 2001;37: 1018-28.

Sloan RS, Kastan B, Rice SI et al. Quality of life during and between
hemodialysis treatments: role of L-carnitine supplementation. Am.J.Kidney Dis.
1998;32:265-72.

Hoppel C, Minkler P, Feronti T, Ricanati E. Plasma Acylcarnitines in Patients
with Chronic Renal Failure. Kidney Int 2000; (abstr).

Ricanati ES, Tsemg KY, Hoppel CL. Abnormal fatty acid utilization during
prolonged fasting in chronic uremia. Kidney Int Suppl 1987;222S145-Sl48.

Labonia WD, Morelli OH, Jr., Gimenez MI, Freuler PV, Morelli OH. Effects of
L—carnitine on sodium transport in erythrocytes ﬁom dialyzed uremic patients.
Kidney Int. 1987;32:754-9.

Matsumura M, Hatakeyama S, Koni I, Mabuchi H, Muramoto H. Correlation
between serum camitine levels and erythrocyte osmotic ﬁagility in hemodialysis
patients. Nephron 1996;72:574-8.

Trovato GM, Iannetti E, Murgo AM, Carpinteri G, Catalano D. Body composition
and long-term levo—camitine supplementation. Clin.Ter. 1998;149:209-14.

Caruso U, Umberto, Leone L, Cravotto E, and Nava D. Effects of L-camitine on
Anemia in Aged Hemodialysis Patients Treated with Recombinant Human
Erythropoietin: A pilot study. Dialysis and Transplantation 27(8), 498-504. 1998.

Hoppel C. Determination of Carnitine. Techniques in Diagnostic Human
Biochemical Genetics: A Laboratory Mannual. Wiley-Liss, Inc. 1991:309-26.

Minkler, P., Ingalls, S, and Hoppel, C. Strategy for the isolation, derivatization,
chromatographic separation and detection of camitine and acylcarnitines. 2004.

Visser R, Dekker FW, Boeschoten EW, Stevens P, Krediet RT. Reliability of the
7-point subjective global assessment scale in assessing nutritional status of
dialysis patients. Adv.Perit.Dial. 1999;15:222-5.

McCann L. Using subjective global assessment to identify malnutrition in the
ESRD patient. Nephrol.News Issues 1999;13:18-9.

130

14.

15.

16.

17.

18.

19.

20.

21.

22.

Moshfegh AJ, Borrud L, Perloff B, et al. Improved Method for the 24-hour
Dietary Recall for Use in National Surveys. FASEB Journal 1999;13:A603
(abstr).

Kutner NG, Brogan D, Fielding B. Physical and psychosocial resource variables
related to long-term survival in older dialysis patients. Geriatr.Nephrol.Urol.
1997;7223-8.

Kutner NG. Assessing end-stage renal disease patients' ﬁrnctioning and well-
being: measurement approaches and implications for clinical practice. Am J
Kidney Dis 1994;24:321-33.

Ware JE, Kosinskia M. SF-36 Physical and Mental Health Summary Scales: A
Mamral for users of version 1. Second Edition. Lincoln, RI: Quality Metric

Incorporated, 2002.

Borum PR Changing perspective of carnitine ﬁrnction and the need for
exogenous camitine of patients treated with hemodialysis. Am.J.Clin.Nutr.
1996;64:976-7.

Evans AM, Faull R, Fomasini G et al. Pharmacokinetics of L-carnitine in patients
with end—stage renal disease undergoing long-term hemodialysis.
Clin.Pharmacol.Ther. 2000;68:238-49.

Vesela E, Racek J, Treﬁl L, Jankovy'ch V, Pojer M. Effect of L-carnitine
supplementation in hemodialysis patients. Nephron 2001;88:218-23.

Rudman D, Sewell CW, Ansley JD. Deﬁciency of carnitine in cachectic cirrhotic
patients. J Clin.lnvest 1977;60:716-23.

F erreira IM. Quantiﬁcation of non-protein nitrogen components of infant

formulae and follow-up milks: comparison with cows' and human milk. Br.J Nutr.
2003;90: 127-33.

131

Descriptive Characteristics at Baseline, 12 and 24 Weeks

Table 6.1

 

 

 

 

 

 

 

 

 

 

 

 

Variable Normal Ranges Baseline 12 Weeks 24 Weeks
for Dialysis n=47 n=41 n=34
Patients
Age (years) 68114 NC NC
Gender 38 NC NC
(%female)
Body Mass >25=overweight, 27.8165 28316.4 27.4196
Index >30=obese
Dietary Intake 30-35 12771443 13361395 14831593
(Kilocalories & kilocalories/kg > 1716
kilocalories/kg) 60years
35 kilocalories <
60years
Dietary Intake 53126 57120 60126
(Protein (g) & 1.2 0,710.4
Elks)
Normalized >1 .2 0910.2 1.0103 0.9103
Protein
Catabolic Rate
Serum >4.0 3.7104 3.7105 3.7104
Albumin
(mg/(1L)
Serum <200 161.7192.7 17251994 18051108.]
Triglycerides
(mg/4L)
Serum 11-12 11.4113 11.6113 12,111.]
Hemoglobin
ML)
‘data shown as mean 1SD

132

 

Table 6.2

Baseline Plasma Total, Free, Short, and Long Chain Acylcarnitine Concentrations

(nmol/L)

 

Whole Sample, Non-fasted‘ Healthy, Fasted Controls

 

 

 

 

 

n=47 n=30

Total Carnitine 36111 46110

Free Carnitine 1917 3718
Short Chain Acylcarnitine 11.1140 5.7135
Long Chain Acylcarnitine 6011.9 3711.5
Acyl-to-Free Ratio 1.0103 0310.2

 

 

 

*radioenzymatic analysis of plasma samples that were shipped to the laboratory on dry

ice and Roma to -80°C until analysis (10)

133

Table 6.3

Baseline Acylcarnitine Concentrations for Total Sample and Healthy, Fasted Controls at

Baseline, 12 and 24 Weeks (nmol/L)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

134

Plasma Healthy Whole Group at
Acylmoiety Controls Baseline
concentrations n=29 n=47
Acetyl 55812.24 53212.06
Propoinyl 0.2710. 10 0.2010. 10
Butynyl 00410.12 0.081013
Isobutynyl 00110.03 00810.18
Isovaleryl 0.011003 0.001001
2- 00010.01 01610.49
methylbutynyl
Hexanyl 0.021005 0.001001
Octanyl 01010.10 00410.10
Decanyl 01210.12 0.031007
Dodecanyl 0.081008 0.041003
Tetradecanyl 0.021001 0.031001
9-hexadecenyl 00310.03 0.021001
Octadecanyl 0.021001 0.021001
9-octadecanyl 01410.06 0.1 110.05
9, 12- 0.091004 0.061004
octadecadienyl
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135

Figure 6.1. Mean Plasma Carnitine Concentrations

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

400
350 I TC treatment
d 300 g: I TC placebo
g 250 :7.- FC treatment
E r
w 200 w :1 FC placebo
F '32.? I SCAC treatment
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U 100 I LCAC treatment
50
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Week 0 Week 12 Week 24

 

 

 

TC — total camitine, FC — free camitine, SCAC — short chain acylcarnitine, LCAC- long
chain acylcarnitine; Radioenzymatic assay technique, between treatment and placebo

groups - p<0.01

136

Figure 6.2. Plasma Free Carnitine at O, 12 and 24 weeks

200
180
160
140
120
1 00
80
60
40

N
O

 

Plasma Free Carnitine (micromolIL)
O

week 0 week 12 week 24

137

Figure 6.3 Chromatograph of One Patient, Baseline, 12 and 24 weeks

Relative Abundance

100

 

 

 

 

 

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Figure 6.4 Treatment Group Baseline, 12 and 24 weeks of Acetyl- through
Hexanylcarnitine

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139

Figure 6.5Treatment Group Baseline, 12 and 24 weeks of Octanyl- through

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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140

Figure 6.6 Treatment Group Baseline, 12 and 24 weeks of Hexadecanyl- through 9, 12-
octadecadienylcarnitine

 

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141

Chapter 7
Sumner:

The prevalence of CKD in the United States is on the rise and with this increase, there are
associated increases in healthcare costs, morbidity, and mortality and decreases in quality
of life for the individuals affected (1). In patients with CKD, quality of life is determined
by multiple factors including adequacy of treatment, the support system, presence of co-
morbidities (e. g. diabetes, cardiovascular disease, anemia, muscle weakness) and
nutritional status. Validated assessment tools such as the Medical Outcomes Short Form-
36 (SF36) can measure perceived quality of life (2). Perceived quality of life has been to
be correlated with mortality (3 ;4).

Carnitine, a low molecular weight molecule found in free and acyl forms, has been shown
to be associated with factors affecting anemia and muscular weakness. Approximately
25% of total body in vivo synthesis of carnitine is ﬁ'om lysine, and methionine in a multi-
step process and 75% is from dietary sources of camitine, lysine, and methionine(5;6).
Due to its small size and water solubility, camitine is lost in urine, typically in the acyl
form, in healthy humans and lost in the dialysate of CKD patients receiving dialysis
treatment. When camitine is lost during dialysis, it is lost in both the free and acyl forms,

thus altering the plasma acyl-to-free camitine ratio (7).

It has been postulated that in patients with altered camitine metabolism treatment with L-

carnitine moves long and medium chain acylcarnitine moieties out of the mitochondria

142

 

and into the plasma where they can be eliminated through urination or in the case of
patients receiving dialysis through the dialysis treatment itself. With the removal of
acylcarnitine moieties from the mitochondria, metabolic ﬁrnction would return to normal.
Normalization of metabolic function could improve clinical parameters such as serum
concentrations of hemoglobin, muscle strength, endurance, and prevalence of muscle

cramping, thereby improving perceived quality of life.

There are a plethora of studies involving camitine in the literature, which give clues to
the possible role, function, and beneﬁt of camitine treatment (8-15). A large majority of
the treatment studies has been done in the CKD population possibly due to the altered
excretion of camitine in dialysis, the poor intake of dietary camitine sources, and the
prevalence of carnitine deﬁciency signs and symptoms (muscle weakness and fatigue).
However, the CKD literature is rampant with small, poorly controlled studies with

conﬂicting results.

It was hypothesized that not all CKD patients had the same degree of alteration in
camitine metabolism as evidenced by plasma camitine values nor did they all react the
same way to L-camitine treatment. Therefore, the purpose of this study was to identify
risk factors associated with plasma camitine concentrations, use these risk factors to
identify a sample of CKD patients with a high level of altered camitine metabolism, treat
the patient sample with intravenous L-carnitine, and monitor for changes in clinical

parameters, perceived quality of life, and plasma acylcarnitine moieties.

143

 

To achieve this purpose a two-phase study was conducted using patient interviews and
examination, medical records review, and blood collection. Plasma total, free and
acylcarnitine concentrations were analyzed with radioenzymatic assay (16) and plasma
acylcarnitine concentrations were analyzed using high performance liquid
chromatography/mass spectrometry (17). Other variables assessed were descriptive (age,
etiology, co-morbidities, treatment and medication information), anthropometric
(MAMC, BMI, weight, and height), laboratory (serum BUN, serum albumin,
hemoglobin, hematocrit, serum parathyroid hormone, serum triglycerides, and high-
density lipoproteins), dietary (energy and protein intake) and perceived quality of life

(SF36).

Baseline results for phase I and 11 indicate that plasma camitine is correlated with the
following parameters: age, dietary protein, nPCR, serum BUN, PTH, hematocrit,
triglycerides, high-density lipoproteins, and albumin, leﬁ arterial dilation, and ejection
fraction percentage. The older patients consuming less dietary protein and thus having
lower nPCR, serum BUN, and serum albumin, with higher arterial dilation and lower
ejection fraction had lower plasma camitine concentrations. After 12 weeks of L-
carnitine treatment, MAMC and BMI were also positively correlated with plasma

camitine.

At baseline, there were statistically signiﬁcant diﬂ‘erences in plasma camitine between

female and male patients, patients who were and were not using aspirin, and patients who

were and were not using mannitol. Females, aspirin users, and non-mannitol users had

144

 

lower plasma camitine concentrations. These correlations and differences in mean values
were used to establish risk factors for identifying patients at increased risk for altered

camitine metabolism.

After 24 weeks of treatment with L-carnitine, the following SF 36 domains were
signiﬁcantly improved: role-functioning, bodily pain, and role-emotional. The physical
composite score was also trending toward signiﬁcant improvement. A limitation of this
data set is the difference at baseline between the treatment and placebo group for the role-
functioning variable. This is a very important variable and the most logical for
improvement with L-carnitine treatment. The multiple linear regression analysis included
group (treatment or placebo), baseline role-functioning and role-functioning change ﬁ'om
baseline to 24 weeks demonstrated that when baseline role-functioning, is controlled for,
the intervention of L-carnitine is a signiﬁcant predictor of role-ﬁrnctioning change from
baseline to 24 weeks. Patients in the treatment group had signiﬁcantly improved scores in
role-functioning while patients in the placebo group did not change signiﬁcantly. The
ﬁndings in the SF36 scores were expected but unique. This is the ﬁrst study to
successﬁrlly demonstrate improvement in these variables. Others such as Ahmad et al (9),
Sloan et al (10), and Brass et al (15) have attempted to demonstrate improvements with

quality of life but have not shown improvements in the SF36 domains.

The second primary outcome of this study was a signiﬁcant improvement in synthetic

erythropoietin doses from baseline to 24 weeks in the treatment group. This is a logical

and expected ﬁnding. Similar ﬁndings have been found in a sample greater than 65 years

145

of age and in dialysis patients supplemented with iron and intravenous L—carnitine

simultaneously (12;18).

The third primary outcome of this study was measurement of plasma acylcarnitine
moieties in this high camitine risk group of patients and subsequent increase of
acylcarnitines in the treatment, but not placebo, group. The data from this study indicate
that the acetylcamitine concentrations were within normal limits. This was unexpected

given the elderly age and high percentage of patients with DM in the sample.

Another unexpected result was the plateauing of long chain acylcarnitine concentrations
between 12 and 24 weeks. It is unknown exactly when homeostasis of long chain
acylcarnitine in the mitochondria occurs. However, our dat suggest that it may occur

between 12 and 24 weeks of treatment.

Strengths
The strengths of this study include establishment of risk factors for altered camitine

metabolism; the fact that a randomized, double-blind, placebo controlled methodology
was used; and statistically signiﬁcant improvements in three domains of the SF36 and in
synthetic erythropoietin doses in the treatment group. The establishment of risk factors
was an original concept in the camitine literature, currently no other reports of risk
factors have occurred. However, there were reports of responders versus non-responders,
indicating that a high level of variety from L-carnitine treatment was documented

(11;18). A randomized, double-blind, placebo controlled study is the gold standard in

146

methodology for clinical trials. Although the sample was small, the methodology was
stringent. Finally, signiﬁcant results were found in the primary outcome variables
indicating the hypothesis and methodology were sound and the aims appropriate.
Another, strength, to this study was the level of volunteerism. The patients, nurses,
pharmacist, and physicians that volunteered to assist with this study did so without any

ﬁnancial compensation. Participation without compensation in clinical trials is very rare.

Limitations

While this study had signiﬁcant strengths, it also had limitations. During both phases 1
and II the sample size was too small for extensive multivariate analysis. This was
unfortunate, especially since a major criticism of previous studies in the literature was
their small sample size. A goal of the study was to obtain a large sample size and thereby
overcome this weakness. This was done via power calculations which suggested a sample
size of 50 in each group or higher. However, patient recruitment was more diﬁicult than
anticipated. Clinical trials are fraught with challenges and this study was no exception.
Access to a sufﬁciently large population from which to screen and recruit the sample was
unquestionably the most challenging. The Dialysis Center of Lincoln had over 200
patients. However, the strict inclusion criteria, especially >1 year on dialysis limited, the
number of patients that could be included in the study. In CKD, patient mortality is
increased compared to non-CKD patients, and co—morbidity is also signiﬁcantly higher.

Even after patients were included into the study, attrition due to death was high.

147

To ensure that patients were blind, L—carnitine for the treatment group was placed in an
infusion bag with normal saline to mask drug odor and all members of the research team
were blind to the randomization process except for Dr. Alan T. Davis who did not collect
any data. However, no questionnaire was used to measure the effectiveness of these

procedures.

Future Directions

The completion of this study is a platform for many other studies in both the clinical and
metabolic areas. The risk factors that were established could be further clariﬁed. Do all of
the factors contribute the same weight in identifying alteration in camitine metabolism or
would do one or two make a larger contribution. Are there other factors that would aid in
identifying altered camitine metabolism that were not accounted for in this study? For
example, would screening for an SF36 physical composite score of less than 35 capture a
population with the same degree of alteration as was found in this study due to their

severely diminished physical functioning?

Muscle biopsies were not feasible in this study.Hhowever, measurement of muscle
camitine content at different time points during L-carnitine treatment would result in
better knowledge of muscle camitine turnover, homeostasis, and tissue saturation.
Measurements of muscle strength and fatigue could be measured in conjunction to

determine associations with tissue camitine concentration and muscle ability.

148

SF36 is a tool used to measure perceived quality of life and although it measures
perceived physical ability, it is not an actual objective measure of physical ﬁrnctioning.
Therefore, physical functioning measures such as muscle strength and fatigue, (sit-to-
stand, six-minute walk, and gait speed) could be measured pre and post intervention in a
randomized, double-blind, placebo controlled trial with intravenous L-camitine
treatment. These items have been shown to have a high correlation to mortality,
hospitalization rates, independent living, and perceived quality of life (19-22). Therefore,
improvement in these parameters would be a signiﬁcant contribution to the CKD

literature.

The increase in plasma acylcarnitine concentrations in the treatment group nwds further
investigation. It was hypothesized that the increase was due to mass action from the
mitochondria. However, this has not clearly been established. Using isotopic tracers,
camitine could be tagged and measured after homeostasis in the plasma is reached. The
tracers would reveal how much of the free carnitine was acylated and what lengths of

fatty acyl moieties were attached.

In summary, in a deﬁned sample of CKD patients receiving hemodialysis, after treatment
with intravenous L-carnitine, there were improvements in total, free and acylcarnitine
moieties, the SF36 domains: role-ﬁrnctioning, bodily pain, and role-emotional, and in
synthetic erythropoietin dosages. Therefore, intravenous L-carnitine might be particularly
beneﬁcal as an adjunct to treatment in patients receiving hemodialysis, especially if

deﬁned risk parameters are present.

149

10.

11.

12.

Reference List

. System URD: USRD 2001 Annual Data Report; Atlas of End Stage Renal

Diseases in the United States. Bethesda, MD, National Institute of Health,
National Institute of Diabaes and Digestive and Kidney Diseases, 2001. 2001.

Curtin RB, Lowrie EG, DeOreo PB. Self-reported functional status: an important
predictor of health outcomes among end-stage renal disease patients. Adv.Ren
Replace.Ther. 1999;6:133-40.

Kalantar-Zadeh K, Fouque D, Kopple ID. Outcome research, mrtrition, and
reverse epidemiology in maintenance dialysis patients. J Ren Nutr. 2004;14:64-
71.

Mapes DL, Lopes AA, Satayathum S et al. Health-related quality of life as a
predictor of mortality and hospitalization: the Dialysis Outcomes and Practice
Patterns Study (DOPPS). Kidney Int 2003;64:339-49.

Rebouche CJ, Lombard KA, Chenard CA Renal adaptation to dietary camitine in
humans. Am J Clin.Nutr. 1993;58:660-5.

Lombard KA, Olson AL, Nelson SE, Rebouche CJ. Carnitine status of
lactoovovegetarians and strict vegetarian adults and children. Am J Clin.Nutr.
1989;50:301-6.

Evans AM, Faull R, Fomasini G et al. Pharmacokinetics of L—carnitine in patients
with end-stage renal disease undergoing long-term hemodialysis.
Clin.Pharmacol.Ther. 2000;68:238-49.

Bellinghieri G, Savica V, Mallamace A et al. Correlation between increased
serum and tissue L-carnitine levels and improved muscle symptoms in
hemodialyzed patients. Am J Clin.Nutr. 1983;38:523-31.

Ahmad S, Robertson HT, Golper TA et al. Multicenter trial of L-carnitine in
maintenance hemodialysis patients. Kidney Int. 1990;38:912-8.

Sloan RS, Kastan B, Rice SI et al. Quality oflife during and between
hemodialysis treatments: role of L-carnitine supplementation. Am.J.Kidney Dis.
1998;32:265-72.

Stefanutti C, Vivenzio A, Lucani G, Di Giacomo S, Lucani E. Effect of L-
carnitine on plasma lipoprotein fatty acids pattern in patients with primary
hyperlipoproteinemia. Clin.Ter. 1998;149:115-9.

Caruso U, Umberto, Leone L, Cravotto E, and Nava D. Effects of L-carnitine on
Anemia in Aged Hemodialysis Patients Treated with Recombinant Human
Erythropoietin: A pilot study. Dialysis and Transplantation l998;27(8), 498-504.

150

13.

14.

15.

l6.

17.

18.

19.

20.

21.

22.

Feinfeld DA, Kurian P, Cheng JT et al. Effect of oral L—carnitine on serum
myoglobin in hemodialysis patients. Ren Fail. 1996;18:91-6.

Trovato GM, Iannetti E, Murgo AM, Carpinteri G, Catalano D. Body composition
and long-term levo-carnitine supplementation. Clin.Ter. 1998;149:209-14.

Brass EP, Adler S, Sietsema KE, Hiatt WR, Orlando AM, Amato A Intravenous
L-carnitine increases plasma camitine, reduces fatigue, and may preserve exercise
capacity in hemodialysis patients. Am.J.Kidney Dis. 2001;37:1018-28.

Brass EP, Hoppel CL. Carnitine metabolism in the fasting rat. I Biol.Chem.
1978;253:2688-93.

Minkler, P., Ingalls, S, and Hoppel, C. Strategy for the isolation, derivatization,
chromatographic separation and detection of carnitine and acylcarnitines.
Publication in process.

Kletzrnayr J, Mayer G, Legenstein E et al. Anemia and camitine supplementation
in hemodialyzed patients. Kidney Int.Suppl 1999;69:893-106.

Painter P, Carlson L, Carey S, Paul SM, Myll J. Physical functioning and health-
related quality-of-life changes with exercise training in hemodialysis patients. Am
J Kidney Dis 2000;35:482-92.

Painter P, Carlson L, Carey S, Paul SM, Myll J. Low-functioning hemodialysis
patients improve with exercise training. Am J Kidney Dis 2000;36:600-8.

Johansen KL, Painter P, Kent-Braun JA et al. Validation of questionnaires to
estimate physical activity and functioning in end-stage renal disease. Kidney Int
2001;59:1121-7.

Johansen KL, Chertow GM, da Silva M, Carey S, Painter P. Determinants of

physical performance in ambulatory patients on hemodialysis. Kidney Int
2001;60: 1586-91.

151

 

Appendixes

 

152

Appendix A

153

INFORMED CONSENT FORM
Phase I
For the participants of the
Carnitine Status in Patients Receiving Hemodialysis Study

Investigators: Alison Steiber MS, RD
(517) 355-9893

Lorraine Weatherspoon Ph.D, RD
(517) 432-0813

Alan Davis, Ph.D
(616) 454-9960

Project Description: This project is designed to assess camitine levels in patients
receiving hemodialysis and the role of carnitine in factors such as: age EPO dose,
Mannitol dose, exercise, SF36 scores and protein intake. You may reﬁrse to continue
participation in this study at any time without any adverse effects on your medical care.
Confidentiality: Your identity will not be reported in any paper or document produced
from this research. During and after this research, your privacy will be protected to the
maximum of the law.

Procedures: Participation in this study will involve you allowing a small sample of the
blood drawn, during your routine blood draw at the beginning of the month, to be used
for analysis of carnitine. It will not cost you anything and it will not require any
additional “pokes” by needles. With your permission the following information will be
taken from your chart and used for analysis: age, EPO prescription, mannitol use,
exercise data, most recent SF36 score, treatment information and health history. This
information will be sent by mail to Michigan State University for analysis by the primary
investigator.

Risks and Benefits: There are no risks or discomforts, beyond routine dialysis,
associated with this research. There are no implied beneﬁts from participating in this
study.

Participant’s rights as Human Subjects of Research:

Research subjects are encouraged to contact Dr. David E. Wright Chair of the University
Committee on Research Involving Human Subjects, at Michigan State University (517)
355-2180, if you have any questions regarding your rights in this study.

Statement of Agreement: Please indicate your agreement with the contents of this
consent form by signing and dating below.

 

Signature of Research Participant Date

154

Consent Form

For Participants in the
Supplementing Hemodialysis Patients with L—Carnitine:
Effect on Clinical Parameters and Quality of Life

Investigators: Lorraine Weatherspoon PhD, RD
(517) 432-0813

Alison Steiber MS, RD
(517) 432-0870

Alan Davis, PhD
(616) 454-9960

Leslie Spry MD
1(800) 927-2618

Jay Wish, MD
1(2161844-3163

PROJECT DESCRIPTION:

We plan to study the effects of taking L-carnitine after dialysis treatments on
quality of life, parathyroid hormone concentrations, hemoglobin and
hematocrit concentrations, Epogen® dose, muscle cramps, and weight gain
between dialysis treatments. Previous studies have suggested the use of
carnitine decreases muscle cramping and promotes muscle functioning,
lowers Epogen® doses and raises serum hemoglobin and hematocrit ,
concentrations, lowers serum triglycerides and increases perceived quality of
life scores. Patients who can participate in this study are those who are
greater than 18 years of age, receive hemodialysis three times per week,
and have not taken L-carnitine within the last three months.

WHAT YOU WILL BE ASKED TO DO IF YOU PARTICIPATE:

Your voluntary participation in this study will entail the following:

1. Allowing the researchers access to your medical records to collect
data on Epogen® dose, weight changes, hypertonic solution use and
monthly laboratory results such as serum blood urea nitrogen,
parathyroid hormone, hematocrit. and hemoglobin.

1<<

2. Allowing a registered dietitian to perform 2 food histories on you at
the beginning of the study and the end, the food histories will take
approximately 15 minutes each,

3. Allowing a registered dietitian to perform a brief physical including
lightly touching your temples, shoulders, clavicle region. knees, and
calves at the beginning and end of the study, this brief physical will

. take approximately 10 minutes each time,

4. Allowing a social worker to administer a quality of life questionnaire at
the beginning and end of the study, the quality of life questionnaire
will take approximately 45 minutes to conduct and will include
questions regarding your feelings on interactions with family
members, friends and co-workers and your feelings on your-abilities to
perform various daily tasks,

5. Allowing a nurse to keep track of your muscle cramps and weight gain
while you are on dialysis and during the study time,

6. Allowing a nurse to take a portion of the routine blood drawn each
month to be analyzed for camitine concentrations.

7. During the time of the study, you would receive either a placebo or L-
camitine after each dialysis treatment. Both the placebo and the L-
camitine will be given in your dialysis arterial-venous access, with no
additional pokes with a needle required

You will be randomized to treatment or placebo. Randomization means

that you will be placed into a group by chance, like flipping a coin.

Neither you nor your study doctor will chose what group you will be in

and you will have an equal chance of being placed in either group.

Placebo refers to the group that will not receive camitine, and instead will

receive an inactive agent. There will be approximately 63 patients in each

group.

The study will take 6 months to complete and it will not cost you or your
insurance anything. The L-camitine will be provided free of charge as will
the laboratory tests for camitine. L-carnitine will given in doses of
20mg/kg of dry body weight/day. The pills will be taken with liquid
immediately following dialysis treatment on dialysis days and in the
morning with breakfast on non-dialysis days.

YOUR PARTICIPATION IN THIS PROJECT IS VOLUNTARY:
You may refuse to continue participation in this study at any time. There is
no penalty and your medical care will not be altered at all for withdrawing

from this study.

YOUR PARTICIPATION IN THE PROJECT WILL BE KEPT PRIVATE:

During and after this research, your privgy will be protected to the
maximum extent of the law. Results of this study will not identify any
individual. Each subject will be assigned a subject number and in analyzing

156

the results from this study the data will be entered by subject number
instead of the patients name to insure privacy. The bags which will be given
to the patients participating in this study will be labeled with the patients
name and subject number. All results will be kept in a locked file in the
primary researchers office where only the primary researchers have the key.
This study will be a double blind study where neither the primary researchers
nor the nurses will know which patient is receiving the L-carnitine and which
is receiving the placebo. Furthermore, the nurses administering the pills will
not be the researchers collecting the data; therefore they will not have
access to the individual results. The records and results of this study may be
accessible to representatives of the Food and Drug Administration and the
Combined Institutional Review Board.

Potential Benefits and Hazards:
RISK AND BENEFITS:

Some‘ potential benefits from the camitine supplementation may be
increased quality of life, decreased parathyroid hormone concentrations,
increased hemoglobin and/or hematocrit concentrations, decreased Epogen® ‘
dose, and decreased muscle cramping and fluid weight gain between dialysis
treatments. If desired, you may learn of the study results by asking the one
of the investigators for the results to available to you when they are

completed.

Taking L-carnitine long term may cause transient nausea and vomiting, body
odor and/or gastritis. Large doses of L-carnitine do not result in toxicity,
however may result in diarrhea. The dialysis process easily removes the L-
carnitine from the serum preventing toxicities. In a study with L-camitine
supplementation in hemodialysis patients 27 oUt of 62 of the patients
reported some flu-like symptoms, 35 out of 62 reported headaches, 27 out
of 62 reported sore throat, and 21 out of 62 reported high blood pressure, -
however it is not known whether these symptoms are related to the L-
camitine supplementation. Mild muscle weakness has been described only in
uremic patients receiving D, L-carnitine which will not be used in this study.
Seizures have occurred in persons with or without pre-existing seizure
activity, and an increase in seizure frequency has been reported. Valproic
acid is a medication given to children prone to seizures. Valproic acid has
been reported to lead to camitine deficiency. Therefore the children receiving
valproic acid for their seizures may also be receiving camitine therapy.
Therefore no reports of seizures by hemodialysis patients receiving camitine
therapy have been documented.

RISK'OF PHYSICAL INJURY:

157

If you are injured as a result of your participation in this research project, the
Dialysis Center of Lincoln will provide emergency medical care if necessary.
You will not be held responsible for any medical expenses incurred as‘a
result of this injury. All such medical expenses will be paid by Michigan State

University.

YOUR RIGHTS AS HUMAN RESEARCH PARTICIPANTS:

Participants in this study are welcome to call any of the study investigators
with questions. Phone numbers for Dr. Weatherspoon, Mrs. Steiber and Drs.
Davis and Spry are listed at the top of the Consent Form. You are
encouraged to contact Dr. Ashir Kumar, Chair of the University Committee
on Research Involving Human Subjects, at (517) 355-2180 or the Combined
Institutional Review Board in Lincoln, NE (402) 486—7144 with any questions
regarding your rights as a human subject raised by participation in this study.
You are encouraged to ask one of the investigators about any specific
questions regarding the research project.

STATEMENT OF AGREEMENT:

Please indicate your agreement with the contents of this consent form by
signing and dating below.

 

Signature Date

Witness Date

 

UCRIHS APPROVAL FOR
THIS project EXPIRES:

AUG 2 7on1

SUBMIT RENEWAL APPLICATION
ONE MONTH PRIOR TO
ABOVE DATE TO CONTINUE

158

Appendix B

159

Data collection Forms I and 11

Phase I

160

Data Collection Form I

 

 

 

 

 

Subject #:

Date:

Total: , Free: , Acyl-carnitine:

DOB: , Sex: (circle) male/female, Wt:__ kg, Ht: _in
Time receiving HD: (circle) months/years,

Treatment Time: (circle) minutes/hours

 

Live in Lincoln: yes / no,

if no: Travel > 1 hour to dialysis: yes / no,
Married: yes / no,

if no: Live alone: yes / no: Work: yes / no,

Latest SF36 Score: , History of ETOH Abuse: (circle) yes/no

 

Exercise for at least 1x/mo.: yes/no,
if yes: Ave RPEfor current week: , Ave PD for current week: ,

Blood Pressu_rg: Systolic: , Diastolic:

 

Labs: Alb: , BUN: , Chol: , Creat: , Ferritiu: ,

Hct: ,Hgb: , PTH: , TG: , URR: , Al:

 

 

 

 

Pre-alb.: , Iron: ,
Meds: circle any that apply:
oral contraceptives, beta-blockers, salicylates, steroids, thiazidic diuretics,

benzodiazepines, clonidine, cimetidene, eparin, thiroyd homes, phenylidantoyne;

161

EPO dose/treatment: , Length of time on EPO ;
List any lipid lowering

drugs:

 

Patient is on an Al-based phosphate binder: yes/no

Mannitol: yes / no,

if yes: Ave. dose over the past month? ,

Co-morbidities: circle any that apply:

Type 1 Diabetes, Type 2 Diabetes, Inflamatory Disease (Rheumatoid Arthritis,
Crohns, Ulcerative Colitis, Asthma, COPD), Lupus, Current Cancer, History of
Cancer, Current Infection, HTN,

other:

 

If yes for cancer,

type:

 

Cardiac dysfrgrctions:

 

CHF: yes/no,

LVH: yes/no; if yes: end diastolic diameter__, left artrial dialation
Ejection Fraction "/0

Other:

 

 

 

162

Dietitian:

Data Form 11

Phase I

Dietary 24 Hour Recall

 

Patient:

 

snacks

Afternoon:

snacks

Pm:

snacks

Totals: Protein:

163

, Kcals:

, Carnitine:

Data Collection Forms

Phase II

164

Subject Number: Date:

Anthropometric data:

TSF: Ave TSF: (cm)
MAC: (cm)

MAMC: MAC - (1r x TSF) =

Height

Weight BMI:

Demographic Data:

Age:
Primary etiology:

 

 

Date:

 

Co-morbldities: Check any that apply
. Hypertension

. Cardiovascular disease
. LVH

. Active Cancer

. History of Cancer

. Active Infection

. Crohn's

. Ulcerative Colitits

. Diabetes: type 1 or 2
10.Lupus

11. HIV/AIDS

12. Other.
EF ‘16 LAD:

 

 

 

 

 

 

 

 

OGNOJMJIwN-e

 

 

 

Dialyzer type: Duration of txr.
Dialysis start date:

Laboratory Values:
nPCR

BUN

PTH

Alb

TG

HDL (most recent draw)
Hct

Hgb

 

 

 

 

 

 

 

 

Medications:
Aspirin yes: no: .. :'
Lipid lowering Medications yes: -‘ > no.
(lipitor.tricor,zocor.questran....) yes:- “no: '
Epogen yes: ' no 1

dose ~ ' .. ‘

 

165

Date:

Daily Nursing flow sheet

Phase II Carnitine Trial

 

Day/Date :

Patient Number:

 

 

 

Patient Name:

Patient exercising? YES_______ NO

Pro-dialysis weight: _, Post-dialysis weight:
Patient receiving hypertonic solution? YES____ NO

 

If YES: Amount
Is patient having MUSCLE CRAMPING? YES , NO

If YES: NUMBER of cramps during dialysis:

 

Were the cramps (circle one):

MILD MODERATE STRONG VERY STRONG

Is patient on Epogen®? YES NO

If yes: Amount per- kg:

 

166

 

SF-36 HEALTH SURVEY

 

INSTRUCTIONS: This survey asks for your views about your health. Thls information wﬂl help keep track
of how you feel and how well you are able to do your usual activities.

Answer every question by marking the answer as indicated. If you are unsure about how to answer a
question. please give the best answer you can.

1. In general. would you say your health is:
' (circle one)

Excellent ................................................... 1
Very good .................................................. 2
Good ...................................................... 3
Fair ....................................................... 4
Poor ...................................................... 5

2. Compared to one year ago. how would you rate your health in general now?

. (circle one)
Much better now than one year ago ............................... 1’
Somewhat better now than one year ago ........................... '. -2
About the same as one year ago .................................. 3
Somewhat worse now than one year ago ........................... 4
Muchworsenowthanoneyearago..............; ................ 5

167

3. The following items are about activities you might do during a typical day. Does your hgeajthmow
limit you in these activities? it so. how much?

(circle one number on each line)

 

 

 

 

 

 

 

 

 

 

 

Yes. Yes. No, [Not
ACTiVlTiES ~ Limited Limited Limited
_ A Lot A Little At All
a. Vigorous activities. such as running. liiting heavy 1 2 3
objects. participatirﬂ in strenuous sports
b. Moderate activities. such as moving a table. pushing a 1 2 3
vacuum cleaner. bowing or playing golf
0. Lifting or carrying groceries 1 2 3
d. Climbing several ﬂights of stairs 1 2 3
e. Climbing one ﬂight of stairs 1 2 3
t. Bending. kneeling. or stooping 1 2 3
9. Walking more than a mile 1 2 3
h. Walking several blocks 1 2 3
1. Walking one block 1 2 3
j. Bathing or dressing yourself 1 2 3

 

 

 

 

 

 

4. During the get 4 wgeks. have you had any of the following problems with your work or other regular
daily activities as g result of your ghysicai health?

(circle one number on each line)

 

 

 

 

 

YES NC
a. Cut down on the amount at time you spent on work or 1 2
other activities
b. Accomplished less than you would like 1 2
c. Were limited in the kind of work or other activities 1 2
d. Had difficulty performing the work or other activities (for 1 2
example. it took extra effort)

 

 

 

 

 

 

168

5. During the past 4 weeks, have you had any of the following problems with your work or other regular
daily activities as a result of any emotional problems (such as feeling depressed or anxious)?

(circle one number on each line)

 

 

 

 

YES NO
a. Cut down the amount of time you spent on work or other activities 1 2
' b. Accomplished less than you would like 1 2
c. Didn't do work or other activities as carefully as usual 1 2

 

 

 

 

 

6. During the [Est 4 weeks. to what extent has your physical health or emotional problems interfered with
your normal social activities with iamily. friends. neighbors. or groups?
(circle one)

Not at all ............. i ...................................... 1
Slightly ..................................................... 2
Moderately .................................................. 3
Quite a bit .................................................. 4
Extremely .......... ' ......................................... 5

7. How much bodily pain have you had during the past 4 weeks?

(circle one)
None ...................................................... 1
Very mild ................................................... 2
Mild ....................................................... 3
Moderate ................................................... 4
Severe ......................................... ‘ ............ 5
Very severe ................................................. 6

169

a. mwmmmmmnwmmmmmwdondwwmmm

outeidethehomeendhoueework)?
(cirolaone)
Notltll ................................................... 1
Aliitlebit ................................................... 2
Moderately .................................................. 3
Oulteeblt .................................................. 4
Barentely ................................................... 5

9. mmmmmmwwmmmmmmw
memmwmmmmmmmmmmmmm.
mmummammm-

 

 

 

 

 

 

 

 

 

 

(cideonenmtberoneachﬂm)
All Iloet AGood Some ALIlile None
oithe ofthe allot oithe attire attire
Time Time theTime Time Time Time
a Dldyouieeinloipep? 1 2 3 4 5 o
b. Haveyoubeenevery 2
7 1 3 4 . 5 6
c. Haveyouielteodownin
thedunpettutnahhg 1 2 3 4 5 a
couldcheeryo'uupt?
d. Haveyouieitcelmand 6
1 2 3 4 6
peasant?
e. Didyouheveelatof
e 7 1 2 3 4 5 6
i. Haveyouielt
d I Hqu 1 2 3 4 5 . 6
g. Dldyouieelwomou’i 1 2 7 3 4 § 6
h. Haveyoubeenaheppy 1 2 3 4 5 5
person? '
l. Didyouiedtlred? 1 2 3 4 5 6

 

 

 

 

 

 

 

 

 

170

 

 

 

 

 

to. Duingthe howmuchoithetlmehaeyour
nederedwlthyou social activities (Ilrevielthgwlhinertdsrelatlves. etc)?
(circleone)
Aloithetkne 1
Moetdtheth‘ie ............................................. 2
Someoithetlme ............................................. 3
Aiittieofthetime ............................................ 4
Noneoithetime .............................................. 5
11. HawTRUEorFALSEIemoitheiolowhgmioryotﬂ
_ (choleonenunberoneaohline)
Deﬁnitely Mostly Don’t Mostly Deﬁnitely
True Ti'ue Know Pelee Pelee
. mamas 1 2 . 4 s.
b lamaeheaithyaearwodyl 1. 2 3 4 5
“~ 'm‘wwww 1 2 s 4 5
worse
d. Myhealthhemceliert 1 2 3 4 5

 

 

 

 

 

 

 

 

171

Date:

24 hour recall ”Multigie Pass Method"

. Sibject mutter:

 

Food Consumed

 

172

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