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The Vitamin E Status of Hemodialysis Patients

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Patricia Smith Brown

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THE VITAMIN E STATUS OF HEMODIALYSIS PATIENTS

By

Patricia Smith Brown

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1980

ABSTRACT
THE VITAMIN E STATUS OF HEMODIALYSIS PATIENTS
By

Patricia Smith Brown

Serum total vitamin E, serum total vitamin E:Serum
total lipid ratio, and erythrocyte hemolysis were used to
compare the vitamin E status of 20 patients with chronic
renal failure being treated with hemodialysis and 20
healthy, age and sex matched controls. Subjects showed no
evidence of nephrotic syndrome, diabetes or androgen therapy
and were within :l5% of ideal weight. Two 24-hour dietary
records for each subject were evaluated for intake of
a-tocopherol and polyunsaturated fat. Values for serum
total vitamin E and the serum total vitamin Ezserum total
lipid ratio were within normal limits and there was little
difference between patients and controls. The erythrocyte
hemolysis test did not appear to be a valid indirect assess-
ment of vitamin E status in hemodialysis patients, probably
because of interference by uremic toxins. Positive
correlations were shown between serum total vitamin E and
serum total lipids and between intakes of a-tocopherol and
polyunsaturated fat. There was little correlation between
dietary intake of aetocopherol and serum total vitamin E.
From this study it appears that the vitamin E status of

individuals with chronic renal failure treated with

Eatricia Smith Brown
hemodialysis is normal as assessed by absolute serum levels
of total vitamin E and the serum total vitamin Ezserum
total lipid ratio. The results suggest that the vitamin
E requirement of these hemodialysis patients can be met by

their usual dietary intakes without the use of supplements.

ACKNOWLEDGEMENTS

I wish to express my Sincere appreciation and gratitude
to Dr. Wanda Chenoweth for her expertise, assistance and
encouragement; to Drs. James A. Greene, III and Hi Sung Park
for their guidance and support of this study; to Drs. Jenny
Bond and Maurice Bennink for their insight and assistance;
and to Dr. John Gill for his statistical advice. I would
also like to extend special thanks to my parents for their
encouraging my education and to my husband, George, for his

understanding and constant support.

ii

TABLE OF CONTENTS

LIST OF TABLES.
INTRODUCTION.
REVIEW OF LITERATURE.

Vitamin E. . .
Chronic Renal Failure.

MATERIALS AND METHODS

Subject Recruitment.
Informed Consent . . .
Subject Characteristics.
Nutrient Intake. .
Collection of Blood Samples.
Blood Analysis . . .
Rat Study. . .

Statistical Methods.

RESULTS
Nutrient Intake.
Blood Analysis
Rat Study.
DISCUSSION.
Subject Characteristics.
Nutrient Intake.
Blood Analysis
Rat Study.
CONCLUSIONS .

LIST OF REFERENCES.
APPENDICES.

Page

iv

Table

450)

A6
A7

LIST OF TABLES

Summary of patient characteristics.
Summary of control subject characteristics.
Average daily dietary intake of subjects.

Erythrocyte hemolysis, serum total vitamin E and
total lipids. . .

Composition of vitamin and mineral supplements
per capsule/tablet. . . . . . . . .

Routine blood analyses for patients

Composition of purified rat diet.

iv

Page
38
39
47

SI

80
8T
88

INTRODUCTION

The nutritional status of a patient with chronic renal
failure who is being treated with hemodialysis plays a
major role in predicting the outcome of the illness. Con-
sequently there is considerable interest in determining
abnormalities in nutrient parameters which are secondary
to the disease process or its treatment. The nutritional
status of hemodialysis patients has been studied with
respect to several vitamins, while other vitamins, such as
vitamin E, have received little attention. Until recently
there was no reason to expect abnormalities in the fat-
soluble vitamins and research centered around the removal
of the water soluble vitamins in the dialysate fluid. With
the discovery of abnormalities in the metabolism of vitamins
A and D in chronic renal failure it is possible that the
metabolism of other fat-soluble vitamins is altered in the
hemodialysis patient.

The function of vitamin E in humans cannot be stated
with certainty but may involve its possible antioxidant
properties or metabolic regulating activities. It is
possible that these functions may have implications for the
hemodialysis patient. There are a number of complications

accompanying uremia and the hemodialysis process, such as

lipid abnormalities, anemia, and a number of nutrient
imbalances that may have some relation to vitamin E status.
The experiment described herein was designed to study
the effect of selected dietary and serum components on the
vitamin E status of free-living adults with chronic renal

failure being treated with hemodialysis.

REVIEW OF LITERATURE

Vitamin E

Function A

The function of vitamin E at the metabolic level espe-
cially in man remains an enigma. After many years of work
in the area, investigators cannot agree on the function of
vitamin E. Some researchers contend that the vitamin is
primarily an antioxidant for polyunsaturated fatty acids
(PUFA) in tissues. Others argue that it must be a component
of an enzyme or transport system and is therefore primarily
metabolic in function (Scott, l978). The action of vitamin
E will continue to be the subject of future research.

The antioxidant theory hypothesizes that unsaturated
lipids are under attack by free radicals and when oxygen
is present the lipids undergo peroxidation. Vitamin E
reacts with the free radicals in vivo and inhibits peroxi-
dation. If vitamin E is not present in sufficient amounts
the peroxidation becomes uncontrolled and leads to damage
of intracellular membranes and enzymes. Therefore the
diverse effects of vitamin E deficiency may be secondary to

lipid peroxidation (Green, l972).

Because of the technical difficulties in measuring the
minute amounts of actual peroxides in tissues, the basis of
proof of the antioxidant theory rests on the in vitro tests
quantifying the susceptibility of red blood cells (RBC) to
hemolysis and malonyldialdehyde (MDA) formation from the
degeneration of lipid peroxides (Green, l972). The crux of
the matter is if lipid peroxidation in vitro can be
considered to be representative of peroxidation in vivo.

The role of vitamin E and selenium in the peroxidation
of lipids is often confused because selenium has been
effective in treating vitamin E responsive diseases in
animals (Burk, l976). However, a requirement of selenium
in some species has been shown even when adequate vitamin
E is supplied in the diet. The functional difference is
that selenium is part of the enzyme, glutathione peroxi-
dase (Rotruck et al., 1973), which reduces lipid peroxides
to hydroxy acids (Christophersen, l968a, 1968b). while the
proposed antioxidant function of vitamin E is to prevent
the formation of lipid peroxides. The biochemical proper-
ties of selenium are similar to those of sulfur (Li and
Vallee, l980). In vitamin E deficiency glutathione peroxi-
dase detoxifies in vivo lipid peroxides. Therefore the
selenium in this enzyme spares vitamin E by preventing the
decomposition of peroxides to free radicals that could rein-
itiate peroxidation. Some signs of vitamin E deficiency

that are dependent on dietary PUFA do not respond to

selenium, such as erythrocyte hemolysis, perhaps because
the aqueous enzyme cannot reach the cellular Site of
peroxidation or the peroxides of PUFA may decompose quickly
before the enzyme can interfere. If glutathione cannot be
formed in RBC, hemolytic anemia will result. Vitamin E
therapy has been successful in improving erythrocyte survi-
val in deficiencies of glutathione synthetase (Spielberg

et al., l979). Therapy with vitamin E in a neonate de-
creased the symptoms of glutathione peroxidase deficiency
without altering the levels of the enzyme (Boxer et al.,
l979).

The RBC hemolysis test has been utilized to demonstrate
the peroxidation of RBC lipids in numerous studies, some of
which will be reviewed later. Horwitt et al. (l968) has
also attempted to verify the antioxidant theory by showing
that adult men receiving 3 mg of toc0pherol per day demon—
strated increasing RBC hemolysis as the fat content of their
diet progressed from 30 gm of lard to 30 gm of oxidized corn
oil and finally to 60 gm of oxidized corn oil over a period
of six years. During that time plasma toc0pherol concen-
trations gradually declined displaying an inverse relation-
ship to the RBC hemolysis.

Further support for the antioxidant theory is found in
the work of Folkers (T974) who Showed epoxidation of lipoi-
dal coenzyme Q in vitamin E deficient rabbits thus suggesting

that vitamin E protects the highly unsaturated lipid,

coenzyme Q, from oxidation.

Lucy (l972) hypothesizes that vitamin E inhibits the
oxidation of non-heme iron-containing proteins found in
the mitochondria and smooth endoplasmic reticulum of rat
livers. Diplock (l974) demonstrated that oxidation-labile
non-heme iron was present in rat livers only when both
vitamin E and selenium were available in the diet. The
function of the non-heme iron-containing proteins in
microsomal electron transfer systems which are responsible
for drug metabolism was investigated. The demethylation
of drugs was altered in vitamin E deficient rats. Diplock
proposed that during vitamin E deficiency the selenium
protein may be replaced by a sulfur protein which would
change the kinetics of the associated demethylase system.
In related work Carpenter and Howard (1974) investigated
the effect of vitamin E and androgens on the enzyme systems
bound to the endoplasmic reticulum in rat livers. They
showed that the effect of vitamin E on hepatic microsomal
hydroxylations of drugs is independent of the androgen

status of the rat.

The lack of evidence of lipid peroxides in tissues has
led to research which openly questions the antioxidant

theory and consequently has resulted in many interesting

discoveries of proposed metabolic functions. Vitamin E is
thought to play a role in heme synthesis (Nair, 1972) by
controlling the induction and repression of the rate
determining enzymes aminolevulinic acid synthetase and
aminolevulinic acid dehydratase. The heme protein, cata-
lase, may catalytically scavenge peroxides created by the
oxidation of lipids. Caasi et al. (1972) support this
idea with their work on the effect of vitamin E deficiency
on heme proteins, which showed that catalase activity
decreased significantly when rats were fed a vitamin E
deficient diet.

Many other metabolic functions for vitamin E have been
postulated recently. Kawai.et a1. (1974) showed that the
addition of vitamin E to brain cell enzyme preparations

inhibited cell membrane ATPaseS in vitro. Their results
suggest the possibility of a vitamin E enzyme function
because the antioxidant, BHT, did not produce a similar
ATPase inhibition when it was added to the cell prepara-
tions. Tocopherol has been shown to promote larger body
sizes of rotifers when included in the culture medium
(Gilbert, 1974). This was the first report of a vitamin E
stimulated growth response. The conversion of vitamin 812
to its coenzyme form may be inhibited by a vitamin E
deficiency (Pappu et al., 1978). Olson (1974) has tested
the hypothesis that vitamin E regulates the synthesis of

specific proteins necessary for muscle function and found

that the turnover of creatinine phosphokinase was twice the
normal rate in vitamin E deficient rabbits.

Another function of vitamin E relates to its interaction
with vitamin A. Green (1972) suggests there are four ways
for vitamin E to exhibit a sparing effect on vitamin A:

1) protect vitamin A in the gut from oxidation, 2) increase
vitamin A absorption, 3) increase vitamin A utilization, and
4) increase storage of vitamin A. The effects of this
complex relationship depend on dosage of the vitamins, diet,
and the length of the experimental period. In studies with
rats fed a diet deficient in vitamins A and E, Sondergaard
(1972) showed that the addition of vitamin E to the diet
maintained approximately 90% of liver stores of vitamin A.
When other antioxidants were given with the vitamin A
deficient diet this effect was not seen. In situations where
the intakes of both vitamins A and E are excessive, vitamin E
tends to alleviate the toxicity of vitamin A (McCuaig and
Motzok, 1970; Jenkins and Mitchell, 1975). The latter
authors have also shown an interaction between vitamin A and
E when given in excess amounts such that each appears to
cancel the negative effects of the other.

While it appears that there is a sizable body of lit-
erature against the antioxidant theory, new research makes
a strong argument to renew the controversy. With the
availability of a new technique, expired air from a subject

can be analyzed by gas chromatography for ethane and

pentane which evolve from the peroxidative decomposition
of linolenic and linoleic acids (Riely et al., 1974).
This is the first in vivo method for determining peroxida-
tion of lipids. Hafeman and Hoekstra (1977a) have vali-
dated the method by showing that supplements of vitamin E,
selenium, or methionine when added to a diet deficient in
all three will protect against peroxidation as evidenced
by ethane evolution when rats are injected with carbon
tetrachloride (CC14). In continued research (1977b) these
authors have shown similar effects with vitamin E and
selenium deficient diets without the use of CC14.
Deficiency

A deficiency of vitamin E is rarely seen in humans
except for premature infants and children or adults with
failure to absorb fats. However, deficiency states can be
easily induced in animals producing avarjety of symptoms
(Bieri, 1975). The contrast between man and animal can be
explained by their respective nutritional intakes. Animals
are restricted in the variety of foodstuffs consumed as com-
pared to man. In addition, animals can be raised from
infancy in the vitamin E deficient state, which would never
be considered in humans. It is highly unlikely to be able
to induce a vitamin E deficiency in healthy human adults
because of the tissue stores of this fat-soluble vitamin.

Fitch (1972) contends that there may be three mecha-

nisms involved in the etiology of the anemia seen in

l0

conjunction with vitamin E deficiency, depending on the
species involved. The mechanisms are: 1) blood loss,
possibly by effusion of blood into tissues that may occur
in poultry, 2) oxidant hemolysis as demonstrated by the

RBC of premature human infants, and 3) ineffective erythro-
poiesis as seen in bone marrow abnormalities of rhesus
monkeys.

The increased susceptibility of RBC hemolysis in
vitamin E deficiency is easily quantified and is reported
frequently in the literature. The RBC hemolysis test
involves the incubation of washed erythrocytes in solution
with an oxidant such as hydrogen peroxide or dialuric acid.
The resulting hemolysis is quantitated by measuring
colorimetrically the hemoglobin released and comparing it
to that released in distilled water by red blood cells
from the same sample. The results are expressed as a
percentage. Erythrocytes incubated in buffered saline
may be incubated also and used as controls (Sauberlich
et al., 1974).

Leonard and Losowsky (1967) compared the results of RBC
hemolysis and plasma vitamin E levels in 233 ill and
healthy subjects and found that approximately 90% of the
subjects with hemolysis greater than 10% had plasma vitamin
E levels of less than 0.3 mg/100 ml. In addition to docu-
menting the effects of hydrogen peroxide (H202) concentra-

tion, its rate of addition, time, and temperature on the

ll

RBC hemolysis test, Horwitt et al., (1956) showed when 19
human males were fed a vitamin E depleted diet over a 20
month period that there was an inverse relationship

between plasma vitamin E and the percent of RBC hemolysis.
Stocks and Dormandy (1971) examined the autoxidation of

RBC from healthy adult humans by measuring the formation of
malonyldialdehyde (MDA), a product of polyunsaturated fat
oxidation. This in vitro technique has been criticized

for not being representative of in vivo reactions; however,
the same criticism can be made for the RBC hemolysis test.
Their results showed that the concentration of Specific
antioxidants such as o-tocopherol or BHA had only a partial
effect on MDA formation when RBC were exposed to oxidative
stress and that therefore there may be a multitude of
intra- or extra-cellular factors affecting the suscepti-
bility of cells to autoxidation.

One of these other factors that has been examined in
detail is the effect on RBC hemolysis of fatty acid compo-
sition, both within the erythrocyte and as taken in the
diet. When rats were fed either corn, cottonseed, or
hydrogenated coconut oils as 30% of the diet or fat-free
diets each with or without toc0pherol supplements, the
rats consuming the unsupplemented corn oil, hydrogenated
coconut oil, or fat-free diets demonstrated RBC hemolysis
greater than 50% (Alfin-Slater et al., 1969). Brin et al.
(1974) studied the effect of erythrocyte fatty acid levels

12

on erythrocyte hemolysis in rats and rabbits. They showed
that in RBC membranes arachidonate is oxidized preferen-
tially over linoleate and because rabbits have lower levels
of arachidonate than rats, the RBC of rabbits were rela-
tively more resistant to RBC hemolysis.

Gyorgy et a1. (1952) have shown that in the normal
newborn infant, some hemolysis (approximately 30%) is seen
in the first few days of life, but the levels of hemolysis
begin to approach normal by the fifth day. In the premature
infant, the supplementation of 25 to 150 mg daily of
a-toc0pherol acetate at 2 to 4 weeks of age changed the
results of the RBC hemolysis test from strongly positive
to negative (Gordon and deMetry, 1952). Gordon et a1.
(1955) in further studies, examined erythrocyte hemolysis
levels in full term and premature infants at birth and
7 weeks of age. There was no significant difference in the
two groups at birth, however at 7 weeks the full term
infants, especially those that had been breast fed, had
significantly less hemolysis than the premature infants.

Because iron catalyzes the autoxidation. of unsaturated
fatty acids (Smith and Dunkley, 1962) its routine use in
the treatment of the anemia of premature infants was inves-
tigated by Melhorn and Gross (1971). Premature infants
were divided into 4 study groups as follows: vitamin E
supplemented, iron supplemented, vitamin E and iron supple-

mented, or no supplementation and followed for 16 weeks.

13

The lower the birth weight the more pronounced was the
effect of increasing RBC hemolysis in any of the four
groups. The group receiving iron supplements had the
greatest degree of hemolysis followed by the group receiving
no supplements, followed by the group receiving vitamin E
and iron supplements, and followed by the group receiving
vitamin E supplements in order of decreasing hemolysis.
Those infants receiving only vitamin E demonstrated the
highest serum vitamin E levels, especially those With

the lowest birth weights. This research was extended

by Williams et al. (1975) who examined the fatty acid
composition as well as the iron levels in formulas being

fed to premature infants. High and low levels of both

PUFA and iron were given in four combinations. Those
infants receiving high levels of both PUFA and iron were

the only group to demonstrate hemolytic anemia as evidenced
by RBC hemolysis. The use of intramuscular vitamin E in

the premature infant has been observed to prevent high
levels of RBC hemolysis in most cases if given in sufficient
amounts (Graeber et al., 1977).

In addition to the anemia associated with the vitamin E
deficiency in premature infants, other conditions may be
related to the Vitamin deficiency in infants. Retrolental
fibroplasia, a disease of growing blood vessels in the eye.
occurs in premature infants as their retinal vessels (which

are sensitive to oxygen tension) are not yet mature.

14

Johnson et a1. (1974) have reported that treatment with
intramuscular vitamin E appears to decrease the incidence
and severity of retrolental fibroplasia when compared with
a placebo treatment. Bronchopulomonary dysplasia is the
toxic result of exposure of high concentrations of oxygen
to the lungs which may occur with the acute respiratory
distress syndrome of premature infants. Intramuscular
injections of vitamin E were reported to have no statistical
effect in decreasing the severity of the disease (Ehren-
kranz et al., 1978), but the infants receiving vitamin E
generally required less respiratory therapy.

Melhorn and coworkers (1971) have demonstrated that
anemias which are considered hemoltyic in nature, such as
sickle cell disease, autoimmune hemolytic anemia, erythro-
blastosis fetalis, and iron deficiency will give abnormal
values for RBC hemolysis. Anemias due to bone marrow
abnormalities, however, such as aplastic anemia, juvenile
pernicious anemia, folic acid deficiency anemia, and
thalassemia are associated with minimal or no RBC hemolysis.
Serum vitamin E levels were reported to be within the normal
range for all anemias in both classifications.

Other workers are in disagreement, however, having
found lower levels of serum vitamin E in some of the
anemias that Melhorn's group classified as hemolytic.

Fujii and Shimizu (1973) found significantly lower values

for serum vitamin E, although still within the normal

l5

range, in subjects with iron deficiency anemia when com-
pared with controls. Natta and Machlin (1979) using a
plasma toc0pherol criteria of 0.8 mg/gm of total lipid
found that 75% of sickle cell anemia patients were deficient
in vitamin E as compared to none of the controls. These
deficient patients responded to oral supplementation of
DL-o-tocopheryl acetate. Additional work (Natta et al.,
1980) showed that supplementation of a-tocopherol increases
plasma levels of the vitamin and decreases by approximately
one half the percentage of circulating irreversibly sickled
cells. Therefore vitamin E may exhibit a stabilizing effect
on the erythrocyte membrane.

Cystic fibrosis is associated with malabsorption of
fats that may result in vitamin E abnormalities. This
hypothesis was investigated by Farrell and coworkers (1977)
who found that cystic fibrosis (CF) patients who were not
supplemented with vitamin E were indeed deficient as
evaluated by plasma o-tocopherol and RBC hemolysis levels.
The percent of hemolysis was related to o-toc0pherol con-
centration in an inverse, sigmoidal manner. In vivo hemoly-
sis was assessed by measuring the decreased survival of
51Cr labeled erythrocytes in the CF patients. Oral supple-
mentation of o-toc0pheryl acetate restored plasma concen-
trations of the vitamin to within normal limits and

improved the lifespan of labeled erythrocytes.

l6

Vitamin E requirement

 

The vitamin E requirement of a species depends on
numerous variables including: efficiency of fat absorption,
age, dietary levels of pro-oxidants, antioxidants, seleni-
um, PUFA, sulfur amino acids and other fat-soluble vitamins
(Scott, 1978). Various authors have hypothesized recommen-
dations for vitamin E requirements, usually in relation to
dietary fat. It has been recommended that the intake of
toc0pherol should be 1 mg for every 60 mg of PUFA consumed
(Harris and Embree, 1963). Witting (1972) has criticized
this level as impractical as it is difficult to attain in
normal mixed diets, although he did agree that the vitamin
E requirement is related to the tissue PUFA content. With
the trend to replace vegetable fat for animal fat in the
American diet, Bieri and Evarts (1973) found that y-toco-
pherol was the predominate form of toc0pherol in typical
American intake. This was due to the increased consumption
of corn and soybean oils which are excellent sources of
y-tocopherol. They suggested that more than the o-content
of diets needed to be considered in the requirement. At the
time the y-form was considered to be poorly utilized, but
shortly thereafter the same authors (1974) investigated the
absorption of the y—form in rats and saw no marked difference
between its absorption and that of the o-form. However,
they did note that its rate of tissue turnover was twice as

great as that for o-tocopherol.

l7

The four tocopherols and four tocotrienols have been
considered as food sources of vitamin E, although o-toco-
pherol has the highest vitamin E activity in man (Bauern—
fiend, 1977). Biological activity of the different vitamers
is difficult to assess in humans due to a lack of technology,
so tests with animals are generally accepted as indicators
of activity. Different species will give a variety of
different values (McLaughlin and Weihrauch, 1979).

According to the Food and Nutrition Board's (FNB) current
Recommended Dietary Allowances (RDA) of 1980 the correct
designation for intake of vitamin E is now milligrams of
o-tocopherol equivalents. This term is derived as the sum
of: the milligrams of o-tocopherol, the milligrams of
B-tocopherol multiplied by 0.5, the milligrams of y-tocopherol
multiplied by 0.1, and the milligrams of o-tocotrienol
moltiplied by 0.3. These are the only vitamers believed to
have significant biological activity which are present in
American diets. If only the o-tocopherol content is known,
the value should be increased by 20% to account for the other
vitamers, resulting in an estimate of<x—tocopherol equivalents.

Horwitt (1974) proposed a formula which recommends that
individuals should receive 4 mg of o-tocopherol equivalents
per day even if they consume no PUFA to compensate for tissue
synthesis of peroxidizable compounds. Horwitt's formula for
the requirement of vitamin E in o-tocopherol equivalents is:

4crtocopherol + 0.25 (% PUFA of total lipids + gm of PUFA)
equivalents

18

This formula is based on earlier research of Horwitt (1962)
where the levels of linoleate in depot fats tended to
approach the percentage in the diet if it was fed over a
sufficiently long period. Witting and Lee (1975a) suggest
that dietary requirement Should be related to tissue
linoleate levels which can be easily obtained and measured.
Based on the 1974 Recommended Dietary Allowances (RDA) a
ratio of 0.6 mg of dietary vitamin E activity per gm of
linoleate in 100 gm of adipose tissue fatty acids would
result in adequate vitamin E nutriture. The current RDA
(1980) are given as 8 and 10 mg of a-tocopherol equivalents
for adult females and males, respectively.
Intake of Vitamin E and Fat

It is often difficult to assess the vitamin E content
of foods if direct analysis is not available and one must
rely on tables of food composition because there is a lack
of published information on this vitamin. In addition,
tables of composition will demonstrate considerable vari-
ability in the nutrient content of different samples of
the same food depending on soil composition, degree of
maturity, trimming of edible portion, variety or strain,
length of storage, milling and other processing such as
canning or cooking (Watt, 1980). In a study of vitamin
E intakes of subjects in the United Kingdom, poor cor-

relation was found between values of intakes estimated

19

from food tables arui values from actual analysis of the
food (Smith et al., 1971).

Recent analyses have updated the literature of vitamin
E content of foods especially for the various tocopherols
in vegetable oils (Yuki and Ishikawa, 1976), canned and
vended foods (Koehler et al., 1977), as well as a wide
variety of food stuffs (Bauernfeind, 1977; McLaughlin and
Weihrauch, 1979).
I Bunnell and coworkers (1965) have analyzed the o-toco-
pherol content of a typical daily intake of food that might
be consumed and found it to range from 2.6 to 15.4 mg with
an average of 7.4 mg. Bieri and Evart (1973) analyzed
cafeteria meals for alpha, gamma, delta and total toco-
pherols, fat, PUFA, and calories. They reported daily ‘
values of 9.0 mg a-toc0pherol, 38.1 mg total toc0pherol,
118 gm fat, 21.2 gm PUFA, and a o-tocopherolzPUFA ratio of
0.42. In a survey of food intakes of dentists and their
wives, the men consumed an average of 45 o-tocopherol
equivalents (converted) with a range of 4 to 146 equivalents
while the women consumed 41 o-toc0pherol eQuivalents with
a range of 3 to 143 equivalents as judged by food frequency
data compiled by computer (Cheraskin et al., 1974).
Witting and Lee (1975b) analyzed typical meals served in
the cafeteria of a women‘s university and found average
intakes of 7.5 mg o-tocopherol, 28.7 mg total tocopherols,
96 gm fat, and 19.9 gm PUFA for a typical 2500 kcal daily

20

intake.

Intake does not necessarily reflect absorption.
Losowsky et a1. (1972) in studies utilizing rats and
humans showed that vitamin E deficiency, amount of fat
intake, or vehicle of administration did not affect absorp-
tion of a-tocopherol, although the percent of absorption
did decrease with increasing doses of the vitamin.

Analysis of Vitamin E

 

To preface the section on vitamin E status,a discussion
of the various methods of analyzing the vitamin E concen-
tration in blood will be presented. The Emmerie-Engel
(1938) colorimetric method later modified by Quaife et al.
(1949) has been used for many years. Fluorometric methods
have been utilized by others (Duggan, 1959; Hansen and
Warwick, 1966; Thompson et al., 1973; and Taylor et al.,
1976). Most recently high performance liquid chromatography
(HPLC) techniques have appeared in the literature (Hatam
and Kayden, 1979; and Bieri et al., 1979). The Emmerie-
Engel reaction depends on the reduction of Fe3+ which may
be non-specific if other reductants are present. The
fluorometric methods have the advantages of sensitivity
and specificity but are not applicable to differential
analysis of tocopherols. When compared with the Emmerie-
Engel reaction, Duggan (1959) found the fluorometric method
to give slightly lower results. Thompson compared the

fluorometric method with both colorimetric and column

21

chromatography methods and also reported the fluorometric
method gave slightly lower results than the Emmerie-Engel
reaction. However, the chromatography method indicated
that both the colorimetric and fluorometric methods had a
tendency to overestimate the concentration of vitamin E.
With the advent of HPLC techniques the separation of the
tocopherols is possible in a method that is claimed to be
rapid, free from interferring impurities and useful with
small samples (Bieri et al., 1979).
Vitamin E Status

There are numerous reports in the literature of blood
vitamin E and lipid levels in American subjects. In 197
adult subjects of both sexes Harris et a1. (1961) found a
mean total plasma toc0pherol level of 1.05 mg/100 ml i 0.32
with a range of 0.36 to 1.80 mg/100 ml using a modified
Emmerie-Engel method. Bieri and coworkers (1964) reported
mean serum tocopherols of 1.06 r 0.25 mg/100 ml for 71 men
and 1.04 i 0.27 mg/100 ml for 61 women by the Emmerie-Engel
reaction where one third of the subjects were Black and two
thirds were Caucasian. Rubenstein et a1. (1969) investi-
gated serum vitamin E and various serum lipid components
in 27 normal subjects and reported mean vitamin E levels
of 1.14 i 0.30 mg/100 ml with no significant difference

between sexes. The vitamin E values showed a correlation

of r 0.85 with serum total lipids. In plotting the

previously mentioned values for patients with hyperlipemia,

22

the authors concluded that a serum vitamin E level exceeding
2.3 mg/100 ml usually indicates an elevated serum total
lipid level. Using identification of o-toc0pherol by
combined gas liquid chromatography, mass spectrometry, and
infrared spectroscopy, Siakotos and coworkers (1974) found
a mean level of 0.49 t 0.08 mg/100 ml a-tocopherol with a
range of 0.39 to 0.64 mg/100 ml in nine normal unsupple-
mented adults. Plasma o—tocopherol determinations by thin
layer chromatography showed mean values of 0.96 mg/100 ml
with a range of 0.66 to 1.50 mg/100 ml in twelve normal
subjects (Chow, 1975), while the mean total vitamin E
value was 1.15 mg/100 ml. Using a fluorometric method,
Hansen and Warwick (1966) reported mean values of total
toc0pherol of 1.85 t 0.53 with a range of 1.00 to 3.10 mg/
100 ml for adults less than 40 years of age and 1.45 t 0.37
with a range of 0.m)to 2.30 mg/100 ml for adults older
than 65 years. Blood samples from 26 female college stu-
dents demonstrated total plasma toc0pherol levels of 1.09 i
0.25 mg/100 ml with a range of 0.73 to 1.76 mg/100 ml when
analyzed by thin layer chromatography (Witting and Lee,
1975). In another study of college women (Yeung, 1976) a
mean value of 0.87 t 0.07 mg/100 ml of vitamin E was seen
in twelve subjects using the spectrofluorometric method
of Thompson et al. (1973).

Horwitt et a1. (1972) examined the nature of serum

tocopherolzserum total lipid relationships. After comparing

23

the plots of serum toc0pherol with serum cholesterol, tri-
glycerides, phospholipids, and total lipid levels, they
concluded that the plot of total toc0pherol versus total
lipid produced the most useful indicator of vitamin E
status. A ratio less than 0.8 mg of total toc0pherol per
gm of total lipid in serum should be considered as an indi-
cator of inadequate vitamin E nutriture. In a sample of
22 healthy, young adults (21 to 35 years of age), Farrell
and coworkers (1978) found concentrations of 0.79 i 0.04
mg/100 ml for total tocopherols by a modified Emmerie-Engel
technique, 549 i 14 mg/100 ml for total lipids by a tur-
bidimetric method and a tocopherol:lipid ratio of 1.38 i
0.08 mg/gm. Twenty adult males on a neurology ward demon-
strated serum o-tocopherol levels of 1.17 i 0.10 mg/100 ml
by liquid chromatography, serum total lipid levels of
685 i 46 mg/100 ml by a turbidimetric method, and a toco-
pherol:lipid ratio of 1.71 s 0.13 mg/gm (Vatassery and '
Chiang, 1979).
Status and Requirement of Vitamin E in Rats

The National Academy of Sciences (Board on Agriculture

and Renewable Resources, 1978) has recommended the require-

ment for vitamin E in the rat can be met by 30 mg of
DL-o-tocopheryl acetate (40.5 mg D-o-tocopheryl acetate)
per kilogram of diet with a linoleic concentration of up
to 5% when adequate Sulfur containing amino acids and

selenium are available in the diet.

24

Plasma levels of toc0pherol in the rat vary depending
on the amount and type of fat in the diet in addition to the
intake of toc0pherol. Rats fed soybean oil containing 15
and 108 mg/100 ml of o- and y-tocopherols, respectively, had
plasma concentrations of 0.17 and 0.10 mg/100 ml of a- and
y-tocopherols, respectively (Bieri and Evarts, 1975a).

Stock rats from the same laboratory had plasma o-tocopherol
concentrations of 0.2 to 0.4 mg/100 ml. The ingredients of
the diet for the stock rats were not reported. Female
Zucker rats that had been vitamin E depleted for two weeks
and then repleted with 40 mg of DL-a-tocopheryl acetate/kg
of diet had plasma a-tocopherol levels of 0.71 mg/100 ml
(Bieri and Evarts, 1975b). Organs that had high concentra-
tions of toc0pherol were the liver, heart, and lungs. Rose
and Gyorgy (1952) have shown that female Sprague-Dawley rats
are sufficiently depleted after two weeks on a vitamin E
deficient diet to show positive results for the erythrocyte
hemolysis test.

Therapy with Vitamin E and Use of Excessive Doses

In addition to the previously mentioned uses of vitamin
E for retrolental fibroplasia, cystic fibrosis, broncho-
pulmonary dysplasia, sicklecell anemia and hypervitaminosis
A, it has been shown effective in improving walking toler-
ance and arterial blood flow to the lower legs of patients
suffering from intermittant claudication (Haeger, 1974).

In a double blind study investigating the effects of

25

megavitamin E therapy on angina pectoris, however, no sig-
nificant effects were seen (Anderson and Reid, 1974).

The possibility of negative effects of megavitamin E
doses have been questioned for some time. Recent studies
continue to Show inconclusive results. In vitro tests of
leukocyte activity have shown depressed results when human
subjects received 300 mg of DL-a-tocopheryl acetate for
three weeks while in vivo tests have shown no suppression
(Prasad, 1980). In a large double blind study with 200
subjects, half of whom received 600 mg of DL-o-tocopheryl
acetate daily, there were no positive or negative effects
of supplementation on general health, although there was a
significant reduction of thyroid hormone levels and an
increase in triglyceride levels in females (Tsai et al.,
1978). In another study (Farrell and Bieri, 1975) 28
subjects who had been ingesting 100 to 800 mg per day of
vitamin E for an average of three years showed no evidence
of toxicity as assessed by a battery of twenty standard

clinical blood assays.

Chronic Renal Failure
Overview
Chronic renal failure (CRF) is a major disease process
characterized by acid-base disturbances, anemia, abnormal
calcium-phosphorus metabolism,electrolyte and fluid imbal-

ances, catabolism, and anorexia due to the reduced capacity

26

of the kidney to perform its functions. Kidney function
is impaired by glomerular, tubular, vascular or inter-
stitial damage to the organ (Geschickter and Antonovych,
1971). When an individual with chronic renal failure has
less than 10% of his kidney function remaining, transplan-
tation of a kidney or dialysis must be instituted to
prevent death. As of 1978 there were approximately 35,000
individuals in the United States receiving dialysis treat-
ments (Wineman, 1978). Glomerulonephritis is the primary
disease leading to dialysis in 42% of the population.
Other major causes are cardiovascular disease and hyper-
tension, 14%; urinary tract disease, 11%; congenital ab-
normalities, 8%; diabetes mellitus, 7%, kidney infections,
6%; and unknown causes, 12%. In recent years the age of
the typical dialysis patient has been gradually increasing
due to the tendency to transplant younger patients as
well as broadened patient acceptance criteria for dialysis
patients. In many dialysis centers the mean age exceeds
50 years of age.
Anemia

As early as the 1950's the dual mechanism of the anemia
of CRF was recognized. Desforges and Dawson (1958) noted
mild abnormalities in RBC fragility, decreased RBC survival
time, and decreased plasma iron turnover in CRF patients
,and concluded that both RBC destruction and the inability

of bone marrow to compensate for it contributed to the

27

anemia. Eklund et a1. (1971) saw a degree of hemolysis in
uremics, especially those with chronic pyelonephritis and
active urinary tract infections, when compared with healthy
controls. In examining iron kinetic data, they saw
depressed erythropoiesis which resulted in a larger circu-
lating iron pool which was not utilized. Wallner et a1.
(1976) have tested for the presence of a serum factor toxic
to erythropoiesis in CRF patients and found that in a

System utilizing dog bone marrow cells that CRF sera but

not normal sera or sera from anemias of other chronic
diseases inhibited heme synthesis. Addition of urea or
creatinine did not impair the marrow system activity;
consequently these authors conclude that there is a factor
in the serum of CRF patients that inhibits erythropoiesis

in vitro. Two explanations for the hemolytic nature of

the anemia of uremia of CRF are given by Jacob and coworkers
(1975) whose results show that in 25% of the dialysis
patients studied,the hexose monophosphate shunt of RBC was
inhibited and that chloramine (a powerful oxidant) from
unpurified water appeared to induce hemolysis. The mechani-
cal trauma that RBC receive during the in vitro dialysis
treatment may also contribute to the anemia. Reitman et al.
(1970) have shown with in vitro studies that when RBCs were
exposed to mechanical trauma (such as a rotary pump) that
RBCS from rabbits injected intraperitoneally with vitamin E

were less susceptible than those from normal rabbits to

28

hemolysis induced by H202 at concentrations greater than
1.5%. The effect was due to vitamin E rather than the
trauma, however, as control RBCS (not pumped) displayed
hemolysis similar to normal RBCS. Another contributing
factor to the anemia of CRF is iron deficiency, which may
be due to frequent blood sampling; limited iron consumption
and absorption from a diet restricted in protein and,
indirectly, in ascorbic acid; occult blood loss; and blood
loss during hemodialysis treatments. Compared to serum
iron or transferrin levels, serum ferritin is the best
indicator of bone marrow iron stores (Aljama et al., 1978).
Consequently serum ferritin should be a useful indicator

of total body iron stores in uremic subjects.

Lipid Metabolism

 

In contrast to the hypercholesterolemia seen in neph-
rotic syndrome, CRF is characterized by hypertriglyceri-
demia (Feldman and Singer, 1974). The mechanism of the
hypertriglyceridemia is controversial. It has been hypo-
thesized that an accumulation of insulin antagonists is
responsible for the lipid disorder. One study showed no
correlation between insulin and triglyceride concentrations
in either uremics or controls. While the concentrations of
insulin were similar for both groups, the uremic subjects
had elevated concentrations of triglycerides not seen in
control subjects (Kaye et al., 1973). Glucose intolerance

is a frequent feature of uremia presumably because of

29

tissue insensitivity to insulin, which would result in
elevated levels of serum insulin. Heuck et al. (1978)

have reported elevated levels of insulin in uremic rats.
When the rats were fed diets rich in either carbohydrate,
fat or protein, triglycerides were most elevated in the
group fed a carbohydrate-rich diet prompting the authors to
recommend low carbohydrate diets for uremic patients
because of the risk of coronary heart disease.

In addition to the insulin-related theories, it has
been suggested that a defect in the removal of triglycerides
from plasma exists in patients with uremia. A significant
relationship has been reported between plasma triglyceride
levels and both very low density lipoprotein (VLDL) secre-
tion rate and post-prandial insulin response in uremic
patients, 50% of whom were receiving hemodialysis treat-
ments (Reaven et al., 1980). The majority of these
patients also demonstrated decreased removal of VLDL from
plasma, higher triglycerides at any given VLDL secretion
rate than controls, and lower triglycerides at any given.
post prandial insulin response than controls, indicating a
decreased ability of insulin to stimulate VLDL secretion,
however, they did not examine the uptake of triglycerides.
Changes in dietary carbohydrate of these patients affected
post prandial insulin response, triglyceride levels and
VLDL secretion showing a strong influence of diet on this

mechanism.

30

Chronic hemodialysis may further aggrevate uremic
hypertriglyceridemia. Dialysate glucose levels may be an
additional source of carbohydrate for the uremic patient.
Dombeck et al. (1973) saw no effect of a glucose-free
dialysate on lipid levels, but they did note that one half
of their non-dialyzed uremic patients already exhibited
hypertriglyceridemia. Swamy et a1. (1977) however, repor-
ted triglyceride concentrations were lowered in 25% of
patients receiving a glucose-free dialysate for 6 months.

Other factors such as type of primary disease, nutri-
tional status, body weight, and medications may also effect
hypertriglyceridemia. Gutman et al. (1973) found hyper-
lipidemia in 70% of dialyzed and nondialyzed uremic patients
and it correlated with sufficient caloric intake and good
nutritional status in both groups. Dombeck and coworkers
(1973) reported that administration of an androgen to male
dialysis patients resulted in marked elevations of trigly-
ceride concentrations in 75% of the patients.

Nutrition

 

Patients that have been treated with hemodialysis for
a period of time show the combined effects of the treat-
ment and the disease process. Many of these effects are
nutrition related. Previously nutritional therapy was a
conservative treatment for CRF, used to prolong life.
Today, however, it is an integral component of the thera-

peutic regimen of maintenance hemodialysis which allows the

31

patient a near normal mode of existence (Burton, 1977).

The balance between the patient's renal excretory capacity
and nutritional intake will determine the patient's health
status. Therefore the goal of nutritional therapy is to
minimize the symptoms of the toxicity and other metabolic
disorders of CRF while promoting optimal nutritional status
(Kopple, 1978).

In CRF many metabolic disturbances require adjustments
in nutritional therapy. Some examples are: retention of
nitrogenous wastes requiring a restriction of dietary pro-
tein; decreased ability to excrete sodium, potassium,
magnesium, and phosphorus necessitating their restriction
in the diet; decreased intestinal absorption of calcium
requiring dietary restriction of phosphorus and possible
supplementation of 1,25 dihydroxycholecalciferol; deficiency
or decreased activity of some vitamins requiring supplemen-
tation; and a tendency toward tissue wasting indicating a
need for careful nutritional monitoring (Kopple, 1978).
Successful nutritional therapy requires a strong team
effort by the physician, dietitian, nurse, and social
worker to reinforce the patient and his family.

Nutritional studies are different from other types of
medical research due to a number of factors including:
whole body response, body stores, bioequivalency of some
nutrients, multiple functions of nutrients, and divergence

of results of short- and long term studies (Burton and

32

Wineman, l978). Nutritional studies in CRF are further
complicated by the type, stage, and progression of the
disease. Under ideal conditions, studies should be done
at a time when the patient is in a steady state (Kopple
et al., 1975). However, with the progressive nature of
CRF the patient is always displaying a gradual, but over
time, significant change in excretory capacity and conse-
quently metabolic and endocrine function.

Vitamins

The many vitamin abnormalities seen in hemodialysis
patients are due to: 1) decreased consumption of nutrients
because of dietary restrictions, 2) removal of water-soluble
vitamins in the dialysate during hemodialysis, and 3)
altered excretory or metabolic functions which may affect
absorption from the gut, excretion from the body, or acti-
vity in the body. Altered metabolic functions may be due
to drug-nutrient interactions (Kopple, 1975). The practice
of reducing the potassium content of vegetables by leaching
may also leach water soluble vitamins which would compound
the decreased intake of vitamins due to dietary restrictions
(Burge, 1974).

The literature regarding vitamin status in CRF is not
extensive. In many cases the number of subjects studied
was small. Other frequent problems include no indication
of the levels of dietary intake of nutrients, no reference

to recent transfusions, no mention if supplements were

33

being given, and no discussion of possible interferring
drugs.

Lasker et al. (1963) reported normal to elevated levels
of vitamin 812, biotin and pantothenic acid, normal to
decreased levels of thiamin, decreased levels of folacin
and niacin in a study of four patients. Stone et a1.
(1975) examined plasma pyridoxal-S-phosphate, plasma glu-
tamic-oxaloacetic transaminase and erythrocyte glutamic-
oxaloacetic transaminase and found lower concentrations in
fourteen hemodialysis patients than in thirteen normal
control subjects. Ascorbic acid concentrations have been
shown to decrease markedly during hemodialysis of sixteen
subjects (Sullivan and Eisenstein, 1970).

Ito et al. (1971) have shown hemodialysis patients in
Japan to have elevated levels of riboflavin. The same
authors reported a mean plasma vitamin E concentration of
1.22 i 0.91 mg/100 ml for six hemodialysis patients as
compared to 2.13 i 0.73 mg/100 ml of total vitamin E for
10 controls (p<0.05). However, the value for the patients
would still be considered within the normal range for
populations in the United States.

Vitamin A metabolism is abnormal in CRF possibly due to
increased amounts of free retinol binding protein (RBP).
The RBP has been shown to have an extended half-life in
CRF (Vahlquist et al., 1973) which may indicate that it is

normally catabolized by the kidney. Yatzidis and coworkers

34

(1975) reported serum vitamin A concentrations two to five
times the normal mean value. They hypothesized that a
decrease in the excretion of vitamin A derivatives might be
possible.

The literature as reviewed indicates the importance of
nutrition in CRF and the need for more studies of vitamin
status with larger numbers of subjects, more emphasis on
the evaluation of the amount of nutrients consumed, and
more critical selection of a homogenous patient population.
In as much as the previously mentioned reference related to
vitamin E status in CRF was only in the form of a letter to
the editor, there is a need for more research in this area
to confirm and expand upon the results.

In the vitamin E literature there are implications for
possible interactions between the vitamin and some condi-
tions present in renal failure. Previous research has
shown a correlation between serum vitamin E and serum
lipids. Absolute serum vitamin E levels have been reported
to be normal in hemodialysis patients. Many CRF patients
are also known to have elevated levels of triglycerides.

If the serum vitamin E levels are considered in relation to
total serum lipids is there the possibility that the ratio
may be below the levels suggested for adequate nutriture?
Other questions may be raised. Is there a relationship
between the iron status of CRF patients and serum levels

of vitamin E? What is the relationship between dietary

35

intake and serum vitamin E in this condition? Is the hemo-
lytic anemia of CRF related to vitamin E status? The
present experiment was designed to examine the vitamin E
status of adults with CRF being treated with hemodialysis
and to study the relationship of vitamin E status and rele-

vant parameters.

MATERIALS AND METHODS

Subject Recruitment

 

Twenty individuals with chronic renal failure were
recruited from the outpatients receiving hemodialysis
treatment at the Michigan Nephrology Center at Borgess
Medical Center in Kalamazoo, Michigan. Patients that met
the following criteria were invited to participate in the
study:

1) No evidence of nephrotic syndrome, diabetes, or
severe anemia (hematocrit <20%)

2) Body weight within :15% of desirable weight
3) No androgen therapy
4) At least 18 years of age

5) Treated with hemodialysis for a minimum of two
months

6) History of compliance to medical treatment regimen

Twenty apparently healthy control subjects that matched
the patients in sex and age (:2 years) were recruited from
the staff of the Michigan Nephrology Center and from the
faculty, staff, and students at Michigan State University.
Control subjects met basically the same criteria as above

with the exception of hemodialysis and medical treatment.

36

37

Informed Consent

 

The project was reviewed and approved by the University
Committee on Research Involving Human Subjects at Michigan
State University and the Human Research and Clinical Inves-
tigation Committee at Borgess Medical Center. All subjects
that participated were fully informed of the nature of the
study and of their involvement in it. Separate consent
forms were prepared for the patients and controls because.
of different blood collection procedures (Appendices A-1
and A-2). The consent forms explained the possible risks,
stated that the subject could withdraw from the study at
any time without penalty, and verified that the results
would be kept in strict confidence.

Subject Characteristics

Subjects ranged in age from 18 to 82 years (mean age:
60 years); 55% of the subjects were male (mean age: 67
years) and 45% were female (mean age: 52 years); see
Tables 1 and 2. There were one male and three female
Blacks in the patient group. The remainder of the subjects
were Caucasian. The patients had been on hemodialysis from
2 to 75 months (mean: 26 months) at the time of the blood
collections. They were receiving hemodialysis treatments
for 8 to 20 hours per week (mean: 13 hours). As seen in
Table l the patients had a variety of underlying kidney
diseases. The primary diseases leading to dialysis in

this p0pulation were glomerulonephritis, 35%; cardiovasular

38

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39

 

 

 

 

 

 

 

 

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40

and hypertensive disease, 20%; kidney infections (pyelo-
nephritis and interstitial nephritis), 20%; unknown etiology,
15%; and congenital diseases (polycystic diseases), 10%.
Approximately 90% of the patients received some form of
vitamin B complex supplementation, including 1.0 mg of

folic acid (Table 1). Vitamin supplements consumed by the
controls are noted in Table 2.

The heights and weights of the subjects were
measured at the time of the blood sample collection. The
weights of the patients ranged from 84 to 111% of the
desirable weight/height for men and from 93 to 125% of the
desirable weight/height for women according to Bray (1975).
For controls, the men ranged from 97 to 129% of the desir-
able weight/height and women ranged from 88 to 124% of the
desirable weight/height. Weight was measured on a beam
balance in light clothing without shoes. Height was
measured with the subjects in stocking feet, eyes straight
ahead, and backs straightened on a beam balance height
bar.

Nutrient Intake

 

All subjects were asked to record on a prospective
basis their total dietary intakes for two consecutive 24
hour periods. The subjects were given Food Diary sheets
(Appendix B) on which to record the food consumed. Sub-
jects were instructed to record: all items actually con-

sumed (including beverages), methods of preparation, and

41

amounts consumed. The items recorded on the Food Diary
sheets were coded and analyzed by the Michigan State Uni-
versity Nutrient Data Bank computer program (1979) (adapted
from the Highland View Hospital - Case Western Reserve
University Nutrient Data Base).
Collection of Blood Samples

Blood samples were collected from mid March through
early May in 1980. A11 blood samples were collected fol-
lowing a 12 hour overnight fast. Blood was taken from the
arterial circulation of patients during the initiation and
during the termination of a routine dialysis treatment.‘
Blood was taken from the controls by venipuncture. All
samples were drawn into a dry syringe and separated into
two aliquots. Approximately 0.5 ml of whole blood was
placed in a polypropylene tube for immediate use in the
erythrocyte hemolysis test and the remainder of the blood
drawn was centrifuged in a non-fluorescent glass tube.
The serum was divided into two portions, the first of which
was refrigerated pending total serum lipid analysis within
48 hours. The second portion was placed in a glass vial,
had the atmosphere within replaced with nitrogen, and was
frozen for later vitamin E analysis. Some patient samples
were known to contain the Australian antigen which is asso-
ciated with serum hepatitis B; therefore infectious material

precautions were exercised when handling samples.

42

Blood Analysis

 

Routine Blood Analysis. As part of their routine medi-
cal care, the patients included in this study have the
following blood tests performed at least every six months:
hematocrit, blood urea nitrogen, and serum creatinine, tri-
glycerides, cholesterol, vitamin A, ferritin, and ascorbic
acid. Means and individual values for each of these para-
meters are listed in Appendix 0.

RBC Hemolysis. The method utilized in the current

 

study was modified from the recent method of Farrell (1977),
as described in Appendix E-l. The erythrocyte hemolysis
test was used in this study to determine if it is a valid
indicator of vitamin E deficiency in hemodialysis patients
in the presence of the dual anemia of CRF. .Two concentra-
tions of hydrogen peroxide, 2% and 3.8%, were used in this
study.

Total Lipids. Total serum lipids were determined by
the sulfo-phosphovanillin method of colorimetric analysis
which was first described by Chabrol and Charonnet in 1938
and modified by Zollner and Kirsch in 1962 (Appendix E-Z).
Good correlation is seen between this procedure and gravi-
metric methods (Postma and Stroes, 1968). Knight et a1.
(1972) have described the reaction as having three steps:
(1) a carbonium ion is formed when concentrated sulfuric
acid reacts with unsaturated lipids, (2) an aromatic phos-

phate ester is formed when phosphoric acid reacts with

43

vanillin, resulting in increased carbonyl group reactivity,
and (3) the carbonyl group of the phosphovanillin reacts
with the carbonium ion to form a colored complex. This
reaction requires a carbon to carbon double bond, but com-
pounds with multiple double bonds are affected by steric
hinderance.

Vitamin E. The fluorometric assay for vitamin E of

 

Thompson et a1. (1973) in which serum proteins are precipi-
tated with ethanol and the vitamin E is extracted into
hexane was modified for this study (Appendix E-3). The
major changes in the method included: increasing sample size
from 0.2 to 0.3 m1, increasing the ethanol:water ratio during
the precipitation from 1:1 to 2:1, and increasing the extrac-
tion mixing from 1 to 3 minutes. Thompson has Shown good
correlation between this fluorometric assay and the method
utilizing the Emmerie-Engel reaction.

All vitamin E determinations were done within a 72 hour
period. A separate standard curve was determined for each
of the six assays run during this period. For any patient,
the pre- and post-treatment samples and the sample from the
age and sex matched control were analyzed in the same run.
The coefficient of variation of the concentration value for
the pooled control serum used for each of the six runs was
2%; therefore the standard curves were not adjusted. Reco-
veries of o—tocopherol standards that were added to rat and

human sera averaged 97.5%.

44

Rat Study

 

The rat study was performed as part of this study to
confirm the reliability of the erythrocyte hemolysis test
as performed in this study. At the onset of the study it
could not be foreseen if any of the subjects would demon-
strate a vitamin E deficiency and hence a positive erythro-
cyte hemolysis test. In addition there is no practical
standard for this test due to the fragile nature of RBC.
Consequently rats were depleted of vitamin E to verify the
results of the RBC hemolysis test.

Five male Sprague Dawley rats weighing 225-250 gm were
obtained from spartan Research Animals, Inc., Williamston,
Michigan. For 13 weeks rats were fed ad libitum a vitamin E
deficient purified diet with the following composition
(gm/100 gm): dextrose, 69; vitamin-free casein, 20; par-
tially hydrogenated soybean oil, 5; vitamins 0.16; and
minerals 5.27 (Appendix E). The soybean oil had been heated
to approximately 375°F for 80 hours over a two week period

when it was used for frying in a foodservice facility. Four
(rats were killed after consuming the diet for thirteen

weeks. The fifth rat was killed in the eleventh week because
of respiratory problems. Blood samples were collected from
the intra-orbital sinus with a heparinized capillary pipet
after the rats had been anesthetized with ether. Some
samples were collected from the heart at the time the rats

were killed. Assays were performed for erythrocyte

45

hemolysis and serum vitamin E on samples collected during
weeks 10 to 13. Blood samples were treated in the same
manner as described for human subjects.

Statistical Methods

 

Pre- and post-treatment serum levels of vitamin E were
analyzed by paired t test. Correlations were computed for
the two levels of hydrogen peroxide and the two treatment
groups. A multiple regression utilizing generalized least
squares was computed using the patients' serum vitamin E
values and selected routine blood analyses. A randomized
block analysis with 3 covariates was applied to the multiple
regression computed for the serum vitamin E levels and
dietary and serum parameters (Gill, 1978). All regressions
were done on a Cyber 750 computer utilizing the Statistical
Package for Social Sciences (Vogelback Computing Center,

1979).

RESULTS

Nutrient Intake

 

The daily intake of calories, carbohydrate, protein,
total fat, polyunsaturated fat, total toc0pherol, o-toco-
pherol, other toc0pherols, total vitamin A, iron, selenium,
and ascorbic acid was tabulated for each subject and the
average intake for the two-day period calculated. The mean
intakes of these nutrients as well as values for the ratio
of o toc0pherol:polyunsaturated fat and the estimated
o-tocopherol equivalents were determined (Table 3). The
average polyunsaturated fat intake values ranged from 0.8
to 17.6 mg per day. Average toc0pherol intakes ranged from
1.1 to 39.7 mg per day for total toc0pherol, 0.5 to 14.7 mg
per day for o-tocopherol, and 0.1 to 16.0 mg per day for
other tocopherols.

The patients participating in this study follow dietary
restrictions for protein, sodium, potassium, and fluid
which are reflected in their lower intakes of protein,
total vitamin A, iron, and ascorbic acid as compared to
controls. These dietary restrictions also contribute,
along with other factors, to the limitation of total energy

intake in the patient group.

46

Table 3. Average daily dietary intakes of subjects

47

1,2,3

 

 

 

 

Patients Controls Patients Controls
(n=11) (n=11) (n=9) (n=9)
Total calories 1637 2245 1404 1690
(kcal) : 96 z 145 z 126 z 105
Total 189 261 152 196
carbohydrate (9m) 1 16 t 19 z 15 z 19
Total protein (gm) 60 92 52 70
1 4 1 6 t 4 z 4
Total fat (9m) 72 98 67 67
z 4 z 9 3 7 2 5
Polyunsaturated 9.6 3.6 9.0 8.7
fat (gm) 1 1.2 1 2.3 : 1.4 2 1.0
Total 9.8 15.1 10.7 13.0
toc0pherol (mg) 1 1.9 1 2.9 : 2.0 : 3.6
Alpha 4.0 6.0 3.7 4.2
toc0pherol (mg) 1 0.9 1.3 : 0.7 2 0.8
Other 5.1 6.9 5.5 5.7
tocopherols (mg) 1 1.1 1 1.6 2 1.1 t 1.2
Estimated alpha 4.8 7.2 4.4 .1
toc0pherol 4 1 1.0 1 1.5 r 0.8 - 0.9
equivalents (mg)
Alpha toc0pherol .5 0.4 0.5 0.5
(mg): polyunsaturated 1 0.2 1 0.1 t 0.1 2 0.1
fat (gm)
Estimated 1558 3677 661 2220
retinol 5 1 507 1 1310 t 100 t 242
equivalents (ug)
Iron (mg) 10 17 9 14
‘ 1 1 2 2 1 t 3
Selenium (ug) 14 10 7 13
1 7 1 2 2 4 z 4
Ascorbic acid (mg) 61 143 42 153
1 12 3 19 t 7 t 22

 

1

2Mean 1 SEM.
3

Analyzed using the Michigan State University Nutrient Data Bank Computer Program.

Vitamin or mineral supplements are not included.

4Calculated by multiplying 1.2 times the two day average value of a-tocopherol
intake for each subject (FNB, 1980).

SCalculated by multiplying 0.3 times the two day average value of total vitamin A
(IU) for each group (FNB, 1980).

48

Blood Analysis

 

Vitamin E and Total Lipids. The mean values for serum

 

total vitamin E, serum total lipids, and the ratio of serum
total vitamin Ezserum total lipids are Shown in Table 4.

The values for total vitamin E ranged from 0.8 to 1.9 mg/
100 ml for female patients, 1.0 to 1.7 mg/100 ml for female
controls, 0.8 to 1.5 mg/100 ml for male patients, and 0.9

to 1.6 mg/100 ml for male controls. Total lipids ranged
from 353 to 589 mg/100 ml for female patients, 423 to 603
mg/100 ml for female controls, 384 to 770 mg/100 ml for male
patients, and 424 to 680 mg/100 ml for male controls.

Separate multiple regressions for each sex were computed
with serum total vitamin E as the dependent variable,
patient-control pairs and patient-control groups as co-
variates, and the total serum lipids, the intake of poly—
unsaturated fats, and the intake of o-tocopherol as inde-
pendent variables. For both males and females, only serum
total lipids had a Significant relationship with serum
total vitamin E levels (p<0.05). There was little statis-
tical evidence of a difference between patient and control
groups for serum total vitamin E.

Using values for patients alone, multiple regressions
were computed for males and females with serum total vitamin
E as the dependent variable and serum triglycerides, serum
cholesterol, serum total lipids, serum ferritin, months of

hemodialysis, and hours of hemodialysis per week as

49

independent variables. For the males, serum total lipids,
serum ferritin, serum triglycerides, and serum cholesterol
contributed significantly to the regression equation at the
level of p<0.05. Serum triglycerides, cholesterol, and
total lipids levels showed a high degree of correlation with
serum vitamin E levels. Ferritin was not correlated with
serum vitamin E. For the females, none of the independent
variables were significant in the regression equation. How-
ever, the sample size for females dropped from nine to six
due to some missing values and with such a small sample and
six variables, statistic significance is difficult to
achieve.

0n the same day that patient samples were collected at
the beginning of a hemodialysis treatment, a blood sample
was also collected at the termination of the treatment for
determination of serum total vitamin E. Mean values i SD
for the post-dialysis samples were 1.09 i 0.38 mg/100 ml for
the female patients and 1.07 1 0.32 mg/100 ml for the male
patients. A paired t test showed no significant change
between pre— and post-treatment values for females. For
males, however, a slight mean decrease of 0.07 mg/100 ml was
statistically significant at p<0.05. For both sexes separate
multiple regressions were done with the difference between
the pre- and post-treatment serum total vitamin E value as
the dependent variable and months of hemodialysis treatment,

hours of hemodialysis per week, post hemodialysis weight,

50

and the difference between pre— and post—hemodialysis body
weight as the independent variables. For both males and
females, none of the variables had a significant effect on
the difference between the pre- and post-treatment vitamin E
values in the regression equation. However, there was a
slight negative correlation (r=-.40 to -.55) when the
difference between pre- and post—treatment serum total vita-
min E was compared with the difference between pre- and post-
hemodialysis body weight. There is no apparent explanation
for the slight mean decrease in serum total vitamin E
following hemodialysis as seen in the male patients.
Erythrocyte Hemolysis

Table 4 shows the mean values1

for erythrocyte hemolysis.
None of the erythrocyte hemolysis values for the controls at
either concentration of hydrogen peroxide exceeded 20%, the
maximum value considered to reflect normal vitamin E status
(Interdepartmental Committee on Nutrition for National
Defense, 1963). At the 2% concentration of hydrogen perox-
ide, two male patients had erythrocyte hemolysis values
greater than 20%, while four patients of each sex had values
of more than 20% at the 3.8% concentration of hydrogen
peroxide. However, all subjects, both patients and controls,

had normal values for serum total vitamin E and serum total

vitamin E:serum total lipid ratio. Because the erythrocyte

 

1One value of 31% for a female control was removed from the

data on the basis of the Grubbs and Beck test for outlier
values (Gill, 1978).

51

Table 4. Erythrocyte hemolysis, serum total vitamin E and
total lipidSIaz

 

 

 

 

Males Females
Patients Controls Patients Controls
(n=11) (n=1l) (n=9) (n=9)

Total 1.1 1.2 1.1 1.3
vitamin E i 0.1 i 0.1 i 0.1 i 0.1
(mg/100 ml)
Total lipids 538 540 500 550
(mg/100 ml) i 24 i 17 i 27 i 19
Total 2.1 2.3 2.2 2.4
vitamin E (mg)/ i 0.1 i 0.2 i 0.2 i 0.1
total lipids (gm)
2% “£02 8 8 0.4 2 5 1,0
eryt rocyte i 5 l i 0.3 i 1 2 i 0 5
hemolysis (%)
3'8% H202 21.3 3.1 21.5 1.53
erythrocyte i 5.6 i 1.1 i 5.7 i 0,5
hemolysis (%)
1

Blood samples were collected following a 12 hour fast.
Samples from patients were collected at the beginning of
a hemodialysis treatment.

2
3

Mean 2 SEM.

n=8

52

hemolysis test is an indirect test, factors other than
vitamin E deficiency may have influenced the results.

After separating the data by group and sex, preliminary
correlations were computed for erythrocyte hemolysis test
values from assays with 2% or 3.8% solutions of hydrogen
peroxide. The test results for the two solutions were not
strongly correlated, although the relationship was positive.
Because of this lack of correlation results for the two
solutions could not be combined; therefore for each of the
concentrations of hydrogen peroxide,correlations were
computed comparing the erythrocyte hemolysis values with
the serum total vitamin E values with separate correlations
for each subject group (patients vs. controls), each sex,
and each of the two concentrations of hydrogen peroxide
for a total of eight correlations. There was little evi-
dence (p<0.05) of correlation between the erythrocyte
hemolysis values and the serum total vitamin E values in
the patients or controls regardless of sex or hydrogen
peroxide concentration, except for the female controls at
the 2% level of hydrogen peroxide. These subjects showed
a positive correlation. It does not appear that the
erythrocyte hemolysis test is useful in hemodialysis popu-
1ations, probably because of interference related to the

hemolytic anemia of uremia.

53

Rat Study

 

The vitamin E content of the soybean oil used as the
source of fat in the rat diet was analyzed by the same
method used for serum. There was a mean content of 0.35 mg/
100 m1 i 0.03 of vitamin E in the oil, so by calculation
there was 0.02 mg of vitamin E per 100 gm of rat diet, or
0.2 mg per kilogram of diet. The diet was not analyzed
for vitamin E content.

The rats consumed an average of 20 to 25 gm of diet
per day and four of the animals weighed an average of 510 gm
when they were killed in the thirteenth week. Due to
illness the fifth rat had been killed in the eleventh week:
it had lost weight and weighed 350 gm.

The values of erythrocyte hemolysis for the rats ranged-
from 60 to 74% at 2% hydrogen peroxide and 33 to 43% at 3.8%
hydrogen peroxide. All samples at the 3.8% level displayed
an uncharacteristic green color, possibly due to contami-
nation in used test tubes. All assays for erythrocyte
hemolysis in the human subjects utilized new test tubes.

In any case, all assays in the rat showed results well in
excess of 20% hemolysis. The serum values of total vitamin
E in the rats ranged from 0.03 to 0.12 mg/100 ml, which is
well below the minimum values associated with adequate
nutriture. Consequently, the erythrocyte hemolysis test in
this study appears to be an accurate predictor of vitamin E

deficiency in the rat.

DISCUSSION

Subject Characteristics

 

The patient population that was sampled in this study
appears to be similar to the national population described
by Wineman (1978) with regard to age and the primary
disease leading to dialysis. The patients in the present
study tended to be older because only patients being
treated during the day shift were selected for participa-
tion. This arrangement was necessary in order that the
twelve hour fast required for the total lipid test would
occur overnight. It was considered an undue hardship to
require a patient to fast for twelve hours during the day.
Consequently younger patients who held day-time jobs were
excluded from the study because they received their hemo-
dialysis treatments in the evening.

Nutrient Intake

One day dietary records may Show large fluctuations in
variability of nutrients consumed (Garn et al., 1978) when
compared with seven day records. It is misleading to use
one day records to ascertain intakes for individuals.
However, Garn et a1. (1976) stated they are useful in

deriving information from population samples if the

54

55

dietary records are carefully taken and analyzed. He
cautioned that these one day records should not be used

to rank individuals or to estimate the proportion of indi-
-viduals at nutritional risk. One day food records were
compared with diet histories for a group of 35 obese,
pregnant women by Van Den Berg and Mayer (1954). Their
results Show that significantly higher intakes of calories,
carbohydrate, protein, and fat were reported with the diet
history than with the one day record. They reasoned that
an obese population may be reluctant to list their true
intakes and that the fact of recording the food eaten acts
as a check on food intake.

It was not feasible to collect seven day dietary
records from the hemodialysis patients if they were to be
interviewed regarding their intake following completion of
the records. It was possible to collect two day dietary
records from all subjects. Most subjects were interviewed
briefly when they turned in their records.

The Michigan State University Nutrient Data Bank con-
tains values for approximately 70 nutrients and 3,500 food
items. However, the values for all nutrients are not
complete for each food item. For the nutrients analyzed
in this study the values for the tocopherols, selenium, and
polyunsaturated fat may be incomplete for some food items.
'Therefore the values shown in Table 3 may be somewhat

lower for those nutrients than actual intake because of

56

missing values.

In general the control subjects consumed greater
amounts of nearly all of the nutrients compiled than did
the patients. Sullivan and Eisenstein (1970) along with
Kopple and Swendseid (1975) have noted that dialysis
patients have decreased intakes of those nutrients found
in potassium and protein rich foods. The control subjects
consumed at least 100% of the RDA (FNB, 1980) for protein,
retinol equivalents, and ascorbic acid. The RDA for iron
was met by all control subjects, except the females under
the age of 50 years who consumed 70% of the allowance.
None of the subjects, either patients or controls, appeared
to consume 100% of the RDA for toc0pherol equivalents.

The intakes of toc0pherol equivalents ranged from 40 to
70% of the allowance. The low intakes of toc0pherol
probably reflect the limitations of the data bank due to
missing values. The patients consumed 100% of the RDA for
protein and male patients consumed 100% of the RDA for
retinol equivalents, iron, and ascorbic acid. Female
patients consumed the following percentages of the RDA:
retinol equivalents, 85%; iron, 55%, and ascorbic acid,
70%.

The values for typical daily intakes of o-tocopherol,
total toc0pherol, and polyunsaturated fat cited in the
literature by Bieri and Evarts (1973) and Witting and Lee

(197Sb)are considerably higher than those seen in this

57

study. Aside from the limitations of the nutrient data
bank, it is important to note that in the two studies
mentioned the investigators did not evaluate food actually
consumed by individual subjects, but rather analyzed typi-
cal cafeteria menus. Cafeteria food items may not resemble
those cooked at home or in other types of food services

in type and method of preparation. Although the average
daily energy level of the diets evaluated in those studies
was 2,400 to 2,500 kcal which is considerably more than the
energy intake of the subjects in this study, it is not
enough to account for the differences in toc0pherol levels.
In this study there was a positive correlation between

the intakes of a-tocopherol and polyunsaturated fat.

Blood Analysis

Vitamin E. The values seen for total vitamin E in

 

this study are similar to values analyzed by the Emmerie-
Engel method cited in the literature for subjects of
similar age. Researchers using a variation of this spec-
trophotometric method found mean values of 1.05 mg/100 ml
of vitamin E in studies of 197 and 132 subjects ranging in
age from 17 to 64 years (Harris et al., 1961; Bieri et al.,
1964). Using a similar spectrophotometric method, Farrell
et al. (1978) found mean levels of 0.78 mg/100 ml in 22
subjects 21 to 35 years of age. Rubenstein et a1. (1969)
found slightly higher levels than the previously mentioned

researchers of 1.14 mg/100 ml using a variation of the

58

Emmerie-Engel method. In twelve females, 19 to 26 years
old, Yeung (1976) reported mean vitamin E values of 0.87 mg/
100 m1 using the same fluorometric method that was modified
for the present study. The value cited by Yeung is lower
than reported here but the age range of his subjects tended
to be much younger. Ito et a1. (1971) reported plasma total
vitamin E levels of 1.2 mg/100 ml for patients in Japan
receiving maintenance hemodialysis which compare very
favorably with the levels reported in this study.

Serum Lipids. Based on assays using the same colori-
metric method as used in this study, Postma and Stroes
(1968) have shown that serum lipid values for a large number
of serum samples will have a range of 500 to 1800 mg/100 ml
with most values falling between 500 and 1000 mg/100 ml.

In studies conducted to evaluate vitamin E status, mean
values for serum total lipids of 549 mg/100 ml for young
adults (Farrell et al., 1978) and 685 mg/100 ml for adult
males (Vatassevy and Chiang, 1979) were reported using the
turbidimetric method. In a study of patients with CRF,
Dombeck et al. (1973) reported that 50% of eighteen pre-
dialysis patients with uremia had abnormal total lipid con-
centrations and that the percentage increased on dialysis
if androgens were given. Although serum cholesterol and
triglyceride concentrations are frequently reported in CRF
(Kaye et al., 1973; Dombeck et al., 1973; and Reaven et al.,

1980) serum total lipids are rarely evaluated, probably

59

because of the availability of other methods for the diag-
nosis of hyperlipoproteinemia.

The results of the present study show very little dif-
ference between the total lipid values of patients and
controls especially for females. This was unexpected as
the hypertriglyceridemia associated with CRF would be
expected to contribute to an increase in total lipids. The
subjects for this study were screened very carefully for
factors such as nephrotic syndrome, diabetes, obesity, and
androgen therapy which are known to contribute to the ele-
vation of lipids. In past reports of lipid abnormalities
these contributing factors may not have been well controlled.
Gutman et a1. (1973) in examining triglycerides did restrict
his sample to non-nephrotic subjects. There also were no
diabetic patients included in the study although it did not
appear they had been deliberately omitted. Gutman defined
obesity as greater than 20% over the ideal weight and
included one slightly obese patient in his sample. Use of
androgen therapy was not evaluated. In the previously
mentioned study by Dombeck, only glucose in the dialysate
and androgen therapy were evaluated in relation to the
lipid status of the subjects. In the present study the
control subjects may have had a tendency to exceed their
ideal weight by a slightly greater degree than the controls
which might account for the lack of difference between

lipid values for patients and controls.

60

Vitamin Ezlotal Lipid Ratio. Because there was little
difference in the values for vitamin E and total lipids
between patients and controls, it is logical that there
would be little difference between the total vitamin E:
total lipid ratio. The ratio computed for this study com-
pares favorably with the minimum ratio of 0.8 mg/gm sugges-
ted by Horwitt et al. (l972) as indicating adequate vitamin
E nutriture. Other authors (Farrell and coworkers, l978;
and Vatassevy and Chiang, l979) have reported vitamin E:
total lipid ratios of 1.38 and l.7l mg/gm respectively in
normal subjects.

Erythrocyte Hemolysis

The erythrocyte hemolysis test has been used as an
indirect indicator of vitamin E deficiency since 1948
(Gyorgy and Rose). Rose and Gyorgy (l952) were the first
to use hydrogen peroxide for the oxidizing agent in this
colorimetric method. Others (Gordon and deMetry, l952;
Horwitt et al., l956) have refined the method. The erythro-
cyte hemolysis test is not specific for vitamin E deficiency,
however, but also is positive in those hemoglobinopathies
where peripheral RBC destruction plays a major role (Melhorn
et al., l97l). The test is normal in states where the
anemia is primarily the result of ineffective erythropoi-
esis. The anemia of CRF is due to both increased hemolysis
(Desforges and Dawson, l958; Eklund, 197l) and a defect in

erythropoiesis (Wallner et al., l976).

6l

An inverse relationship would be expected between
erythrocyte hemolysis values and serum total vitamin E
(Horwitt et al., l968; Leonard and Losowsky, 1967). In the
case of the patients, erythrocyte hemolysis was not inver-
sely related to serum vitamin E levels probably because
the hemolysis is aggrevated by factors unrelated to vitamin
E status such as uremic toxins or metabolic defects (Jacob
et al., l975; Desforges and Dawson, l958). However, in the
control group, an explanation for the absence of an inverse
relationship between erythrocyte hemolysis and serum
vitamin E levels was not readily apparent.

Rat Study

 

In contrast to the results seen in human subjects, the
values for erythrocyte hemolysis and serum vitamin E seen
in five rats demonstrated the expected inverse relation-
ship. The rats had mean serum vitamin E levels of 0.07 mg/
lOO ml and erythrocyte hemolysis values exceeding 30%.

The diet consumed by the rats contained no vitamin E in

the vitamin mix and no other major contributing source of
the vitamin except the oxidized soybean oil. Analysis of
the oil showed that it contributed 0.2 mg of vitamin E per
kg of diet. In comparison, rats fed l7 mg of a-tocopherol/
kg of diet, which is below the 30 mg of DL~a-tocopheryl
acetate/kg recommended to meet the requirement of rats,

had plasma a-tocopherol levels of approximately 0.4 mg/lOO
ml after eight months on the diet (Yang and Desai, l977).

62

Rats that had been fed at the level of 200 mg of DL-a-toco-
pheryl acetate per kg of diet, however, had plasma toco-
pherol levels of l.3 mg/lOO ml (Machlin et al., l977).

Statistical analysis failed to show a significant rela-
tionship between serum vitamin E and intake of PUFA or
a-tocopherol. Because of the storage potential of vitamin
E as a fat soluble vitamin, dietary intake would be expected
to have little effect on circulating plasma levels unless
there had been a long term deficient intake.

Examining patient data only, serum vitamin E for both
males and females was not related to the number of months
on dialysis or the number of hours of treatment per week.
Those variables were chosen as indicators of the severity
and length of the disease process. In the small sample
of female patients serum vitamin E was also not related
to serum levels of total lipids, cholesterol, triglycerides,
or ferritin. For males the serum lipid components, choles-
terol, triglycerides, and total lipids, in addition to
ferritin were significantly related to serum vitamin E
with serum lipids being the most prominent. The relation-
ship between serum vitamin E and the serum lipid components
has been demonstrated by others such as Horwitt et al.
(l972). Serum ferritin may be related to vitamin E status
because of the oxidation promoting activities of iron.

There was no significant difference between pre- and

post-dialysis levels of vitamin E for female patients.

63

The change in concentration of vitamin E in males from
pre- to post-treatment was very slight, 0.07 mg/lOO ml;
however, it was statistically different. Because vitamin
E is fat soluble, it would not be expected to cross the
membrane during dialysis. Its molecular weight is small
enough to allow it to pass if it were free and not bound
to low density lipoproteins. The difference between the
pre- and post-treatment levels of vitamin E was not
related to the amount or length of hemodialysis treatment,
body mass, or weight loss during hemodialysis for both
males and females. Body mass is somewhat representative
of plasma volume and weight loss during dialysis is due
partially to plasma volume lost during treatment. The
decrease in serum vitamin E which was observed in males
might not be seen if a larger number of subjects were

evaluated.

CONCLUSIONS

The vitamin E status of individuals with CRF in this
study was normal as assessed by absolute serum levels and
the serum vitamin E:serum lipid ratio. The erythrocyte
hemolysis test did not appear to be an accurate predictor
of vitamin E status in patients with CRF receiving hemodi-
alysis. The nutrient intake for a two day period as com-
piled by the Michigan State University Nutrient Data Bank
also did not have any relation to the vitamin E status of
these patients.

This study indicates that the vitamin E requirements
of the hemodialysis patients studied are being met by their
current dietary intakes without the use of vitamin E sup-
plements. These results, however, should not be inter-
preted as showing that the requirements of hemodialysis
patients are the same as normal individuals. The vitamin E
requirements of CRF patients on hemodialysis have not been
addressed in this study, only the vitamin E status has
been examined. Additional research would be needed to
determine the requirement for vitamin E. Because the
function of vitamin E is not well understood in normal

individuals, it would be difficult to assess the requirement

64

65

in a chronic disease state such as CRF.

REFERENCES

REFERENCES

Alfin-Slater, R.B., R.B. Morris, L. Aftergood, and D. Mel-
nick. Dietary Fat Composition and Tocopherol Require-
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Vegetable Oils of Different Ratios of Unsaturated
Fatty Acids and Vitamin E. J. Am. Oil Chem. Soc. 46:
657-66l, l969. _

Aljama, P., M.K. Ward, A.M. Pierides, E.J. Eastham, H.A.
Ellis, T.G. Feest, G. Conceicao, and D.N.S. Kerr.
Serum Ferritin Concentration: A Reliable Guide to Iron
Overload in Uremic and Hemodialyzed Patients. Clin.
Neph. lO:lOl-lO4, l978.

Anderson, T.N. and D.B.w. Reid. A Double-blind Trial of
Vitamin E in Angina Pectoris. Am. J. Clin. Nutr. 27:
ll74-ll78, l974.

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APPENDICES

77

Appendix A-l

CONSENT FORM FOR HEMDDIALYSIS PATIENTS
DEPARTMENT OF FOOD SCIENCE AND HUMAN NUTRITION
MICHIGAN STATE UNIVERSITY
EAST LANSING, MICHIGAN

I, . agree to participate in a

study of the status of vitamin E (toc0pherol) in renal patients on
hemodialysis being conducted by Pat Smith Brown, R.D. (M.S.U.) under
the direction of Dr. J.A. Green (Borgess Hospital). Dr. H.S. Park
(Borgess Hospital) and W. Chenoweth, Ph.D. (M.S.U.). I understand

that the purpose of this study is to determine if the levels of serum
toc0pherol are low, normal, or high when compared to a control group.

I understand the relationship of vitamin E with serum lipids (fats),
and erythrocyte hemolysis (red blood cell breakage) will also be
examined. I understand that I will be asked to keep a 2 day food diary
listing everything I eat. I realize that on one day only. following

a 12 hour fast, a 7 m1 sample of blood will be taken at the beginning
of a dialysis treatment and a 3 m1 sample of blood will be taken at

the end of the same dialysis treatment. I have been told that the
following tests will be performed on the blood samples: serum toco-
pherol. total serum lipids, and erythrocyte hemolysis. I know that

the potential risks are minimal since the blood will be drawn in con-
junction with a dialysis treatment and will not require any additional
venipunctures. I understand the only risk will be the loss of 10 m1 of
blood which is approximately equal to 2 teaspoons. I understand that
the results of this study will provide information concerning the
effects of hemodialysis and renal disease on vitamin E and its relation-
ship with serum lipids and erythrocyte hemolysis which will be of
benefit to hemodialysis patients in general. I understand that there
are no guaranteed benefits to me as an individual. I give my consent
to have my medical records released to the researcher to obtain infor-
mation concerning results of laboratory tests, length of dialysis
treatment, body weight and height, and medications. I have had all my
questions regarding this study answered. I know that I may withdraw
from this study at any time without penalty or jeopardy to my medical
treatment. I understand that all information gathered in this study
from me. my medical records. or assays (tests) of my blood will be

kept in strict confidence and that I will remain anonymous in all reports
of the results. A summary of the results will be provided to me at my
request.

 

DATE
RESEARCHER/PHYSICIAN
SIGNED

WITNESS

 

 

 

 

78

Appendix A-2

CONSENT FORM FOR CONTROL SUBJECTS
DEPARTMENT OF FOOD SCIENCE AND HUMAN NUTRITION
MICHIGAN STATE UNIVERSITY
EAST LANSING. MICHIGAN

I, , a ree to participate in a
study of the status of vitamin E'(tocopherol in renal patients on
hemodialysis being conducted by Pat Smith Brown. R.D. under the direction
of Dr. J.A. Greene, M.D.. Dr. H.S. Park, M.D. and W. Chenoweth. Ph.D. I
understand the purpose of this study is to determine if the levels of
serun tocopherol are low. normal. or high when compared to a control
group. I understand the relationship of vitamin E with serum lipids
(fats), and erythrocyte hemolysis (red blood cell breakage) will also be
examined. I understand that I will be asked to keep a 2 day food diary
listing everything I eat. I realize on one day only, following a 12 hour
fast. a 7 m1 sample of blood will be taken. I have been told that the
following tests will be performed on the sample: serum tocopherol. total
serun lipids. and erythrocyte hemolysis. I know that the potential risks
are those associated with venipuncture such as: fainting, minor pain, or
a small purplish swelling at the site of venipuncture. and the loss of

7 m1 of blood which is approximately 1% teaspoons. I understand that the
results of this study will provide information concerning the effects of
hemodialysis and renal disease on vitamin E and its relationship with
serum lipids and erythrocyte hemolysis which will be of benefit to hemo-
dialysis patients in general. I understand there are no guaranteed
benefits to me as an individual. I have had all my questions regarding
this study answered. I know that I may withdraw from this study at any
time. I understand that all information gathered in this study from me
or assays (tests) of my blood will be kept in strict confidence and that
I will remain anonymous in all reports of the results. A sunmary of the
results will be provided to me at my request.

DATE
RESEARCHER/PHYSICIAN

SIGNED
WITNESS

 

 

 

 

I wish to have the results of these tests sent to my physician:__yes __ho

Name of physician
Address

 

 

 

79

Appendix B

FOOD DIARY

Please write down everything you eat_and drink for the next two days.

Be sure to put dowfi’how the food was prepared (For example, if you ate
potatoes write down if they were boiled. mashed. fried or however they
were fixed.) Try to guess how much you ate in common household measure-
ments such as measuring cups, tablespoons and teaspoons. If you have
sandwiches write down how many slices of bread and whether or not you
used butter, margarine, mayonnaise or anything else. Don't forget to
write down what you drink.

 

 

Time Food (how was it prepared?) Amount

 

Morning

 

 

 

 

 

Afternoon

 

 

 

 

 

 

Evening

 

 

 

 

 

 

 

 

 

80

APPENDIX C

Table 5. Composition of vitamin and mineral supplements per capsule/tablet1

 

 

Becotin One-A- Allbee Abdol Vicon
Nutrient Becotin with Day with with 2 forte
vitamin C vitamin C minerals
Thiamin
HCl (mg) 10 10 2 15 2.5 10
Riboflavin
(mg) 10 10 2.5 10.2 2.5 5
Pantothenic
acid (mg) 25 25 10 2.5 10
Niacin
(mg) 50 SO 20 50 20 25
Pyridoxine
(mg) 4.1 4.1 l 5 0.5
Ascorbic acid (mg) 150 50 300 50 150
Cyanoco-
balamin (ug) 1 1 10
Vitamin A
(ID) 5000 5000 8000
Vitamin D
(W) 400 400
Vitamin E 40
(Iu)
Zinc
sulfate (mg) 0.5 80
Magnesium
sulfate (mg) 1 7O
Manganese
chloride (mg) 1 4
Iron
(«19) 15
Folic
acid (mg) 0.1 1

 

1Composition was not available for High Potency B Complex with Minerals manufac-
tured by Michigan Pharmical as it has been discontinued for several years.

2Also contains: iodine, 0.15 mg; potassium, 5 mg: copper. 1 mg: calcium. 44 mg:
and phosphorus, 34 mg.

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82

Appendix E-l

Erythrocyte Hemolysis Determination

Reagents

1. 1% sodium citrate in 0.9% saline

2. Phosphate buffered saline (PBS) made with equal
parts of 0.9% saline and phosphate buffer
Phosphate Buffer

250 ml of 0.2M potassium phosphate, monobasic
(KH2R04)

197 ml of 0.2M sodium hydroxide (NaOH)

Distilled water to make 1000 m1

Adjust with 1.2N sodium hydroxide to pH 7.4

3. 30% hydrogen peroxide (H202) diluted with PBS to
concentrations of 2% and 3.8% (made fresh each time)

Procedure

1. Add approximately 0.5 m1 of fresh whole blood to
0.2 ml of 1% sodium citrate in a capped. calibrated,
15 m1, polypropylene. conical. centrifuge tube and
gently shake to mix.

2. Incubate the tube at 25°C for 2 hours.

3. Add 10 ml of PBS at 37°C. Invert gently several
times to mix the contents.

4. Centrifuge the tube at 1800 rpm for 10 minutes and
then remove the supernatant.

5. Dilute the remaining red blood cells (RBC) to a 2.5%
solution with PBS at 37°C. Gently invert the tube
several times.

6. Centrifuge the tube at 1800 rpm for 10 minutes and
then remove the supernatant.

7. Dilute the remaining RBCs to a 5% solution with PBS
at 37°C. Gently invert the tube several times.

8. Label new, disposable, polypropylene, 12x75 mm cul-
ture tubes with caps from 1-10 for each sample.

9. Pipet 0.3 ml aliquots of the 5% RBC/PBS solution

with a plastic pipette tip into each of the 10
labeled tubes.

10.

11.

12.
13.
14.

15.
16.
17.
18.

19.

83

Add 0.3 m1 of freshly made 2% H202 to tubes 1-3
with a plastic pipette without contacting the sides
of the tube.

Add 0.3 m1 of freshly made 3.8% H202 to tubes 4-6
with a plastic pipette without contacting the sides
of the tube.

Add 0.3 m1 of PBS to tubes 7 and 8.
Add 3.8 ml of distilled water to tubes 9 and 10.

Cap all tubes, invert twice and incubate at 37°C
for 3 hours.

Add 3.5 ml of PBS to tubes 1-8.
Centrifuge all tubes at 1800 rpm for 10 minutes.
Transfer the supernatant to cuvettes.

Read the absorbance of the supernatant at 577 nm
on a spectrophotometer.

Average the values for tubes 7 and 8. Subtract

this average from all other values. Average the
values for tubes 1-3 and tubes 4-6 and divide by
the average of tubes 9 and 10 to get the percent
of hemolysis with the two concentrations of H202.

Reagents

1. l.3x10'2M vanillin in 11.8M phosphoric acid

2. Total Lipid Standard (750 mg/dl) - 2.09x10-2M oleic
acid. 3.22x10‘3M palmitic acid, and 2.63xlO'3M
stearic acid in specially denatured alcohol

3. Concentrated sulfuric acid

4. Absolute ethanol

Procedure

1. Prepare dilutions of the standard as follows:

Tube 750 mg/dl Absolute Value
Number Standard Ethanol (mg/d1)
(ml) (m1)

1 0.5 1.0 250
2. 1.0 0.5 500

2. In duplicate pipet: 0.1 ml water into a 15 m1 pyrex

test tube
0.1 m1 serum into an identical
tube
0.1 ml of the standards into 3
identical tubes
(250. 500, and 750 mg/dl
total lipid)

3. Add 5 ml concentrated sulfuric acid to each tube.
Seal with parafilm and mix thoroughly using a
mechanical mixer for 15 seconds.

4. Place all tubes in a boiling water bath for 10
minutes.

5. Remove and cool to room temperature in a cool
(running) water bath.

6. Pipet 0.2 m1 aliquots of the surfuric acid digests
into appropriately labeled dry 18x150 mm test tubes.

7. Add 3 m1 of the vanillin-phosphoric acid solution
to each tube.

8. Mix thoroughly on a mechanical mixer for 30 seconds.

84

Appendix E-2

Total Serum Lipid Determination.I

 

85

9. Place tubes in the dark at room temperature for 45
minutes (:15 minutes).

10. Transfer to cuvettes and tap to remove bubbles.

11. Read absorbance at 525 nm against the water blank
set for zero absorbance.

12. A linear regression equation was prepared from the
absorbance and concentration values of the 3 total
lipid standards and used to calculate the concentra-
tion of the serum from its absorbance value.

 

1The phosphovanillin reagent and the method used are those
specified in the package insert, CH44-4921E. revised 1/79,
from the Dade Division of the American Hospital Supply
Corporation. The method was first described by Chabrol, E.
and R. Charonnet. Presse med. 45:1713, 1937 and was later
modified by Zollner, N. and K. Kirsch. Zeitschrift fur
die Gesamte Experimentelle Medizin 135:545, 1962.

Reagents

86

Appendix E-3

Vitamin E Determination

UV Grade Hexane. glass distilled
KOH pellets. reagent grade

Ethanol 95%. Distill over KOH pellets (ca 50 g/
liter)

Quinine sulfate U.S.P. Prepare a 1% solution of
quinine (10 mg/ml) by dissolving 1.05 gm of the
salt in 100 ml 0.1N H2504. Dilute with 0.1N H250
to prepare a quinine solution containing 0.1 ug/mI.

Vitamin E. Dissolve detocopherol (Eastman Kodak
Co.) in ethanol to give a solution containing
approximately 100 ug/ml. Check the concentration
by reading in a spectrophotometer at 292 mu
1% -

(Elcm - 72).
Dilute with ethanol to make standard dilutions with
the following concentrations: 40 ug/ml. 20 ug/ml.
10 ug/ml, 5 ug/ml. and 2.5 ug/ml.

Acid Wash. Combine one liter of 3N HCl in distilled
water with one liter of 50% ethanol in distilled
water.

Procedure

1.

All glassware to be used in the preparation for or
in the actual determination should be prewashed in
the acid wash.

In triplicate. dilute 0.3 ml of each of the following:
serum, water, and the five toc0pherol standards with
1 ml of distilled water in 12 ml conical glass
stoppered centrifuge tubes and mix on a mechanical
mixer for 5 seconds.

Add 2 m1 of ethanol and mix on a mechanical mixer
for 10 seconds.

10.

11.

87

Add 5 m1 of hexane. stopper the tubes, and mix
thoroughly for 3 minutes with a mechanical mixer.

Centrifuge the tubes for 10 minutes at 1000 rpm.
Keep the tubes stoppered and protected from light
until the hexane extracts can be read in a spectro-
fluorometer.

Using a Varian SF 330 spectrofluorometer set the
following controls:

Read Mode 0.25 sec Excitation 295 mv

Time Constant 1 Emission 320 mv

Selector 10 Slits lO mv excitation
Sensitivity 1 20 mv emission

Insert the 0.1 mg/ml quinine sulfate solution first
and adjust the variable control so that the quinine
solution reads 2.5.

Do not alter the controls after this point.

Read the fluorescence of the water blank, standards
and serum.

A linear regression equation is prepared from the
fluorescence and concentration values of the toco-
pherol standards and used to calculate the concen-
tration of the serum from its fluorescence.

88

APPENDIX F

Table 7. Composition of purified rat diet

 

 

Ingredients g/8 kg diet
Dextrose 5565.00
Casein, vitamin free 1600.00
Soybean oil, oxidized1 400.00
Monosodium phosphate 160.00
Calcium carbonate 160.00
Potassium chloride 40.00
Iodized sodium chloride 40.00
Choline chloride 12.00
Vitamin mix2 21.60

3

Mineral mix 0.53

 

1Heated to approximately 3750 for 80 hours over a two week

period.
2Composed of (in mg/kg of diet): vitamin A (500.000 IU/gm).

10; vitamin 03 (40,000 IU/gm), 7.5; menadione. 0.4; thiamin
hydrochloride. 3; riboflavin. 3; Pyridoxine hydrochloride,

3; calcium pantothenate. 6; niacin, 30; folic acid, 1; and
vitamin 812 (0.1% in mannitol). 2.
3Composed of (gm/100 gm of mineral mix): magnesium sulfate,
anhydrous. 73.82; zinc sulfate (ZnSO4-7H20). 19.66; man-
ganous sulfate, monohydrate, 5.73; cupric sulfate (CuSO4-
5H 0). 0.73; potassium iodate. 0.06; and ferrous sulfate,
0. 6.