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ABSTRACT

A PAPER AND GAS CHROMATOGRAPHIC ANALYSIS
OF URINARY CARBOHYDRATES
IN A MENTALLY RETARDED POPULATION

by

Michael A. Abruzzo

A metabolic screen of institutionalized mentally retarded
individuals in Michigan was begun in 1968. One portion of that
screen tested for the presence of reducing substances in the
urine. The present study, using paper chromatographic
techniques, analyzed all the positives detected in the original
screen. Also, all the known diabetics in the institutions were
studied using paper chromatography. In addition, urines from
a selected sample of normal and retarded individuals were
analyzed by gas chromatography. No identifiable carbohydrate,
except glucose, was excreted in excess. There was no detectable
difference in the carbohydrate excretion patterns of normal
or retarded individuals. The excretion of an unknown compound
was detected in both pOpulations. The distribution, concen-

tration, and significance of this unknown compound is discussed.

A PAPER AND GAS CHROMATOGRAPHIC ANALYSIS
OF URINARY CARBOHYDRATES
IN A MENTALLY RETARDED POPULATION

by

Michael A. Abruzzo

A Thesis

Submitted to

Michigan State University

in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Zoology

1971

To
Dee, Mike and Alison
and

my Parents

ACKNOWLEDGMENTS

I would like to express Special thanks to my professor,
Dr. James V. Higgins, for his guidance and patience during
the course of the experimental work.

Also, I would like to thank Dr. Hermen Slatis and Dr.
William Wells for their advice and constructive criticism.

Without the friendship and encouragement of the following
individuals the completion of this thesis would not have been
possible: Dr. Ajovi Scott-Emuakpor, Terry Hassold, Larry
Yotti, Pat Alvord, Pat Jolly, and Lou Betty Richardson.

Thanks are also due my typists, Chris and Debbie.

TABLE OF CONTENTS

Page
ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . .
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . iii
LIST OF FIGURES . . . . . . . . . . . . . . . . .
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . . 3
MATERIALS AND METHODS . . . . . . . . . . . . . . .
RESULTS . . . . . . . . . . . . . . . . . . . . . . . . 21
DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . 31

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . 39

ii

Table

10

11

LIST OF TABLES

Inborn Errors of Carbohydrate Metabolism
Disorders Associated with Diabetes mellitus

Disorders with Isolated or Generalized
Mellituria

Descending Rg Values of Selected Sugars on
Whatman No. 3MM Paper

Relative Retention Times for TMS Derivatives
of Urinary Carbohydrates

Recovery of Added Quantities of Glucose to
Human Urine

Results of the Original Screen for Urinary
Carbohydrates

Glucose Excreters Detected Using Paper
Chromatography

Carbohydrate Concentrations in Fasting
Urines from Retarded Individuals

Normal Carbohydrate Concentrations in
24 hour Urine

Serum Glucose Concentrations in Difficult to
Control Diabetics

iii

Page

10

ll

16

19

20

22

24

26

29

3S

Figure

LIST OF FIGURES

Paper Chromatogram of Urinary Carbohydrates

Paper Chromatogram of Different Concentrations
of Glucose

Gas Chromatogram of Urinary Carbohydrates

Distributions of Excreters of Ribose,
Glucose, and Fructose

Distribution of Excreters of an Unidentified
Compound

iv

Page

15

15

18

27

3O

INTRODUCTION

It has been known since 1666 that carbohydrates may be present
in the urine of individuals affected with diabetes mellitus, inborn
errors of carbohydrate metabolism, liver dysfunction, kidney failure,
etc. Recently, however, improved chromatographic techniques have
shown that as many as thirty carbohydrates or carbohydrate like com-
pounds may be found in the urine of normal individuals (Jolley and
Freeman, 1968, Butts and Jolley, 1970). The sugars which have been
identified in human urine to date are glucose, galactose, fructose,
xylulose, xylose, ribose, ribulose, sucrose, lactose, raffinose, N-
acetylglucosamine, maltose, arabinose, fucose, mannoheptulose, and
glucose-l-phOSphate (Marks and Samols, 1968, Jolley and Freeman,
1968, Butts and Jolley, 1970). The above carbohydrates, except
glucose, are usually found only in trace amounts.

It has been found in a screen of the general pOpulation (Malina,
1966) that from 1 to 3 per cent of the individuals tested with a
glucose oxidase test strip react positively ( >’30 mg per cent).

Fox et al., (1962) found a slightly higher frequency using both a
glucose oxidase test strip and a reducing substance test (positive if
greater than 100 mg per cent). The most common carbohydrate found in
excess is glucose and is often times indicative of diabetes mellitus

which is clinically recognized in l to 2 per cent of the pOpulation

(Keen, 1966). Fructose, galactose, and pentoses are found in
excess very infrequently (Wright, 1961, Hanson et al., 1964,
Thalhammer et al., 1968, Mayes, 1969, Hall et al., 1970, Schwarz
et al., 1961, Hsia, 1966) but are never the less important
clinically because of the various inborn errors of carbohydrate
metabolism associated with each (Table 1). Lactose is found in

8 to 9 per cent of the pOpulation but only in very small amounts
insufficient to cause a positive reaction with reducing tests
(Darling et al., 1960). Only in lactosuria and late in pregnancy
are significant amounts of lactose excreted.

In 1968, a screen of the institutionalized mentally retarded
in the State of Michigan was begun. One portion of that screen
tested for reducing substances in the urine and 2.3 per cent of
5,200 tested reacted positively. The present study was begun to
qualitate and quantitate the carbohydrate excretions in these

mentally retarded individuals.

REVIEW

Urinary excretion of metabolic products, eSpecially car-
bohydrates, is highly variable. It is often dependent on many
intrinsic and extrinsic conditions. Specific enzymatic errors
in carbohydrate metabolism may lead to the excretion of a car-
bohydrate. The quantity of sugar excreted is also affected by
factors other than the enzymatic activity such as diet, disease,
age, time of sample collection, and sex. Due to these variables
it is difficult to define, quantitatively, what constitutes
normal and abnormal mellituria. Fortunately, in most cases which
reflect a significant metabolic defect,. the amount of sugar
excreted is excessive and easily distinguished from that of normal
subjects.

INBORN ERRORS

 

Inborn errors of carbohydrate metabolism are the most sig-
nificant abnormalities associated with urinary excretion of sugars.
The sugars present in the urine of an individual suffering from either
galactosemia, galactokinase deficiency, essential fructosuria,
hereditary fructose intolerance, or essential pentosuria are rep-
resentative of Specific enzymatic errors.

Galactosemia is an inborn error of matabolism characterized by

the absence of activity of galactose-l-phOSphate uridyl transferase

(Kalckar et al., 1956). There is a slight amount of trans-
ferase activity in galactosemic Negroes (Rogers et al., 1970,
Segal, 1969) . The disease manifests itself after a few days

of milk feeding, showing increased concentrations of galactose

and galactitol in the blood and urine (Egan et al., 1965). If
galactose is not removed from the diet in the first few weeks

of life the child becomes malnourished and dwarfed, mental re-
tardation and cataracts usually present themselves, and cirrhosis
of the liver may occur months or years after the acute phase of
the disease (Hsia, 1966, Isselbacher, 1966, Thalhammer et al.,
1968, Sidbury, 1969). However, removal of galactose from the diet
within the first few weeks of life results in normal physical de-
velOpment but below average mental development with an average 1.0.
of 80 (Donnell et al., 1969, Komrower and Lee, 1970). The urinary
galactose disappears after galactose is removed from the diet but
galactitol continues to be excreted in diminishing concentrations
for several days (Egan et al., 1965).

The disorder is inherited as an autosomal recessive with
heterozygotes having an enzymatic activity between the two
homozygotes (Mayes, 1969, Isselbacher, 1966, Rogers et al., 1970).
Beutler (1965) has described a variant of galactosemia (Duarte
variant) resulting in a 50 per cent reduction in activity of galactose-
l-phosphate uridyl transferase in homozygotes and a 25 per cent
reduction in heterozygotes. Recently, a method for the determination

of the various genotypes of galactosemia, including the Duarte

variant, has been described (Tedesco and Mellman, 1969). This
method measures the ratios between transferase and galactokinase

in cultured human fibroblasts and peripheral blood leukocytes and
they state this analysis eliminates the heterozygote overlaps which
occur with a direct analysis of transferase activity.

As a galactosemic child grows older, some have shown an increased
ability to tolerate small amounts of milk products. It has been
suggested (Hsia, 1966, Tedesco and Mellman, 1969, Segal, 1969) that
this slight tolerance of galactose could be due to a liver enzyme,

UDP galactose perphOSphorylase which catalyzes the following reaction:

 

UTP + gal-1--P.R \'UDP galactose + PPi
The presence of galactitol in many tissues and also in the urine of
galactosemics (wells at al., 1964b, 1965, Quan-Ma et al., 1966, Egan
and Wells, 1966, Wells, 1969) is also evidence for an alternate
metabolic pathway for galactose. Galactose is converted to galactitol
by the enzyme aldose reductase. Because of this alternate pathway
the levels of galactose-l-phosphate within the tissues are reduced.
The activity of aldose reductase has been shown to increase with age
(Wells, 1969).

Another error of galactose metabolism, galactokinase deficiency,
was first described by Gitzelmann, (1965) in a brother and sister of
a gypsy family. Only two other cases have been reported, an Austrian
infant, also a gypsy, (Thalhammer et al., 1968) and the first non

gypsy described by Dahlgvist et al., (1970). This disease is thought

to be due to an autosomal recessive gene. Affected homozygotes
excrete galactose, galactitol, and glucose in varying concen-
trations depending upon the time of sample collection,

(Gitzelman, 1965, 1967) (Table 1). Removal of galactose from

the diet will cause urinary concentration of galactose, galactitol,
and glucose to dr0p to a normal level (Thalhammer et al., 1968). The
major physical problem is the presence of juvenille cataracts and
these can be alleviated if dietary treatment begins at an early age
(Gitzelman, 1967). There is no reported impairment in mental de-
velopment with or without dietary treatment.

Essential fructosuria, a disorder of fructose metabolism, is
a benign condition. Affected individuals show no activity for the
hepatic enzyme fructokinase (Froesch, 1966). If fructose is present
in the diet the urinary concentration can exceed 2g/100 ml following
a meal or 67-270 mg per cent in a fasting sample (Marks and Samols,
1968, Froesch, 1966) (Table 1). There is no treatment required. The
disorder is inherited as an autosomal recessive.

Hereditary fructose intolerance was first described by Froesch
et al., (1957). The metabolic disorder is a deficiency of l-phospho-
fructose aldolase (Kranhold et al., 1969). Ingestion of fructose
causes severe symptoms of anorexia, nausea, vomiting, profound mal-
aise, and abdominal pain (Marks and Samols, 1968). The urinary
excretion of fructose is usually not marked and almost always found
after an ingestion of fructose-containing foods (Cornblath et al.,

1963). Abnormalities in amino acid metabolism, especially tyrosine

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metabolism, have been noted (Grant et al., 1970, Lindman et
al., 1970). Complete exclusion of fructose-containing foods
is all the treatment that is necessary, but early diagnosis
is important in view of the possible cerebral damage which may
follow hypoglycemia (Hsia, 1966).

Essential pentosuria is a benign biochemical anomaly in
which one to four grams of L-xylulose is excreted in the urine
each day. The enzymatic lesion so far has not been demonstrated
by direct assay of liver mitochondria but evidence is strong that
the disorder is caused by a deficiency of NADP xylitol dehydrogenase,
an enzyme of the glucuronic oxidation pathway (HSia, 1966). The
condition is inherited as an autosomal recessive and found almost
exclusively in pe0ple of Jewish ancestry (Hiatt, 1966). The ex-
cretion of L-xylulose is unaffected by diet but may be increased
by loading the patient with glucuronolactone. This also serves as
a method for the detection of heterozygotes (Hiatt, 1966) (Table l).
The disorder is usually not age dependent, but one report (Politzer
and Fleischmann, 1962) suggests that L-xylulose excretion may not
occur until puberty in some individuals. There is no treatment

necessary; the condition is compatable with health and well being.

DISEASE AND MELLITURIA
Diabetes mellitus is probably the most common of all disorders
associated with glucosuria. In untreated individuals glucose is

usually excreted, in concentrations exceeding 150 mg per cent.

Diabetes mellitus is also found in association with many other
diseases (Table 2) but it is not known if the diabetes is a
primary cause or a secondary result of the various diseases
(Sotos, 1970).

Renal glycosuria, a disease characterized by the presence
of glycosuria without hyperglycemia, often times can cause an
excretion of glucose exceeding 100g/24 hr. independent of diet
(Krane, 1966, Milne, 1966). Glucosuria is sometimes associated
with Vitamin D resistant rickets (Burnett et al., 1964, Williams
et al., 1966), and The Fanconi Syndrome, (McCune, 1943, Leaf,
1966, Milne, 1966). It also is present in a glucose transport
defect of the intestine and kidney (Lindquist, 1963).

Fructosuria, glucosuria, and galactosuria are found in some
acute cases of tyrosinosis (Lindmann et al., 1969) and lactosuria
has been noted in infants and children with neonatal jaundice,
septicemia, steatorrhea, and severe dyspepsia (Haemmerli et al.,
1965). In parenchymal liver disease galactose clearance from the
blood is less efficient than normal and after galactose ingestion
blood galactose rises which leads to an excretion of galactose
(Tygstrap, 1961).

In general, individuals with disorders which affect the liver,

kidney, thyroid or intestine may exhibit a mild mellituria (Table 3).

VARIATION WITHIN "NORMALS"
The dietary intake of carbohydrates considerably affects the

excretion pattern of sugars in a patient with metabolic defects.

10

 

TABLE 2

Disorders Associated with Diabetes Mellitus*

 

Alstrom's Syndrome

Ataxia Telangiectasia

Total LipodystrOphy

Optic Atrophy and Nerve Deafness
Werner's Syndrome

Friedreich's Ataxia

Photomyoxlonic Seizures, Progressive Nerve
Deafness, Nephropathy, and Cerebral Dysfunction

Thyroid disorders
Gonadal Dysgenesis

Prader-Willi Syndrome

 

* from Sotos, (1970)

11

 

TABLE 3

Disorders with Isolated or Generalized Mellituria
(excluding disorders from tables 1 and 2)

 

 

KIDNEY INTESTINE
renal glycosuria steatorrhea
tubular intoxication severe dyspepsia
Fanconi syndrome scleroderma**
glucose transport defect enteritus**
nephrosis* acrodermatitis
nephritis* enteropathica**
Lowe syndrome* lymphangiectasia**

giardiasis**
LIVER OTHER

neonatal jaundice

acute tyrosinosis

parenchymal liver disease cystinosis*
Wilson's disease* septicemia
hepatitis* hypophyseal
cirrhosis* dysfunctions*

adrenal dysfunctions*
hiatus hernia*
cystic fibrosis**

 

*from Bickel, (1961)
**from Menzies and Seakins, (1969)
all others found in text

12

However, healthy infants, children, and adults have been shown to
have an increased sugar excretion when the diet is rich in car-
bohydrates (Haworth, 1960, Bickel, 1961, Haemmerli et al., 1965).
Subjects loaded with sucrose, maltose, or lactose can be shown

to have increased urinary concentrations of glucose, galactose,
fructose, sucrose, and lactose.

Age of the individual can also contribute to the overall ex-
cretion pattern of carbohydrates. Infants born at term exhibit a
physiological mellituria which lasts for the first ten days and
reaches its maximum on the third, fourth, and fifth days (Bickel,
1961). Increased glucose and fructose excretion is evident from the
first day and galactose and lactose are excreted when milk feeding
starts. The highest values are for lactose, usually 150 mg per cent
(Haemmerli et al., 1965). Premature infants have even greater
mellituria due to higher fructose and lactose concentrations in the
urine (Dahlquist and Svenningsen, 1969, Haemmerli et al., 1965).
After two weeks of life the carbohydrate excretion stabilizes and
age has no effect except in the type of lactose intolerance which
manifests itself only in adults (Haemmerli et al., 1965).

Sex has no effect on sugar excretion except in two instances:
diabetes mellitus occurs more frequently in women than in men (Sotas,
1970), and excretion of lactose is common in the late stages of

pregnancy (Haemmerli et al., 1965, Marks and Samols, 1968).

MATERIALS AND METHODS

The subjects in this study were institutionalized mentally
retarded individuals. The individuals selected were:

1. those who had reacted positively to either
Clinitest or Labstix.

2. a random sample of mildly retarded in-
dividuals (10 males, 10 females).

3. a random sample of severly retarded
individuals (10 males, 10 females).

4. all individuals reported to be affected
with diabetes mellitus by the institution's
staff members.2
Early morning (fasting) urine samples were collected from each

subject and stored at -20°’C. Each urine was then thawed and
spotted in concentrations of 10 and 30))on Whatman No. 3MM chromato-
graphy paper (56 x 23 cm) and run in a descending direction in a
glass air tight chromatography tank (60 x 30 x 30 cm) for 14 hours
or until the solvent front migrated the length of the paper. The
solvent was a mixture of 3 parts iSOprOponol, 1 part acetic acid,
and 1 part water. A mixture of standards was Spotted on each

Chromatogram. This included glucose, galactose, fructose, ribose,

xylose, glucuronic acid, lactose, and sucrose each in a concentration

 

1Ames Company

2See Discussion for eXplanation of "reported".

13

14

of 100 mg per cent. After completion of the run, the chromato-
grams were dried in a fumehood for four hours and dipped in
aniline diphenylamine location reagent, prepared according to the
method of Menzie and Seakins (1969). The stained chromatograms were
heated in an oven at 95-1000C for 2-3 minutes.

Figure 1 is a sample chromatogram showing the migration pattern
of lactose, sucrose, glucuronic acid, galactose, glucose, fructose,
xylose, and ribose. Each sugar was dissolved in urine in a concen-
tration of 100 mg per cent. Figure 2 shows the difference in
densities of Spots for five samples of glucose in concentrations of
100, 200, 300, 400, and 500 mg per cent. Approximations of urinary
carbohydrate concentrations were made by comparing the unknown
amount of sugar with two known concentrations of the same carbohydrate.
Table 4 lists the Rg values for the various carbohydrates studied.

Selected urines were prepared for gas chromatography by a variation
of the method described by Wells et al., (1964a). A 1 m1 aliquot of
urine along with 1 ml of internal standard (‘1 -methy1-D~mannoside in a
concentration of 1 mg per ml) were evaporated in vacuo at 50c’C. Tb
this dried sample was added 1-2 ml of TMS reagent (Sweely et al., 1965)
and allowed to react at room temperature for 10 minutes. A 1 ul sample
was then injected into a Beckman G-C 45 gas chromatograph equipped with
a flame ionization detector. The glass column (6 ft., 2mm I.D.) was
packed with OV-I, 1% on gas chrom Q, 80-100 mesh. Carrier (N2) flow
rate was 30cc per minute and column temperature was 135c>C isothermal.

Figure 3 is a sample chromatogram of TMS derivatives of some

 

15

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R
)C
W
p
C “L
3 ca.
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Sets
Lac.
Lre-~fl~~—e-w ~-~—«-g-~ ~~« r -» - - ~ ~g~~~~-»«*”~--*“~r
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Fig. 1 Paper chromatogram illuatrating the migration of
acne urinary carbohydratea in a aoivent of iaoprOpanolo
acetic acid. and water (331:1). Mixture 1 containa
(in order from the origin) lactoee, glucuronic acid.
glucose. and ribcae. Mixture 2 containa aucroee.
galactoae. fructose. and xyloae. A. B. C. and D. "0
normal urinea with added carbohydrate.

 

15a

 

100 zoo 300 400 500
age

in

Pi . 2. Pa r chranatogr- illuetrating the difference

9 defiity of apota tor increaeing concentrations of
glucoae.

16

 

TABLE 4

Descending Rg Values of Selected Sugars
on Whatman No. 3MM Paper

 

 

CARBOHYDRATE Rg COLOR
arabinose 1.12 green
xylose 1.30 green
ribose 1.37 green
fructose 1.17 yellow-brown
glucose 1.00 blue-grey
galactose 0.95 blue-grey
sucrose 0.85 tan-brown
lactose 0.44 blue
glucuronic acid 0.90 red-brown

 

solvent - isOpropanol, acetic acid, water
location reagent - aniline diphenylamine

17

urinary carbohydrates and Table 5 lists the relative retention
times of the urinary sugars considered in this study. Each
carbohydrate was examined as an equilibrium mixture except fructose
which was added to the reagent as a solid. Table 6 shows the
results obtained when glucose was added to five urines (which had
previously been shown to have no detectable glucose) and the urine
was then prepared and chromatographed according to the method des-
cribed and the concentration of glucose calculated. The mean per

cent recovery of added glucose was 97.4.

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TABLE 5

Relative Retention Times for TMS Derivatives

of Urinary Carbohydrates*

 

 

CARBOHYDRATE ov-1, 135°C
Ribose forms 0.45, 0.48, 0.49
ooxylose 0.63
~£-xylose 0.88
fructose 1.25
aigalactose 1.31
o6galactose 1.56
B—galactose 1. 95
ooglucose 1.74
fi-glucose 3.12
oomannose 1.17
.B-mannose 1. 87
galactitol 2.44
mannitol 2.37

 

* all retention times are relative to
cx-methyl-D-mannoside, which had a

retention time of 7.25 minutes

20

 

TABLE 6

Recovery of Added Quantities of Glucose

to Human Urine

 

SAMPLE NO.

GLUCOSE(ug)

RECOVERY (%)

 

added found
1 1000 967 96.7
2 1000 970 97.0
3 1000 1002 100.2
4 1000 986 98.6
5 1000 948 94.8

mean 97.4

 

RESULTS

SCREENING STUDIES

In a previous study 5,200 retarded individuals were screened
for urinary carbohydrates using a glucose oxidase test strip
(Labstix) and a reducing test (Clinitest). The labstix is Specific
for glucose and a positive reaction occurs if the urinary glucose
concnetration exceeds 30 mg per cent (Sacre, 1970). Clinitest will
react with any reducing substance in the urine in excess of 100 mg
per cent. A total of 120 urines gave positive reactions on one or
both tests for a frequency of 2.3% (Table 7). Positive reactions
with labstix occurred in 95 urines, a frequency of 1.8%, and clinitest
reacted positively in 63 urines, a frequency of 1.2%. This frequency
of 2.3% is a minimum estimate for many of the diabetics in the in-
stitutions are controlled by either diet or medication and therefore
were not excreting glucose. All urines tested were random samples

collected by the institutions.

PAPER CHROMATOGRAPHIC ANALYSIS

The present study collected fasting (overnight) urines from all
the individuals who reacted positively (traces excluded) in the pre-
vious study. Also, all individuals who were said to be diabetics by

the physicians at the state institutions, were included in the study

21

22

 

TABLE 7

Results of the Original Screen for Urinary Carbohydrates

 

 

TEST SENSITIVITY POSITIVES FREQUENCY
Clinitest 2>100mg% 63 1.2%
Glucose oxidase 21 30mg% 95 1.8%

combined ---- 120 2.3%

 

23

and fasting urines were collected from each. Paper chromatograms
were run for each urine. The carbohydrates which could be identi-
fied were; glucose, galactose, fructose, xylose, ribose, lactose,
and sucrose. The total number of individuals tested was 195.
Twenty-five individuals were detected who excreted glucose
ranging from loo-6,800 mg per cent with a mean excretion of
1,980 mg per cent (Table 8). Of these twenty-five glucose ex-
creters, eighteen were known diabetics and the remaining are
presently being tested by the institutions for abnormal glucose
tolerance. The range of glucose excretion in the individuals not
known to be diabetics is loo-5,000 mg per cent with a mean ex-
cretion of 2,143 mg per cent. Four of these individuals had
excretions of glucose greater than 1,000 mg per cent. Six
individuals had a generalized mellituria excreting both lactose
and sucrose plus a compound with an Rg value near ribose. These
generalized melliturias were not consistent and on re-testing
with a new urine sample were not repeatable. The concentrations
of the sugars inthese individuals was very minimal and at the lower
end of the sensitivity of the paper chromatographic technique,
approximately 30 mg per cent.

The paper chrOmatographic technique was also used in screening
112 individuals from the Mfichigan State University campus as part of
another study (Richardson, 1970). The urine chromatograms of these
individuals did not reveal any Spots correSponding to the standard

sugars of the present study.

24

 

TABLE 8

Glucose Excreters Detected Using Paper Chromatography

 

 

PATIENT AGE CONCENTRATION (mg%)

3 61 3000

7 63 1000
9* 58 4500
16* 42 100
25 -------------- 28 --------------- 200
27* 59 3000
29* 53 500
40 34 6800
42 40 150
45 -------------- 36 -------------- 1200
51 32 1400
56 50 225
58* 50 370
60 18 580
67 ------------- 39 -------------- 4600
134 35 5000
135 33 500
141 22 1000
144 18 300
154* -------------- 56 -------------- 5000
182 63 5000
190 -- 300
191 -- 1200
194* 46 1500
199 17 300

 

* individuals not listed as diabetics by the
state institutions

25

GAS CHROMATOGRAPHIC ANALYSIS

 

Fasting urines were collected from forty retarded in-
dividuals, twenty who were mildly retarded, and twenty who
were severly retarded. There were ten males and ten females
in each group. Gas-chromatography was used to analyze each
urine. Ribose was detected in 26 urines in minor amounts.
Nineteen of these individuals excreted less than 1 mg per cent
(trace concentration). The other seven urines had ribose concentra-
tions ranging from l-5.4 mg per cent (Fig. 4). Fructose was
found in 28 urines. Nineteen urines had trace* (<:3.5 mg %)
amounts of fructose and nine urines had concentrations ranging
from 3.5-26.7 mg per cent (Fig.4). Glucose was detected in the
urine of 28 non-diabetic individuals. Fifteen urines had trace*
(<:5.0 mg %) amounts of glucose and 13 urines had glucose con-
centrations ranging from 5.0-27.0 mg per cent. Peaks corres-
ponding in retention time to mannitol and galactitol were
found in 3 and 6 urines respectively, in trace amounts (<110 mg %).
Xylose and galactose were not found in the forty urines tested.
No differences were seen between males and females or between
severly and mildly retarded individuals. No individual was found
who had an excessive excretion of any of the carbohydrates studied.
Table 9 summarizes the data obtained from the urine samples of the
retarded individuals.

Eleven individuals, with normal intelligence who donated 24

 

*As retention time increases, sensitivity decreases

26

 

TABLE 9

Carbohydrate Concentrations in Fasting'Urines
from Retarded Individuals

 

 

CARBOHYDRATE RANGE (mg%) DETECTED IN
ribose 0.0-5.4 26 urines
xylose ---- 0 urines
fructose 0.0-26.7 28 urines
galactose ---- O urines
glucose 0.0-27.0 28 urines
mannitol* 0.0-Tr 3 urines
galactitol* 0.0-Tr 6 urines

 

* correspond in retention time to mannitol and galactitol

27

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28

urine samples, were analyzed in the same manner, in an attempt
to determine normal excretion patterns. Table 10 summarizes

the data obtained from the urine samples of the normals. Ribose
was detected in all urines, fructose in nine, and glucose in ten
(Fig. 4). Six individuals excreted a compound correSponding in
retention time to mannitol. Of these six, three had trace
amounts, and the remaining three urines had concentrations of
18.2, 28.4, and 105.8 mg per cent. Trace amounts of galactose
were detected in two urines and no individual excreted xylose.

A compound with a retention time between ci-methyl-D-
mannoside and fructose was found in 17 of the retarded individuals
urines in concentrations ranging from trace to 26 mg per cent and
in all of the normal urines ranging in concentration from 15.5-130.0
mg per cent (Fig. 5). The one individual who excretes greater than
100 mg per cent of this unknown compound is known to be a high
excreter of.£?, amino isobutyric acid (BAIB), but BAIB does not
react with the TMS reagent and therefore could not be detected on

the chromatograph.

29

 

TABLE 10

Normal Carbohydrate Concentrations in 24 hour Urine

 

 

CARBOHYDRATE RANGE (mg%) DETECTED IN
ribose Tr-2.7 ll urines
xylose ---- O urines
fructose 0.0-28.0 9 urines
galactose 0.0-Tr 2 urines
glucose 0.0-11.4 10 urines
mannitol* 0‘0-105.0 6 urines
galactitol* 0.0-Tr 3 urines

 

* correspond in retention time to mannitol and galactitol

30

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DISCUSSION

SC REEN ING STUDIES

 

Malins, (1966), using a glucose oxidase test strip, tested
18,532 subjects from a normal p0pulation and detected 610
possible sugar excreters, a frequency of 3.3%. This frequency,
also included all the known diabetics in the p0pulation. The
present study detected 95 possible sugar excreters using a glucose
oxidase test strip, a frequency of 1.8%. This frequency does not
include diabetics who did not spill glucose. When the known
diabetics are included in the present study the frequency becomes
3.8%. This frequency compares well with that of Malins. Fox et al.,
(1962), also tested a normal pOpulation (19,403 individuals),but
used Benedicts reagent as well as a glucose oxidase test strip. With
both of these tests he found a frequency of reducing substance
excreters of 4.5%. The present study using Clinitest and Labstix
found 2.3% positive.

The results of the present study when compared with those
obtained in a normal pOpulation show that there is not a detectable
increase in the frequency of sugar excreters or in diabetes mellitus
in this population of retarded individuals.

The only published study of sugar excretion in a retarded

pOpulation is that of Hall et al., (1970). Hall studied a p0p-

31

32

ulation of 1200 retarded individuals and found galactosuria in

59 of the patients. There was no indication of what urinary
concentration of galactose was present in these individuals.

In the present study no galactose was detected in either the
original screen, the paper chromatographic or the gas chromato-
graphic analysis. This discrepancy in results can be eXplained
by the fact that most of the urines in the present study were
fasting samples while those analyzed by Hall et a1. were samples
collected after a test meal which contained milk and milk pro-

ducts.

PAPER CHROMATOGRAPHIC ANALYSIS

 

In the original screen of 5,200 retarded individuals, 120
possible sugar excreters were detected. Upon careful analysis
of these individuals no significant excretion of any carbohydrate,
other than glucose, was found. This can be eXplained by the rare
occurence of the inborn errors of carbohydrate metabolism, the
random time of urine collection, and the level of sensitivity
of the clinitest reducing test.

Because galactosemia is associated with mental retardation,
you would eXpect a higher frequency of galactosemics in a
mentally retarded p0pulation than in a normal population, but
at the present there are no known galactosemics in any of the
State institutions in Michigan (A large percentage of the blind
patients remain to be screened). The other inborn errors of

carbohydrate metabolism are not known to be associated with mental

33

retardation and therefore the rare frequency of occurrence of
these disorders in the normal population is also the frequency
of occurrence one would eXpect in a mentally retarded p0p-
ulation.

The original urine collections were random, and there-
fore some urines were collected after a meal. This could
cause an increase in urinary carbohydrates in some instances.
The repeat analysis was done using fasting urines, which
would not be subject to as great a dietary variation.

The clinitest reducing test can be shown to be a general-
ized and relatively insensitive method for the detection of
reducing substances in the urine. Carbohydrates will not
cause a positive reaction in quantities less than 100 mg per
cent. Although this level of sensitivity is often sufficient
to detect the excretion of carbohydrates found in individuals
with many of the inborn errors of carbohydrate metabolism it
is not sensitive enough to detect the minor melliturias, which
occur in many individuals. Sucrose and myo-inositol, non-
reducing sugars, will not be detected at any concentration.

In addition, clinitest is known to react with many non-sugar
reducing substances in urine such as urates, ascorbic acid,
glucuronides, etc. Because of this general lack of specificity
and sensitivity in screening for non-glucose excreters it was
not surprising that the original frequency of positive reducing

substance excreters was drastically reduced when each individual

34

was tested with the more Specific and sensitive paper chromato-
graphic technique.

The glucose excreters detected in the paper chromatographic
portion of the study included eighteen known diabetics and
seven patients who were not known to be diabetics. These seven
individuals are presently being tested by the State institutions.
However, many of the patients that were said to be diabetics by
the state institutions do not seem to fit the "classical pattern".
Of the known diabetics who are supposedly under control, we
detected ten individuals who had concentrations of glucose in their
urine in excess of 1000 mg per cent. Data were also available
on serum glucose concentrations of four diabetics who were
difficult to control. Table 11 lists the serum glucose concen-
trations of these four individuals in successive weeks. It is
evident from the data that either the control procedures are not
being followed carefully or that these individuals are not what
we normally consider diabetic. These two findings, excessive
glucose excretion in diabetics under control, and uncontrollable
serum glucose concentrations in diabetics, point out the need for
a detailed study of diabetics in the homes for the retarded. It
is possible that diabetes mellitus, not unlike muscular distrOphy,

is a sympton of many different disorders.

GAS CHROMATOGRAPHIC ANALYSIS
The gas chromatographic analysis of urines from retarded and

normal individuals is a preliminary study and was undertaken to

35

 

TABLE 11

Serum Glucose Concentrations in Difficult to Control

 

 

Diabetics
GLUCOSE CONCENTRATION (mg%)
PATIENT week 1 week 2 week 3 week 4
F.W. 372 ——- 48 208
N.S. 420 106 226 69
J.M. 274 300 252 292

J.T. 168 261 134 ---

 

36

determine the carbohydrate excretion patterns of the two groups.
Although the samples at present, are too small for any statistical

analysis it appears as though there are no, or only minor, differ-

ences between the two pOpulations. One retarded individual is

excreting 26.7 mg per cent of fructose and two retarded individuals
are excreting glucose in concentration greater than 20 mg per cent.

Although these concentrations of sugars are not considered sig-

nificant amounts, they may well be Significant in that the
samples were fasting urines. TWenty-four hour urine specimens
will be collected from these three individuals for the purpose
of determining if there actually is a significant excretion of
carbohydrates.

Three urines from normal individuals had large amounts of
a compound correSponding in retention time to mannitol, but
there is some question as to whether this compound is mannitol
or sorbitol, the two compounds being difficult to separate.
There was no sorbitol available for standardization and there-
fore no definite identification of the compound can be made

using the gas chromatograph.

An unknown compound which is consistently excreted by both

groups sampled is still under investigation. Attempts are being

made to identify this compound and determine its significance

in human urine. It is interesting to note that the compound was

detected in its greatest concentration in a normal individual.

 

fining-en‘l

w......-,‘- ....,

37

CONCLUDING REMARKS

 

Fasting urines were used in this study because of the
numbers of individuals involved. It was not feasible to
collect 24 hour urines from over 200 patients. Fasting urines
were considered better than random urine collections because
they give more consistent results and approximate more closely
a 24 hour urine (Watson and Langford, 1970). It is true, how-
ever, that because of the fasting urines minor melliturias were
probably missed.

0f the analytical methods used in this study, the paper and
gas chromatographic techniques proved to be the most useful.
Paper chromatography is relatively ineXpensive and efficient,
and allows for easy identification of urinary carbohydrates.
However, quantitation with extreme accuracy is not possible
with this technique. The level of sensitivity in the system
was only 30 mg per cent. Carbohydrates in concentrations below
this were not detectable without concentration of the urine.
The gas chromatographic technique was extremely sensitive,
allowing for detection of carbohydrates in concentrations as
small as 1 mg per cent. It was possible to analyze a minimum
of five urines daily by this method. A combination of paper or
thin layer chromatography with gas chromatography being used for

the detailed analysis of abnormal urines is an accurate and

efficient screening technique, which could be used in place of the

traditional reduction tests which are useful only for detecting
severe melliturias. If glucose is the only carbohydrate being

tested for, then the labstix or any glucose oxidase test strip

_ ,_.___. f]

 

 

38

proves to be precise, sensitive, and efficient for qualitative
analysis.

This study, although not large enough to make any
statistical interpretation does, I believe, show that the
general carbohydrate excretion patterns of retarded and normal
individuals are similar. It is evident that future studies
should deal with the defining of normal carbohydrate excretion
patterns, of determining what actually constitutes diabetes
mellitus and of identifying all of the carbohydrate or
carbohydrate-like compounds which are found in urine. There
are, at present, 5000 additional retarded individuals being
screened and it is after these tests are completed that more

definite conclusions can be made.

it“? ' '1le a!
I

 

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mm 11111111 1“