INBORN ERRORS OF METABOLISM

I. THE PRESENCE OF AN INACTIVE ENZYME
AND flUTERQ TOXICITY
IN GALACTC‘SEMIA

II. URINARY MUCOPOLYSACCHARIDES IN
PATIENTS WITH HURLEE‘S $YNDRQME,
THEIR FAMILIES AND NORMAL MAN

Thesis Ior II'no Degree of DI}. D.
MICHIGAN STI‘LTE UNIVERSITY
Jary S. Mayes
1965

 

”3‘3 LIBRARY

Michigan State
University

This is to certify that the

thesis entitled
lnhurn erors of Tetaiolisn
I. The Presence of an Inactive Limi’t'ie and in ‘1tero
Toxicity in Calactosenia .-
II. 'Jrin‘mjr icowolvsaccharides in Patients with
'ri'irler's "1;.'r..;1rorqe, Their- Fanilies and Torte-l ‘I.;,n
presented by

9 "
AI.

. ‘3 u,
Jk‘ii “V 'J 0 AJ V93

has been accepted towards fulfillment
of the requirements for

7‘33- degree inwm ' 1"fury

    

Major professor

Datea VC/C/ /C{/ /QgS——

 

0-169

ABSTRACT

INBORN ERRORS OF METABOLISM

I. THE PRESENCE OF AN INACTIVE ENZYME AND IN_N:EBQ
TOXICITY IN GALACTOSEMIA

II. URINARY MUCOPOLYSACCHARIDES IN PATIENTS WITH
HURLER'S SYNDROME, THEIR FAMILIES AND NORMAL MAN
by Jary S. Mayes

EBBLL

Galactosemia is a disorder of galactose metabolism in
humans in which Gal—l—P uridyl transferase activity is
very low or completely absent in these individuals. Experi-
ments were designed to determine if the protein is completely
lacking or altered to an inactive form.

Gal-l-P uridyl transferase was purified about 500 fold
over a crude extract from calf liver and some of its proper—
ties were studied. Antibody against the purified protein was
prepared by injecting it into rabbits. The serum from these
rabbits precipitates the calf liver and human erythrocyte
enzymes. A fraction from the blood of patients with galacto-
semia precipitates the antibody suggesting an inactive
Galcl-P uridyl transferase protein in galactosemia.

The heterozygous carriers of galactosemia are usually

asymptomatic. However, two children were observed who had

Jary S. Mayes

some of the symptoms of galactosemia, but the transferase
levels in the erythrocytes were in the heterozygous range.
In the mothers of these individuals, transferase levels
were in the low heterozygous range suggesting that lfl.2£§£9

toxicity could have occurred during pregnancy.
25132-1;

Hurler‘s syndrome is a disorder of mucopolysaccharide
metabolism in which chondroitin sulfate B and/or heparitin
sulfate are excreted in the urine and accumulate in the
tissues. The heterozygotes do not show any symptoms, but
small abnormal amounts of mucopolysaccharides may be excreted
in the urine. It is also important to know the normal types
and amounts of mucopolysaccharides when studying mucopoly-
saccharide disorders.

Therefore, it was of interest to measure the mucopoly-
saccharides in urine samples from Hurler‘s syndrome. their
families and normal man. A marked decrease in the amount of
total mucopolysaccharides and chondroitin sulfates per unit
of creatinine was observed with decreasing age in normal
children, but the amount became constant with age after the
late teens. In the urine samples from Hurler's syndromes,
abnormal amounts of heparitin sulfate were found in five
patients and abnormal amounts of heparitin sulfate and

chondroitin sulfate B were found in one patient. It is

Jary S. Mayes

suggested that the type of Hurler's syndrome that excretes
only heparitin sulfate may be more common than previously
thought. It was possible to obtain urine samples from

some of the families of these patients. The mucopoly—
saccharides in these samples were within the normal range by

all criteria employed.

II.

INBORN ERRORS OF METABOLISM

THE PRESENCE OF AN INACTIVE ENZYME AND IN HTERO
TOXICITY IN GALACTOSEMIA

URINARY MUCOPOLYSACCHARIDES IN PATIENTS WITH
HURLER'S SYNDROME, THEIR FAMILIES AND NORMAL MAN

BY

. Jary S. Mayes

A THESIS

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

DOCTOR OF PHILOSOPHY
Department of Biochemistry

1965

ACKNOWLEDGMENTS

The author wishes to express his appreciation to
Dr. R. G. Hansen for his guidance and interest throughout
the course of this project. The support of a NIH predoctoral
fellowship during this study is also appreciated.

The author also wishes to thank Dr. Meyer, Dr. Castor
and Upjohn Company for supplying some of the muCOpoly-
saccharides,Dr. Harrigan and Dr. Rennell, who supplied the
urine samples from Hurler's syndrome, and Dr. Wells and
Dr. Donnell, who supplied the blood samples from galactosemic
patients.

The author is especially grateful to his wife, Faye,
and his sons, Jeff and Kevin, for their encouragement and

love throughout the course of this work.

ii

To Faye

iii

VITA

Jary S. Mayes was born on October 19, 1938 at Walters,
Oklahoma. He graduated from Walters High School in 1956.
After attending Cameron Junior College, he transferred to
Oklahoma State University and received the Bachelor of
Science degree in 1960 from the Department of Agricultural
Chemistry. His graduate studies were pursued at Michigan
State University in the Department of Biochemistry with the
aid of a graduate assistantship and later a National Institute
of Health predoctoral fellowship. He received the Master of
Science degree in 1965 and will complete the requirements
for the degree of Doctor of Philosophy in 1965. He is a
member of the American Chemical Society, American Association

for the Advancement of Science, and Sigma Xi.

iv

TABLE OF CONTENTS

Page
PART I
THE PRESENCE OF AN INACTIVE ENZYME AND
IN_UTERO TOXICITY IN GALACTOSEMIA
INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW. . . . . . . . . . . . . . . . . . 5
Galactose Metabolism . . . . . . . . . . . . . 5
Galactosemia . . . . . . . . . . . . . . . . . 6
Purification and Properties of Gal—1-P Uridyl
Transferase. . . . . . . . . . . . . . . . 15
Molecular Genetics of Human Inborn Errors of
Metabolism . . . . . . . . . . . . . . . . 16
MATERIALS AND METHODS. . . . . . . . . . . . . . . . 20
Chemicals. . . . . . . . . . . . . . . . . . . 2O
Tissues. . . . . . . . . . . . . . . . . . . . 21
Immunological. . . . . . . . . . . . . . . . . 22
Assay of Gal-1-P Uridyl Transferase. . . . . . 22
RESULTS 0 O O O O O O O O O O O O O O O 0 O 0 O O O O 25
I. Purification of Gal-1-P Uridyl Trans-
ferase from Calf Liver. . . . . . . . 25
Purity. . . . . . . . . . . . . . . . . . 51
Stability . . . . . . . . . . . . . . . . 31
Effect of Enzyme and Substrate Concen-
tration . . . . . . . . . . . . . . . 55
Substrate Specificity . . . . . . . . . . 56
Effect of Sulfhydryl Reagents . . . . . . 36
Effect of Metal Ions. . . . . . . . . . . 59
II. Purification of Gal-1-P Uridyl Trans-
ferase from Human Erythrocytes. . . . . 39

TABLE OF CONTENTS - Continued
Page
III. Immunological Studies . . . . . . . . . . . 45
Precipitation of Enzymes from Calf Liver
and Human Erythrocytes by Antiserum . . 45
Precipitation of the Antibody by Blood
Extracts from Galactosemics . . . . . . 49
IV. Symptoms of Galactosemia in the Hetero-
zygote and Possibility of in utero
Toxicity in These Individuals . . . . . 52
DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 55
SUMMARY. . . . . . . . . . . . . . . . . . . . . . . . 60

REFERENCES . . . . . . . . . . . . . . . . . . . . . . 61

PART II

URINARY MUCOPOLYSACCHARIDES IN PATIENTS WITH HURLER'S
SYNDROME, THEIR FAMILIES AND NORMAL MAN

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 66

LITERATURE REVIEW. . . . . . . . . . . . . . . . . . . 68

Chemistry and Metabolism of Mucopolysaccharides. 68
Mucopolysaccharides in Normal Human Urine. . . . 72
Mucopolysaccharide Metabolism in Hurler's

Syndrome . . . . . . . . . . . . . . . . . . 75

MATERIALS AND METHODS. . . . . . . . . . . . . . . . . 78

Mucopolysaccharides. . . . . . . . . . . . . . . 78
Quantitative Measurements. . . . . . . . . . . . 78
Urine Samples and Isolation of Mucopolysac-

charides from Urine. . . . . . . . . . . . . 79

RESULTS. . . . . . . . . . . . . . . . . . . . . . . .. 80

Mucopolysaccharide Excretion in Urine of Normal
Man O O O O O O O O O O O O O O O O O O O O O 80

vi

TABLE OF CONTENTS - Continued

Page
Mucopolysaccharides in Urine from Families with
an Affected Child . . . . . . . . . . . . . 87
Mucopolysaccharides in Urine from Patients with
Hurler's Syndrome . . . . . . . . . . . . . 87
DISCUSSION. . . . . . . . . . . . . . . . . . . . . . 91
SUMMARY . . . . . . . . . . . . . . . . . . . . . . . 94
REFERENCES. . . . . . . . . . . . . . . . . . . . . . 95

vii

TABLE

II.

III.

IV.

VI.

VII.

VIII.

IX.

X.

XI.

LIST OF TABLES

Purification of Gal-1-P Uridyl Transferase from
Calf Liver. . . . . . . . . . . . . . . . . . .

pH Stability of Gal-1-P Uridyl Transferase. . .

Substrate Specificity of Gal-1—P Uridyl Trans-
ferase. . . . . . . . . . . . . . . . . . . . .

Effect of Sulfhydryl Reagents on Gal-1-P Uridyl
Transferase . . . . . . . . . . . . . . . . . .

Purification of Gal-1—P Uridyl Transferase from
Human Erythrocytes. . . . . . . . . . . . . . .

Precipitation of Human Erythrocyte Enzyme with
Rabbit Antiserum. . . . . . . . . . . . . . . .

Precipitation of Antibody by a Fraction of
Galactosemic Blood. . . . . . . . . . . . . . .

Gal-1-P Uridyl Transferase Levels in Erythro-
cytes of Families with Possible in utero
Toxicity. . . . . . . . . . . . . . . . . . . .

Constituents of Acid Mucopolysaccharides. . . .

Mucopolysaccharides in Urine from Parents and
Siblings of Hurler's Syndrome . . . . . . . . .

Mucopolysaccharides in Urine from Patients with
Hurler's Syndrome . . . . . . . . . . . . . . .

viii

Page

28

32

57

58

46

5O

51

54

69

88

89

LIST OF FIGURES

FIGURE Page
1. Elution pattern from DEAE cellulose . . . . . . 50

2. Effect of substrate concentration on the re-
action rate . . . . . . . . . . . . . . . . . . 55

5. Effect of magnesium on the reaction rate in the
one step assay. . . . . . . . . . . . . . . . . 41

4. Effect of metal ions on the reaction rate in
the two step assay. . . . . . . . . . . . . . . 45

5. Precipitation of calf liver enzyme with anti-
serum . O O O O O O O O O O O O O I O O O O O O 48

6. The amount of chondroitin sulfates relative to
creatinine in urine samples from humans of
various ages. 0 O O O O O O O O O O O O O O O O 82

7. The total quantity of mucopolysaccharide rela-
tive to creatinine in urine samples from humans
of various ages . . . . . . . . . . . . . . . . 84

8. Ratio of chondroitin sulfates to mucopoly-

saccharides in urine samples from humans of
various ages. . . . . . . . . . . . . . . . . . 86

ix

PART I

THE PRESENCE OF AN INACTIVE ENZYME AND
I11 UTERO TOXICITY IN GALACTOSEMIA

INTRODUCTION

The number of known human enzyme deficiencies has in-
creased at a phenomenal rate within recent years. Although
clinical and other studies have been carried out in these
disorders, it has been difficult to do molecular studies,
because of the inaccessibility of appropriate tissue for
laboratory analysis. One of the important questions in
patients with these disorders is whether the enzyme protein
is completely lacking or altered to an inactive form. The
results in individuals with acatalasemia would indicate that
the catalase protein is very low and not present as an in-
active molecule, but in Glu-6-P dehydrogenase deficiency an
inactive form of the enzyme protein may be present.

One of the deficiencies that has been extensively

studied is galactosemia. In this disorder Gal-1—P uridyl

 

1The following abbreviations are used: ADP, adenosine
diphosphate; ADPG, adenosine diphOSphate glucose; ATP, adeno-
sine.triphosphate; CDPG, cytidine diphOSphate glucose; DEAE,
diethylaminoethyl; EDTA, ethylenediamine tetraacetate;
Fru-1—P, fructose-l-phosphate; Gal-1—P, galactose-l-phosphate;
Gal-6-P, galactose-6-phosphate; GDPG, guanosine diphosphate
glucose; Glu-1-P, glucose-1-phosphate; Glu—6-P, glucose-6-
phosphate; Man-l-P, mannose-l-phOSphate; TDPG, thymidine di—
phosphate glucose; TPN, triphosphopyridine nucleotide; TPNH,
triphosphopyridine nucleotide, reduced; UDP, uridine diphos-
phate; UDPG, uridine diphosphate glucose; UMP, uridine mono-
phosphate; UTP, uridine triphosphate; Xyl-1—P, xylose—1-
phosphate.

 

'H!

 

1g .5. d.:..,..«,.:%.. . .

transferase activity is extremely low or completely missing.
Therefore, the galactosemics are unable to effectively
metabolize galactose, a normal constituent of milk, and
symptoms develop at an early age. The heterozygotes for
this disorder have a half level of the enzyme and can
usually metabolize galactose rapidly enough to avoid compli-
cations and symptoms of the disease, but in certain cases
the heterozygote may be affected.

The objectives of these experiments were twofold:

1) Purification of the Gal-1-P uridyl transferase, and

2) examination of the galactosemic for the possible presence
of an inactive protein. Procedures for the partial purifi-
cation of the enzyme from calf liver and human erythrocytes
were developed. Antibodies were prepared against the purified
calf liver enzyme and used to detect cross-reacting material
in galactosemia. The cross—reacting material can be in-
terpreted as an indication of the presence of an inactive
Gal—1-P uridyl transferase molecule.

During the course of this investigation an opportunity
was available to examine some unusual heterozygotes and
their families. These heterozygotes had some of the symptoms
of galactosemia, suggesting the possibility of in_utg£g

toxicity during pregnancy.

LI TERAT URE REVI EW

Galactose Metabolism

 

The aldohexose galactose is found throughout nature,
but its occurrence in milk is especially significant where
it is coupled with glucose as the disaccharide lactose.
Since milk is the chief nutrient for young mammals, it would
seem necessary to metabolize galactose for energy. Thus in
the early 1900's (1) it was shown that galactose could be
converted to glycogen. However, it was not until after 1950
that the conversion of galactose to glucose was elucidated.

In 1945 Kosterlitz (2) showed that rabbits fed a high
galactose diet accumulated Gal—1-P in their livers. He (5)

also showed that a galactose-adapted strain of Saccharomyces

 

cerevisiae could ferment Gal-1—P at a rate equal to that of

 

Glu-1-P. Therefore, he postulated that galactose was metabo-
lized through Gal-1-P which could be converted to Glu-1—P.

A few years later Leloir and his associates estab-
lished the enzymatic steps and cofactors in this conversion.
They (4) were able to Show a galactokinase (reaction a) in

galactose-adapted Saccharomyces fragilis. They next dis-

 

covered (5) and identified UDPG (6) as the key coenzyme in
the interconversion of galactose and glucose. Leloir (7)

later demonstrated in galactose—adapted s, fragilis an

 

enzyme, UDPGal 4-epimerase (reaction c), that catalyzed the
conversion of this coenzyme to a galactose derivative which
he identified as UDP Gal. From this observation Leloir
postulated two steps in the conversion of Gal-1-P into
Glu-l-P: 1) The hyopthetical transfer of UMP from glucose
phosphate to galactose phosphate, and 2) the conversion of
UDPG to UDPGal by the reaction he had described. Kalckar
.EE.§£- (8) later demonstrated an enzyme,Gal—1-P uridyl
transferase (reaction b), in S. fragilis that catalyzed the
reaction postulated by Leloir in step 1.

This pathway for galactose metabolism has become known
as the Leloir pathway (9) or the Gal-1-P uridyl transferase
pathway (cf. 17) and appears to be the major pathway for
galactose metabolism in all organisms. It can be formulated
as follows:

a) Galactose + ATP -+>Gal-1-P + ADP
b) UDPG + Gal-1-P :UDPGal + Glu—1-P
c) UDPGal :UDPG

Sum b and c: Gal-1-P :Glu-1-P

In the overall process galactose is converted to
Glu-1—P which can be further metabolized by known pathways.

Other minor pathways have been described for the
metabolism of galactose and they may play an important role
in certain situations. A UDPGal pyrophosphorylase (reaction

d) has been described in mammals (10,11), yeast (8), and

plants (12) which with UDPGal 4-epimerase (reaction c) and
UDPGlu pyrophosphorylase (reaction e) could convert Gal—1-P
to Glu-1-P. This alternate pathway can be formulated as
follows:

d) Gal-1-P + UTP :UDPGal + PPi

c) UDPGal :UDPG

e) UDPG + PPi : Glu-1-P + UTP

Sum d, c, and e: Gal-1-P :Glu-l-P

The enzyme for reaction d has been shown to increase
with age in rat liver (10,11), but even in adult rat liver it
is much less active than Gal-1-P uridyl transferase.

It is possible that galactose can be metabolized via a
route that doesn't require a uridine coenzyme intermediate.
Posternak and Rasselet (15) have shown that phosphoglucomutase
can convert Gal—1-P to Gal-6-P and there is some evidence
that Gal-6—P can be oxidized by the hexose monophOSphate
shunt (14). Under normal conditions this oxidative pathway
probably has no role in galactose metabolism, but it is of
interest that Inouye §t_§l, (14) have identified Gal—6-P in
erthrocytes from galactosemic patients who accumulate
Gal-1-P in their tissues.

Wells §t_§l, (15,16) have recently identified galactitol
in the urine and brain of galactosemic patients and they
suggest that the reduction of galactose to galactitol could

be an alternate route of galactose metabolism.

Galactosemia'

The term galactosemia has come to mean a hereditary
condition characterized by a lack of the enzyme Gal-1-P
uridyl transferase (reaction b). Several review articles
have recently appeared on the subject (17,18,19,20).

Affected infants usually appear normal at birth, but after

a few days or weeks of milk ingestion vomiting, diarrhea

and general listlessness may develop. Other consequences are
jaundice, enlargement of the liver and spleen, cataracts,

and mental retardation if galactose is continued in the diet.
Death is common in the first few weeks of life. These clini—
cal features vary from individual to individual. The common
laboratory findings are elevated blood galactose and Gal—1-P,
abnormal galactose tolerance, deficiency of Gal—1-P uridyl
transferase in red cells, and galactose, amino acids, and
protein in the urine.

The first breakthrough in elucidating the biochemical
basis of the disease was the discovery by Schwarz gt El: (21)
that the erythrocytes of galactosemics on a milk diet
accumulated Gal-1-P and that erythrocytes from galactosemic
patients incubated_in vitro with galactose also accumulated
Gal-1-P. This indicated that either the transferase or the
epimerase was the block in the pathway of galactose metabol-
ism in galactosemia. Shortly after this finding, Kalckar

and co—workers (22,25) were able to show that erythocytes

from galactosemia patients were devoid of Gal-1-P uridyl
transferase activity while the other enzymes of galactose
metabolism were within the normal range. In galactosemia
they (24) subsequently showed that the enzymatic activity
was missing in the liver, where most of the galactose is
metabolized (24). If normal blood is mixed with galactosemic
blood, it retains its ability to metabolize galactose show—
ing that there is not an inhibitory substance in the galacto—
semic blood.

Although the absence of Gal-1-P uridyl transferase is
certainly the cause of galactosemia, it is difficult, at
present, to explain all the varied biochemical and clinical
features of the disease by the absence of this one enzyme.
Holzel §t__l, (25) suggested that the accumulation of Gal—1-P
in the various tissues produces a disturbance of cell
metabolism which gives rise to the clinical findings in
galactosemia, and Gal-1-P has been shown to inhibit phospho-
glucomutase (26,27), Glu-6-P phOSphatase (28) and UDPG
perphosphorylase (29). This concept of Gal-1—P toxicity in
galactosemia appears to be well established and is widely
accepted. Galactitol has recently been isolated and identi~
fied in the urine (15) and brain (16) of galactosemic indi-
viduals. This accumulation of galactitol could possibly cause
some of the clinical symptoms, especially cataracts, seen in
the disorder. Another interesting report on the possible

cause of idiocy of galactosemia has been made by Woolley

and Gommi (50). They suggest that the Gal—1-P which
accumulates:higalactosemianmy'inhibit the formation of a
galactolipid which functions as a serotonin receptor.
Therefore, a functional deficiency of serotonin can arise
and possibly cause mental defects.

Although the Gal—1-P uridyl transferase pathway is
the major means of galactose metabolism and galactosemia re—
sults from the absence of one of the enzymes in this pathway,
it soon became apparent that some galactosemic patients could
metabolize significant amounts of galactose. Eisenberg §t_al.
(51) administered radioactive galactose to a galactosemic
patient and concurrently fed menthol to permit the formation
of the menthol glucuronide. They estimated that from the
1 gm of galactose administered 75 to 80 percent was not
metabolized beyond Gal-1-P which accumulated in the tissues,
5 percent was found in the menthol glucuronide and 20 to 25
percent was not accounted for. The radioactivity in the men-'
thol glucuronide demonstrated that some of the galactose can
be utilized by galactosemic patients and that the metabolism
probably occurred through uridine mucleotide intermediates.

Segel gt al. (52) have observed galactose metabolism
in galactosemic patients by injecting intravenously radio-
active galactose and measuring the radioactive carbon dioxide
in the expired breath. They examined twelve patients who

showed the clinical symptoms of congenital galactosemia,

absence of Gal-1-P uridyl transferase in the erythrocytes

and inability of hemolysates to oxidize radioactive galactose
to carbon dioxide in vitro. Three patients could metabolize
1 gm of galactose to a normal extent while the other nine
patients were found to metabolize the sugar poorly. There
was no evidence that the subgroup who could metabolize
galactose differed in symptoms, severity, or clinical
histories, and the only correlation in the subgroup was that
the three patients were Negro.

Weinberg (55) found, using whole blood or leucocytes
‘in vitro, that the blood from one individual was able to con-
vert radioactive galactose to carbon dioxide at a substantially
greater rate than observed with other patients in the group.
By employing a modification of this in vitro procedure, Ng
.££.§l- (54) have more recently shown that blood from three
galactosemics could produce carbon dioxide from radioactive
galactose at a higher rate than the other ten patients.

The two most widely discussed views on why the amount
of galactose metabolized by galactosemics are the presence
of alternate pathways or only partial block in transferase
activity. For an alternate pathway the UDPGal pyrophos-
phorylase has received attention, since this enzyme has
been claimed to occur in mammalian liver (11). Several
authors have suggested an incomplete block and recently Ng

§t_§l, (54) have concluded that the enzymatic block in some

10

galactosemics is incomplete. The basis for this latter con-
clusion was that some galactosemics could form more UDPHexose
than the usual galactosemic when whole blood was incubated
with radioactive galactose, but hemolysates could not form
labeled UDPHexose from UTP and labeled Gal-1-P which would
indicate no UDPGal pyrophosphorylase activity.

A number of investigators have reported a small amount
of Gal-1-P uridyl transferase in some galactosemics.
Schwarz §t_§l, (55),employing a manometric procedure, have
reported some transferase activity in 15 of 25 galactosemic
children. Donnell §£_§I, (56), employing a UDPG consumption
test,have also found transferase activity in some galacto-
semics. Hsia and co-workers (57,58L employing various methods
for measuring transferase, also reported activity in some
galactosemics. They (57) showed that one-third of the
galactosemics tested had activity by the oxygen uptake or
the UDPG consumption test, but not by both. In a few cases
enzymatic activity was found by both methods and in about half
the cases neither method showed any enzymatic activity. The
discrepancy in the low amount of activity in some galacto-
semics can also be attributed to variation in the analytical
methods.

The frequency of galactosemia has been difficult to
assess because many cases die shortly after birth and may

not be properly diagnosed or they may not develop symptoms

11

and go undetected. However, it appears to occur frequently
enough that most hospitals with a large children's depart-
ment will see an appreciable number of cases. After a survey
by pediatricians and pathologists in Great Britian it was
estimated that 1 in 70,000 children is born with the disorder
(55). Hansen 3: al. (59) have determined the number of
heterozygotes in a given population and then by the Hardy-
Weinberg equation they calculated the frequency of galacto~
semia to be 1 in 18,000 births. This frequency is more in
the range of other inborn errors which can be determined more
accurately such as phenylketonuria (cf. 40). Beutler §£.§l~
(41) have also determined the frequency of the carrier state
of galactosemia and calculated an expected birth frequency
of about 1 in 17,500 births which is similar to that calcu-
lated by Hansen gt al. (59). Beutler 35 al. place a different
interpretation of their results and state that the calcu-
lation is in error in the case of galactosemia. They propose
a new genetic abnormality (Duarte variant) for Gal—1-P
uridyl transferase deficiency which can not produce true
galactosemia and therefore, determined estimates of the birth
frequency of galactosemia based on the heterozygote frequency
will be too high, unless correction is made for the Duarte
variant.

The basis for treatment of galactosemia is to feed a

galactose free diet and this diet should be given at the

12

earliest possible age. There have been several diets
described and some have become commercially available (cf.
17,42). The treatment of young infants have had varying
degrees of success (cf. 17,42). The amount of galactose
ingested and the time of starting of the diet appears to be
important. In general, improvement is usually observed but
in some cases all the symptoms do not disappear. The treat—
ment of older patients has usually been with an ordinary
mixed diet, avoiding only milk, dairy products, and lactose
which is sometimes used as filler in vitamin tablets, etc.
Since in utero toxicity has been suggested (cf. 17) and a
possible case reported (59) it would seem desirable that the
expecting heterozygous mother should not consume a large
amount of galactose during pregnancy.

Since the diagnosis of galactosemia needs to be made
as early as possible so the infant can be placed on a galac-
tose free diet, methods for early detection of the disorder
are desirable. A general reducing sugar test can be pre-
formed on the urine in expected cases, and if positive,
galactose can be identified by one of several methods.

The presence of galactose in the urine is not necessarily
diagnostic of galactosemic as other disorders can cause
galactose in the urine. Other methods are therefore needed
to confirm the disorder. The direct assay for Gal-1-P uridyl

transferase in erythrocytes is the best test for diagnosis.

15

The UDPG consumption test (45), mamnometric assay (44), and
radioactive techniques (45,46,47) have been widely employed
to measure transferase in erythrocutes. However these pro-
cedures require equipment which is not found in every hospital
laboratory. Nordin et al. (48) have modified the UDPG con-
sumption test, Schwarz (49) has simplified the manometric
assay, and London et al. (50) have developed a simple, inex~
pensive radioactive test, so they can be used on equipment
found in most hospital laboratories and possibly for screening.
Recently Beutler et al. (51) have developed a new method for
the detection of galactosemia. It is visual and based on a
dye-linked system for the estimation of Gal-1-P uridyl
transferase. The test appears to be simple, economical, and
precise enough to be used for a screening procedure.

Early indications from classical human genetic tech-
niques, such as absence of the disease in parents, high
incidence in the offspring of consanquineous marriages and
in siblings, and more or less equal occurrence in both sexes,
suggested inheritance by a single autosomal recessive gene.
However, with the development of techniques for detecting the
heterozygote, galactosemia has become one of the best docu-
mented examples of a single autosomal recessive gene in
human genetics.

Holzer and Komrower (52) used the galactose tolerance
test to try to detect the heterozygous carriers of galacto-

semia and an abnormal tolerance was observed in some

14

parents but the method did not detect all heterozygous indi-
viduals as expected. Later Donnell §t_al, (55) re-evaluated
the galactose tolerance test and identified the heterozygous
state in 59 members of galactosemic families. However, the
overlap between heterozygous and normal is large and the
method must be considered unsuitable for the detection of

the heterozygous individual. Hsia g; l. (54), employing the

original UDPG consumption test of Anderson g£__l. (45), found
a difference between normal and heterozygotes, but consider-
able overlap was observed.

The first clear-cut demonstration of the detection of
the heterozygous carrier of galactosemia was reported by
Bretthauer ggigl. (55). They modified the UDPG consumption
test of Anderson g; al. (45) to quantitatively assay the
Gal-1-P uridyl transferase in erythrocytes and were able to
show that parents had transferase levels that were inter—
mediate between the normal and galactosemic. Donnell 3: al.
(56), using the procedure of Bretthauer g£_al. (55) found a
maximum overlap of only 5 percent of the area under the
normal distribution curves in a thorough study of the genetics
of galactosemia. This was the first clear—cut demonstration
of autosomal recessive inheritance in galactosemia.

Concurrently with the study of Bretthauer and co-
workers, Kirkman and Bynum (44) developed a manometric method

for measuring Gal-1-P uridyl transferase in hemolysates and

15

achieved a sharp separation between heterozygous carriers
of galactosemia and normal. With improved techniques they
observed (56) no overlap between heterozygotes and normals.
Schwarz E£.2l- (55), using a manometric technique giving
maximum activity of Gal-1-P uridyl transferase, found a
slight overlap of enzyme levels in the control and hetero-
zygote groups.

Hsia and co-workers (57,58,55,57), using various
methods, have sometimes found good discrimination between
the heterozygote and normal groups.

From these data it is clear that galactosemia is in—
herited as a autosomal recessive and that the diseased have
none or very little transferase activity and that the
heterozygotes have one-half the enzyme activity of normal
individuals.

Purification and Prgperties of Gal-1-P
Uridyl Transferase

 

 

Although Gal-1-P uridyl transferase is one of the key
enzymes in galactose metabolism and its activity is absent
in galactosemia, the enzyme has not been extensively puri~
fied or studied. Kurahashi and Anderson (58) have purified
the enzyme about fifty—fold over the crude extract from calf
liver and applied the partially purified enzyme to the
determination of Gal-1-P. Kurahashi and Sugimura (59) have

purified the enzyme 60-70 fold above the crude extract from

16

a galactokinase-less strain of Escherichia coli, examined

 

some of its properties and applied the partially purified
enzyme to the determination of Gal-1—P. The Km values for
all the substrates were determined and found to be between
1.5 and 4.1 x 10-4. The equilibrium constant was 1.1 in

the direction of UDPGal formation. The purified enzyme
required cysteine for activity and showed no requirement for
Mg, but was inhibited by high Mg concentrations. The pH
optimum was between 8.5 and 8.0. Similarly, the pH optimum
of the calf liver enzyme has been reported in this range (9).

Molecular Genetics of Human Inborn
Errors of Metabolism

 

 

The term, inborn errors of metabolism, was coined by
Garrod in the early 1900's to explain several disorders that
he had observed. Garrod clearly defined biochemical ex~
pressions of inherited variability and set forth the idea of
inherited blocks in metabolism. His insight was so remark-
able for that time that his work was overlooked and it wasn‘t
until the classical work of Beadle and Tatum that the gene—
enzyme relationship was well-established. The number of
known inborn errors of metabolism has now increased into
the hundreds. Their delineation has helped to establish
pathways of metabolism, to explain the ig.yiy9_role of
enzymes, and to understand the physiology and biochemistry

of normal individuals.

17

The question of whether an enzyme is totally absent or
is merely in an inactive state in these disorders is of
primary concern. The work with microorganisms show that in
some cases the mutant enzymes are present but are inactive
because of some structural alteration, and even though they
are catalytically inactive the proteins will cross react with
antibodies prepared against the normal enzyme. In many
cases, such as abnormal hemoglobins in humans and tryptophan
synthetase in microorganism, the structural alteration has
been traced to a single amino acid substitution which would
indicate that a change has occurred in the nucleotide code
in the DNA and is passed on to the mRNA and finally to the
protein.

Striking genetic effects on qualitative characteristics
of human enzymes have been found in recent years, although
the nature of their possible structural differences is'dif—
ficult to determine because of the inaccessibility of signifi—
cant quantities of the appropriate tissue. Nevertheless,
attempts have been made to determine the absence or presence
of mutant enzymes. Robbins (60) prepared antiserum against
crystalline rabbit muscle phosphorylase a and b by in~
jecting them into roosters. The antibodies were found to
cross-react well with normal human muscle phosphorylase,
but no precipitation occurred with extracts from the muScle

tissue of a patient lacking phosphorylase. He concluded

18

that a phosphorylase-like protein in the individual was
either completely lacking or that it was modified so
extensively that it was both enzymatically and antigenically
inactive.

Nishimura §t__l, (61) produced an antiserum against
catalase by injecting a purified preparation of human erythrOa
cyte catalase into rabbits and precipitin reactions were
negative with acatalasemic hemolysates. They also concluded
that the biochemical defect in acatalasemia appears to be
either an absence of erythrocyte catalase or a faulty molecu»
lar configuration of it. Ogata and Takahara (62) also could
not find any immunological evidence for a mutant protein in
hemolysates or acetone extracts of acatalasemic blood by us-
ing a quantitative precipitin test. They (65) verified the
absence of catalase protein in a spectrophotometric study.
Purified catalase from normal individuals showed three ab-
sorption bands, at 505, 520, and 625 mu, but in acatalasemic
blood the catalase bands were not detected. Aebi (64) has
recently obtained sufficient quantity of catalase from an
acatalasemic subject to compare its properties with the
enzyme from normal individuals. The catalase from the
acatalasemic subject was identical by its mobility on
Sephadex G-100 columns, electrophoresis on starch gel,
sensitivity towards azide and precipitation by anti-catalase

serum. However the concentration of antigenic protein in

19

the acatalasemic subject differed from that of the normal
by a factor of about 200 which is similar to the ratio of
catalase activity. It was concluded that true catalase is
present in the blood of acatalasemic subjects but only in
very small amounts. Thus it is possible that this enzyme
defect is not due to a structural gene but rather to a
change within the operator gene system.

Kirkman and Crowell (65) have concluded from electro-
phoresis and titration with TERI that the deficiency of
activity of Glu—6-P dehydrogenase in erythrocytes of
primaquine sensitive Negroes is due principally to a decrease
in the number of molecules of Glu-6-P dehydrogenase. In con-
trast, Ramot and Bauminger (66) have shown that an inactive
Glu-6-P dehydrogenase fraction from erythrocytes of enzyme
deficient human subjects could absorb antibody prepared
against normal human erythrocyte Glu—6-P dehydrogenase.
Since normal enzyme diluted to an activity similar to that
of the mutant enzyme was not able to remove the antibody,
they suggest that the removal of the antibody was not due to
a small amount of active enzyme in the mutant fraction, but
due to the presence of an inactive form of Glu—6-P dehydro~
genase in enzyme deficient subjects which is immunologically

similar to the normal enzyme.

MATERIALS AND METHODS

Chemicals

 

The biochemicals were purchased as follows: UDPG
(isolated from yeast), DEAE cellulose, and Glu-6-P dehydro-
genase from the Sigma Chemical Company; TPN from P—L Bio-
chemicals, Incorporated; crystalline phosphoglucomutase and
synthetic nucleotide sugars (UDPG, CDPG, GDPG, and ADPG)
from CalBiochem. TDPG was synthesized in the laboratory
according to the method of Michelson (67,68). Gal-1~P and
Xyl-1-P were made in the laboratory according to the method
of Hansen g___l. (69). Complete Freund adjuvant was ob-
tained from Difco Laboratories.

Calcium phosphate gel was made in large batches by a
modification of that given by Colowick (70). One hundred
seventy-seven gm of CaC12:2H20 was dissolved in about 6
liters of distilled water. Three hundred and four gm of
Na3PO4:12H20 was dissolved in about 2 liters of distilled
water and was poured with stirring into the CaC12 solution.
After adjusting the pH of the mixture to pH 7.4 with 6N acetic
acid, the gel precipitate was washed with 50-40 liters of

distilled water for four times and three times with deionized,

distilled water. Concentration of the gel was achieved by

20

21

sedimentation by gravity and it was stored in the cold until
ready for use.

DEAE cellulose was washed according to the method of
Peterson and Sober (71). The fresh or used DEAE cellulose
was washed with 1N NaOH until the filtrate was clear. After
removing the excess NaOH by washing 5 or 6 times with dis~
tilled water, the DEAE cellulose was then converted to the
phosphate salt by washing with 0.5N Na3P04 for columns or
0.5N phosphate buffer pH 7.0 for removing hemoglobin from
blood. The excess phosphate was removed by washing 5—6
times with distilled water. A thick slurry was kept in the

cold until ready for use.
Tissues

Calf liver was obtained from a local slaughterhouse.
The calves usually ranged from 1é-to 5 months in age. Human
erythrocytes were obtained from the Lansing Regional Blood
Center. Both fresh rejected and outdated samples were ob~
tained, but the fresh rejects were higher in enzymatic
activity, so these were usually used. Blood samples for the
_in utero toxicity study were obtained from families living
in Michigan. Galactosemic blood samples were supplied by

Dr. Donnell and Dr. Wells.

22

Immunological

 

The purified calf liver Gal-1-P uridyl transferase was
emulsified with a syringe with an equal volume of complete
Freund adjuvant and injected subcutaneously into Dutch
rabbits. A total of three injections were made at weekly
intervals. A week after the last injection the rabbit was
bleed by making a small nick in the marginal ear vein with a
razor blade. Usually, 10 to 20 ml of blood was obtained.
The blood was left overnight to clot, centrifuged, and the
serum decanted. The gel diffusion test was performed by the
method of Ouchterlony (72). The inhibition of the enzyme
was performed by a procedure which was similar to that

described by Ramot and Bauminger (66).

Assay of Gal-1-P Uridyl Transferase

 

For the i2_2£g£g toxicity studies the Gal-1-P uridyl
transferase in erythrocytes was measured by the method of
Bretthauer E£.§l: (55). .For the other studies the enzyme
was measured by methods similar to those described by
Kurahashi and Sugimura (59). In a one step assay of trans-
ferase Glu-1—P formation is followed spectrophotometrically
by TPNH reduction at 540 mu in a coupled reaction with phos-
phoglucomutase and glucose-6-P dehydrogenase. The reaction
mixture consists of 0.4 umole of M9C127 0.2 umole of TPN,

UDPG, and Gal-1-P; 5 ugm of crystalline phosphoglucomutase;

25

0.025 units of glucose-6-P dehydrogenase; appropriate amount
of Gal-1-P uridyl transferase and 0.1 M glycine buffer pH
8.7 which contained 0.01 M mercaptoethanol to a total volume
of 0.50 ml. The assays were performed at 250 C on a Beckman
DU SpectrOphotometer equipped with a Gilford changer and
recording attachment (75,74).

A unit is defined as the amount of enzyme that causes
the formation of 1 umole of product per min. under the
conditions described above. The formation of 1 umole of
product is based on the molar extinction coefficient of
6.22 x 103 for TPNH at 540 mu. Specific activity is defined
as units per mg of protein. Protein was determined by the
method of Lowry 22.3l- (75).

When some of the properties of transferase was studied,
a two step assay method was employed so the effects would be
on transferase and not on the coupling enzymes. The appro—
priate amount of transferase was incubated at 250 C with
0.4 umole of UDPG and Gal-1-P, various sulfhydryl compounds
(2umole) and metals and 0.2 M glycine buffer pH 8.0 to a total
volume of 0.2 ml. After eight min. of incubation the re-
action was stOpped by placing the tubes in a boiling water
bath for 1.5 min. The amount of Glu-1—P formed was measured
by adding TPN, MgClg, phosphoglucomutase and Glu—6-P dehydro—
genase and observing the total change in optical density at

540 mu.

24

In crude preparations 6-P gluconic acid dehydrogenase
was present and interfered with the calculation of the trans-
ferase activity. 6-P gluconic acid dehydrogenase was there-
fore measured according to the principle of Horecker and
Smyrniotis (76) with 0.2 umole of TPN and 6-P gluconic acid,
0.4 umole MgClg and 0.1 M glycine buffer pH 8.7 in a total
volume of 0.50 ml. This procedure was employed for most of
the one step assays for Gal-1-P uridyl transferase and taken
into account in calculating the activity. Also in crude
preparation a pyrophosphatase was present which hydrolyzed
UDPG to Glu-1-P. Therefore, a control without Gal-1-P was

necessary to measure this activity.

RESULTS

I. Purification of Gal-1-P Uridyl Transferase
from Calf Liver

 

The livers from calves were obtained as soon as possible
after death, sliced into small pieces and packed in dry ice.
They were transported to the laboratory and kept frozen until
ready for use. Purification of the enzyme was conducted in
the following manner with all steps being carried out at 0-40
C and with distilled, deionized water. Centrifugation, unless

otherwise stated, was for 20 min at 25,500 x g.

Step 1. One kilogram of frozen calf liver was diced
and extracted with 2000 ml of 0.05 M KOH-0.005 M EDTA Na4 in
a large Waring blender for 2 min. The extract, which was
originally pH 8.6, was adjusted to pH 6.8 with 6.0 N acetic
acid. Four hundred ml of 2% protamine sulfate was added and

after stirring for 15 min, the mixture was centrifuged.

Step 2. To the supernatant from step 1, solid ammon-
ium sulfate was added slowly with stirring until 50% satur-
ation (0.298 gm/ml). After stirring 50 min the solution
was centrifuged. The supernatant was discarded and the.
precipitate dissolved in a minimum of cold water and dialyzed

for 5 hrs against 2 changes of about 10 liters of water.

25

26

Step 5. To the dialysate of step 2, calcium phosphate
gel was added until the gel to protein ratio was 0.8. After
stirring for 10 min the solution was centrifuged. To the
supernatant four times the original amount of calcium phos~
phate gel was added. After stirring for 2 hrs the solution
was centrifuged and the supernatant discarded. The precipi-
tated gel was suspended in 1500 ml of 0.02 M phosphate buffer
pH 8.0 and after eluting overnight the mixture was centri-

fuged.

Step 4. The supernatant from step 5 was placed di-
rectly on a DEAE cellulose column (50 x 2 cm). The activity
was eluted with a gradient of phosphate buffer pH 7.0.

A gradient system similar to that described by Hurlbert pp al.
(77) was employed. Starting with water in a 500 m1 mixing
flask and 0.05 M buffer in the reservoir, the elution was
continued until the enzyme started to come through. The elu~
tion of the enzyme was completed with 0.08 M buffer in the

reservoir.

Step 5. Tubes from step 4 containing the enzyme were
combined and diluted with 1.5 volumes of cold water. The
enzyme was adsorbed on a small column (7 x 1 cm) of DEAE
cellulose, then eluted with 0.16 M phosphate buffer at pH

7.5.

27

Step 6. Tubes from step 5 containing the enzyme were
combined and solid ammonium sulfate added until 50% satur-
ation (0.168 gm/ml). After stirring for 15 min the solution
was centrifuged. Ammonium sulfate was again added to the
supernatant until 50% saturation (0.119 gm/ml). After stir~
ring for 15 min the solution was centrifuged. The supernatant
was discarded and the precipitate dissolved in a small amount
of 0.05 M phosphate pH 7.0 which contained 0.01 M mercapto~
ethanol.

A summary of the procedure is given in Table 1. An
overall purification of about 500 fold was achieved with a
yield of about 25 percent. The purification is probably
higher because the protamine sulfate usually resulted in
precipitation of some protein. The procedure has been re-
peated several times with similar results. In step 5 the
protein concentration should be about 50 mg/ml and the gel
concentration should be about 25 to 50 mg/ml for best results.
After the DEAE cellulose column the activity should be con~
centrated as soon as possible because the enzyme is very
unstable in the dilute solution.

The elution pattern from the DEAE cellulose column is
given in Figure 1. A large amount of protein was not absorbed
while the sample was being placed on the column. The peak of
protein was eluted with 0.05 M phOSphate buffer, while the
activity was eluted in a sharp peak with 0.08 M phOSphate buffer.

Tubes number 206 through 246 were combined for the next step.

28

Table I. Purification of Gal-1-P Uridyl Transferase from
Calf Liver

 

 

Step Ml Units/ml TOIal Protein S.A.
Units

Protamin sulfate 2540 0.852 2000 54.2 .025

Ammonium sulfate 785 2.58 1870 55.4 .044
(o-so%)

Ca3(PO4)2 gel 1595 0.700 1120 0.57 1.25

DEAE cellulose 655 1.25 780 0.14 8.79
(column)

DEAE cellulose 55 15.1 x. 550 1.4 10.8
(concentration)

Ammonium sulfate II 2.2 217.0 480 17.7 12.4

(so-50%)

 

Assay conditions, unit of enzyme, and specific activity are
described in text. Protein is given as mg/ml.

 

29

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Ioumou mafia zmmp onu pam .15 0mm um UEEAEHouop
mm Camuonm mucmmoumou ocfla pflaom .uxou mnu CH

poflfluomop mum EOHDSHT mo ponuma paw EESHOU one

.omoHDHHoU mdmn Eoum cumupmm COHDSHM

.H onsmflm

("') WW/SIIND

¢.O

md

N._

m._

O.N

mmmZDZ wma...

CON Om. ON. Om ov O

 

0.... Ia
I «on s. 89 Teen. .2 mod I I mnazqm

 

 

JIIIIIIJ: I
.

 

 

ow Ia

 

 

00. «0.
O O

(—) dw 082

0.

‘AlISNBO “Ivoudo

51

Purity

On cellulose acetate electrophoresis the enzyme from
the last step of purification gave one major protein band.
If the cellulose acetate strip was cut in half and one—half
stained for protein and the other half cut into small pieces,
eluted and assayed for transferase, the peak of activity
corresponded to the protein band. At this stage the enzyme
was thought to be fairly homogeneous. However, when anti—
bodies were prepared against the enzyme, multiple precipitin
lines were observed on gel diffusion and in the analytical
ultracentrifuge the pattern became dispersed as the protein
moved down the cell. When the protein was electrophoresized
on the more resolvable polyacrylamide gel, several major and
minor bands were observed. When one of the gel columns was
eluted and assayed for transferase, the activity corresponded
to one of the major bands. The purity can be estimated

roughly at 10 to 50%.

Stability

 

For pH stability 100 gm of calf liver was extracted in
the usual manner and purified through step 2. One ml of the
enzyme was mixed with 1 ml of each of pH 6.0, 7.0 and 8.0
phosphate buffer 0.1 M. The mixtures were incubated at 250
C and assayed at the various times which are given in Table II.

The enzyme was stable at pH 7.0 and 8.0, but it was unstable

52

Table II. pH Stability of Gal-1-P Uridyl Transferase

 

 

Activity units/ml

 

 

Time pH 6.0 pH 7.0 pH 8.0
0 1.06 1.00 1.05
1 hr .96 1.00 1.00

15 hr .71 1.06 1.05

18 hr .60 1.05 .95

 

55

at pH 6.0 where it lost almost half of its activity in 18
hours. This trend in pH stability was also observed in
early purification procedures since if the calcium phosphate
gel or DEAE cellulose steps were carried out at acid pHs, a
loss in enzyme activity was observed.

Sulfhydryl reagents also appeared to stabilize the
enzyme.

Effect of Engyme and Substrate
Concentration

 

 

In the one step procedure the response was linear with
respect to enzyme concentration until a change in optical
density of about 0.02 per min. A range of 0.008 to 0.02
change in optical density per min was usually employed in
assaying the enzyme. Since in the two step assay the reaction
was linear up to 12 min, eight min was employed for routine
determinations.

The effects of concentration of various substrates on
the reaction rate are given in Figure 2. The optimum
concentration for UDPG is 0.4 umole/ml and at higher concen-
trations of UDPG the reaction was inhibited. Therefore, a
concentration of 0.4 umole/ml was used to measure the
enzyme. The optimum concentration for Gal-1-P was between
0.4 and 1.0 umole/ml. 0.4 umole/ml of Gal—1-P was usually
employed in the reaction mixtures. The Optimum concen—

trations for TDPG and CDPG were much higher.

54

Figure 2. Effect of substrate concentration on the
reaction rate.

 

 

240

I60

 

80

A 0. D./MlN./ML

0

l I

0 0.8 IS

24

I6

A 0. D./MlN./ML

0

UDPG ()JMOLE/ML)

 

l l

0 ' 0.8 |.6
TDPG (IJMOLE/ML)

A 0.0./M|N./ML

A O.D./MlN./ML

240

ISO

80

0

 

 

I I

r

0 0.8 l.6

0.6

0.4

0.2

Gal-l-P (UMOLE/ML)

 

l l I

0 4 8. l2

CDPG (pMOLE/ML)

56

Substrate Specificity

The substrate Specificity of purified Gal-1-P uridyl
transferase is given in Table III. The enzyme was somewhat
7 active in catalyzing the hexose transfer reaction with the
pyrimidine molecules, but there was no detectable activity
with the purine diphosphate glucoses or other sugar phos—
phates. After the natural substrates, Gal-1-P, Glu—1-P,
UDPG, and UDPGaI, TDPG was the most active. This probably
is a non-specific catalysis of the enzyme since the UDPG/TDPG
activity ratio remained constant throughout purification.
Also, the activity was not a small contamination of UDPG in
the TDPG because in an end point assay with high enzyme‘
concentration the TDPG showed 95.5% of the expected value.
The catalysis with CDPG as substrate is less certain. At a
concentration of 0.4 umole/ml (Table III), the reaction is
barely detectable, but at higher concentrations of substrate
the reaction rate is increased to about 0.25% that of UDPG

and appears to be non-specific.
Effect of Sulfhydryl Reagents

The effect of sulfhydryl reagents on the catalysis rate
is given in Table IV. All three of the compounds tested
stimulated the enzyme to about the same extent, although
mercaptoethanol always appeared to be slightly more active
than glutathione or cysteine. Therefore, mercaptoethanol

was regularly used to stabilize the enzyme.

57

Table III. Substrate Specificity of Gal-1-P Uridyl
Transferase

 

 

Substrate Percent of Activity

 

1. Nucleotide sugars

UDPG 100

TDPG 8.5
CDPG .07
ADPG < .01
GDPG < .01

2. Phosphate sugars

Gal-1-P 100

Man-1-P < .01
Fru-1-P < .01
Xyl-1-P < .01
Gal—6-P < .01

 

58

Table IV. Effect of Sulfhydryl Reagents on Gal-1-P Uridyl

 

 

 

 

Transferase
Change in O. D.
Reagent Expt. 1 Expt. 2
None .084 .072
Mercaptoethanol .160 .112
Glutathione .142 .100
Cysteine .158 .104

 

The assay is the same as described in the text for the two
step assay, except no metal ions were used.

59

Effect of Metal Ions

The effect of magnesium on the catalytic activity of
the transferase in the one step assay is shown in Figure 5.
Magnesium was stimulatory at low concentration but was in-
hibitory at high concentration. In order to check if this
effect was directly on transferase or the coupling enzymes,
a two step procedure was performed and the results are
shown in Figure 4. Under these conditions only the inhibition
was observed. Since phosphoglucomutase is known to require
a small amount of magnesium, the stimulation was probably due
to this reaction. Several other divalent metal ions were
tested and all were inhibitory with copper being the most

affective.

II. Purification of Gal-1-P Uridyl Transferase
from Human Erythrocytes

 

The erythrocytes from four pints of blood were washed
twice with physiological saline and held frozen overnight to
rupture the cells. The frozen cells were thawed and diluted

with an equal volume of water to form the hemolysate.

Step 1. The purpose of this step was to remove the
hemoglobin. The procedure adopted was modified from that
described by Hennessey pp pl, (78). An equal volume of'a
thick slurry of DEAE cellulose was added to the hemolysate,

and after stirring for 1 hour, the mixture was poured into

40

Figure 5. Effect of magnesium on the reaction rate in
the one step assay.

UNITS/ML

I20

I00

80

60

40

20

 

 

0 IO
MgCI 2

I l
2.0 5.0

()JMOLE/ML)

42

Figure 4. Effect of metal ions on the reaction rate in
the two step assay.

 

A 0.0.

240

200

I60

120

80

40

 

 

I I

0 5.0 I0.0_ I5.0 20.0

pMOLE/ML

44

a large fritted glass funnel. The DEAE cellulose on the
funnel was washed with 0.0025 M phOSphate buffer pH 7.0
until the red color was removed. The DEAE cellulose was
then removed from the funnel and the enzyme eluted with

0.5 M KCl for one hour and filtered from the DEAE cellulose.

Step 2. To the filtrate from step 1, solid ammonium
sulfate was added until 50% saturation (0.298 gm/ml).
After stirring for 20 min the solution was centrifuged.
The supernatant was discarded and the precipitate dissolved
in water. The solution was dialyzed for 5 hours against

two changes of water of about 10 liters each.

Step 5. To the dialysate of step 2, calcium phOSphate
gel was added to reach a geltx) protein ratio of 1.2.
After stirring for 15 min the solution was centrifuged. Eight
times the original amount of calcium phosphate gel was added
to the supernatant. After stirring for 2 hours the solution
was again centrifuged. The precipitated gel was eluted with
0.05 M phosphate buffer pH 8.0 for 2 hours and centrifuged

to remove the gel.

Step 4. To concentrate the supernatant of step 5,
solid ammonium sulfate was added until 60% saturation and
after stirring for 15 min the solution was centrifuged.

The supernatant was discarded and the precipitate dissolved

in a small amount of water.

45

A summary of this purification procedure is given in
Table V. An overall purification of about 500 fold with a
yield of 24% was achieved. Several additional steps, such
as DEAE cellulose column and AlCV gel, were attempted with-
out success. The main problem was that the enzyme became
too dilute and lost activity before concentration. The
above procedure gave a transferase that was free of enzymes,
such as 6—P gluconate dehydrogenase and pyrophOSphatase, that
interfere with the measurement of the enzyme. Thus this
preparation procedure can be used to study the enzyme from

human erythrocytes.

III. Immunological Studies

 

Precipitation of Enzymes from Calf Liver and
Human Egythrogytes by Antiserum

In order to measure the amount of antibody-antigen
interaction, the antigen and antibody were mixed with Tris~
HCl buffer pH 7.8 at a concentration of 0.1 M, incubated for
50 min at 250 C, and centrifuged. From the amount of
enzymatic activity in the supernatant, the amount of pre-
cipitation or percent inhibition could be calculated.
A duplicate tube of control antibody was always run.

The percent inhibition of the calf liver enzyme at
various concentrations of serum is shown in Figure 5. The
inhibition at low levels was proportional to serum concen—

trations, but became nonlinear at high serum concentrations.

46

Table V. Purification of Gal-1-P Uridyl Transferase from
Human Erythrocytes

 

 

 

Total
Ml U/ml Units Protein S.A.
Hemolysate 2020 .0177 55.9 160 .00011
DEAE cellulose 2145 .0105 22.4 5.5 .0020
Ammonium sulfate 268 .071 19.0 17.7 .0040
(0-50%)
Ca3(PO4)2 gel 540 .055 11.9 .66 .055
Ammonium sulfate 9.5 .90 8.5 15.6 .058
(O-60%)

 

Assay conditions, units (U), and specific activity (S. A.)
are described in text. Protein is given as mg/ml.

L__.mw.vfl

Figure 5.

47

Precipitation of calf liver enzyme with anti-
serum.

0.01 ml of a 1-10 dilution of enzyme (0.014
units) was incubated at 25 C for 50 min with
0.025 ml of Tris-HCl buffer 1.0 M, pH 7.8 and
water and/or serum to a total volume of 0.555
ml. The mixture was centrifuged and an aliquot

(0.025 ml) of the supernatant assay for activity.

The difference between the activity in control
serum and antibody serum was used to calculate
the percent inhibition.

 

“Mwfi. _

 

 

 

_ _ r

O . O O . O
2 4 6

ZOEhEEé.§

80 —
I00

05

02

OJ

ML SERUM

49

This is probably due to a small amount of Gal-1-P uridyl
transferase in the antiserum which would not be precipi-
tated and could become meaningful at high concentration of
serum.

The inhibition of the human erythrocyte enzyme by the
antibody to the calf liver enzyme is given in Table VI.
The human erythrocyte enzyme was not as effective as the calf
liver enzyme in precipitating the antibody, but did as ex-
pected show some inhibition or cross reaction. 0.05 ml of
antiserum would precipitate about 0.007 units of calf liver
enzyme but only 0.0005 units of human erythrocytes enzyme.

Precipitation of the Antibody bijlood
Extracts from Galactosemics

 

Blood from galactosemic patients was fractionated
through the second step in the purification procedure for
erythrocytes. This fraction (0.06 ml) was then incubated
with antigenic and control serum (0.06 ml) and buffer for
50 min at 250 C and centrifuged. An aliquot (0.1 ml) was
incubated with calf liver enzyme for 50 min at 250 C as
described in Figure 1, and the activity in the supernatant
was measured. The results are summarized in Table VII.

The fraction from galactosemic blood appeared to precipitate
rabbit antibody prepared in response to the calf liver trans-
ferase. Thus it is probable that a protein is present in

the blood of the galactosemic which is immunologically similar

to the normal transferase but catalytically inactive.

_
‘ I

50

Table VI. Precipitation of Human Erythrocyte Enzyme with
Rabbit Antiserum

 

 

 

A O.D./min % I
Experiment 1
Control serum .0116
20
Antiserum .0088
Experiment 2A
Control serum .0172
16
Antiserum .0144
Experiment 2B
Control serum .0188
28
Antiserum .0156

 

In experiment 1, 0.1 ml of the enzyme (0.0048 units) was
incubated for 50 min at 25 C with 0.1 ml of serum and
0.02 ml 1.0 M Tris-HCl buffer pH 7.8. The mixture was
centrifuged and the supernatant assayed for activity. The
difference between the activity in the control serum and
antibody was used to calculate the percent inhibition.
Experiment 2 was similar to experiment 1, except 0.05 ml
of antibody and 0.05 ml of antigen in 2A and 0.10 ml of
antigen in 2B were used.

51

Table VII. Precipitation of Antibody by a Fraction of
Galactosemic Blood

 

 

Percent Inhibition of
Calf Liver Enzyme

 

Control 1 41
Patient 1 21
Control 2 47
Patient 2 26

 

In the control, 0.06 ml of water was added to each of two
tubes. 0.06 ml normal rabbit serum was added to one tube,
and 0.06 ml of immune rabbit serum was added to the other
tube. To two other tubes, 0.06 ml of a fraction of galacto-
semic blood was added. 0.06 ml of normal rabbit serum was
added to one tube, and 0.06 ml of immune rabbit serum to

the other tube. After incubating for 50 min, the mixtures
were centrifuged; and an aliquot (0.1 ml) of each was incu—
bated with calf liver enzyme for 50 min. After centrifuging,
the supernatants were assayed for activity. The difference
between the activity of samples incubated with normal rabbit
serum and immune rabbit serum was used to calculate the per-
cent inhibition.

52

IV. Symptoms of Galactosemia in the Heterozygote and
Possibilipy of in utero Toxicipy in
These Individuals

 

 

 

In the study of the frequency of galactosemia (40),
a patient was observed in a hospital for the mentally re—
tarded which had some of the symptoms of galactosemic, for
example, eye cataracts and mental retardation. Upon measur~
ing the Gal-1-P uridyl transferase in the erythrocytes, the
level was found to be within the range of heterozygous indi-
viduals. It was possible in this case to do a family study
which revealed that the mother and several siblings had
transferase levels within the heterozygous range while the
father and other siblings had levels within the normal range.

In a second case, the child was thought by pediatricians
to be a galactosemic since jaundice was present and reducing
substances were found in the urine. The jaundice disappeared
upon the administration of a galactose-free diet. However,
upon measuring the Gal-1-P uridyl transferase in the erythro~
cytes, the value was within the range of heterozygous indi~
viduals. In a family study it was again the mother who had
transferase levels in the heterozygous range. The infant
was also thought to have biliary atresia which complicates
the symptoms in this case, but since biliary atresia has been
described (cf. 17) in galactosemia it is possible that this

complication resulted from galactose toxicity.

55

A summary of the transferase levels in the two families
is given in Table VIII. Since transferase levels below 4.0
have been shown to be indicative of heterozygous individuals
(55), the patient and mother in both families were within
this range while the fathers were clearly within the normal.
Thus it is possible that these two children are unusual
heterozygotes that show some of the symptoms of galactosemia.
The symptoms in the children may have resulted from inade_

quate metabolism of galactose by the mother during maternity.

54

Table VIII. Gal-1-P Uridyl Transferase Levels in Erythro~
cytes of Families with Possible ;p_utero

 

 

 

Toxicity
um of UDPG consumed'
per ml erythrocyte per hr
Family 1
Patient 2.4
Mother 2.4
Father 5.5
Siblings 6.8, 6.5, 2.4, 2.9
Family 2
Patient 5.8
Mother 2.0

Parents of Mother
Mother 5.4
Father 5.4

Father 6.4

 

DISCUSSION

It is well-established that galactosemia is an inborn
error of metabolism in which the enzyme Gal-1—P uridyl
transferase is very low or completely missing in the tissues.
It is of interest to know if the enzyme protein is only
modified to an enzymatically inactive form or is completely
lacking. If the mutation is in the structural gene an
altered inactive enzyme would be expected, but if the mu—
tation is in the regulator gene a decrease or complete loss
of activity would occur. Of the human enzyme deficiencies
studied, both types of mutations appear to exist. In phos-
phorylase deficiency (60) and acatalasemia (61,62,65,64) the
enzyme proteins appear to be either completely lacking or
very low and in acatalasemia (64) the mutation has been sug-
gested to occur within the Operator gene system. However,
in Glu-6-P dehydrogenase deficiency, an inactive Glu-6-P
dehydrogenase fraction appears to exist (66).

In consideration of an inactive Gal-1-P uridyl trans-
ferase the approach was to purify the enzyme, prepare
antibodies against it, and then see if the antibody would
cross-react with a fraction from erythrocytes from galacto-

semic patients.

55

56

After preliminary purification of transferase from
galactose—adapted yeast, calf liver and human erythrocytes,
calf liver was finally chosen as the tissue for purification
of the enzyme, because it was readily available, had high
levels of activity, and was expected to cross react with
human.Gal-1-P uridyl transferase.

The enzyme from calf liver was purified 500 fold from
the crude extract with a 25% yield (Table 1). The purified
enzyme was fairly specific (Table III). For the nucleotide
moiety the enzyme only reacted to a slight extent with TDPG
and very slightly with CDPG in addition to UDPG as the major
substrate. For the sugar moiety the enzyme appeared to be
Specific for glucose and galactose. The calf liver enzyme
was stimulated 50 to 50% with sulfhydryl reagents. This
stimulation is not as much as that reported for the E, 99;;
enzyme which had an absolute requirement for sulfhydryl
reagents (cysteine) for activity (59). The purified enzyme
had no requirement for divalent metals but all the metals
tested were inhibitory. This phenomena has been reported
for magnesium for the E, ppl;_enzyme (59). The purified
enzyme was inhibited with high concentrations of UDPG.

Antibody was prepared against the purified Gal-1-P
uridyl transferase by injecting the enzyme into rabbits.
Since the enzyme showed several lines of precipitation on
gel diffusion with the antibody and several lines on disc

electrophoresis, an inhibition method was used to determine

57

the amount of antibody—antigen interaction. The calf liver
enzyme (Figure 5) and normal human erythrocyte enzyme
(Table VI) were both precipitated with the antibody.
However, the human erythrocyte enzyme did not react as
effectively as the calf liver enzyme. A fraction from the
erythrocytes of patients with galactosemia purified through
step 2 of the fractionation procedure for erythrocytes
(Table V), precipitated the antibody (Table VII). It is
assumed that the substance in the erythrocytes responsible
for the precipitation of the antibody is inactive protein.
A small amount of active Gal-1-P uridyl transferase could
give this effect but no activity of Gal-1-P uridyl trans~
ferase could be detected in the fraction and by using the
purification procedure to concentrate the enzyme, less than
5% of the normal amount should be detectable. Therefore,
the substance in the erythrocytes of the galactosemic that
precipitates the antibody appears to be catalytically in-
active Gal-1-P uridyl transferase.

Two possible cases of ip_ppp£p toxicity of galactose
have been observed. The first case was found in a school
for the mentally retarded. The child, his mother and some
Siblings had levels of Gal-1-P uridyl transferase in their
erythrocytes which indicate that they were heterozygous
while the father and other siblings had values which indi-

cate that they were normal for the galactosemia trait.

58

It is of interest why the one child, which was heterozygous,
had symptoms of galactosemia and was severely affected while
the other heterozygous siblings were not affected. This
could have possibly been due to the diet, especially milk
and dairy products, of the mother during pregnancy or
possibly the other siblings did Show some of the symptoms at
one time or another, but they weren't as severe as the
mentally retarded child. In the second case the mother and
child also had levels of transferase in their erythrocytes
indicative of heterozygous individuals.

In both cases it was the mother with transferase levels
which were low and in the heterozygous range as was the
child's. Since women are usually encouraged to drink milk
during pregnancy, it is possible that in mothers heterozygous
for galactosemia the galactose is increased significantly in
the blood and a toxic level of Gal-1-P or some other product
may reach the fetus.

Other authors have suggested ip utero toxicity.

Since improvement of galactosemia patients on a galactose-
free diet is sometimes limited, even when the diet was
started fairly early in infancy, Clay and Potter (79) sug—
gested that this may be due to damage occurring ip_utero.
Schwarz pp_pl. (80) found an increased level of Gal—1-P in
the blood of one galactosemic infant who had been given no

milk suggesting ip_utero toxicity. The fact that the

59

galactose tolerance curve tends to be abnormal in late
pregnancy (45), lower birth weight for galactosemics than
for their normal sibs (57), and induction of cataracts in
fetal rats by feeding the pregnant mothers a high galactose

diet (81), are also suggestive of ip_utero toxicity.

SUMMARY

Gal—1—P uridyl transferase has been purified about
500 fold from extracts from calf liver and some of its
properties have been studied. The enzyme is fairly specific
for substrates reacting slightly with TDPG and very slightly
with CDPG. The enzyme was inhibited with divalent metals
and high concentration of UDPG and stimulated with
sulfhydryl compounds.

Antibodies were prepared against the purified protein
and the antibody precipitated both the calf liver and
human erythrocyte Gal-1—P uridyl transferase. A fraction
from galactosemia blood would also precipitate the antibody
suggesting the presence of an inactive Gal-1—P uridyl
transferase protein in the galactosemic.

Two possible cases of ip_ppp£p_toxicity in individuals
heterozygous for galactosemia were found. In both cases
the mother and the affected child had transferase levels
in the low heterozygous range and the child showed some of
the symptoms of the galactosemic suggesting ip_ppp£p

toxicity.

60

10.

11.

12.

15.

14.

15.

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PART II

URINARY MUCOPOLYSACCHARIDES IN PATIENTS WITH HURLER'S
SYNDROME, THEIR FAMILIES AND NORMAL MAN

INTRODUCTION

Several hereditary disorders have been reported in
which there are abnormal metabolism and excretion of muc0poly-
saccharides. Of these,Hurler's syndrome is the only severe
mucopolysaccharide disorder that has been conclusively docu-
mented. It is well-established that chondroitin sulfate B
and/or heparitin sulfate are excreted in the urine and
accumulate in the tissues in abnormal amounts in these indi-
viduals. Hurler's syndrome is not a single entity as several
different types appear to exist. From genetic studies, it is
clear that two hereditary forms, an autosomal recessive
and a sex-linked recessive, exist. It now becomes clear,
from mucopolysaccharide excretion patterns, that four or five
different types may exist.

The heterozygotes of Hurler's syndrome are asympto-
matic and the reports concerned with the detection of hetero-
zygotes by abnormal urinary mucopolysaccharides are not
consistent.

The excretion of mucopolysaccharides in the urine of
normal man appears to be more complicated than originally
observed. Chondronitin sulfate A or C was first demonstrated
to be the major mucopolysaccharide in normal human urine,

but more recently heparitin sulfate, kertosulfate,

66

67

chondroitin sulfate B, and products of partial and complete
desulfation of chondroitin sulfate A or C have been identi-
fied in normal human urine. The total amount of urinary
mucopolysaccharides has also been shown to vary with age

in children.

In this study the urinary excretion of mucopolysac—
charides in Hurler's syndrome, their families, and normal
man was examined. The chondroitin sulfates and total muco-
polysaccharides were measured in urine specimens from
humans of various ages and of both sexes. Mucopolysaccharides
were also measured and identified in Hurler's syndrome and
it was possible in some cases to measure the urinary

mucopolysaccharides in families with affected children.

LITERATURE REVI EW

Chemistry and Metabolism of the
Mucopolysaccharides

The mucopolysaccharides are linear polymers of high
molecular weight. They usually consist of a disaccharide
repeating unit of a hexosamine and a hexuronic acid moiety
joined by a glycosidic bond. Currently eight distinct
connective tissue acid mucopolysaccharides are known and
Table IX gives a survey of the compounds and their com-
ponents. Some recent reviews discuss their chemistry and
biology (1,2,5).

Only two of the mucopolysaccharides found in connective
tissue are non-sulfated, e.g., hyaluronic acid and chon-
droitin. Hyaluronic acid is widely distributed and has
been extensively studied being the first mucopolysaccharide
for which a complete structure was well-established. It has
alternating glucuronic acid and glucosamine units. Chon-
droitin is less common in nature, and can be obtained by
the removal of sulfate from the chondroitin sulfates. It
has the same structure as hyaluronic acid except that it
contains galactosamine instead of glucosamine.

Among the sulfated mucopolysaccharides, three types

of chondroitin sulfates, A, B, and C, are recognized as

68

69

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70

distinctive isomers and chondroitin sulfate—D had been iso-
lated from shark cartilage, but its characterization is

less firmly established. The repeating units of chondroitin
sulfate-A are glucuronic acid and N-acetylgalactosamine-4-
sulfate. Chondroitin sulfate-B, which is also referred to
as B—heparin and dermatan sulfate, differs from A in that
the glucuronic acid moiety is replaced by its C-5 epimer,
L-iduronic acid. Chondroitin sulfate-C differs from A in
that it is the 6-0-sulfate instead of the 4-0-sulfate.

The other sulfated mucopolysaccharides of connective
tissue are heparin, heparitin sulfate, and kertosulfate.

The characteristics of these compounds are not as firmly
established as the chondroitin sulfates or hyaluronic acid.
Heparin contains glucosamine, glucuronic acid and both 0-
and N-sulfate. Heparitin sulfate is probably related to
heparin but appears to have less sulfate, especially
N-sulfate. Keratosulfate is a polysaccharide that contains
equal proportions of N-acetylglucosamine, galactose, and
sulfate.

These mucopolysaccharides form an important part of
the amorphous ground substance which lies between the extra-
cellular fibers and the cells in connective tissue. Several
biological functions have been described (Cf. 5) for these
compounds, such as calcification, control of electrolytes

and water in extracellular fluids, wound healing, lubrication

71

of joints, blood coagulation, clearing activity, main-
tenance of the stable transport medium of the eye, and hair
growth. Participation in most of these functions is
undoubtedly due to the polyanionic nature of the compounds.
The general outline (Cf. 5) of the biosynthesis of
the mucopolysaccharides has been fairly well-established
with the use of radioactive compounds and the discovery of
the role of the nucleotide sugars in polysaccharide syn-
thesis. The mechanism and extent of removal from tissues
is still quite obscure. The rate of turnover of the
various constituents of connective tissue varies.
Collagen has a very slow turnover whereas the ground sub-
stance is relatively rapid. The half—life of hyaluronic
acid as 2 to 4 days and chondroitin sulfate as 7 to 16 days
has been calculated from isotopic studies of rabbit skin
(4,5) and rat cartilage (6). The rate of turnover changes
considerably with age (7). The half-life of keratosulfate
appears to be very long and probably exceeds 120 days (8).
The removal of mucopolysaccharides from tissue may
be accomplished both by extensive degradation and by ex-
cretion of the unchanged polymers. Skin extracts have been
reported to promote the degradation of chondroitin sulfate
B, but the mechanism of such breakdown is unknown (9).
Reports have also appeared indicating the existence of

hyaluronidase-like activities in mammalian tissues (10).

72

Mucopolysaccharides are also excreted in the urine (discussed
in the next section). Kaplan and Meyer (11) studied the
fate of injected chondroitin sulfates A, B, and C and
heparitin sulfate in man and dog. After injection of chon~
droitin sulfate A and C there was a rapid disappearance

from the blood but no significant amount was demonstrable in
the urine. On injection of these mucopolysaccharides in
heavier doses into a dog a small amount was recovered in the
urine, but the blood level remained high for several hours.
The fate of the unrecovered polysaccharides is unknown and
it is difficult from this data to explain the normal ex-
cretion of chondroitin sulfate A. After injection of chon-
droitin sulfate B and heparitin sulfate they disappeared
from the blood stream and a large percent could be recovered
unchanged in the urine. On long continued injection of
chondroitin sulfate B, a polysaccharide was excreted with
the properties of heparitin sulfate.

Mucopolysaccharides in Normal Human
Urine

 

In early studies, Astrup (12) precipitated a substance
from urine with benzidine hydrochloride which was non—
dialyzable, metachromatic with toluidine blue, alcohol—
insoluble and possessed low anticoagulant activity. _Kerby
(15), using Astrup's method of precipitation, showed that the

major component of the fraction migrated with commercial

75

chondroitin sulfate on paper chromatography and produced
a single spot when mixed with the commercial chondroitin
sulfate. Hamerman pp_51, (14) also found by paper chroma-
tography an acidic substance migrating at a rate similar
to that of chondroitin sulfate from the non-dialyzable uri-
nary solids. DiFerrante and Rich (15), using quaternary
ammonium salts as precipitant, were able to isolate suf-
ficient amounts of urinary mucopolysaccharides for purifi-
cation and identification. Their data concerning composition
and properties indicated that the major mucopolysaccharide
was most likely chondroitin sulfate A, but from their
methods it was impossible to clearly differentiate between
chondroitin sulfate A and C and minor constituents would
not have been detected by their methods.

It soon became evident that chondroitin sulfate A was
not the only mucopolysaccharide in normal human urine.
From paper chromatography, paper electrophoresis, analytical
and enzymatic data, DiFerrante (16) suggested that in addi-
tion to chondroitin sulfate A normal urine also contains
hyaluronic acid. Heremans pp 51. (17) demonstrated by
electrophoresis three different acid mucopolysaccharides in
urine. The most abundant compound migrated at the same
speed as commerCial chondroitin sulfate. The other two peaks
migrated at somewhat slower rates. King pp 51, (18) pre-
sented evidence for a glucosamine—containing acid mucopoly-

saccharide in normal human urine in addition to chondroitin

74

sulfate. From the presence of glucosamine, electrophoresis,
and enzymatic data, they suggest that it is heparitin sul—
fate. Linker and Terry (19), using carbazole-orcinol color
reactions for uronic acid, paper chromatography, and specific
enzymatic hydrolysis, identified chondroitin sulfate A,
chondroitin sulfate B, heparitin sulfate, and possibly a
small amount of kertosulfate in human urine. The percent
of each was estimated to be 80% chondroitin sulfate A, 10%
chondroitin sulfate B, and 10% heparitin sulfate.
DiFerrante (20), by fraction of urinary mucopolysaccharides
on ECTEOLA cellulose and using enzymatic and chemical
analysis of the peaks, identified chondroitin sulfate B,
heparitin sulfate, and products of partial and complete
desulfation of chondroitin sulfate A or C in addition to
chondroitin sulfate A.

Kerby (15) also showed that the mean 24-hour excretion
of mucopolysaccharides was 5.0 mg of glucurOnic acid_equiva-
lent material in white females and 4.9 mg in white males.
The difference between the two values was significant.
DiFerrante and Rich (20) obtained slightly higher values
using a different method for isolation. Their average per
24—hour urine collection was 5.77 mg of glucuronic acid
material for women, 6.00 for men, 5.80 mg for girls (7—8
years), and 7.45 for boys (7-16 years). Since it was ob—
served that the amount of mucopolysaccharides was higher

in children, Rich 55_51. (21) investigated more thoroughly

75

the excretion in the urine of normal children. They found
a gradual increase in mucopolysaccharide excretion during
childhood, but a regular decrease occurred in the ratio of
mucopolysaccharide excretion to creatinine excretion.
Teller pp_51. (22) have also investigated urinary mucopoly-
saccharides in normal children and established the normal
95% probability range of mucopolysaccharide excretion in
these Children. Their results were similar to that describ-
ed by Rich and co-workers with slightly lower values and
they were unable to demonstrate a significant difference

in excretion between girls and boys.

Mucppolysaccharide Metabolism in
Hurler's Syndrome

 

 

Hurler's syndrome is a heritable disorder of a severe
disturbance of mucopolysaccharide metabolism. Several
names, such as Hurler's syndrome, gargoylism and lipo-
chondrodystrophy, have been applied to this disorder.
Several recent reviews have appeared (5,25,24,25).

Brante (26) was the first to suggest that the dis~
iorder was a mucopolysaccharidosis. By histochemical and
isolation procedures he showed the presence in the tissues
and urine of large quantities of a sulfated mucopoly-
saccharide which he believed to be a chondroitin sulfate.
This was further clarified by Stacey and Barker (27) and

Brown (28) who isolated a dextrorotatory sulfated

76

mucopolysaccharide which contained glucosamine from the
liver of patients. Dorfman and Lorincz (29) and Meyer

1. (50) isolated and identified two sulfated mucopoly-

g
saccharides, chondroitin sulfate B and heparitin sulfate,
from the urine of patients with Hurler's disease. Meyer
‘gplg1. (51) later reported the presence and distribution of
these two mucopolysaccharides in various organs of patients
with Hurler's disease. The livers contained predominantly
heparitin sulfate while in other organs, especially in the
spleen, chondroitin sulfate B is the main polysaccharide.
Since the identification of mucopolysaccharidosis as the
defect in Hurler's syndrome,numerous procedures and methods
have appeared for their determination in urine which appear
to be of diagnostic value.

It soon became clear that there were several types
of Hurler's patients. From genetic studies (cf. 24), there
was identified a sex—linked and an autosomal type of
inheritance. In the report by Meyer 55_51, (50), two cases
of sex—linked and two cases of autosomal inheritance were
reported and there was no obvious difference in the pattern
of urinary mucopolysaccharides between the two groups.
However, Terry and Linker (52) have distinguished four
types by their mucopolysaccharide excretion pattern:
classical autosomal recessive, classical sex-linked re—
cessive, a type that excretes only heparitin sulfate, and

an adult form. The autosomal recessive form was shown to

77

excrete significantly more chondroitin sulfate B in propor—
tion to heparitin sulfate than the sex-linked cases.

Meyer pp_51, (50) also reported two cases with only
heparitin sulfate in the urine, but they did not classify
them as distinct types. Harris (55) published data from an
analagous case in which the chemical study was performed
by K. Meyer and suggested a possible new genotype of
Hurler's syndrome. Sanfilippo pp 51, (54) confirmed these
findings with eight patients with chemical, clinical,
morphological and radiological studies. Maroteaux and
Lamy (55) have recently presented a detailed study of this
type. They observed seven patients that excreted only
heparitin sulfate in the urine. They present clinical,
radiological, genetic, and biochemical evidence and propose
the term oligophrenia polydystrophy for this disorder.

From the available data it would seem that this type is
inherited as an autosomal recessive (52,55) and appears to
differ clinically from the other types of Hurler's syndrome
(55). Maroteaux pp 31. (56) have also studied a new type
of Hurler's patient which excretes only chondroitin sulfate

B in the urine.

MATERIALS AND METHODS

Mucopolysaccharides

 

Crude chondroitin sulfate and heparin were obtained
from General Biochemical Incorporated. Chondroitin sulfate
A was purified from the crude chondroitin sulfate by
fractionation with cetyl pyridinium chloride and by column
chromatography on Dowex-1-X2-Cl according to the method of
Schiller et al. (57). It was further purified by alcohol
fractionation as the calcium salt according to the method
of Meyer pp _1. (58). Chondroitin sulfate B was a gift of
Dr. Karl Meyer and chondroitin sulfate C was a gift of
Dr. C. W. Castor. Hyaluronic acid was obtained from the

Worthington Biochemical Company. Heparitin sulfate was a

gift from the Upjohn Company.

Quantitative Measurements

Uronic acid determinations were performed by the
carbazole method of Dische (59) with added borate (40) as
modified by Bitter and Muir (41) and by the naphthoresorcinol
method of Pelzer and Staib (42). Creatinine was determined
by the method of Folin and Wu (45). Chondroitin sulfates

were determined as previously described (44)°

78

79

Urine Samples and Isolation of Mucopoly—
saccharide from Urine

Urine samples from patients with Hurler's syndrome
were obtained from state schools for the mentally retarded
in Michigan. Specimens were also obtained from three
families each with an affected child. For the control
studies, urine was obtained from the state schools for the
mentally retarded and a local hospital. Controls were of
both sexes and of various ages. The amount of urine for
isolation of the mucopolysaccharides varied from 5 to 40
ml depending upon the availability, age, and whether from
Hurler's patients or normal.

The mucopolysaccharides were isolated from urine by
a slight modificatiOn of the method of DiFerrante and Rich
(20) as previously described (45). Paper chromatography of
mucopolysaccharides was carried out by the method of Spolter

and Marx (46) employing the type III solvent system.

RESULTS

Mucopo1ysaccharide Excretion in Urine
of Normal Man

There was some variation in the amount of mucopoly-
saccharides in different urine samples, but the data
revealed a marked decrease in mucopolysaccharide excretion
with advance in age in children and a close correlation
with creatinine levels. A uniform excretion pattern be-
comes evident when the amount of chondroitin sulfates
(Figure 6) or total mucopolysaccharides (Figure 7) per unit
of creatinine are plotted against age. The marked decrease
with age is apparent until the late teens after which the
level of mucopolysaccharide per unit of creatinine remains
constant. Total mucopolysaccharides per unit of creatinine
varied from 60 to 140 in the very young and became constant
at a value of about 6 in adults. The chondroitin sulfates
per unit of creatinine varied from 50 to 80 in the very
young and became constant at a value of about 2.5 in adults.

In addition to the decrease in the amount of mucopoly-
saccharide with age, a decrease in the chondroitin sulfate/
mucopolysaccharide ratio is also apparent (Figure 8).
This ratio varied from 0.60 to 0.80 in children to 0.50 to

0.50 in adults.

80

 

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Mucopolysaccharides in Urine from Families
with an Affected Child

Family 1 had three affected children and all excreted
heparitin sulfate. Likewise the affected child in family 2
excreted hepartin sulfate. While the affected child in
family 5 excreted both heparitin sulfate and chondroitin
sulfate B. The data concerning mucopolysaccharides in urine
samples from families with affected children are given in
Table X. By all criteria employed the mucopolysaccharides
in urine samples from all parents and siblings were within
the normal range. When the samples from parents and siblings
of affected patients are compared with the controls, the
amount of chondroitin sulfates (Figure 1) and total mucopoly-
saccharides (Figure 2), and the chondroitin sulfate/mucopoly~
saccharide ratio (Figure 5) appeared to be in the same range.

Mucopolysaccharides in Urine from Patients
with Hurler's Syndrome

 

The data on the mucopolysaccharides in urine samples
from Hurler's syndrome are given in Table XI. Five of the
six patients showed above normal C/N ratios, a normal amount
of chondroitin sulfates, a high level of total mucopoly—
saccharides and a low chondroitin sulfate/mucopolysaccharide
ratio. All these criteria would indicate that these
patients excreted heparitin sulfate. The other patient

excreted both chondroitin sulfate B and heparitin sulfate

88

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as indicated by a low C/N ratio and excessive amounts of
chondroitin sulfates and mucopolysaccharides.

On paper chromatography the one sample (No. 6) showed
three spots with mobilities similar to chondroitin sulfate B,
heparitin sulfate and chondroitin sulfate A. The other
samples showed only two spots with mobilities similar to
heparitin sulfate and chondroitin sulfate A. Our standard
of heparitin sulfate streaked in this solvent system and it
was not completely clear that the spots from the samples
were heparitin sulfate. Judging from the mobility reported
by Spolter and Marx (46), the chemical evidence, and the
established fact that heparitin sulfate occurs in urine
from Hurler's patients, it was concluded that the spots
were heparitin sulfate. The solvent system for chroma-
tography does not separate chondroitin sulfates A and C,
but again it is well-established that chondroitin sulfate A

is the major mucopolysaccharide in normal human urine.

DISCUSSION

In this study a marked decrease in the amount of total
mucopolysaccharide per unit of creatinine was observed
with age in children which is in general agreement with what
has been published (21,22), but after the late teens the
amount of mucopolysaccharides per unit of creatinine becomes
constant with age. The amount of chondroitin sulfates in
the urine of various age groups was also measured and a
similar pattern to the total mucopolysaccharides was ob-
served with age. A decrease in the ChS/MPS ratio with age
was also observed which would indicate that a decrease in
the amount of chondroitin sulfates relative to total muco-
polysaccharides occurred with age. Therefore, the types of
mucopolysaccharides at various ages as well as the total
amount should be considered in control urine samples.
A similar pattern of total mucopolysaccharide and chon-
droitin sulfates per unit of creatinine with age may occur
in other mammals (45).

Abnormal excretion of mucopolysaccharides in the urine
of heterozygous carriers of Hurler's syndrome has been
seen by Teller pp_51, (47). They observed a low C/N ratio
in suspected heterozygous carriers as compared to normal

urine samples which would indicate an abnormal excretion of

91

92

chondroitin sulfate B. They also reported a normal mean

C/Cr ratio in carriers which would indicate that the increase
in total mucopolysaccharides was not detectably different
from the normal. However, Linker and Terry (19), employing
the C/N ratio and isolation of the mucopolysaccharides,
observed no significant differences in urinary mucopoly—
saccharides in the carrier and normal individual.

In this study the mucopolysaccharides have been measured
in urine samples from three families who have at least one
child with Hurler's syndrome. In two families the affected
children excreted heparitin sulfate and mucopolysaccharide
excretion studies have not previously been reported in parents
and siblings of this type of Hurler's syndrome. Since this
defect is probably inherited as an autosomal recessive trait,
the parents and possibly some of the siblings might excrete
an excess of heparitin sulfate. The ChS/MPS ratio and total
mucopolysaccharides appeared to be within the normal range.

In the other family the affected child excreted both

heparitin sulfate and chondroitin sulfate B. The mucopolyc
saccharides from the urine of both parents and a sibling
showed a normal C/N ratio and normal amounts of mucopoly-
saccharides and chondroitin sulfates in the urine. In general,
the mucopolysaccharides from urine of parents and siblings

of Hurler's patients were within the normal range by all
criteria. Possibly the methods are not sensitive enough to

detect a very small amount of a given mucopolysaccharide, but

95

the parents and siblings do not appear to excrete a large
amount of abnormal mucopolysaccharides.

In this survey of Hurler's syndrome in the state schools
for the mentally retarded in Michigan, we have found that five
out of six Hurler's patients excrete heparitin sulfate.
Although, three of these patients were in the same family,
still a large percentage of the patients excrete heparitin
sulfate. Now that methods are available to distinguish the
various types of Hurler's syndrome this latter type may be
more prevalent than previously thought.

The determination of the normal excretion of mucopoly-

saccharides in urine is important because it may reflect the
metabolism of these compounds in the body and the normal
pattern is most likely used when comparing excretion in
mucopolysaccharides disorders. The occurrence of mucopoly~
saccharides in normal human urine appears to be more compli-
cated than originally observed. The chemical nature of the
acid mucopolysaccharide normally present in urine is by no
means settled, but it is Clear that chondroitin sulfate B
and heparitin sulfate occur in the urine in addition to the

major mucopolysaccharide, chondroitin sulfate A.

SUMMARY

Mucopolysaccharides have been measured in urine
samples of normal persons, patients with Hurler's syndrome
and their relatives. A marked decrease was observed in the
samples from controls in the amount of mucopolysaccharides
and chondroitin sulfates per unit creatinine with age until
the late teens after which the amount became constant.

A decrease in the ChS/MPS ratio with age was also observed.
After making appropriate allowances for age and creatinine,
mucopolysaccharides excretion in parents and siblings of
Hurler's syndrome was found to be similar to normal indi-
viduals by all criteria used. Several patients with the
heparitin sulfate uria type of Hurler's syndrome were found

in this survey.

94

10.

11.

12.
15.

14.

15.

16.

REFERENCES
Proceedings of a Symposium on Connective Tissue. Biophys.
J. 4, No. 1 Part 2 (1964).

Stacey, M., and Barker, 8. A., Carbohydrates of Living
Tissue. D. Von Nostrand Co., Ltd., New York, N. Y., (1962).

Brimacombe, J. S., and Stacey, M., Adv. Clin. Chem. 1, 199
(1964).

Schiller, S., Mathews, M. B., Cifonelli, J. A., and
Dorfman, A., J. Biol. Chem. 218, 159 (1956).

Davidson, E. A., and Small, W., Biochim. Biophys. Acta
§9, 455 (1965).

Bostrom, H., in Springer, G. E., (editor) Polysaccharides
in Biology. Josiah Macy, Jr. Foundation, N. J. (1959).

Davidson, E. A., Small, W., Perchemlides, P., and Baxley,
W., Biochim. Biophys. Acta 15, 189 (1961).

Davidson, E. A., and Small, W., Biochim. Biophys. Acta
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Davidson, B. A., and Riley, J. G., Biochim. BiOphys.
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Bollet, A. J., Bonner, W. M. Jr., Nance, J° L., J. Biol.
Chem. 258, 5522 (1965).

Kaplan, D., and Meyer, K., J. Clin. Invest. 11, 745
(1962).

Astrup, P., Acta Pharmacol. Toxical. 5, 165 (1947).
Kerby, G. P., J. Clin. Invest. 55, 1168 (1954).

Hamerman, D., Hatch, F. T., Reife, A., and Bartz, K. W.,
J. Lab. Clin. Med. 15, 848 (1955).

DiFerrante, N., and Rich, C., Clin. Chim. Acta 1, 519
(1956).

DiFerrante, N., J. Clin. Invest. 55, 1519 (1957).

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17.

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20.

21.

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