Focus on Homocysteine

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Christina Bolander-Gouaille
Edvin Berlings Gata 32
SE-252 50 Helsingborg
Sweden
[email protected]

ISBN 978-2-287-59682-7 ISBN 978-2-8178-0741-6 (eBook)

DOI 10.1007/978-2-8178-0741-6
© Springer Verlag France 2001
Imprime en France

Library of Congress Cataloging-in-Publication Data

Gouaille, Christina, 1939.
Focus in homocysteine / Christine Gouaille.
p. em.
Includes bibliographical references.
ISBN 978-2-287-59682-7
I. Homocysteine-- Pathophysiology. 2. Homocysteine--Metabolism--Disorders.
3. Homocysteine--Metabolism. 1. Title.
RC632 H65 G68 1999
616.1071--dc21
99-052050

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Preface

A relation of homocysteine to human disease was first suggested in 1962 in

the classical paper of Carson and Neil, reporting on elevated homocystine
in the urine of mentally retarded children. Homocystinuria was later
found to be associated with occlusive cardiovascular disease in early life
and even in childhood. These observations raised the question whether
moderately elevated plasma homocysteine is a risk factor for cardio-
vascular disease in the general population.
Since the mid eighties, massive amounts of data from about
12 000 patients and controls have documented that moderately elevated
homocysteine in serum/plasma is a strong and independent risk factor for
occlusive arterial disease in the coronary, cerebral and peripheral vessels.
Notably, high homocysteine seems to provoke the acute events, particular-
ly in subjects with an underlying disease.
Recently, elevated homocysteine has also been established as a risk fac-
tor for venous thrombosis. Hyperhomocysteinaemia is also associated
with neural tube defects, pregnancy complications, and cognitive impair-
ment in the elderly and various neuro-psychiatric disorders.
An important aspect is the involvement of several B-vitamins, in partic-
ular folate and cobalamin, in homocysteine metabolism. Homocysteine in
plasma or serum is a useful marker of folate and cobalamin status both for
diagnosis of deficiency and during follow-up.
Furthermore, supplementation with folate, alone or in combination
with cobalamin and vitamin B6 , are efficient means to reduce plasma ho-
mocysteine, even in subjects without overt vitamin deficiencies.
The existence of safe and inexpensive homocysteine lowering regimens
has opened up for several planned and ongoing intervention trials which
address the question whether reduction of homocysteine will affect the
occurrence or progression of homocysteine associated diseases. Such data
may support causality and also provide therapeutic or preventive
measures with great health implications. Results of these trials will,
4

however, not undermine the current status of plasma homocysteine as an

extremely useful predictor of cardiovascular and other common diseases.
The homocysteine literature is growing and now includes about
5500 articles filed in the MEDLINE database. This book by Christina
Bolander-Gouaille - who has earlier written much appreciated reviews on
vitamin B12 - is an updated, systematic and concise review of this rapidly
expanding field, with emphasis on the medical and diagnostic aspects. It
will be valuable background for the general practitioner as well as the
specialist using plasma/serum homocysteine in laboratory diagnostics.

PER MAGNE UELAND, PROFESSOR, MD,

Haukeland Hospital, Bergen,
NORWAY
Acknowledgements

I am grateful for the constructive comments, criticism and advice of many

researchers and clinicians, particularly Assistant Professor Teodoro Bot-
tiglieri, MD, Metabolic Disease Center, Baylor Research Institute, Dallas,
USA. Benedicte Christensen, MD, PhD, Ulleval University Hospital, Oslo,
Norway. Professor Jean-Pierre Nicolas, MD, and Abalo Chango, MD, IN-
SERM U-308, Biochemical Laboratory A, CHU Nancy, Vandoevre-les-
Nancy, France. Assistant Professor Bjorn Regland, MD, Department of
Psychiatry and Neurochemistry, Institute of Clinical Neuroscience, Uni-
versity of Gothenburg, Gothenburg, Sweden. Associate Professor, Joern
Schneede, MD, PhD, Department of Pharmacology, University of Bergen,
Bergen, Norway.
I also wish to express my gratitude to all authors and journals for gen-
erously granting me permission to reproduce tables and figures.
Contents

Introduction 9

What is homocysteine? 11

The homocysteine metabolism 14

The methylation cycle 14
Common enzyme defects 18

How can hyperhomocysteinaemia be harmful? 20

Vascular damage 20
Neurological damage 24
Pregnancy complications, neural tube defects, 30
and other congenital malformations

Why do homocysteine levels increase? 32

Lifestyle factors 32
Age-related factors 37
Hormonal changes 39
Diseases 40
Drugs 44

Clinical conditions associated 47

with hyperhomocysteinaemia
Vascular disease 47
8 Focus on homocy teine

Cognitive impairment and neuro-psychiatric disorders 54

Teratogenicity and pregnancy outcome 62

When and how to check the homocysteine levels 69

When can hyperhomocysteinaemia be suspected? 69
Blood sampling 71
Methionine loading 71

How to interpret the test result and

how to handle hyperhomocysteinaemia 74
Reference ranges 74
Interpretation 76
Intervention 76

References 80

Abbreviations 95
Introduction

Homocysteine is becoming a familiar concept. Since the early 1990S a

continuously increasing number of studies have been published on ho-
mocysteine. It has also been the topic of two international conferences, in
1995 and 1998.
Hyperhomocysteinaemia, generally defined as fasting plasma homocys-
teine levels above 1511mol/L, has been shown to be an independent risk fac-
tor for cardiovascular disease and for complications in pregnancy and
congenital malformations. Many studies have also found a link between
impaired homocysteine metabolism and neuro-psychiatric disorders and
with cognitive impairment in the elderly.
Within the next few years, the results of several large homocysteine-low-
ering intervention studies will become available. There are strong reasons
to believe that they will confirm the predicted effects of homocysteine-low-
ering treatment on neural tube defects, other complications of pregnancy
and cardiovascular disease. Normalization of homocysteine levels may
also have a positive impact on cognitive impairment and several neuro-
psychiatric disorders.
Moderate elevations of homocysteine may often be the result of one or
more modifiable lifestyle factors, like smoking, a low nutritional intake of
vitamins, a high coffee consumption, and too little physical exercise.
Diagnosing hyperhomocysteinaemia could thus be an important incentive
for the patient to opt for a healthier lifestyle. In addition, successful
treatment of hyperhomocysteinaemia may, in most cases, be accomplished
by a simple vitamin supplementation.
This book is an attempt to summarize current knowledge. It aims at
giving an overview of physiological and pathophysiological aspects of the
homocysteine metabolism. It outlines the factors that can influence the
homocysteine metabolism and clinical situations where hyperhomocys-
teinaemia should be suspected. Lastly, it gives some advice on blood sam-
pling procedures, handling and storage of samples, interpretation of the
test results, and treatment of hyperhomocysteinaemia.
10

The book offers a kind of "map" of the homocysteine field. If most "plac-
es" are indicated, they are described rather briefly. Many data had to be left
out to not to make the text too extensive and the "map" too hard to read.
Hence, the reader will have to consult the original publications for more
ample information on his or hers "places" of interest.
There is a slight overlap of the different headings. This was done inten-
tionally in order to make each chapter independent and thus allow reading
in any order.
The exponentially increasing interest in the homocysteine metabolism
is illustrated by the fact that of 468 publications cited, 429 are from the
1990S and 243 from 1997-1999. The number of references still had to be re-
stricted. Hence, reviews are sometimes referred to in which the original
references can be found.

August 1999 CHRISTINA BOLANDER-GOUAILLE

 Chapter

What is
homocysteine?

Homocysteine was originally identified in 1962 in the urine of mentally re-

tarded children (Gerritsen et al. 1962, Carson and Neill 1962). A couple of
years later the genetic defect of cystathionine ~-synthase (CBS), causing
homocystinuria and very high plasma levels of total homocysteine (tHcy),
was identified (Mudd et al. 1964). These patients were found to have fre-
quent thrombo-embolic events. More than 50% of the patients had cardio-
vascular events and 25% died before the age of 30 (Gibson et al. 1964,
Schimke et al.1965).
In 1969 McCully described the vascular pathology in these patients, in-
cluding smooth muscle proliferation, progressive arterial stenosis, and
haemostatic changes. Severe defects in other enzymes, methionine syn-
thase (MS) and methylenetetrahydrofolate reductase (MTHFR), were later
discovered; these also caused homocystinuria and vascular pathology, as
well as mental disturbances (Mudd et al.1972, Rosenblatt et al.1990, Rozen
1996).
Epidemiological studies in the general population have later de-
monstrated an association between moderately elevated levels of tHcy in
the circulation, and not only vascular diseases but also pregnancy
complications, neural tube defects, other congenital malformations, various
neuro-psychiatric disorders and cognitive impairment in the elderly. These
studies are outlined on pages 47-68. Two recent prospective case-control
studies also show that overall mortality is correlated to tHey levels,
independent of the classical risk factors. (Hoogeveen et al. 1998, Bostom
et al. 1999).
Homocysteine is a sulphur-containing amino acid that is closely related
to methionine and cysteine. There is no DNA-coding for this amino acid,
and it is not present in naturally occurring proteins. All homocysteine
found in organisms is formed in the metabolism of the essential amino
acid methionine, in the methylation cycle, page 14. This is the only known
source of homocysteine.
12

Reduced homocysteine has a highly reactive free thiol group. which is

susceptible to auto-oxidation at physiological pH. thereby forming disul-
phide bonds between two molecules or mixed disulphides with other thiols.
In plasma only about 1% of homocysteine normally exists in the free re-
duced form. About 70% is bound to albumin. The rest forms low molec-
ular weight disulphides. predominantly with cysteine. The sum of all the
forms is termed total homocysteine. Homocysteine is sometimes written
homocyst(e)ine. since this term more clearly designates all the molecular
species that are measured.
The abbreviations Hcy for homocysteine and tHcy for total homocys-
teine are used in the following. where tHcy generally refers to plasma or se-
rum levels.
The assays generally measure the tHcy in plasma or serum. sometimes
in the CSF. rarely in urine. Analysis of the different fractions of tHcy is
complicated and only used for research purposes.
During the last ten years. several assays for measuring homocysteine in
plasma. serum. and CSF have been developed. A further step forward is the
recent introduction of enzyme immunoassays. which will allow determi-
nation of homocysteine in most routine laboratories.
Three enzymes are directly involved in the Hcy metabolism: methion-
ine synthase (MS). betaine homocysteine methyltransferase (BHMT). and
cystathionine ~-synthase (CBS). Vitamin B6 • Bl l• and folate are co factors
to these enzymes. The metabolism of Hcy is described on pages 14-19 and
illustrated in fig I. page 15. If this metabolism is disturbed, because of some
enzymatic defect or intracellular deficiency of some cofactor to the men-
tioned enzymes, Hcy accumulates in the cell and is then exported to the
circulation where levels rise.
Hcy is mainly eliminated by renal catabolism. Only about 1% of the Hcy
filtered by the glomeruli is normally found in the urine (Guttormsen et al.
1997). The rest is reabsorbed and metabolized. Thus, the kidneys are Hcy-
metabolizing rather than Hcy-excreting (Bostom et al.1995a, van Guldener
1998. Refsum 1998a).
The plasma levels of tHcy in the circulation found in the general popu-
lation vary with age and sex, as shown in table 6, page 75.
Plasma tHcy increases throughout life in both sexes. Before puberty,
children of both sexes have low and similar levels (about 5 )lmollL). Dur-
ing puberty, levels markedly increase, more in boys than in girls. At the
same time, tHcy values start to show a skew distribution in populations.
Throughout life, the mean tHcy increases by 3-5 )lmoI/L. At the age of 40-
42, there is a difference of about 2 )lmollL between men and women, with
mean values of about 11 and 9 )lmollL. respectively (NygArd et al. 1995).
What Is homocy telne? 13

During pregnancy, tHcy is reduced by up to a half. The tHcy returns to

previous values within 2-4 days of delivery (Andersson et at. 1992a, Kang
et at. 1986, Bonnette et at. 1998, Walker et at. 1999).
More muscle mass in men may explain some of the differences between
sexes, as the formation of creatine generates Hcy (Mudd 1995, Brattstrom
1994). Nutritional habits may also sometimes differ between the sexes
(Selhub et at. 1993, Refsum et at. 1996, Thcker et at. 1996a), which could
contribute to lower tHcy levels in women. After the menopause, as oestro-
gen levels decrease, the tHcy levels increase, but remain lower in women
than in men (Andersson et at. 1992b, NygArd et at. 1995).
The higher tHcy concentrations seen in the elderly may be a conse-
quence of a general slowdown of the metabolism, malabsorption or insuf-
ficient nutritional supply of folate, vitamin B12 , and B6 , reduced kidney
function, and other physiological age-related changes. These and other
factors known to increase tHcy levels are described in more detail on
pages 32-46.
 Chapter

The homocysteine
metabolism

Homocysteine is metabolized through two major pathways: transsulphu-

ration and methylation. Normally, about 50% is catabolized in the trans-
sulphuration pathway, where Hcy and serine form cystathionine, which is
cleaved into cysteine and a-ketobutyrate. The other 50% enters the meth-
ylation cycle, which is part of the one-carbon metabolism. S-adenosylme-
thionine (SAM) is an important regulator of the remethylation and
transsulphuration of Hcy.

The methylation cycle

In the methylation cycle, Hcy is formed by demethylation of the essential

amino acid methionine, fig 1. Methionine is derived from dietary proteins.
It contains a methyl group, which is activated by conversion to S-adeno-
sylmethionine (SAM). This reaction is mediated by methionine adenosyl
transferase (MAT) and adenosine triphosphate (ATP).
SAM is the principal biological methyl group donor. It is required for
numerous methylation reactions, of which about 100 have been identi-
fied. A product of all methylation reactions is S-adenosylhomocysteine
(SAH), which is hydrolyzed to Hcy in a reversible reaction. In most
tissues, Hcy may be remethylated to methionine by the enzyme methion-
ine synthase (MS). A few tissues, predominantly the liver, express the
enzyme betaine homocysteine methyltransferase (BHMT) that functions
as an alternative pathway for the remethylation of Hcy. However, most
tissues, like the CNS, are entirely dependent on the MS-mediated recy-
cling of Hcy. The role of SAM as a methyl group donor was recently
reviewed (Chiang et al. 1996).
SAH is a potent competitor to SAM at different binding sites and can
therefore inhibit methylation (Mudd et al.1989). The SAM/SAH ratio may
be use,d as an indicator of methylation status.
The homocysteine metaboli m IS

The Homocysteine etabolism

!
Dietary protein

ATP

Methionine~
Dimethylglyclne
<§
""'re e rylation
cycle

serine ~
<§>l Vit. Bs
~ Hcy-thiolactone

T,"rio" -+- Cysteine

Glutathione
S,liate +CO,

S
®
Cystathionine ~-synthase
S-Adenosylmethionine

ad~ne transferase S-Adenosylhomocysteine

Methionine
@
Methionine synthase
o
Tetrahydrofolate

8
5,10 Metyhlenetetrahydrofolate reductase Methyltetrahydrofolate

8
Betaine homocysteine methyltranferase
Q
5,10 Methylenetetrahydrofolate

Fig. 1 The homocysteine metabolism

16 Focus on homocysteine

If the methionine balance is negative, and in the presence of low concen-

trations of SAM, Hcy is primarily directed towards the remethylation path-
way to form methionine by the MS-mediated reaction. Vitamin B12 is a
cofactor and methyltetrahydrofolate (methyITHF) a substrate in this reac-
tion. MethylTHF is formed in a reaction catalyzed by methylenetetrahy-
drofolate reductase (MTHFR). This enzyme has therefore a strong,
indirect influence on the Hcy remethylation (Engbersen et al. 1995).
Conversely, if SAM concentrations are high, more Hcy is directed to-
wards the transsulphuration pathway to form cystathionine and - except
in the CNS - cysteine by two vitamin B6 -dependent reactions, the first of
which is catalyzed by cystathionine p-synthase (CBS). (A rare, severe, ge-
netic defect in CBS leads to homocystinuria with very high levels of tHcy
in the circulation.)
Decreased remethylation of Hcy to methionine and SAM may impair the
methylation reactions required, for instance, for normal brain function.
The methyl group of the methionine moiety of SAM is activated by a
positive charge of its sulphur atom. This methyl group is very reactive and
can easily be transferred to a large variety of acceptor substrates, including
nucleic acids (DNA and RNA), proteins, phospholipids, myelin, polysac-
charides, catecholamines, and a large range of small molecules.
Considering the critical role of methylation in various cellular process-
es, it is understandable that any alteration in the availability of SAM may
have profound effects on cellular growth, differentiation, and function.
This may be critical, especially in the aging brain, where neurochemical
processes related to methylation may be declining, and also in psychiatric
and neurological diseases and for the rapidly growing fetus and infant.
Another consequence of a block in the conversion of Hcy to methionine
is reduced recycling of methylTHF into the pool of active folates. Forma-
tion of THF, required for the synthesis of purines, DNA, and RNA, is re-
duced, fig 2. The accumulation of methylTHF caused by vitamin B12
deficiency is called the "folate trap". Serum levels of folate may then be
high despite an intracellular deficiency.
The conversion of Hcy into methionine may be severely blocked in some
rare, genetic defects in MS or MTHFR. Such patients have high levels of
tHcy, low levels of methionine, neurological symptoms, and mental retar-
dation, as well as vascular pathology (McCully 1969, Mudd et al. 1972,
Rosenblatt et al.1990, Rozen 1996). Brain magnetic resonance imaging has
revealed demyelinated lesions. Low CSF levels of SAM were also found
(Hyland et al. 1988, Surtees et al. 1991, Kischi et al. 1994).
Nitrous oxide inactivates MS by interaction with methyl-cobalamin, and
has been used for experimental inactivation of this enzyme in animals to
 17

Tetrahydrofolate Metabolism
DNA/RNA

t
Purines

dTMP ~ DNA

Cell membrane

ET~~ @
Methylenetetrahydrofolate reductase Methionine synthase

Methyl-
G
5.10 Methylene-
o
Tetrahydrofolate
tetrahydrofolate tetrahydrofolate

Fig. 2 The tetrahydrofolate metabolism.

18

study the importance of methylation reactions for various neurological

functions (McKeever et al. 1995a and b, Scott et al. 1994, Weir et al. 1988 and
1992). The consequences of such experimental inactivation of MS on the
nervous system, and also of severe genetic deficiencies impairing the rem-
ethylation pathways of Hcy, were reviewed in 1992 by Metz.
Any disturbance ofHcy remethylation may result in hyperhomocystein-
aemia. An enzyme defect, deficiency or disturbed distribution of one of
the involved vitamins, an overload of the enzyme system or a combination
can thus impair the methylation cycle and increase tHcy levels. The most
important causes of hyperhomocysteinaemia are unhealthy life-style, poor
nutrition with low intake of folate, vitamin B6 and Bl2 , malabsorbation of
vitamins, and common enzyme defects.

Common enzyme defects

Mutations causing hyperhomocysteinaemia can now be directly assayed.

During the last few years, the enzymes, CBS, MS, MTHFR, and MAT have
been cloned. Many mutations have been identified.
Some of the less severe enzyme defects affecting the Hcy metabolism are
quite common, such as the C677T mutation of the MTHFR gene. A specific
C~ T substitution converts an alanine to a valine residue and results in a
thermolabile variant. The impact of this polymorphism has been investi-
gated in a large number of clinical studies. The mutation shows ethnic dif-
ferences. It is almost absent in Afro-Americans. In Caucasians the mean
prevalence of homozygous subjects - TT variant - is about 12% and over
40% are heterozygous. In the homozygous form the enzyme has only 30-
50% of its normal activity (in vitro).
Homozygosity for the mutation, and to a some extent heterozygosity, is
associated with moderately increased fasting tHcy levels (Kang et al. 1991,
Engbersen et al. 1995, Frosst et al. 1995, Guttormsen et al. 1996, Harmon et
al. 1996, Jaques et al. 1996, Kluijtmans et al. 1997, Ali et al. 1997, Clarke et al.
1998a, Verhoef et al. 1998).
Enzyme defects affecting the remethylation of Hcy, however, do not al-
ways lead to increased fasting levels of tHcy. It is sometimes necessary to
perform a methionine loading (PML) test to detect the defects (page 71) .
The enzyme system is loaded and the transsulfuration pathway in partic-
ular. If the enzymatic capacity is reduced, the increase in tHcy will be ab-
normally high and particularly in vitamin B6 deficiency (Ubbink et al.
1996) or mild CBS defects.