|Year : 2012 | Volume
| Issue : 3 | Page : 139-147
Homocysteine- an amino acid culprit in ill health and disease
Satyanarayana Uppala, Vijayalakshmi Udipi Badikillaya
Department of Biochemistry, Dr. Pinnamaneni Siddhartha Institute of Medical Sciences and Research Foundation. (Dr PSIMS and RF), Chinoutapalli, Gannavaram, Andhra Pradesh, India
|Date of Web Publication||15-Oct-2012|
Dr. Pinnamaneni Siddhartha Institute of Medical Sciences and Research Foundation, Chinoutapalli-521 286, Gannavaram (Mdl), Krishna Dt, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
Homocysteine (Hcy) is a sulfur-containing amino acid with its only source in the human body being methionine. Three B complex vitamins (B 6 , folate or B 9 , and B 12 ) participate in the metabolism of Hcy. Deficiency of these vitamins and the enzymes involved in its metabolism leads to elevated plasma Hcy levels (normal 5 - 15 μmol/l). Hyperhomocysteinemia (HHcy) is associated with several disorders. Hcy levels above 50 μmol/l is thought to be a risk factor for recurrent heart attacks and a value between 150 and 200 μmol/l may cause ischemic stroke. Levels above 300 μmol/l may induce mental deficiency. Several factors contributing to HHcy have been identified. These include lifestyle factors (smoking, alcoholism), drugs (phenytoin, valproate) and genetic causes (cystathionine β synthase deficiency). The various manifestations of HHcy are explained on the basis of oxidative damage and protein homocysteinylation, which cause damage to most tissues/organs leading to health complications and occurrence of various diseases. HHcy may affect major systems in the body like cardiovascular (coronary artery disease, myocardial infarction), central nervous (cerebrovascular accident, Alzheimer's, dementia) skeletal (increased risk of fractures), renal (low GFR), reproductive (risk of infertility), besides increasing the risk of hypothyroidism and cancer. The measures to prevent HHcy include consumption of nutritious foods, lifestyle changes (avoidance of smoking and alcohol) and supplementation of diet with vitamins - B 6 , folate or B 9 , and B 12 .
Keywords: Cobalamin, folate, hyperhomocysteinemia, oxidative stress, vascular diseases
|How to cite this article:|
Uppala S, Badikillaya VU. Homocysteine- an amino acid culprit in ill health and disease. J NTR Univ Health Sci 2012;1:139-47
| Introduction|| |
Homocysteine (Hcy) has in the recent past become a biomolecule of great importance for biochemists and clinicians alike. Its role in causing vascular diseases has been recognized. Hcy has been found to be a marker or a causative agent of organ dysfunctions such as of cardiovascular, skeletal, renal, reproductive, ocular and nervous systems. Hcy has been implicated in the causation of cancer and ischemic changes in diabetes mellitus.
| Chemistry of Homocysteine|| |
Homocysteine is sulfur containing and considered to be a non-protein amino acid, derived from methionine, an essential amino acid. It was discovered as early as in 1930s by Du Vigneaud, an American biochemist who named it Hcy, as its structure was found to be similar to cysteine with an extra carbon atom.  Hcy exists in different forms. It is present as the reduced form with a free sulfhydryl (SH) group, as homocystine or the oxidized form with the disulfide (S-S) linkage, as Hcy thiolactone - a cyclic thioester, as a dimer with cysteine and as adducts (Latin for a product of 2 or more molecules). The adducts are formed by N-homocysteinylation where Hcy thiolactone binds to lysine residues of proteins. , Hcy thiolactone is formed by methionine tRNA synthetase when Hcy is mistakenly selected by the enzyme in place of methionine during protein biosynthesis.  The different forms namely Hcy, homocystine, Hcy dimer, Hcy thiolactone and Hcy adducts form the total Hcy (tHcy). The concentration of the free form is low and that of Hcy adducts (Hcy thiolactone form bound to proteins) is very high in the plasma.
| Metabolism of Homocysteine|| |
The only source of Hcy in the body is methionine. Hcy can enter into 2 pathways. One is the remethylation pathway that depends on vitamin B 12 to form methionine and the other the trans-sulfuration pathway that depends on vitamin B 6 to form cysteine. 
Under normal conditions, most of the Hcy is remethylated back to methionine or converted into cysteine by the trans-sulfuration pathway. The latter pathway is inactive in the brain and the vascular endothelial cells. Therefore, the brain and the vascular endothelium are mainly dependent on adequate supplies of folate and cobalamin in order to maintain normal levels of Hcy [Figure 1].
|Figure 1: An overview of the metabolism of Hcy: Methionine forms S-adenosyl-methionine (SAM), which is converted to S-adenosyl homocysteine (SAH) after donating its methyl group to acceptors. SAH is hydrolyzed to homocysteine [THF-tetrahydrofolate, MSmethionine synthase, CBS- cystathionine β synthase, BHMT-betaine Hcy methyl transferase, MTHFR-methylene THF reductase N5, N10_MTHF – methylene THF]|
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A different remethylation pathway in the kidney and liver uses betaine as the methyl donor to methylate Hcy. Another enzyme that is indirectly related to Hcy metabolism is N 5 , N 10 - methylene tetrahydrofolate reductase (MTHFR), which reduces N 5 , N 10 -methylene tetrahydrofolate to N 5 -methyl tetrahydrofolate, which in turn methylates vitamin B 12 that is a cofactor for methionine synthase. Three B complex vitamins are involved in Hcy metabolism viz. B 6 , folate or B 9 , and B 12 . Deficiency of these vitamins and/or of the above-mentioned 3 enzymes involved in the metabolism increases plasma Hcy levels.
| The History of Hcy in Relation to Disease|| |
Kilmer McCully largely contributed to the present day knowledge of the biomedical significance of Hcy. He described the history of evolution of Hcy from being an innocuous amino acid to a causative factor of disease. Some important milestones in the history of Hcy in relation to the disease are briefly described.  In 1953, Frederick Stare and his colleagues found that plasma cholesterol concentration and experimental atherogenesis in monkeys were inhibited by taking diets rich in methionine. This suggested a relationship between arteriosclerosis and sulfur-containing amino acid metabolism. In 1962, investigators in Ireland and Philadelphia while screening urine of children with mental retardation found that many of these children had homocystine in their urine. These children had in addition other features like ocular dislocation of lens, accelerated growth, osteoporosis and a tendency to develop thrombi. Later, it was found that they were deficient in the enzyme CBS. In 1968 at a Massachusetts hospital, a 2-month-old baby was found to have homocystinuria and cystathionine in urine, and was found to be deficient in the enzyme methionine synthase. McCully found on studying the slides of these patients that there was arteriosclerosis scattered throughout the body in both the cases and concluded that Hcy had a role in causing arteriosclerosis. In 1972, a third major enzyme deficiency, MTHFR was found in a child having homocystinuria who also had arteriosclerotic plaques in arteries. Thus, the relationship between hyperhomocysteinemia and arteriosclerosis was established.
| Quantification, Reference Range and Hyperhomocysteinemia (HHcy)|| |
Plasma homocysteine elevation is associated with several disorders; hence, its quantification assumes significance. Hcy plasma levels can be measured by enzyme assays, enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography (HPLC). Homocystinuria can be detected by the simple silver nitrate nitroprusside test.  The free Hcy levels or the total Hcy levels can be estimated. Usually the tHcy levels are measured.  The normal plasma tHcy is about 5 - 15 μmol/l.  The age-wise distribution of plasma Hcy levels is given in [Table 1]. 
In general, males have a higher plasma Hcy level (around 2 μmol/l) when compared to females.  In pregnancy, the upper reference range is 10 μmol/l. It is observed that the plasma Hcy levels in individuals on a folate-fortified diet are around 2-10 μmol/l lower than those not on such diets. 
A methionine loading test is done for research purposes to identify the hyperhomocysteinemias due to CBS and MTHFR gene polymorphisms. , Plasma homocysteine thiolactone (normal levels 0 - 34.8 nmol/l) can also be measured by HPLC, though it is not done routinely.  Hyperhomocysteinemias are graded as follows: 
Mild 15-30 μmol/l
Moderate 30-100 μmol/l
Severe >100 μmol/l
Hcy levels above 50 μmol/l is thought to be a risk factor for recurrent heart attacks and a value between 150 and 200 μmol/l may cause ischemic stroke. Levels above 300 μmol/l may induce mental deficiency. 
| Factors Contributing to Hyperhomocysteinemia|| |
Hcy levels in general are found to be higher in men, postmenopausal women, in those with increased muscle mass and in the elderly. ,
Smoking, excess alcohol (chronic alcoholics, intoxication) and coffee consumption (>5 cups/day) are known to cause HHcy. ,, It is theorized that all these factors may affect the remethylation pathway thus raising plasma Hcy levels. A vegetarian diet may cause a marginal rise in Hcy levels probably due to insufficient cobalamin in the diet, which prevents Hcy remethylation.  Vegans (those who do not consume eggs and milk) are at a greater danger of having elevated Hcy levels, as they have extremely low levels of cobalamin. 
Antiepileptics like phenytoin, phenobarbitone and valproate, others like metformin, niacin, cholestyramine raise plasma Hcy levels. ,,
Homocystinurias due to the deficiency of one or more of the following enzymes viz. cystathionine β synthase, MTHFR and methionine synthase lead to high plasma Hcy levels. Deficiency of cystathionine β synthase increases methionine levels that lead to mental retardation, seizures, ectopia lentis, skeletal abnormalities including disproportionate growth, osteoporosis and vascular changes. The aforementioned features lead to premature death. The major symptoms due to a deficiency in the latter 2 enzymes are vascular changes and neuropsychiatric sequelae. Apart from these enzymatic deficiencies, absorption defects in the pathway of remethylation of Hcy to methionine cause psychomotor retardation, lethargy, megaloblastic anemia and failure to thrive. , MTHFR polymorphisms are common and if the mutation causes reduced activity of the enzyme, it leads to HHcy. These polymorphisms are found to be associated with cardiovascular and cerebrovascular diseases and dementia. Studies have shown that in people with these polymorphisms, dietary folate and cobalamin supplementation reduces plasma Hcy levels. 
Countries such as Japan, France and Spain with higher intake of pyridoxine and folate have lower Hcy concentration, averaging 6-8 μmol/l when compared to countries like Finland, Scotland and Northern Germany with lower B vitamin intakes and correspondingly higher Hcy concentrations of 10-12 μmol/l.  Indians are known to be at a greater risk for HHcy due to poor intake of B vitamins and also due to polymorphism of the MTHFR gene.  Community survey-based studies for HHcy in India have reported an incidence of 52- 84%, which is higher when compared to the western countries.  In general, vegetarians especially vegans are found to have a higher Hcy level when compared to non-vegetarians due to deficiency in cobalamin, which causes HHcy.  Indians are prone to diabetes and therefore heart diseases and one of the causes of coronary artery disease is HHcy. A typical Indian diet is mainly vegetarian and often deficient in cobalamin and folate and the food is not fortified with these vitamins leading to the higher incidence of HHcy among Indians. 
Hypohomocysteinemia is seen physiologically in pregnancy probably due to hemodilution and folate supplements.  It is also seen in hyperthyroidism, which may be due to elevated glomerular filtration rate (GFR). A low plasma Hcy level is also seen in patients on drugs like simvastatin, adenosine analogs, sulfhydryl compounds and tamoxifen. ,
| Mechanism of Hyperhomocysteinemia Induced Damage|| |
The mechanism of action of Hcy in causing ill health and disease appears to be complex and not clearly known. The various manifestations of HHcy are explained on the basis of oxidative damage and protein homocysteinylation.
Hcy (with -SH group) continuously gets oxidized resulting in homocysteine (S-S group) and homocysteinylated proteins. , This leads to the formation of reactive oxygen species (ROS) such as super oxide, hydrogen peroxide and hydroxyl free radicals. The damaging effects of ROS on biomolecules (proteins, lipids) are well known. Further, superoxide converts nitric oxide (NO) to peroxynitrite thereby depleting tissue NO levels  (adequate NO is required for various biochemical functions including vasodilatation and inhibition of platelet aggregation).
Protein homocysteinylation 
Hcy thiolactone is highly reactive and it can acylate free amino groups (e.g., lysine) of different proteins to form adducts under physiological conditions. This process is referred to as N-homocysteinylation. The degree of protein homocysteinylation increases with increased plasma tHcy. Homocysteinylated proteins may become inactive or result in modified proteins that are detrimental to health e.g., modified hemoglobin, LDL, plasma proteins, to name a few. It appears that the conversion of Hcy to Hcy thiolactone followed by protein N- homocysteinylation largely contributes to manifestations of Hcy toxicity. Homocysteinylation causes immune activation, autoimmune inflammatory response, cellular toxicity and cell death, and enhanced protein degradation. Thus, excess Hcy is detrimental to the body as it can damage most of the tissues/organs leading to health complications and occurrence of various diseases.
Karolczak and Olas recently reported that elevated levels of plasma Hcy and its thiolactone disturb the hemostasis (flowing properties of blood) that may ultimately lead to blood clotting and cardiovascular diseases .  Hyperhomocysteinemia causes several biochemical changes, some of which are listed in [Table 2]. 
| Clinical Conditions Associated with Hyperhomocysteinemia|| |
The existing epidemiological data clearly indicate that moderately elevated plasma Hcy levels increase the risk of cardiovascular events. About 30% of cardiovascular diseases that cannot be explained by traditional risk factors are attributable to HHcy.  In the United States (US), after fortification of flour and other refined grain foods with pyridoxine and folate in the 1960s, it was found that mortality from vascular diseases, particularly of the heart was considerably reduced when compared to the early 20th century.  There are studies to suggest that Hcy may be a better predictor of recurrent cardiovascular events than the primary cardiovascular disease. In addition Hcy was found to be a better predictor of congestive heart failure in patients without prior cardiovascular events.  Hcy induces oxidative stress resulting in increased production of 3-hydroxy 3-methyl glutaryl coenzyme A (HMG CoA) in the endothelial cell that leads to increased cholesterol production. Statins are helpful in such cases as they decrease cellular cholesterol and increase NO. Further silent coronary artery disease was found to be associated independently with HHcy.  An increase by 1 μmol/l of plasma Hcy increases the risk of cardiovascular disease by 10%. 
In diabetes mellitus (DM), there is accelerated atheroscleropathy as the endothelium has to face additional oxidative stress due to HHcy along with the other risk factors of DM. Hayden and Tyagi have described the Framingham Offspring study where it was found that insulin resistance is associated with HHcy.  It was also reported that Hcy levels decreased with improved diabetic control as assessed by HbA1c levels.  HHcy was associated with microvascular complications due to its atherogenic potential, as already described. There is an evidence that Hcy acts as a competitive inhibitor of rosiglitazone, pioglitazone and fibric acid derivatives (drugs that are used in the treatment of DM to improve insulin sensitivity, decrease inflammation and reduce lipid levels) and impairs their activity. 
| Central Nervous System|| |
Hcy metabolism in the brain is different from other organs. The trans-sulfuration pathway is not active and the remethylation pathway using betaine is absent.  Thus the capacity for Hcy metabolism is largely dependent on the supplies of folate and cobalamin. The glial cells have very low stores of vitamin B 12 that are quickly depleted during its negative balance. In addition, adenosyl cobalamin is a cofactor for methyl malonyl CoA mutase, so there is methyl malonic aciduria due to deficiency of B 12 . Methyl malonic acid is neurotoxic and the symptoms range from mild irritability, mood swings, and forgetfulness to depression and dementia. The effect of HHcy on vasculature has already been described. It causes cerebral ischemia leading to cerebrovascular accidents. Folate and cobalamin supplementation was found to decrease the risk of secondary stroke by reducing the Hcy levels.
Alzheimer's dementia is associated with amyloid precursor protein and neurofibrillary tangles, which are due to improper gene expression caused by deficient DNA methylation. The reason for deficient methylation is decreased SAM and increased SAH that inhibits DNA methylation. Folate and cobalamin supplementation was found to improve mild cognitive impairment and dementia.  Depression is associated with HHcy and supplementation of folate and SAM appear to alleviate this condition.
| Skeletal System|| |
HHcy is also associated with an increased risk of fractures. It is hypothesized that increase in plasma Hcy levels prevent the formation of collagen cross links. The bone matrix is not proper and this leads to fragile bones. The enzyme affected is lysyl oxidase and its function is impaired due to homocysteinylation. , Long-term studies (results expected in second half of 2013) are now in progress on the effect of supplementation of cobalamin and folate on fracture incidence with a particular reference to plasma Hcy levels. 
| Renal System|| |
Hyperhomocysteinemia is seen in end stage renal disease patients (ESRD). HHcy causes endothelial dysfunction. Plasma Hcy level is dependent on glomerular filtration rate (GFR). , Therefore, a low GFR is associated with increased Hcy levels and this causes further damage to the glomerular endothelial cell through oxidant stress. Trials are going on with supplementation of folate, cobalamin and pyridoxine in patients with ESRD undergoing dialysis for a period of 6-8 years. The results of these studies may prove that there will be lowering of Hcy levels and decreased risk of coronary artery disease. 
| Reproductive System|| |
0Hyperhomocysteinemia is also associated with reproductive abnormalities in males and females. Forges et al. summarized the damage to the reproductive system caused by HHcy in males and females. 
The increased plasma Hcy level may lead to infertility. HHcy causes decreased spermatogenesis, penile erection, sperm motility and fertilization. Although a small amount of reactive oxygen species (ROS) is required for sperm-oocyte fusion and normal sperm function, an excessive amount of ROS causes sperm DNA damage adversely affecting sperm function, fertilization and early embryo development, and may also lead to apoptosis. The relation between defective DNA function and impaired DNA methylation is known.
Studies have shown that increased plasma estrogens decrease Hcy levels. This explains the low Hcy levels in women compared to men, in premenopausal women when compared to postmenopausal women and also in women on hormone replacement therapy. Hyperhomocysteinemia is also associated with polycystic ovarian disease. In women with recurrent pregnancy loss it was found that folate supplementation reduced fetal mortality. The pathophysiology of HHcy on the female reproductive physiology is comparable to males. It causes defective ovulation, improper embryonic cleavage and poor implantation. Further, HHcy affects arterial pressure regulation, uterine quiescence, contractions and cervical ripening. HHcy-induced oxidative stress and apoptosis are involved in follicular development, cyclical endometrial changes, and in pathological conditions like endometriosis, infertility, spontaneous abortions, preeclampsia, gestational diabetes, embryopathies and preterm labor. Studies have also shown that impairment in methylation of DNA, proteins and lipids lead to poor proliferation and differentiation of granulosa cell layer inhibiting follicular maturation and steroidogenesis.
| Thyroid Function|| |
Hypothyroidism is associated with elevated plasma Hcy levels. Hypothyroidism decreases the conversion of riboflavin into its coenzyme forms i.e., FMN and FAD. FAD is required as a coenzyme for MTHFR, which reduces methylene THF to methyl THF that acts as a donor of methyl groups for cobalamin, a coenzyme for MS. MTHFR cannot function normally in hypothyroidism because of a deficiency of the coenzyme FAD, therefore remethylation of Hcy is impaired and this leads to HHcy.  Hyperthyroidism is associated with low levels of Hcy probably due to increased glomerular filtration. 
| Ocular System|| |
HHcy is implicated in ocular vascular disease. Impaired optic nerve head perfusion due to HHcy may play a role in glaucomatous optic neuropathy. Raised levels of Hcy lead to increased production of free radicals, intimal damage, digestion of elastin and collagen in the tunica media leading to vascular diseases. HHcy is associated with retinal artery occlusion, diabetic retinopathy, age related macular degeneration, choroidal neovascularization, and glaucoma. Supplementation of folate and cobalamin in these patients has been found to reduce plasma Hcy levels. ,
| Cancer|| |
HHcy is associated with increased cancer risk. It is postulated that HHcy affects carcinogenesis via estrogen-induced pathways or by DNA damage.  Studies have also found folate supplementation reduces the risk of colorectal cancer. However, for other carcinomas the data related to folate supplementation are contradictory.
| Population at Increased Risk for HHcy|| |
0Special care must be taken regarding the people more prone for the vitamin deficiencies and increased risk for HHcy. These include pregnant women, patients with inflammatory gastric conditions, people taking distinct drugs, alcoholics and the elderly. The latter are at increased risk for neuropsychiatric complications following folate and B 12 deficiency due to poor intake or absorption defects. ,
| Measures to Prevent Hyperhomocysteinemia|| |
Elevated plasma Hcy levels have been implicated in the increased risk of various diseases. The best way to decrease Hcy level is primary prevention. Hence the measures to prevent HHcy should be significant. This consists of the following:
- Consuming healthy nutritious foods rich in folate, cobalamin and pyridoxine.
- Lifestyle changes like abstaining from smoking and alcohol and reducing coffee consumption.
- Supplements to lower Hcy levels : Studies on prevention of cardiovascular disease using the vitamin supplements like B 9 , B 12 are equivocal.  Further several studies have demonstrated the benefits of using these vitamin supplements in alleviating neuropsychiatric ailments, healing of osteoporotic fractures and improving reproductive health. ,, Folic acid, folinic acid and methyl tetrahydrofolate are known to reduce the levels of Hcy.  There are wide variations in the recommended doses of the vitamins for supplementation to prevent HHcy.  The doses recommended for supplementation of the 3 B complex vitamins in neuropsychiatric disease are vitamin B 6 2 - 25 mg/day, B 9 0.3 - 1 mg/day and cobalamin 0.01 - 0.1 mg/day. 
- Recent studies indicate that administration of betaine,  carnosine  and N-acetyl cysteine (NAC) have beneficial effects in lowering plasma Hcy.  However, this has to be proved beyond doubt in humans.
| Conclusion|| |
Hcy, considered as an insignificant sulfur-containing amino acid in the early 19th century, has now been found to be a part of protein  and a risk factor in the causation of several diseases. Lifestyle changes, supplementation of the required vitamins and correction of the disease process lead to lowering of plasma Hcy levels and improvement in the health of the patient. Fortifying common foods with the vitamin trio of B 6 , B 9 and B12 will help prevent morbidity and mortality due to HHcy especially in developing countries where malnutrition is rampant.
| Acknowledgment|| |
We are thankful to Siddhartha Academy of General and Technical Education, Vijayawada for providing basic facilities to write this review article. We are grateful to Dr C.Nageswara Rao, Director General and Dr Sudha. D. Deshpande, Principal of Dr PSIMS and RF, for their constant support and encouragement.
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[Table 1], [Table 2]