Current knowledge on Hyperhomocysteinemia
Definition and forms of homocysteine
Homocysteine (Hcy) is an endogenous sulfure-containing, non protein-forming amino acid (Figure 1). Hcy is produced from the essential amino acid methionine. It is a small molecule (molecular weight MW=135 Dalton) that’s water solubility is extremely low. Methionine (Meth) can also be generated from the methylation of Hcy. Cystathionine (CYS), is a by-product in the catabolism of Hcy which is formed by the addition of serine molecule to Hcy. Only trace amount of Hcy circulates in blood as free thiol form; the reminder is present mainly as disulfide complexes in which Hcy binds to (-SH) group supplied by sulfhydryl amino acids of plasma proteins (mainly Albumin). Furthermore, both mixed disulfides (i.e., with cysteine) and symmetric disulfide (homocystine= Hcy-Hcy) complexes have been reported. The concentrations of non-protein bound Hcy are only 1-2% of the total Hcy.
The physiologic significance of Hcy arises from its central location between three metabolic pathways (remethylation, transmethylation and transsulfuration). Methionine is the only known precursor of Hcy in humans. Methionine supplied by food proteins is converted in presence of adenosine three phosphate (ATP) into S-adenosylmethionine (SAM). SAM is the novel methyl donor in many biological reactions. After donating its methyl group, SAM is converted into S-adenosylhomocysteine (SAH), which can be hydrolysed into Hcy. The transmethylation pathway is the sole metabolic pathway that is known to produce Hcy in the body. Hcy is catabolized either into cystathionine (CYS) or into methionine. The catabolism of Hcy into methionine is catalysed by the enzyme methionine synthetase (MS) and its cofactor methylcobalamin. A methyl group provided by 5-methyltetrahydrofolate (5-MTHF), is transferred to methionine synthase, which then transfer it to Hcy producing methionine. Homocysteine catabolism via the transsulfuration pathway is mediated by the enzyme cystathionine-beta-synthetase (CBS), which requires vitamin B6 (pyridoxal 5- phosphate) as a cofactor. Cystathionine is then produced, and converted into cysteine and alpha-ketobutyrate in presence of cystathioninase, another vitamin B6 dependent enzyme. Cysteine can be further transformed into glutathione, the major constituent of the antioxidant defence in humans. Additionally, HCY has a key role in the one-carbon metabolism. This role is shared with 5-MTHF. Both HCY and 5-MTHF are substrates for the production of tetrahydrofolate (THF) as well as methionine. Tetrahydrofolate (THF) is the form of folate that can be used to synthesize purins (for DNA synthesis). Hcy catabolism via the transsulfuration pathway is favoured under methionine overload situation (after meal). On the other hand, Hcy-remethylation to methionine is favoured during the relative methionine shortage within the cells (fasting conditions). The integrity of Hcy metabolism depends on the availability of vitamin B12, B6 and folate in addition to several key enzymes. Important steps in the metabolism of Hcy are illustrated in (Figure 2).
Regulation of Hcy metabolism
Homocysteine metabolism is tightly regulated under normal physiological situations. Upon SAM can enhance the transsulfuration pathway and inhibit the remethylation pathway. When folates in the form of 5-MTHF are available, the production of SAM will be enhanced and SAM can inhibit the formation of 5-MTHF from 5,10-methylenetetrahydrofolate by inhibiting the enzyme methylene tetrahydrofolate reductase (MTHFR). Unlike in folate deficiency, cobalamin deficiency is accompanied by slight elevation of Hcy because the role of SAM in enhancing the transulfuration pathway is not affected by cobalamin deficiency. SAM is the main methyl donor in many biochemical reactions in humans like, in the synthesis of methylated phospholipids (phosphatidylcholine), nucleic acids, amino acids, and neurotransmitters. Above all, the ratio SAM/SAH indicates the methylation potential of the cell and is more important than the absolute concentrations of each of these compounds. For example, low SAM/SAH ratio causes DNA-hypomethylation thus affecting gene expression.
Hyperhomocysteinemia as a risk factor for human diseases
HHCY is subdivided in three concentration ranges – moderate, intermediate and severe. The normal and pathological ranges of homocysteine are given in (Table 1).
Homocysteine and coronary vascular diseases
Large metaanalysis studies reported that HHCY could be causally implicated in coronary diseases. Each 5 µmol/L increase in plasma tHcy is associated with 1.32 odds ratio for ischemic heart disease, 1.60 for thrombosis, 1.59 for stroke. Lowering plasma concentrations of tHcy by 3 µmol/L is expected to reduce ischemic heart diseases by 16%, thrombosis by 25%, and stroke by 24%. Despite the association between HHCY and cardiovascular diseases, it remains unclear whether lowering tHcy with a vitamin therapy can reduce the incidence of cardio-vascular diseases. Worldwide about 52,000 people are currently included in intervention studies for determining the possible benefits of vitamin therapy. In such studies, the vitamins are added to the conventional treatment (including LDL-lowering, aspirin, ec). Therefore, it is not possible to evaluate the sole role of the vitamins. Dietary supplementation with B-vitamins may lower plasma tHcy by about 25% to 30%, and in populations with folate fortification by about 10 to 15%. A decrease of 25% in tHcy has been associated with an estimated 10% lower risk for CVD and 20% lower risk for stroke. In a very recent publication 12 randomized trials assessing the effects of lowering HCY with B vitamins were reviewed. A power analysis of the available studies demonstrated that the available subject’s number is far from being sufficient to make a valid conclusion. Therefore it is not surprising that some of these studies do not show positive results in terms of prevention. The VISP study included 3860 stroke patients who were treated with conventional medication over 2 years and additionally, in times of fortification, with “low or high dosages” of B vitamins [low (high) 20 (2500) µg folic acid, 6 (400) µg B12, 0.2 (25) mg B6]. The tHcy level only decreased by 2µmol/l in the high-dosage therapy. There was no significant effect on the end points (stroke, coronary episodes or death), even though there was a significant link between the baseline tHcy level and the end points. Possible reasons for the lack of therapeutic effect are, folate enrichment in grain products in the USA during the study, the short observation period, and the fact that the vitamin B12 status and kidney function were also not taken into consideration. A post-analysis of the VISP study was conducted on a sub- group (2155 patients) excluding patients with low (< 250 pmol/l) or high (>637 pmol/l) vitamin B12 levels and kidney patients. In this sub-group, the combined end points (stroke, coronary heart disease and death) were significantly lowered by 21% in the high-dosage vitamin B12 patient group. The NORVIT study, also reported no detectable decrease in risk according to present analysis. The NORVIT study included 3749 patients, who had suffered a myocardial infarction at most 7 days before inclusion in the study and were treated with B vitamins (divided into 4 therapeutic groups or placebo, a two by two factorial design) in addition to conventional medication for 3 years. The tHcy level was lowered significantly, by 28%, in the group that received folic acid and vitamin B12. There was no risk reduction regarding the end points (heart attack, stroke). This study also did not eliminate numerous possible co-variables that may affect the end points (stroke and myocardial infarction) and potentially mask any therapeutic effect. Obviously, looking at the Kaplan-Meir estimates, another weak point of this study is, that half of the primary end-points occurred in the first half year of treatment. “Homocysteine lowering with folic acid and B vitamins in vascular disease” is the title of the very recent HOPE 2 study publication which is somewhat misleading. This treatment study (n=5522) with B-vitamins includes patients with vascular disease or diabetes. The results reported were that supplements combining folic acid and vitamins B6 and B12 did not reduce the risk of major cardiovascular events in patients with vascular disease. However, elevated tHcy levels were not considered as part of the inclusion criteria. Moreover, tHcy levels were only assessed in 581 treated patients and 588 controls, in a consecutive manner, limiting the analysis of treatment on tHcy to ~ 1/5th of the cohort, therefore any effect of the B-vitamins on tHcy is only seen in these patients. These patients treated with B-vitamins, had no elevated tHcy, and had no folate-, B6- or B12- deficiencies. Why should these patients be treated with B-vitamins? Nevertheless there was a tendency towards a lowered risk of 5% induced by vitamin treatment even though these patients were neither hyperhomocysteinemic nor did they have a vitamin deficiency. Furthermore, sub-group analysis of the HOPE 2-study revealed that vitamin treatment lowered the risk of stroke by about 25% (95%CI 0.59-0.97 p=0.03).
B-vitamins lower the risk of stroke – data from a recent meta-analysis
An important meta-analysis by Wang et al. (9) that included eight randomised trials with 16,841 patients provided convincing evidence that lowering of plasma HCY by folic acid supplementation reduces significantly the stroke risk by about 18% in total. The reduction of stroke risk was even higher when the treatment exceeded 36 months (-29%), when the HCY lowering was more than 20% (-23%), when the patients had no history of stroke (-25%), or when the patients consumed no folic acid enriched grain products (-25%). It is concluded that folic acid supplementation can effectively reduce the risk of stroke in primary and secondary prevention. This is in line with results from the USA and Canada where food fortification with folic acid since 1998 has been contributed to a significantly decreased stroke risk (10). The meta-analysis has also demonstrated that only intervention studies lasting longer than three years could show a significant lowering of stroke risk. The mean observation period of the VISP and NORVIT study was only 2 and 3 years, respectively. The HOPE-2 study lasted 5 years. Final statements about the efficacy of B-vitamins in risk reduction regarding cardiovascular diseases have to be postponed until a meta-analysis which includes about 50,000 patients is on the table.
HHCY and dementia
B-vitamin deficiency and HHCY are common in the elderly and coincide with a high incidence of dementias at older age. Dementias are progressive illnesses with manifestations that include memory loss, cognitive dysfunction, attention deficit and other behavioral and emotional disturbances. The prevalence of dementias will double every 20 years to more than 80 million cases by 2040. HHCY and Alzheimer disease (AD) could be linked by stroke or microvascular disease, because, 25% of dementia cases are attributed to stroke. AD constitutes 60 to 90% of all dementias. AD is characterized by depositions of extracellular senile plaques in brain, comprising amyloid beta (Abeta), lipids, and other cellular components. Abeta is a 37 to 42 amino acid fragment originating from proteolysis of a precursor protein (amyloid precursor protein; APP). Another protein contributing to AD is tau protein. Tau has a very important physiological function that is stabilizing neuronal microtubules. Structural modifications of tau protein facilitate self aggregation of this protein. For example, hyperphosphorylated tau protein (P-tau) self aggregates and participates in neurofibrillary tangle [NFTs] formation in the brain of patients with dementia. NFTs cause impaired axonal transport, synaptic dysfunction, and axonal degeneration. Data from prospective studies suggests a causal role for HHCY in the etiology of AD. Plasma concentration of tHcy is a strong predictor of cognitive decline with age. Patients with dementia have lowered CSF-SAM or increased CSF-tHcy or SAH. Patients with late onset AD have lower concentrations of CSF-folate than control subjects. Therefore, disordered Hcy metabolism can negatively influence some biological pathways in the brain, and increase the risk of dementia. Vascular dementia is the second commonest cause of dementia in elderly people after AD. Vascular dementia (or multi-infarct dementia) constitutes 10% to 40% of all dementias. Diffuse periventricular white-matter abnormalities and lesions in the central lacunar result from atherosclerotic changes in small cerebral arteries and arterioles. An elevated plasma tHcy concentration is an independent risk factor for cerebrovascular damage and vascular dementia. Additionally, HHCY is associated with carotid artery disease and macroangiopathic and microangiopathic central nervous system diseases. Mild cognitive impairment (MCI) is an intermediate state between normal aging and dementia. Approximately 20% of patients with MCI develop AD or progressive dementia within 2 years of follow up. HHCY is associated with an increased risk of MCI [OR (95% CI) 3.1(1.2-8.1)]. In the Framingham study, a relationship between tHcy and cognitive function was found in older adults. Subjects with elevated concentrations of tHcy at baseline were more likely to develop dementia after several years compared with subjects with normal tHcy. Similar studies also confirm that higher concentrations of tHcy correlate with some measures of cognitive function in elderly subjects. The correlation between serum concentrations of B-vitamins (B12, B6, folate) and cognitive function is documented even in healthy elderly subjects. The Hordaland study, a follow up study extending over 6 years, showed that tHcy plasma concentration can predict memory decline with age in elderly people. Approximately 7-8% of the variations in cognitive function between elderly people could be explained by plasma concentrations of tHcy. Numerous studies report a marked and a dose-dependent correlation between concentrations of tHcy or B-vitamins and cognitive decline (Table 2). On the other hand, there are several studies that failed to detect an association between tHcy and cognitive domains. The association between elevated tHcy and cognitive decline can be confounded by other factors such as, age, gender, ethnic origin or associated diseases.
Effect of treatment on cognitive function
There is interest in whether folate and/or vitamin B12 supplementation may improve cognitive function or delay the progression of cognitive decline, whether this effect is related to lowering tHcy or to an independent effect of the vitamins. Several intervention studies are underway with the aim of improving symptoms or disease progression in patients with dementia. Several, but not all, vitamin intervention studies document improvements in some measures of cognitive function (Table 3). In general, there is no strong evidence that folate and / or B12 had significant effects on cognitive function in subjects who had already developed dementia. There are several major limitations for all available studies. Firstly, most included only a small number of subjects. Secondly, their duration is too short to achieve a marked improvement. Thirdly, because baseline tHcy concentrations are related to a decline in cognitive function with age, a halting of such decline would mean a protective effect. It is currently believed that ensuring sufficient B-vitamin intake might be more effective in disease prevention rather than in disease treatment.
HHCY and depression
Folate deficiency is very common in patients with depressive disorders. In a cross sectional study of 883 elderly Latino females (> 60 years), only 1% were folate deficient (serum folate <6.8 nmol/L). However, serum folate < 25.24 nmol/L (i.e.in the ‘low-normal’ range) was associated with an OR for depressive symptoms of 2.04 (95% CI; 1.38-3.02). The association between vitamin B12 deficiency and depression was observed in the Women’s Health and Aging study of 700 community dwelling non-demented disabled women (age = 65 years). In another study, increased plasma MMA (> 400 nmol/L) was associated with a high prevalence of depression; however, vitamin B12 injection (1 mg/wk, for 1 month) did not improve symptoms. In a cross sectional study on middle age men (46-64 years) from Finland, tHcy > 11.4 µmol/L was associated with a higher risk of being depressed. In the Rotterdam study involving 112 subjects with depressive disorders, low vitamin B12 (< 258 pmol/L) or HHCY (tHcy > 15 µmol/L) were related to an increased risk of depression. Moreover, subjects with a lifetime diagnosis of major depression had lower blood folate than subjects with no history of depression. In a study of patients with severe depression, more than half of the patients had concentrations of tHcy > 11.9 µmol/L. Because of the role of folate in maintaining methylation status, disturbed methylation may underlie mood disorders. Recent clinical and experimental studies link folate, SAM and monoamine metabolism, probably via the biopterin pathway. Early reports documented an antidepressive effect of SAM. SAM is as effective as tricyclic antidepressants in treating depression, particularly endogenous depression. SAM appears to lift mood faster than antidepressants. Folate deficiency can also be a result of poor diet in patients with mood disorders. Nevertheless, whether folate deficiency is primary or secondary in depression, folate administration enhances recovery of the mental state. On the basis of available data, oral doses of folic acid (0.8–2.0 mg daily) and vitamin B12 (1 mg daily) are recommended to improve outcome in depression.
HHCY and osteoporosis and bone metabolism
There is convincing clinical data showing that HHCY and a low B vitamin status increase fracture risk. Patients with homocysteinuria show accelerated bone growth, skeletal deformities, flattened vertebral bodies and a decreased bone density. Prospective, population-based studies demonstrated a strong link between moderate HHCY and the frequency of osteoporotic fractures in elderly persons. Analysing data from 2406 persons in the LASA and Rotterdam study, showed a 1.4-fold increase in total fracture risk per 5.5 µmol/l increase in plasma tHcy. Findings from patients with pernicious anemia have suggested a link between bone density and B-vitamin deficiency in elderly people. A 1.9-fold increase in the risk of proximal femur fractures, a 1.8-fold increase in the risk of vertebral fractures and a 2.8-fold increase in the risk of distal forearm fractures were detected in patients with pernicious anaemia compared with controls. Studies analysing Hcy in relation to bone mineral density (BMD) found no or only a weak association between these two variables. However, the equivocal studies regarding the correlation between Hcy and BMD do not conflict the homogeneous findings considering Hcy and fracture risk because BMD depicts mainly bone mineralization and provides only an integral measure of bone metabolism over time. A correlation between tHcy and circulating concentrations of the bone resorption marker carboxyterminal telopeptide of human collagen I (ICTP) has been reported. A significant correlation has been reported between tHcy level and serum ß-crosslaps which is another bone resorption marker. The bone resorption markers desoxypyridinoline crosslinks (DPD) and tartrate resistant acid phosphatase 5b (TRAP5b) revealed only weak correlations with tHcy concentration. In the Longitudinal Ageing Study Amsterdam (LASA), higher urinary DPD concentrations were found in women with elevated tHcy concentrations but not in men. In a study on post- and perimenopausal women the tHcy concentration correlated positively with DPD, but not with the bone formation marker osteocalcin in serum, suggesting a shift of bone metabolism towards bone resorption. The association between bone markers and tHcy was more pronounced in women than in men. Beside an increased bone resorption, a reduced bone formation could also contribute to the relation between tHcy and fracture risk. Taken together, the majority of available studies indicate significant correlation between tHcy and bone resorption markers but no association between tHcy and bone formation markers.
HHCY and pregnancy
HHCY or impaired status of B-vitamins are risk factors for pregnancy complications and poor outcomes. Homocyteine itself is a cytotoxic compound that adversely affects the endothelial system, including the placenta. HHCY has been related to preeclampsia, since endothelial dysfunction is one major complication in this disease. Concentrations of tHcy decrease during normal pregnancy for reasons largely independent of the decrease in serum albumin. Reasons behind tHcy decrease during pregnancy are largely unknown, but this could be related to the active metabolism of this compound via the remethylation pathway to insure delivering sufficient methyl groups and tetrahydrofolate for DNA-syntheiss and many methylation reactions. Homocysteine is usually lower in pregnant women receiving folate supplementation when compared to non-supplemented women.
HHCY and birth defects
Several congenital anomalies may be prevented by folic acid supplement before and during early pregnancy. The exact mechanism by which folic acid exerts its protective effect is unclear. However, the role of the vitamin in one-carbon metabolism, nucleic acids synthesis and the integrity of DNA are plausible explanations. Neural tube defects (NTDs) are the most common congenital malformations that have been linked to maternal-folate deficiency and HHCY. Concentrations of tHcy were higher in mothers of NTDs children compared to mothers of normal children. Low blood folate was associated with an odds ratio of 2.6 for having NTD and of 3.1 for being a mother for NTD child. Recent strategies for primary prevention of NTDs included preconceptional folic acid supplementation. In 1998, the U.S. Food and Drug Administration has mandated that all grain products be fortified with folic acid (140 µg/per 100 gr grain). Similar policy has also been put in place in Canada, Chile and Hungary. This national health policy is proposed to provide a daily intake of at least 400 µg/day folic acid in women of reproductive age. Folate status and concentrations of tHcy have been improved in women of childbearing age after the fortification. This metabolic improvement has also been associated with a marked reduction in birth defects (between 15-30%).
Acquired causes of mild to moderate hyperhomocysteinemia
Most studies have focused on the modifiable factors that are known to induce hyperhomocysteinemia. As presented in (Table 4), lifestyle factors like smoking, high coffee consumption, low physical activities and alcoholism all have been reported to convey hyperhomocysteinemia.
On the population level, the most important factors that have been extensively investigated for their influence on tHcy levels are the B-vitamins deficiencies, age, renal function, and lifestyle factors. Importantly, tHcy concentrations may be severely increased in case of impaired renal function. A causal relationship between increased tHcy levels and the coronary risk in patients with renal insufficiency have been suggested. A substantial decrease in renal function may account for increased coronary risk in elderly subjects. Sex-differences in tHcy levels and metabolism have been reported. Females have usually lower tHcy levels than males. The differences may be disappeared between male and post menopausal women. Ethnic differences in tHcy levels and metabolism have been also reported. Blacks have lower tHcy levels than whites. The last notice was due to a more effective tHcy metabolism via the transsulfuration pathway in blacks. Target populations where screening for HHCY has been strongly recommended are shown in (Table 5).
tHcy estimation, methods and pre-analytical conditions
Most researchers agree that tHcy should be measured in EDTA-plasma that has been separated from the red blood cells within 45 min at most. This is related to the fact that red blood cells continue to release tHcy in vitro, and this process is time and temperature dependent. Therefore, placing samples on ice and centrifuging at 4°C are also recommended. Several compounds have been described and utilized to stabilize concentrations of tHcy up to 48 hours after blood collection. Fasting concentrations of tHcy is preferred and allow comparing levels from various studies, because tHcy is affected by methionine content of the diet. There are today several available methods for estimating tHcy (Table 6). Moreover, there are several confounding factors that should be considered because they influence interpretation of tHcy concentrations. Other methods and pre-analytical conditions concerning the other metabolic markers are also presented in (Table 6).