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المجلد 4 , العدد 3 , رمضان 1427 - تشرين الأول (أكتوبر) 2006
 
Importance of Hyperhomocysteinaemia
with Special Reference to Syria
أهمية فرط هوموسيستئين الدم مع مرجع خاص بسورية
Wolfgang Herrmann
د. ولفغانغ هيرمان
Clinical Chemistry and Laboratory Medicine, Central Laboratory
Saarland University Clinical Centre, 66421 Homburg, Germany
الملخص Abstract
إن فرط هوموسيستئين دم hyperhomocysteinemia (HHCY) معتدل هو عامل اختطار risk لأمراض قلبية وعائية cardiovascular (CVD) وأمراض تنكسية عصبية neurodegenerative diseases، وكسور تخلخل العظام ومضاعفات complication أثناء الحمل. وينتشر فرط هوموسيستئين الدم. لقد وجدنا في سوريه تواتراً عالياً لعوز فيتامين B المنخفضة و HHCY في سوريه بالأمراض القلبية الوعائية، والخثار thrombosis الوريدي ومقدمات الارتعاج pre-eclampsia.
تؤكد دراسات استباقية peospective واستعادية retrospective العلاقة السببية بين HHCY واختطار CVD. لكن تجارب تداخل بعض الفيتامينات لم تخفض اختطار CVD. ومن حساب القوة يمكن للشخص أن يستنتج أن هذه التجارب لم تشتمل على أعداد كافية من المرضى لتأكيد مصدوقية الاستنتاجات إحصائياً. لكن إعادة تحليل دراسة VISP (باستبعاد الفشل الكلوي والعوامل المؤثرة على حالات فيتامين B12)، كشفت نقصاً بنسبة 21% في اختطار السكتة stroke. ولقد تم التأكد من هذا الرقم بنتائج من تجربة تداخل فيتامين HOPE2. لقد أدت منتجات الحبوب الغنية بحمض الفوليك Folic acid في كندا والولايات المتحدة (US) إلى تناقص يعتد به significant في معدل الوفيات بالسكتة، فمنذ عام 1998 قل الموت بالسكتة سنوياً إلى أقل من 12900 في US وأقل من 2800 في كندا. على الرغم من النتائج السلبية من تجارب الوقاية الثانوية المتعلقة بتراجع اختطار CVD فإن هنالك بينة مقنعة حول فعالية الإمداد بفيتامين B- في تخفيض اختطار السكتة. حسب الانخفاض العام في اختطار السكتة في تحليل رجعي من دراسات استباقية ووجد أنه يبلغ 20% تقريباً في تجارب التداخل. بالإضافة إلى ذلك فإنه تم حديثاً ربط HHCY بحدوث ووخامة severity فشل القلب المزمن. إن HHCY عامل اختطار أيضاً لكسور تخلخل العظام وخفضت المعالجة بالفيتامينات من اختطار الكسر بصورة يعتد بها. وبالإضافة إلى ذلك هنالك ارتباط بين HHCY والاضطرابات المعرفية أو مرض الزهايمر Alzheimer's disease. إن HHCY متثابتة تكهنية لانخفاض الوظيفة المعرفية. ونقص إدخال جذر الميثيل hypomethylation هو من بين الآليات المركزية التي يفعل من خلالها HHCY كسام خلوياً Cytotoxically.
إن HHCY وانخفاض الفولات هما عاملان مسببان لمضاعفات الحمل. وتشير نتائج الأبحاث لدى السوريين إلى استقلاب HCY متبدل بشكل واضح في حالات الحمل المصحوبة بمضاعفات. وينبغي التأكيد أكثر على وضع المدخول الزائد من فيتامينات B- خلال الحمل. فبالإضافة إلى الإمداد المنصوح به بالفولات، فإن الإمداد بالفيتامين B12 يمكن أن ينقص مضاعفات الحمل إلى حد أكبر.
يكون HCY واصمة marker اختطار هامة حين الإمداد بكليهما معاً. وينبغي أن يكون تعيينه جزءاً من مرتسم (بروفيل) profile اختطار الشخص، وبشكل خاص في الأشخاص المسنين والناس لآخرين المعرضين للاختطار. إن الإمداد بفيتامينات B-، اعتماداً على المعطيات الموجودة، يكون مفيداً في الوقاية من السكتة وربما يخفض اختطار الأمراض التنكسية العصبية الأخرى، مثل CHF، تخلخل العظام، الخلل الوظيفي المعرفي والخرف dementia، مضاعفات الحمل.  
Moderate hyperhomocysteinemia (HHCY) is a risk factor for cardiovascular (CVD) and neurodegenerative diseases, osteoporotic fractures and complications during pregnancy. Elderly have a high prevalence of HHCY. Vitamin deficiency is by far the most common cause of HHCY. In Syria we found a high frequency of HHCY and B-vitamin deficiency, which could be mainly explained by Syrian lifestyle. Low B-vitamin status and HHCY in Syria were related to cardiovascular diseases, venous thrombosis and pre-eclampsia.
Retrospective and prospective studies emphasise a causal relationship between HHCY and the CVD risk. Some vitamin intervention trials, however, did not lower the risk of CVD. From power calculation one can conclude that these trials may not involve sufficient numbers of patients to assure statistically valid conclusions. Reanalysis of the VISP study (excluding renal failure and vitamin B12 status tampering factors), however, detected a 21% decrease in the risk of stroke. This number has been confirmed by results from the HOPE 2 vitamin intervention trial. Folic acid enrichment of grain products in the US and Canada has led to a significant decline of stroke-mortality, since 1998 annually 12900 fewer stroke death in the US and 2800 fewer stroke death in Canada. Despite negative results from secondary prevention trials regarding the CVD risk reduction there is convincing evidence about the effectiveness of B-vitamin supplementation in lowering the stroke risk. The overall decline in stroke risk calculated in metaanalysis from prospective studies and found in intervention trials is around 20%. Additionally, HHCY was recently linked to the occurrence and severity of chronic heart failure. HHCY is also a risk factor for osteoporotic fractures and vitamin treatment lowered the fracture risk significantly. Furthermore, there is a correlation between HHCY and cognitive disorders or Alzheimer’s disease. HHCY is a predictive parameter for the decline in cognitive function. Hypomethylation is among the central mechanisms through which HHCY acts cytotoxically.
HHCY and low folate are causal factors for pregnancy complications. The findings in Syrians suggest a markedly altered HCY metabolism in complicated pregnancies. More emphasis should be placed on increasing the intake of B-vitamins throughout the pregnancy. In addition to the recommended folate supplementation, vitamin B12 supplementation could further decrease pregnancy complications.
Taken together, HCY is an important risk marker. Its determination should be part of the individual risk profile, especially in elderly people and other populations at risk. Based on existing data B-vitamin supplementation is beneficial in stroke prevention and may lower the risk for other degenerative diseases, like CHF, osteoporosis, cognitive dysfunction and dementia, or pregnancy complications.
Key words: homocysteine, coronary vessel disease, chronic heart failure, renal disease, osteoporosis, cognitive dysfunction, thrombosis, pregnancy complications.  
1- Introduction 
Homocysteine (HCY) is a non-protein forming, sulphur-containing amino acid of the methionine metabolism. In addition to severe hyperhomo-cysteinaemia (HHCY), moderate HHCY is today considered an independent risk factor for cardiovascular diseases and is held responsible for about 10% of the total risk (1-3). HHCY is also a risk factor for stroke, venous thrombosis, neurodegenerative diseases (e.g. vascular dementia, Alzheimer’s disease or cognitive disorders), osteoporotic fractures or pregnancy complications. In particular, elderly people have a high prevalence of HHCY due to frequent vitamin deficiency.
Vitamin deficiencies are by far the most common cause of HHCY (4-6). Deficiency can develop by lack of supply, decreased absorption in the gastrointestinal tract, increased requirements (pregnancy, AIDS) and by interaction with some medications. People who ingest a vegetarian diet, older people, and pregnant women, patients with kidney diseases, malabsorption (inflammatory intestinal diseases) and patients with tumours are among the risk groups for vitamin deficiencies of clinical relevance. Folate deficiency is the most common vitamin deficiency in central Europe (7). Vitamin B12 deficiency is very frequent in older people. Vitamin B12 mal-absorption can affect 30-40% of older people and is due to age related disorders like, decrease of stomach acid secretion, a lack of intrinsic factor, or pernicious anaemia (8-10).
Renal patients have higher HCY levels compared to people with normal renal function (11, 12). HHCY in renal patients can be explained by impaired remethylation and transsulfuration of HCY to methionine (11,13). In addition, renal patients also have significant intracellular B vitamin deficiencies indicated by abnormal metabolite concentrations [high HCY and methylmalonic acid (MMA) which is a metabolic marker of vitamin B12 deficiency] while serum concentrations of B-vitamins are frequently normal (11, 14, 15).
The 677C?T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene is the most common enzyme mutation in the folate cycle. Around 5-15% of the population in Central Europe are homozygous carriers of this thermolabile enzyme variant which is associated with about 70% reduction in the enzyme activity (16). Carriers of this MTHFR mutation are sensitive to a folate and/or vitamin B12 deficiency and their HCY rises by about 25% (corresponding to about 2.6 µmol/l) when compared to carriers of the wild type variant of the enzyme (17, 18). Recent meta-analyses reported about 20% higher risk for degenerative vascular disorders for the homozygous genotype (19, 20). About 1% of the population has mutations in the gene of the cystathionine-?-synthase (CBS) in heterozygous form. Carriers of this mutation frequently have a normal fasting-HCY in the plasma, but higher HCY values after an oral methionine loading test. Those individuals are also at a higher risk for vascular diseases.  
2- Epidemiology of hyperhomo-cysteinaemia and intervention studies 
Since HHCY met in large studies the statistic criteria of strength, consistency, specifity, temporality, biologic gradient, plausibility and coherence, HHCY has been classified as an independent risk factor for cardiovascular diseases (CVD) (21). Slight to moderate increases of the HCY-plasma concentration can be caused by aging, B vitamin deficiency, renal insufficiency or frequent genetic polymorphisms (22). HHCY is particularly a cardiovascular risk factor for diabetics. The 5-year mortality rate was about twice as high in diabetics with HCY level > 14 µmol/l than in diabetics with HCY lower than this level (23, 24). Recent meta-analyses (3, 25) from more than 90 retrospective and prospective studies emphasize the causal connection between HHCY and degenerative vascular disease. According to the meta-analysis a decrease in the HCY-plasma level by 3 µmol/l would lead to a 16% decrease of the risk of an ischemic heart disease, a decrease of the thrombosis risk by 25% and a decrease of the stroke risk by 24% (3). Despite the causal connection between HHCY and cardiovascular diseases the last link in the causality chain, which is still open, is reducing the incidence of CVD by lowering the HCY level with a vitamin therapy. Worldwide about 60,000 people are currently included in intervention studies for determining the possible benefits of a HCY-decreasing therapy within the scope of secondary prevention. The problems of studies like these are, among other things, that treatment is not compared to non-treatment, but only conventional treatment to conventional treatment plus vitamins. Therefore, finding the truth about the effectiveness of HCY lowering treatment another approach would be helpful. Dietary supplementation with B-vitamins may lower plasma HCY by about 25% to 30%, in populations with folate fortification about 10 to 15%. A decrease of 25% in HCY has been associated with an estimated 10% lower risk for CVD or 20% less stroke. The strength of the association of HCY with the risk of CVD may be weaker than has been expected earlier. In a very recent publication 12 randomized trials assessing the effects of lowering HCY with B vitamins were reviewed (26). From this power calculation one can conclude that these trials may not involve sufficient numbers of patients with vascular events or they may not last long enough to assure statistically valid conclusions. Therefore it is not surprising that some of these studies do not show positive results in terms of prevention. A prospective meta-analysis of the ongoing HCY lowering trials, including up to 50.000 patients, should provide reliable information about the effects of such interventions on CVD outcomes.
The VISP and NORVIT studies initially did not observe a drop in the stroke and/or cardiovascular risk with B vitamins therapy (27, 28). 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 HCY level only decreased by 2 µmol/l in the high dosage group. There was no significant effect on the end points (stroke, coronary episodes or death), even though there was a significant link between the baseline HCY level and the end points. Possible reasons for the lack of therapeutic effect are, among other things, beginning folate enrichment in grain products in the US 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 with a focus group (2155 patients) excluding patients with vitamin B12 malabsorption, with additional vitamin B12 supplementation and with renal dysfunction (29). The combined end points (stroke, coronary heart disease and death) were now significantly lowered by 21% in the high-dosage vitamin B12 patient group. The post-analysis of the VISP study made clear that in order to detect therapeutic effects the results affecting co-variables should be excluded as much as possible.
Another intervention study with B vitamins, the NORVIT study, has also not detected any decreases in risk according to present analysis (28). 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 HCY 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 co-variables significantly affecting the end points (stroke and myocardial infarction) before the analysis; whereby possible therapeutic effects may have remained hidden. 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, (comments on NORVIT study at www.dach-liga-homocystein.org).
“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 (30). This treatment study (n=5522) with B-vitamins is on patients with vascular disease or diabetes and the result was 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 HCY levels were not considered as part of the inclusion criteria. Moreover they were only assessed in 581 treated patients / 588 controls, in a consecutive manner, limiting the analysis of treatment on HCY to ~ 1/5th of the cohort, so the effect of the B-vitamins on HCY is only seen in these patients. These patients treated with B-vitamins, had no elevated HCY, and no folate-, B6- or B12- deficiencies. Why should these patients be treated with B-vitamins? Nevertheless there is a tendency towards a lowered risk by 5% in the treatment group even though these patients were neither hyperhomocysteinemic nor did they have a vitamin deficiency. We are not aware of a publication stating that every patient at risk for atherosclerosis should take B-vitamins, no matter whether HCY is elevated or not, in order to prevent MI or a stroke. Furthermore, subgroup analysis of the HOPE 2-study revealed that vitamin treatment lowered the risk of stroke by about 25%.
Another study on secondary prevention, the Swiss Heart Study, detected a clear decrease in the restenosis rate (on average around 50%) through vitamin supplementation over 6 months in patients who had undergone a coronary angioplasty (31). This result was not confirmed in a similar study (FACIT trial), which resulted in a conflicting situation (32). More specific analysis revealed that both studies used different vitamin dosages, particularly regarding vitamin B12 and B6 and that the drop in the HCY level in the Swiss Heart Study was higher at 35% than in the FACIT study at 26%. Furthermore, the therapeutic procedures (balloon dilation compared to stent dilation) or the prevalence of other risk factors (diabetes, smoking) were also different in the studies and could have contributed to the difference in the results. Both studies, however, detected that high-risk patients with an HCY level > 15 µmol/l, diabetics and women benefit from a decrease in HCY through vitamin therapy and that the restenosis rate dropped. In both studies B vitamin treatment lowered the risk by 20 to 30% in diabetics and women.
The studies on secondary prevention do not allow conclusions on the success of primary intervention. The CDC (Centre of Disease Control and Prevention, Atlanta) initiated in the US and in Canada nationwide folate enrichment in wheat products in 1998 as a primary-prophylactic measure to reduce neural tube defects. A secondary effect, to some extent, was the significant decrease in strokes and also, though less pronounced, in myocardial infarctions since 1998 (33). Since 1998 there have been about 12,900 fewer annual deaths from stroke in the US, and 2800 fewer cases of stroke death in Canada. The ongoing decline in stroke mortality observed in the US between 1990 and 1997 accelerated in 1998 to 2002 in all population strata, with an overall change from -0.3% to 2.9% per year. In Canada the drop in stroke mortality averaged -1.0% per year in the period 1990 to 1997 and accelerated to 5.4% per year in 1998 to 2002. The decline in stroke mortality in the US and Canada during the time period after folate fortification correlates with the fall in the HCY level and is considered as causal link. The improvement in stroke mortality observed after folic acid fortification in the US and Canada but not in England and Wales where no folate fortification has been introduced supports the hypothesis that folic acid fortification helps to lower the death rate from stroke.
Taken together, negative results from secondary prevention trials regarding the risk reduction of CVD have been reported which, however, does not mean that intervention with B-vitamin has no positive effects in lowering the CVD risk. A meta-analysis of the presently still ongoing and already finished intervention trials would help to clarify the effectiveness of HCY lowering therapy by B-vitamins in reduction of CVD risk. Furthermore, there is convincing evidence about the effectiveness of B-vitamin supplementation in lowering the stroke risk, which starts with results from meta-analysis from prospective studies (24% decline in stroke risk by 3µmol/L HCY lowering) (3); continues with secondary prevention trials, the VISP study subgroup analysis (21% decrease in stroke risk) (29) and the HOPE 2 study (25% lower stroke risk by vitamin intervention), and ends with folic acid fortification as primary prevention measure in the US and Canada (since 1998 anually 12900 fewer stroke death in the US and 2800 fewer stroke death in Canada) (33). The overall decline in stroke risk obtained in these intervention trials is around 20%.  
3- Hyperhomocysteinaemia and chronic heart failure 
Blacher et al. published first evidence for an association between HHCY and CHF in 1999 (34). This study on patients with terminal renal insufficiency showed a positive correlation of HCY and the left ventricular mass index (LVMI). Moreover, Ventura et al. reported a high prevalence of CHF in hospitalised patients with HHCY (35). Recent prospective data from the large epidemiologic Framingham Heart Study demonstrate that even a moderate elevation in HCY (women > 11.0, men > 11.7 µmol/l/) increases the incidence of CHF about twofold (36). In addition, own results suggest that circulating HCY is also linked to the severity of the disease [e.g. NYHA stages, N-terminal pro-brain natriuretic peptide (BNP), ejection fraction (EF) and the left ventricular diameter (LVDD)] (37).
In a recently published double blind placebo controlled intervention study with micronutrients (rich in vitamin B6, B12 and folic acid) after 9 months treatment a significant improvement in clinical parameters of CHF was observed (38). After introduction of nationwide folic acid supplementation in foods in the US, the incidence of CHF remained on the same level as before, but the survival time of afflicted patients was much longer (39). Regarding vitamin B12, the ejection fraction in patients with vitamin B12 deficiency was found to be lower compared to controls (40). Moreover, further intervention trials supplementing CHF patients with B-vitamins will have to clarify if CHF patients benefit from this easy, safe and cheap therapeutic option.  
4- Hyperhomocysteinaemia and osteoporosis 
About 50 years ago HCY was first linked to pathological osseous mutations in patients with homocystinuria (41). These patients exhibited accelerated bone growth, skeletal deformities (genu valgus, kyphoscoliosis, chicken breast, etc.), flattened vertebral bodies and a decreased bone density (42). Recently, two prospective, population-based studies demonstrated a strong link between moderately elevated HCY plasma concentrations and the frequency of osteoporotic fractures in elderly persons (43, 44). Van Meurs et al. analysed 2406 persons (55 years old and older) in the LASA and Rotterdam study, who were monitored over a period of 11,253 man years. The study showed that there was a 1.4-fold increase in total fracture risk per standard deviation increase in HCY (5.5 µmol/l) [161]. In a subgroup of the Framingham study McLean et al. obtained similar results [162]. The fracture risk for men (in average 70 years old) with an HCY level in the 3rd quartile (mean value 13.4 µmol/l) was 2.07 times higher than for men with an HCY level in the first quartile. Men in the 4th quartile (mean HCY value 20.8 µmol/l) exhibited a 3.84 times higher fracture risk than men in the 1st quartile. In women this relation was less pronounced.
Studies analysing HCY in relation to bone mineral density (BMD) found no or only a weak association between these two variables (45, 46). BMD is only an integral measurement of bone metabolism over a longer period of time and reflects mainly bone mineralization. Therefore BMD does not provide information on the current metabolic status or the bone’s microarchitecture. Dhonukshe-Rutten et al. reported an increase of bone formation and resorption markers in patients with hyperhomocysteinaemia (47). In an own investigation on postmenopausal and perimenopausal women the HCY concentration correlated positively with the bone resorption marker urinary deoxypyridinoline crosslinks (DPD), but not with the bone formation marker osteocalcin in serum, suggesting a shift of bone metabolism towards bone resorption (45).
The strongest evidence for a crucial role of B-vitamins in bone health comes from a prospective intervention trial, including 600 patients with osteopenia and osteoporosis (48). Patients were treated with 5 mg of folic acid and 1500 µg of vitamin B12 or placebo for 2 years. The incidence of fractures dropped by about 75% in the treatment group. In conclusion, there is clinical data showing that HHCY and a low B vitamin status increase fracture risk. However, the underlying mechanisms have barely been investigated to date.  
5- Hyperhomocysteinemia and neurodegenerative diseases: cognitive dysfunction and dementia 
Epidemiological studies showed a clear and independent association between HCY and cognitive disorders as well as Alzheimer’s disease (AD) (49). Direct cerebrovascular and neurotoxic mechanisms are discussed in this respect (50). It is unclear whether HCY causes the development of dementia or whether it is a surrogate marker for other causes, such as folate or vitamin B12 deficiency or defective transmethylation reactions. B vitamins (folate, vitamin B12 and vitamin B6) are essential for maintaining nerve function in adults (51). The neurological symptoms in older people, whose high HCY and/or low vitamin B12 levels were normalised through substitution, tended to improve during follow-up treatment. Also, low folate and vitamin B12 absorption and blood concentrations were associated with neuropsychiatric diseases; the severity and symptoms of which improved through treatment with B vitamins (52). Examinations on more than 2000 subjects of the Framingham cohort demonstrated a link between cognitive function and the HCY level in older, but not younger persons (53). Studies on more than 3000 subjects from three ethnic groups reported similar results (54). A high HCY level correlated with a lower “minimal mental state examination score (MMSE)” in persons over 65 years old. The changes in the HCY concentration determined the decline in the “memory score” over a follow up period of 6 years in the Hordaland study (49). Additionally, the risk of developing depression was twice as high for persons with HCY in the upper than the lower tertiles (55, 56). The fasting plasma HCY level is considered an independent predictive parameter for the decrease of cognitive capacity in older people (53) and HHCY is being discussed as a causal factor for the development of cognitive disorders.
Numerous publications reported that HHCY was also a risk factor for developing AD (53, 57- 59). It was observed that the risk of developing AD was 4.5 times higher in patients with a HCY level ? 14 µmol/l compared to those with a level ? 11 µmol/l (58). The data from the Framingham study stated that the risk of AD is doubled in patients with a plasma-HCY > 14 µmol/l (53). A recent study stated that HCY was linked to the development of late-onset, but not early-onset AD (57). HCY is therefore considered a marker for a long-term process that begins with the decrease of cognitive capacity and later ends in dementia. From risk calculation follows that HHCY is linked significantly to mild cognitive disorders (odds ratio 3.1:1.2-8.1), vascular dementia (odds ratio 4.3:1.3-14.7) and Alzheimer’s dementia (odds ratio 3.7:1.1-13.1) (60). However, current evidence does not support a causal role of HHCY in the development of dementia.  
6- Hyperhomocysteinaemia and pregnancy complications 
HHCY and a low folate status are being discussed as causal factors for the development of complications during pregnancy, such as neural tube defects, pre-eclampsia, HELLP syndrome, prematurity or intrauterine growth retardation (61, 62). Folate concentration in children with spina bifida was lower than in healthy children (63). In the US all wheat products are enriched with folic acid (140 µg of folic acid per 100 grams of grain) to avoid the risk of neural tube defects. The goal of this is for all women of child-bearing age to ingest at least 400 µg a day. The folate status in women in the US has significantly improved through this and it has led to a 15 and 30% decrease in neural tube defects (64). The vitamin B12 levels in women who gave birth to a child with NTD were lower than in women who gave birth to children without NTD and it was therefore concluded that mothers of children with NTD had a defective vitamin B12 metabolism (65). For this reason vitamin B12 supplementation, in addition to folate supplementation, could lead to a further reduction of NTD. Based on current information folic acid supplementation is recommended throughout the pregnancy and not only around the time of conception (66). 
7- Hyperhomocysteinaemia and defective methylation capacity  
HHCY is also an indicator for defective methylation capacity. Intact interaction between folate and vitamin B12, as it occurs with normal HCY levels, is very important for the synthesis of DNA and different methylation reactions (methylation of DNA, RNA, myelin, phospholipids, receptors and neurotransmitters). It was reported that patients with neuropsychiatric disorders had reduced levels of S-adenosyl-methonine (SAM) as the universal methyl group donor (67). Disorders of the methylation capacity are considered important for ?-amyloid formation in brains of AD patients (68). Hypomethylation is among the central mechanisms that explain the toxicity of HHCY. Defects in the one carbon metabolism affect the genomic integrity through changing the methylation pattern or increased uracil mis-incorportaion in the DNA. Alteration in DNA methylation are being discussed within the scope of the biology of ageing, the development of tumour or the development of degenerative vascular processes (69). Recent studies have reported a negative correlation between global DNA methylation and HCY concentration (70). However, it may be more useful to study promotor methylation in a specific gene to obtain greater insight in a pathological process (71). In renal patients we have seen hypomethylation of the promotor region of the p66Shc gene (unpublished data). 
8- Hyperhomocysteinemia and B-vitamin deficiency in Syria 
In our studies on Syrians we investigated the prevalence as well as the role of HHCY as risk factor for the development of degenerative diseases. Furthermore we looked for determining factors because they are not fully understood. We enrolled 273 Syrian patients with angiographically confirmed stenosis, along with 159 Syrian and 75 German controls (72). Plasma HCY, cystathionine, methylmalonic acid (MMA), vitamin B6, B12, folate, lipids, apolipoproteins and methylenetetrahydrofolate reductase (C677T-MTHFR) mutation were analysed. There was a very high prevalence of HHCY (>12 µmol/l) in Syrians (patients 61%, controls 44%, Germans 16%). Additionally, a very high prevalence of functional vitamin B12 deficiency was found, indicated by elevated MMA (49% in patients, 47% in controls, Germans 3%) and low holotranscobalamin (HoloTC) (53% in CAD patients, 44% in controls, Germans 10%), which was in contrast to the low frequency of decreased serum vitamin B12 (12% in CAD patients, 7% in Syrian controls, Germans 0%). The vitamin B12 deficiency induces folate trap; higher levels of folate are needed to prevent HHCY. Smoking habits explained to some extent the lower folate status found in Syrian smokers (especially in males). We could confirm that the reasons for HHCY in Syrians were in fact mostly related to a relative folate deficiency, which is due to a vitamin B12 shortage. The profile of cardiovascular risk factors seems to be different between males and females. Syrian lifestyle seems to be responsible for the reported high prevalence of HHCY and vitamin B12 deficiency in this population. Further research (e.g. vitamin supplementation trials) is required which may clarify the causes of vitamin B12 deficiency in this population and its implications on the public health.
HHCY, a proxy measure for the nutritional status of the B-vitamins, may be involved in the etiology of preeclampsia by inducing endothelial dysfunction. We investigated serum concentrations of folate, vitamin B12, B6, HCY and related metabolites in 139 Syrian preeclamptic women and 93 asymptomatic pregnant women of comparable age, gestational age and socioeconomic status (61). Higher concentrations of HCY, cystathionine and MMA were closely linked to a lower status of the B-vitamins. Higher concentrations of HCY and cystathionine were observed in the preeclamptic group than in the matched controls (median HCY 9.3 vs. 6.0 µmol/L; median cystathionine 284 vs. 232 nmol/L). Serum folate was significantly lower in patients than in controls (16.4 vs. 36.0 nmol/L). Folate supplementation was less likely to be used in preeclamptic women. Concentrations of MMA were elevated in patients and controls and did not differ significantly between the two groups. Median plasma concentrations of asymmetric dimethylarginine (ADMA), an inhibitor of NO-synthase that may adversely affect the endothelium, were significantly lower in asymptomatic women than in those who developed preeclampsia before the 37th week of gestation (0.61 vs. 0.68 µmol/L).
In conclusion, the findings suggest a markedly altered HCY metabolism in complicated pregnancies in Syrians, which is related to B-vitamin deficiency. In preeclampsia HCY was, compared with normal pregnancy, significantly elevated in all trimesters of gestational age. Elevated serum concentrations of HCY, cystathionine and MMA indicate poor status of the B-vitamins during pregnancy. The findings underline that HHCY resulting from B-vitamin deficiency is an important contributing factor for pregnancy complications in Syrians. The adverse effect of HCY on endothelial function might be related to ADMA in early-onset preeclampsia. More emphasis should be placed on increasing the intake of B-vitamins in pregnant women from developing countries.
Venous thromboembolism is a multi-factorial disease involving numerous genetic and environmental risk factors. In our study on Syrians we investigated the occurrence and the risk associated with factor V Leiden, HHCY and low folate and vitamin B12 levels in young patients with thrombosis (73). We studied 78 patients (33 females/45 males, mean age 33 years) with a history of thrombosis in a lower limb. Additionally, 98 healthy subjects (45 females/54 males, mean age 44 years) were included. Factor V Leiden was highly prevalent in the patients (39% heterozygous, 10% homozygous vs. 6.3% heterozygous in controls). An increase in the risk of idiopathic venous thrombosis was associated with HCY levels > 15 µmol/l [odds ratio (OR) 2.8], folate < 15 nmol/l (OR 7.5) and vitamin B12 < 182 pmol/l (OR 12). Low levels of folate or vitamin B12 were independently and strongly associated with the risk of venous thrombosis in a multivariate model (OR for idiopathic thrombosis 16 and 11, respectively). 53% of patients with factor V Leiden had low levels of vitamin B12, compared to 28% of patients who were non-carriers of the mutation. In contrast, none of the control carriers of the mutation had a low level of vitamin B12. The risk of venous thrombosis associated with lower levels of vitamin B12 and folate was stronger than that introduced by elevated HCY levels. The increased risk of venous thrombosis, accompanied by factor V Leiden, may be related to confounding environmental factors. This result is of special interest for regions with high prevalence of factor V Leiden mutation and B-vitamin deficiency. Because the risk for venous thrombosis introduced by factor V Leiden mutation is strongly modified by simultaneous B-vitamin deficiency and HHCY.
MTHFR 677 polymorphism may provoke HHCY when folate status is low. The influence of MTHFR 677 mutation on HCY levels in relation to vitamin B12 and folate status was investigated in our study on Syrians compared with Germans (17). The study included 117 omnivorous Syrians, 113 German and Dutch vegetarians, and 123 omnivorous Germans. Syrians had compared with omnivorous Germans and vegetarians the highest HCY level (median 11.3, 8.0, and 10.4 µmol/l, respectively). When the data was pooled, the OR (95% CI) for HCY > 12 µmol/l was 3.8 (1.5-9.3) in TT compared with CC subjects. The OR increased to 29 (4.6-179) in TT subjects who had folate in the lowest tertile, and to 22 (4.8-99) in TT subjects who had MMA in the highest tertile. It is concluded that MTHFR 677 TT individuals are more liable to HHCY under vitamin B12 deficiency than the other two genotypes. This is of special importance for Syria because of the high prevalence of vitamin B12 deficiency. In such a case, relative folate shortage may progressively increase HCY levels. TT individuals may have increased folate and vitamin B12 requirements compared to the other CC and CT genotypes.  
Conclusion 
Resulting from retro- and prospective studies high plasma HCY has been established as independent risk factor for CVD. However, this relation has not been confirmed in secondary prevention trials. The study design in secondary prevention has many weak points which do not allow far-reaching conclusions and the statistic power of presently ongoing vitamin intervention trials is to low to assure statistically valid conclusions. A prospective meta-analysis of the ongoing HCY lowering trials could provide reliable information about the effects of B-vitamin interventions on CVD outcomes. Although lack of proof of benefit, it does not prove no benefit. All there is to say so far is that we cannot advocate the use of B vitamins in post myocardial infarction subjects at this time.
Furthermore, there is convincing evidence about the effectiveness of B-vitamin supplementation in lowering the stroke risk, which starts with results from meta-analysis from prospective studies; continues with secondary prevention trials (VISP study subgroup analysis, HOPE 2 study) and ends with folic acid fortification as primary prevention measure in the US and Canada. The overall decline in stroke risk achieved in intervention trials is around 20%.
Additionally, HHCY was recently linked to the occurrence and severity of CHF and vitamin therapy improved significantly clinical parameters of CHF. HHCY is also risk factor for osteoporotic fractures and vitamin treatment lowered the fracture risk significantly. Furthermore, there is a correlation between HHCY and cognitive disorders or Alzheimer’s disease. HHCY is a predictive parameter for the decline in cognitive function.
In Syria we found a high prevalence of HHCY and B-vitamin deficiency, which could be mainly explained by Syrian lifestyle. Low B-vitamin status and HHCY in Syria were related to cardiovascular diseases, venous thrombosis and pregnancy complications. It should be noticed that the high risk of venous thrombosis, accompanied by factor V Leiden, might be related to confounding environmental factors like B-vitamin deficiency. This result is of special interest for regions like Syria with high prevalence of factor V Leiden mutation and B-vitamin deficiency. Our findings also suggest a markedly altered HCY metabolism in complicated pregnancies in Syrians. More emphasis should be placed on increasing the intake of B-vitamins throughout the pregnancy.
In summary, HCY is an important risk marker and its determination should be part of the individual risk profile, especially in elderly people and other populations at risk. Based on existing data B-vitamin supplementation is beneficial in stroke prevention and may lower the risk for other degenerative diseases, like CHF, osteoporosis, cognitive dysfunction and dementia, or pregnancy complications.  
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المجلد 4 , العدد 3 , رمضان 1427 - تشرين الأول (أكتوبر) 2006

 
 
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