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Sample #2
This is a doctoral-level, pathobiological epidemiology
research paper of 29 pages (8,100 words) in APA style.
Homocysteine and Atherosclerosis
Cardiovascular disease is a serious public health problem, responsible for vast numbers of hospitalizations and deaths each year. The costs of treatment--including drugs and invasive procedures--have increased dramatically, indicating an urgent need for the development of prevention and treatment measures for atherosclerosis.
In order to develop effective treatments and prevention strategies, potential causes of atherosclerosis must be addressed. Recent research has marked the distinct possibility that homocysteine may play an important role in the presence and progression of atherosclerosis (van den Bosch et al., 2003; Aronow, 2003; Geisel et al., 2003; Lawrence de Koning et al., 2003; Hackham & Anand, 2003; Stuhlinger et al., 2003). Homocysteine is an amino acid found in the blood in different concentrations. Evidence has indicated that increased levels of homocysteine in the blood are associated with a higher risk of cardiovascular disease, especially atherosclerosis.
Research evidence has shown considerable correlations between elevated homocysteine levels and an increased risk of atherosclerosis. Homocysteine appears to be implicated in the early changes in structure and function of the artery wall. Prolonged exposure to homocysteine detrimentally affects the dilation of the blood vessels, and as a result the development of atherosclerosis ensues (Stuhlinger et al., 2003). Increased levels of homocysteine may be caused by a number of biological and lifestyle factors. This report aims to thoroughly examine the role that homocysteine plays in atherosclerosis, and the extent to which certain biological and lifestyle factors, such as vitamin status and diet, affect levels of homocysteine and the presence and progression of atherosclerosis.
Evidence of the relationship between homocysteine and atherosclerosis
The majority of cross-sectional and case control studies have indicated that an increased level of plasma total homocysteine is an independent risk factor for cardiovascular disease (Bozkurt et al., 2003). Bozkurt et al. (2003) set out to investigate the relationship between plasma total homocysteine levels and the presence, extent, and severity of cardiovascular evidence. The study involved three hundred and forty-one patients who underwent coronary angiography, of which 195 had cardiovascular disease and 146 had normal coronary arteries. The results of the study indicated that the mean total homocysteine level was higher in patients with significant cardiovascular disease. The group of patients with cardiovascular disease also had a higher rate of hyperhomocysteinemia. Furthermore, this study found positive relationships between total homocysteine levels and male gender, smoking, hyperlipidemia, and hypertension. Regression analysis of the data set determined that hyperhomocysteinemia was an independent risk factor for cardiovascular disease (odds ratio [OR]=3.69, CI 95% 1.51-9.06, P=0.004). However, in patients with low cardiovascular risk, hyperhomocysteine was not found to be an independent risk factor.
Furthermore, this study demonstrated no relationship between the level of total homocysteine and the severity and extent of cardiovascular disease. Overall, this study provided evidence that increased levels of total homocysteine is related to the presence of cardiovascular disease, but not the severity or extent of it. The implications of this study lie in the development of improved diagnostic tools that may use homocysteine levels as an important indicator of the presence or potential of cardiovascular disease.
Woo et al. (2002) assert that atherosclerosis is a crucial medical problem facing the Western world in the 21st century, but that it is possible that traditional risk factors usually associated with cardiovascular disease may only account for 50 % of the problem. These researchers recognize the emergence of hyperhomocysteinemia as an independent risk factor for atherosclerosis, associated with renal failure, folate deficiency, and relative deficiency of MTHFR (C677T polymorphism) or alternate enzymes dependent on age, gender, and smoking status. These researchers state that hyperhomocysteinemia has been reported to occur in 11-22% of Western people, which is an alarming statistic due to the association between hyperhomocysteinemia and an increased occurrence of atherosclerosis.
There is a growing body of evidence indicating the association between homocysteine and cardiovascular disease in case-control observations and prospective cohort studies, as well as in vitro experiments and in vivo experimental models utilizing both human and animal subjects (Woo et al., 2002). Evidence for the improvement of endothelial function observed as a result of lowered homocysteine levels, and its effects on atherosclerosis are further indicative of the possible causal role that homocysteine may play in relation to atherosclerosis (Woo et al., 2002). Woo et al. suggest that this evidence could play an important role in the development of secondary prevention strategies to decrease the re-occurrence of cardiac events in patients who have suffered from cardiovascular disease in the past.
Hyperhomocysteinemia has been determined to be a robust and independent risk factor for atherosclerosis, and this has been demonstrated through several epidemiological studies (Lawrence de Koning et al., 2003). Lawrence de Koning et al. (2003) explains how the cause of hyperhomocysteinema lies in a deficiency in the enzymes or vitamin co-factors that are a requirement for the metabolism of homocysteine. These authors describe the hypotheses that have been proposed to explain the cellular mechanisms involved in the promotion of cardiovascular disease by hyperhomocysteinemia. These cellular mechanisms include oxidative stress, endoplasmic reticulum stress, and the activation of factors that promote inflammation. Lawrence de Koning et al. (2003) discuss how genetic and diet-induced animal models have recently demonstrated a direct causal relationship between hyperhomocysteinemia, endothelial dysfunction and accelerated atherosclerosis. These recently developed animal models allow researchers to gain a greater understanding of the cellular mechanisms involved in the contribution the relationship between hyperhomocysteinemia, endothelial dysfunction and atherosclerosis. These models may also contribute to the development of effective therapeutic agents to be used in the treatment of cardiovascular disease.
Further evidence for the causal role that elevated homocysteine levels may play in the occurrence of atherosclerosis is provided by Geisel et al. (2003), who investigated the stimulatory effect that homocysteine has in interleukin-8 expression in endothelial cells in humans. These researchers recognized that a crucial early stage of atherosclerosis is the adhesion and infiltration of monocytes to the site of the lesion. Furthermore, the inflammation-promoting cytokine called interleukin-8 has been shown to rapidly cause rolling monocytes to adhere firmly onto monolayers that express E-selectin.
The aim of these researchers was to examine the effect that homocysteine has on the production of interleukin-8 in human endothelial cells. In the experiment, cells were incubated with varied concentrations of homocysteine for 20 hours. The researchers determined the gene expression by real time PCR and measured the interleukin-8 protein using immunoassay analysis. Results of the study indicated that homocysteine indeed enhanced interleukin-8 expression manner (181% of controls at 2.5 mmol/l homocysteine). In addition, stimulation of gene expression was associated with a parallel increase in the synthesis of interleukin-8 protein (160% of controls at 5.0mmol/l homocysteine). A further increase of interleukin-8 expression was observed due to co-incubation of endothelial cells with homocysteine and copper sulfate (251% of controls). Based on the results of this study, Geisel et al. (2003) concluded that homocysteine did indeed alter endothelial cell function via the stimulation of interleukin-8 protein expression. Moreover, these results demonstrate that homocysteine plays a contributing role in the appearance and progression of atherosclerosis, and the researchers also propose that the formation of oxidation products induced by homocystein might be one of the underlying mechanisms of this effect.
Hackam & Anand (2003) recognize the growing health concern imposed by atherosclerosis, and emphasize the necessity for effective means of identifying those at risk for cardiovascular disease. These researchers sought to review the epidemiological, basic science, and clinical evidence in regards to emerging risk factors for cardiovascular disease, including C-reactive protein, lipoprotein(a), fibrinogen, and homocysteine. In this study, the MEDLINE database was extensively searched from January 1990 to January 2003 using several key terms such as atherosclerosis, risk factors, and homocysteine. Also, conference proceedings, abstract booklets, bibliographies of relevant books and articles, as well as personal files were all hand searched in order to identify additional sources of information. Studies were selected for inclusion in the review if they were original investigations or reviews of the epidemiology of atherosclerosis, and the association of various risk factors with cardiovascular disease. Three hundred seventy-three studies were identified for inclusion in the study, which included randomized control studies, prospective cohort studies, systematic overviews,case-control, cross-sectional, and mechanistic studies.
Results of the data synthesis indicated that the available basic science and epidemiological evidence supports independent associations between the four risk factors and atherosclerosis, to varying degrees. However, the researchers remark that there is little data concerning the added value of screening for these factors over that of the validated global risk assessment strategies currently utilized. Moreover, there are few controlled intervention studies that investigated the efficacy of proven risk-reduction therapies with individuals exhibiting these risk factors. In other words, there may be value in screening for homocysteine levels as part of a global assessment strategy, but the effects have yet to be determined through research.
The evidence that plasma homocysteine is an important risk factor for atherosclerosis, as well as cardiovascular disease in general, is pervasive and widespread. Research has shown that homocysteine is related to cardiovascular disease in several demographics, including the elderly (Aronow, 2003) and women (van den Bosch et al., 2003). Van den Bosch et al. (2003) recognized the need for more information regarding the relationship between hyperhomocysteinemia and cardiovascular disease in young women, and sought to assess whether or not there is a significant association.
The study by van den Bosch et al. (2003) aimed to assess hyperhomocysteinemia as a risk factor for cardiovascular disease in young women. An evaluation of joint exposure to hyperhomocysteinemia and other traditional risk factors was also conducted. Two hundred and twenty women, within the age range of 18- 49 years, with cardiovascular disease and a control group of 629 healthy women from a population-based case-control study completed the same structured questionnaire and donated blood samples for the determination of plasma homocysteine levels. For the purposes of the study, hyperhomocysteinemia was defined by a non-fasting, total plasma homocysteine level above the 90th percentile of the control range. Results of this study indicated that young women with hyperhomocysteinemia had a 2.5-fold (95% confidence interval [CI], 1.7-3.9) increased risk for cardiovascular disease. Furthermore, when the presence of hyperhomocysteinemia was combined with the presence of a traditional risk factor, relative risk was shown to significantly increase in smokers (OR, 18.9; 95% CI, 8.3-42.9), and in women with hypertension (OR, 10.3; 95% CI, 5.4-19.8), hypercholesterolemia (OR, 8.5; 95% CI, 4.2-17.1), and diabetes (OR, 8.9; 95% CI, 1.7-46.9). Based on these results, the researchers concluded that hyperhomocysteinemia is a significant risk factor for cardiovascular disease. In addition, there is a strong associative effect between hyperhomocysteinemia and all traditional vascular factors. These findings may have important implications for the screening and management of hyperhomocyateinemia and cardiovascular disease.
Now that the evidence in support for the relationship between homocysteine and atherosclerosis, as well as cardiovascular disease in general, has been discussed, it is necessary to explore the mechanisms by which homocysteine levels affect the initiation and progression of atherosclerosis. The effects that homocysteine has on atherosclerosis are mediated by endothelial function, so it is therefore necessary to investigate exactly how homocysteine effects endothelial function. Stuhlinger et al. (2003) explain how it has been shown that homocysteine inhibits the production of NO by cultured endothelial cells by causing the accumulation of asymmetric dimethylarginine (ADMA) in animal models. These researchers conducted a study designed to determine whether this same mechanism exists and is operative in humans.
The study involved 9 patients (6 men and 3 women) with documented peripheral arterial disease, who were between the ages of 61 and 67 years, as well as 9 age-matched people at risk for atherosclerosis, who were older men, aged 64-66 years. The study also involved 5 younger men as control subjects, who had no evidence of or risk factors for atherosclerosis. The researchers measured endothelial function by flow-mediated vasodilation of the brachial artery before and after a methionine loading test. Also, blood was drawn along with these measurements in order to assess homocysteine and ADMA concentrations.
Results from the study indicated that plasma homocysteine increased significantly (P<0.001) after the methionine-loading test in each group. Levels of plasma ADMA also rose in all subjects, and flow-mediated vasodilation was reduced in all subjects. The researchers also discovered positive correlations between plasma homocysteine and ADMA concentrations (P=0.03, 4=0.450), as well as positive correlations between ADMA and flow-mediated vasodilation (P=0.002, r=0.623). Based on these results, the researchers concluded that hyperhomocysteinemia, at least in experimental conditions, leads to an accumulation of the endogenous NO synthase inhibitor ADMA. Also based on the results, hyperhomocysteinemia leads to different degrees of endothelial dysfunction, which is contingent to the pre-existing state of cardiovascular health in the patient. This provides and understanding of the mechanisms underlying homocysteines effects on endothelial function, and furthermore, on its involvement in the initiation and progression of atherosclerosis.
Although the bulk of available research investigations regarding the impact that homocysteine has on the presence and development of atherosclerosis provides evidence in support of its possibly causal role, some studies have produced evidence to the contrary. For instance, Guilland et al. (2003) acknowledge the fact that elevated plasma total homocysteine is considered a risk factor for cardiovascular disease, and comment that this concept is grounded in observations of early cardiovascular disease in patients with hyperhomocysteinemia, as well as the association between plasma total homocysteine and an increased risk of cardiovascular disease in prospective studies. However, these researchers explain how some other observations put plasma total homocysteine as a risk factor for cardiovascular disease into question. The researchers outline three observations that conflict with the evidence in support of elevated homocysteine levels as a risk factor for cardiovascular disease.
First, Guilland et al. (2003) claim that low risk population-based prospective studies usually tend to indicate a weak association or no association at all between plasma total homocysteine and cardiovascular disease. However, the point could be argued that increased homocysteine levels may only elevate the risk of cardiovascular disease if other risk factors, such as smoking or diabetes, are present. The existence of a possible causal relationship between homocysteine and cardiovascular disease should not be completely denied based only on studies conducted with low risk populations.
Second, Guilland et al. (2003) explain how several traditional risk factors for cardiovascular disease, such as smokins status, obesity and diabetes, are in fact associated with plasma total homocysteine, and may, in turn, confound the relationship between homocysteine and cardiovascular disease. These possibility of confounding factors could be prevented in future research by controlling for traditional risk factors in the analysis of data, and focus solely on the statistical associations between homocysteine and atherosclerosis.
Third, and finally, Guilland et al. (2003) describe how research has found that the C667T trasnsition of the methylenetetrahydrofolate reductase causes a moderate increase in plasma total homocysteine, but it only causes minor, if any, increased risk of cardiovascular disease. Guilland et al. (2003) emphasize the necessity of placebo-controlled intervention studies in order to determine more definitely whether or not a causal relationship exists between elevated levels of plasma total homocysteine and cardiovascular disease.
A study that challenges evidence that supports a causal relationship between homocysteine and cardiovascular disease, was conducted by Mehrabi et al. (2002). These researchers aimed to investigate whether elevated serum levels of homocysteine, which was predisposing to endothelial dysfunction during the progression of atherosclerosis, were accompanied by increased concentrations of homocysteine in human coronary arteries. In order to execute the study, paraffin sections of coronary arteries were taken from explanted hearts of patients suffering from cardiovascular disease, who had received cardiac transplants, as well as from heart donors where transplantation was not performed to administration-related circumstances. These sections of coronary arteries from healthy hearts and those with cardiovascular disease were then characterized immunohistochemically for homocysteine, CD68, as well as smooth muscle alpha-actin.
Results from this study indicated that the cardiovascular diseased group demonstrated high serum homocysteine levels, but the media and intimal layers of the coronary arteries that contain the endothelium displayed the lowest concentration of homocysteine. Conversely, and to the surprise of the researchers, the control group demonstrated an extensive homocysteine concentration, which was co-localized with vascular smooth cells. Based on the findings from this study, the researchers concluded that this is in fact evidence for a reverse relationship between homocysteine serum concentration and the concentration of homocysteine in coronary arteries of patients with severe cardiovascular disease. This therefore may suggest that homocysteine does not have a direct relationship in the progression of atherosclerosis in coronary arteries. However, it could be argued that the scope of this study is limited due to the small sample size. This could especially be argued in regards to the sample of healthy hearts, which only totalled six. Furthermore, this study still does not discount the possibility of a relationship between elevated homocyteine levels and cardiovascular disease mediated by other lifestyle factors, such as smoking, which has been shown to affect homocysteine levels as well as the initiation and progression of atherosclerosis.
The effects of diet and vitamin supplementation on homocysteine levels
There is a growing body of evidence indicating that elevated levels of homocystiene are associated with atherosclerosis, and that this increased levels are related to deficiencies of folate and B-12 (Schroecksnadel et al., 2003). Zhou et al. (2003) investigated the effects of vitamin supplementation and increased levels of homocysteine on the progression of atherosclerosis in apolipoprotein E-deficient mice in order to further understand the impact that vitamin supplementation may have in humans. These researchers explain how it has been demonstrated that hyperhomocystenemia accelerates the progression of atherosclerosis in apolipoprotein E-deficient mice, and they sought to investigate possible mechanisms of pathogenesis by inducing hyperhomocysteinemia in these mice through the administration of a vitamin-defined chow diet. Different diets were administered to seven groups of mice. These diets included a vitamin-defined purified chow diet alone (control), the same diet supplemented with L-homocystine or L-homocysteine, diet high in L-methionine, diet high in B-vitamins, diet deficient in folate or deficient in vitamin B6.
After eighteen weeks, the researchers measured plasma total homocysteine, lipids and atherosclerotic plaque burden. Results indicated that plasma total homocysteine levels were similar in the diet high in B-vitamins, diet deficient in folate, diet deficient in vitamin B6, and control groups. Plasma total homocysteine levels were mildly elevated in the group that received the L-homocystine supplemented diet, and were moderately elevated in the group that received the diets that were high in L-methionine and supplemented with L-homocysteine. Mice in the groups receiving these latter two diets demonstrated consistently more atherosclerosis. Based on these findings, the researchers concluded that the development of atherosclerosis is enhanced by dietary methionine and homocysteine, but not by dietary homocystine. Also, the researchers remarked that it seems B vitamins present protection against atherosclerosis that is independent of homocysteine. Further more, the researchers suggest that there may be a threshold level for homocysteine below which it does not affect the development or progression of atherosclerosis. They also suggest that the effects that elevated levels of homocysteine have on atherosclerosis, may be mediated by an intracellular pathway, and that the preventative and diminishing effects that B vitamins have in mice with normal homocysteine expression are independent of plasma total homocysteine levels.
Animal models provide an understanding of some of the biological mechanisms possibly involved in the effects that vitamin supplementation has on homocysteine levels and, furthermore, atherosclerosis. However, in order for causal relationships to be determined, research must be conducted with human populations. Renal-transplant recipients have been shown to have a high prevalence of hyperhomocysteine, which makes them a useful sample for research purposes (Marcucci et al., 2003). The elevated levels of homocysteine observed in this population might account for their increased risk for cardiovascular disease. Marcucci et al. (2003) conducted a study to investigate the effects that vitamin supplementation has on carotid intima-media thickness in renal-transplant recipients with elevated levels of homocysteine. Carotid intima-media thickening is an early sign of atherosclerosis.
This study consisted of 56 renal-transplant recipients, which were randomly assigned either to vitamin supplementation or a placebo group for a time period of six months. The participants all underwent assessment for cardiovascular risk factors, which included fasting homocysteine levels assay, as well as high resolution B-mode ultrasound in order to measure the intima-media thickness of common carotid arteries. These assessments took place at the time of enrolment in the study and after six months.
Results of this study by Marcucci et al. (2003) indicated that after treatment, fasting homocysteine levels significantly decreased in the vitamin supplementation group (P<0.0001). There were no significant changes observed post-treatment in the placebo group. Also, in the vitamin supplementation group, carotid intima-media thickness also significantly decreased after treatment (P<0.0001). Conversely, the placebo group demonstrated a significant increase in carotid intima-media thickness after the six-month treatment period (P<0.05). Based on the findings of this study, it could be concluded that vitamin supplementation may provide beneficial effects in the treatment of hyperhomocysteinemia, which would therefore reduce risk of cardiovascular disease, including atherosclerosis.
The majority of evidence points toward the beneficial effects that vitamin supplementation has on homocysteine levels and, therefore, risk of cardiovascular disease. Lindeman et al. (2003) further investigated this evidence by examining the relation that serum homocysteine concentrations have to serum folate and vitamin B-12 concentrations and cardiovascular disease in an urban, bi-ethnic community. These researchers compared fasting serum total homocysteine concentrations in a randomly selected sample of elderly Hispanic and non-Hispanic White men and women, that were 65 years of age or over. The researchers also examined associations of total homocysteine with vitamin B12 and folate, and then correlated these factors with the prevalence of cardiovascular disease in these 4 gender/ethnic groups, while adjusting for other cardiovascular disease risk factor, such as age, smoking, and diabetes.
Results of this study indicated that men and Hispanics demonstrated higher serum total homocysteine concentrations in comparison to women and non-Hispanic Whites, respectively. Furthermore, after adjusting for lower concentrations of serum folate and vitamin B12 among Hispanics, the differences between Hispanics and non-Hispanic Whites was no longer found to be significant. In addition, there was a direct relationship between serum total homocysteine concentrations and the prevalence of cardiovascular disease after other known risk factors were adjusted for. This association was shown to be most significant in Hispanic women. In conclusion, the researchers describe how the higher serum concentrations of total homocysteine observed in Hispanics compared to non-hispanic Whites, could be explained by lower demonstated levels of folate and vitamin B12. These findings further indicate the important role that vitamins, in particular vitamin B12 and folate, play in homocysteine levels and cardiovascular health.
The effects that vitamin supplementation has on homocysteine levels and atherosclerosis were investigated by Haynes (2002). This researcher describes that elevated levels of plasma total homocysteine are most commonly caused by deficiencies in B-vitamins, folic acid and vitamins B6 and B12 in particular. Also, other factors may contribute to increased levels of homocysteine, including genetic factors, renal impairment, and certain pharmaceutical agents. Elevated homocysteine levels can lead to serious cardiovascular effects, such as increased oxidant stress, impaired endothelial function, stimulation of mitogenesis, and induction of thrombosis, as well as increased arterial pressure (Haynes, 2002).
The use of methionine loading to experimentally induce hyperhomocysteinemia in humans causes extensive impairment of endolethium-dependent dilation in both conduit and resistance arteries (Haynes, 2002). However, this endothelial dysfunction can be reversed through the administration of antioxidants. Haynes (2002) emphasizes, based on the established evidence that elevated homocysteine is an independent risk factor for atherosclerosis among other cardiovascular diseases, that it is appropriate to screen for and treat hyperhomocysteinemia in individuals with progressive atherosclerosis. Furthermore, this author stesses the importance of folic acid and vitamins B6 and B12 as mainstays of treatment for hyperhomocysteinemia. However, this author questions the utility of treating moderately elevated homocysteine levels in patients that do not exhibit symptoms of atherosclerosis, and asserts that treatment of this population should be deferred until randomized outcome trials have been completed.
Folates have been proven to play an important role in the extent to which homocysteine levels are elevated. Wasilewka et al. (2003) acknowledge that low levels of serum folates, cobalamin and pyridoxine are associated with increased risk of cardiovascular disease. Also, most dietary products contain cholesterol and methionine, so these authors suggest that hyperlipidemia could be related to an elevated level of homocysteine, and inversely related with lower levels of B vitamins. Wasilewka et al. (2003) conducted a study to investigat the differences in levels of lipids and vitamins that affect the metabolism of homocysteine in different groups of patients. These researchers observed 38 healthy people, 55 patients that were hospitalized for cardiac surgery, as well as 62 patients that had no clinical evidence of atherosclerosis, but did exhibit one of the risk factors for atherosclerosis, which were high cholesterol, NIDDM or chronic renal dysfunction. The levels of total cholesterol, triglycerides, vitamin B12 and B6, and folic acid were assessed for each group.
Results of this study by Wasilewka et al. (2003) indicated no significant association between lipids and B vitamins in any of the groups under examination. However, significant differences were found between concentrations of analyzed parameters in all groups of patients in comparison to the healthy control group. Based on these findings, the researchers conclude that the lack of significant association between the levels lipid parameters and B vitamins in serum may indicate that these are additional, independent risk factors for atherosclerosis. These researchers emphasize that patients with increased risk for atherosclerosis must be permanently monitored for B vitamin levels. Oh & Brown (2003) explain how the diagnosis of vitamin B12 deficiency is usually based upon the measurement of serum vitamin B12 levels, but approximately 50 percent of patients with subclinical disease may have normal vitamin B12 levels. Therefore, these researchers explain that a more sensitive method of screening for vitamin B12 deficiency is measurement of homocysteine levels, which are elevated early in vitamin B12 defiiciency. These researchers also promote supplementation with oral vitamin b12 as the safest and most effective remedy for vitamin B12 deficiency.
Further evidential support for the impact that folate has on homocysteine is provided by Taylor (2003). This researcher expresses that there is an abundant amount of evidence indicating that increased plasma homocysteine is independently associated with both the presence and the progression of atherosclerosis, and that the vast majority of studies have all established that the administration of vitamins, especially folate, reliably results in decrease in homocysteine levels, without the dangerous or irritating side effects sometime experienced from certain medications. It is explained by Taylor (2003), that the beneficial effects of folate treatment have been demonstrated in two small, randomized clinical trials, which showed significant reductions in homocysteine levels as a direct effect to folate administration. This researcher stresses the importance of further research into the possible benefits of folate treatment as well as other vitamin therapies intended to lower homocysteine levels, since these could be the first effective therapies for atherorsclerosis that does not require expensive and potentially harmful medications or difficult lifestyle changes.
The mechanisms by which folates act to decrease homocysteine levels and, furthermore, decrease risk of atherosclerosis were explored by Melenovsky et al. (2003). These researchers explain how therapies that lower lipids with fibric acid derivatives, or fibrates, act to increase plasma total homocysteine levels. This study was conducted to determine whether folic acid, which has the potential to significantly lower total homocysteine levels, could modify the elevation of plasma aminothiols induced by fenofibrate. Patients in this study presented with hyperlipidemia, and were randomized to receive 9 weeks of treatment with micronized fenofibrate at a dosage of 200mg per day, or treatment with fenofibrate at 200mg per day plus folic acid at a dosage of 10 mg every other day. The patients' total homocysteine levels were assessed using high performance liquid chromatography both before and after the treatments.
Results of this study byMelenovsky et al. (2003) indicated a significantly increased level of total homocysteine in the group that received the fenofibrate-only treatment, at an elevation of 51.3%. Total homocysteine levels increased also in the group that received folic acid treatment, but only at a level of 14.6%. The difference between these two treatment groups was found to be significant (P<0.001). The researchers interpreted these findings as conclusive evidence that folic acid effectively reduces elevations of total homocysteine induced by fenofibrate, and that folic acid has no effect on the lipid-reducing action of fenofibrate. Furthermore, the possible increase in risk of cardiovascular disease posed by the elevated homocysteine levels induced by fenofibrate may be negated through the co-administration of folic acid.
In order to contribute to the wealth of research regarding the effects that vitamin levels have on total homocysteine levels, Folsom et al. (2003) analyzed the cross-sectional association of homocysteine, plasma and dietary B vitamin levels with multiple markers implicated in inflammation, endothelial dysfunction, or thrombogenesis, such as C-reactive protein, fibrinogen, white blood cell count, intrcellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, factor VIII, and von Willebrand factor. Results of this study indicated that no marker was significantly associated with homocysteine. Furthermore, the researchers concluded that levels of B vitamins were not a strong indicator of circulating levels of inflammatory markers, cellular adhesion molecules, or thrombogenic factors in healthy adults. Therefore the assessment of vitamin B levels may only be beneficial for patients with symptoms of atherosclerosis, or exhibiting risk factors for cardiovascular disease.
Schroeksnadel et al. (2003) explain that folate and vitamin B-12 are essential co-factors in the remethylation of homocysteine to methionine, and that immune activation seems to be significantly involved in the development of atherosclerosis. Since evidence has pointed to a causal role of homocysteine in the progression of atherosclerosis, these researchers sought to investigate a possible influence of immune stimulation of the metabolism of homocysteine. To execute this study, the researchers performed in vitro experiments using peripheral blood mononuclear cells upon stimulation with phytohaemagglutin, concanavalin A, and pokeweed mitogen.
Results of this study by Shroecksnadel et al. (2003) indicated a dose-dependent increase of homocysteine concentrations were found in stimulated cells. Furthermore, when cells were kept in a medium that was supplemented with methionine, homocysteine concentrations were found to increase even further. However, supplementation of the cells with folate only slightly affected the homocysteine levels. The researchers conclude that, based on the findings, homocysteine is accumulating in supernatants of stimulated peripheral blood mononuclear cells. Furthermore, the researchers remark that T cell activation could be involved in the development of increased levels of homocysteine, and therefore, increased risk of atherosclerosis, and this may play a greater role in the determination of homocysteine concentrations than folate does.
However, it has been determined through numerous studies that folate does affect homocysteine levels and the risk of cardiovascular disease. Martinez (2003) explains that effects of folic acid are a prime example of the interaction between genes and nutrients. Folates play an integral part in DNA synthesis due to their intervention in deoxythymidine and deoxyuridine methylation. The absence of folates instigates severe disorders in DNA replication and synthesis that often prove to be pre-cancerous. Martinez (2003) recognizes how homocysteine is a prominent endothelial toxin that is derived from methionine metabolism, and that folate is crucially involved in homocysteine metabolism. This author stresses the importance that a diet rich in vitamins, including folate, is important, and that pediatricians should address childhood nutrition issues with future health in mind, since risk factors for conditions such as cardiovascular disease can sometimes emerge at a young age.
The effects of homocysteine levels on mortality
Since numerous research studies have established the influential role that homocysteine levels have on the initiation and progression of atherosclerosis, and since atherosclerosis is responsible for en enormous number of hospitalizations and deaths each year, it is reasonable to suggest that homocysteine levels may have a direct association to mortality. Furthermore, diet, and in particular vitamin B and folate levels, may also directly influence mortality via their effects on homocysteine levels.
Liem et al. (2003) sought to explore the effectiveness of folic acid as a secondary prevention measure in a patient population with stable cardiovascular disease. These researchers discuss the favourable effects that folic acid has on vascular endothelium, and how folates have been found to decrease plasma homocysteine levels, which may decrease risk of cardiovascular disease. In light of this established knowledge, Liem et al. (2003) investigated the value that folic acid has in the secondary prevention of cardiovascular disease. Five-hundred and thirty nine patients were included in the study, of which 300 were randomized to folic acid administration, and 293 were used as control subjects. The average follow-up time for the study was 2 years.
The findings after two years indicated that patients treated with folic acid experienced an 18% decrease in plasma homocysteine levels, while homocysteine levels in the control group remained unchanged. The difference in total homocysteine levels between the treatment and control group was found to be significant (P<0.001 between groups). The primary end-point, which encompassed all-cause mortality and a composite of vascular events, was experienced in 31, or 10.3% of patients in the folic acid group, and in 28, or 9.6%, of patients in the control group (relative risk 1.05; 95% confidence interval: 0.63 to 1.75). The difference in end-points between the treatment and control group did not reach significance. Furthermore, the researchers found that in a multi-factorial survival model that made adjustments for clinical factors, homocysteine was the third most significantly predictive laboratory parameter after levels of creatinine clearance and plasma fibrinogen.
Liem et al. (2003) interpreted these findings as indication that folic acid does not appear to effectively reduce clinical end points in patients with cardiovascular disease, which further points to the possibility that homocysteine levels may only be a modifiable indication of disease. These researchers also suggest that supplementation with low-dose folic acid should be carefully considered and treated with the utmost reservation until the outcomes from more definitive studies involving folic acid become available.
Patients with end-stage renal disease are an effective sample for investigations of the effects that homocysteine has on mortality, due to the fact that in this population, cardiovascular disease is highly prevalent and is a major cause of mortality, and plasma total homocysteine levels are often three to four times greater than in the general population (Suliman et al., 2003). Suliman et al. (2003) remark that several other risk factors, such as diabetes, inflammation and malnutrition are also common and contribute to the occurrence of cardiovascular disease in patients with end-stage renal disease. In addition, strong associations between inflammation, malnutrition and hypoalbuminemia have been observed in these patients. Vitamin status has been shown to play an important role in regards to plasma total homocysteine, and these researchers aimed to investigate the influence that nutritional status has on total homocysteine, and hypoalbuminemia was of key interest because a considerable portion of total homocysteine, greater that 70%, is bound by protein, predominantly to albumin.
Suliman et al. (2003) reported that in studies of patients with end-stage renal disease that had very high levels of homocysteine (greater than 90%), total homocysteine levels were strongly associated to serum albumin levels. Also, patients with malnutrition had lower levels of total homocysteine and serum albumin than people with normal nutritional status. In addition, inflammation, diabetes and cardiovascular disease were all found to be associated with hypoalbuminia, and therefore, with lower levels of hyperhomocysteinemia. Furthermore, these researchers demonstrated with different groups of patients with end-stage renal disease, which should be noted, have inherently higher levels of homocysteine, that increased total homocysteine levels are associated with lower cardiovascular disease mortality. These researchers argue that these paradoxical results do no negate the idea that hyperhomocysteinemia is an important risk factor for cardiovascular disease. Conversely, a possible detrimental effect of elevated homocysteine levels on cardiovascular disease in patients with end-stage renal disease may be obscured by the influence of hypoalbuminemia, because it and its causes are significant predictors of mortality. According to these findings, it is implied that nutritional status and serum albumin levels, along with the presence of diabetes and inflammation, should be carefully considered when exploring total homocysteine as a risk factor for cardiovascular disease in patients suffering from end-stage renal disease. These findings may also hold implications for all populations at risk of cardiovascular disease due to increased levels of homocysteine.
More evidence regarding the impact homocysteine levels may or may not have on mortality, was addressed by Bro et al. (2003). These researchers recognized how cardiovascular mortality is 10 to 20 times as great in patients with chronic renal failure, and furthermore, that risk factor for atherosclerosis are commonplace among patients with this condition. This study sought to examine the effects of chronic renal failure on the development of atherosclerosis in apolipoprotein E-deficient mice in order to further understand the processed underlying the presence and progression of cardiovascular disease in chronic renal failure. Mice that were seven weeks old underwent 5/6 nephrectomy, unilateral nephrectomy, or no surgery, and were observed twenty-two weeks later. At this point, mice that underwent 5/6 nephrectomy showed increases in aortic plaque area fraction, aortic cholesterol content, and aortic root plaque area in comparison to the mice that did not undergo surgery. Mice that underwent unilateral nephrectomy demonstrated intermediate values.
Based on the results of this study, the researchers concluded that uremia significantly accelerates the development and progression of atherosclerosis in apolipoprotein E-deficient mice, and that this effect could not be adequately explained by changes in blood pressure, plasma homocysteine levels, or total plasma cholesterol concentrations. Therefore, the role that homocysteine plays in mortality due to cardiovascular disease is put into question since a direct relation was not indicated between atherosclerosis and homocysteine based on this model. However, it must be acknowledged that these findings were based on an animal model, which may only have limited transference to humans. Also, the evidence provided here is based on a specific population (chronic renal failure), and the effects observed may not extend to the general population.
Cardiovascular disease is also a major cause of mortality in patients undergoing maintenance hemodialysis (Kato et al., 2003). Kato et al. (2003) prospectively tested the predictive values of atherosclerosis-related parameters for all-cause and cardiovascular outcomes in 219 patients undergoing hemodialysis. The patients were assessed for blood homocysteine, carotid artery intima-media thickness, and percentage of aortic wall calcification at the L2/3 region. These patients were all followed for a time period of five years. After the five-year period was over, 54 patients, or 25%, had died, 40 of them, or 74%, died of cardiovascular-related problems. It was found that intima-media thickness was significantly higher in patients who died than those who survived, and that intima-media thickness was significantly related to age. Furthermore, the survival rate during the observation was significantly lower in the final intima-media thickness third (58%) than in the first and middle thirds (90% and 80%, respectively). Also, diabetes and intima-media thickness were determined to be independent risk factors of all-cause and cardiovascular death, while percentage of aortic wall calcification and total homocysteine did not affect the patients' 5-year mortality. These findings provide evidence against homocysteine's influence on mortality, and suggest that intima-media thickness is a better predictor of cardiovascular mortality in patients receiving maintenance hemodialysis.
However, homocysteine may have a key role in mortality in patients with acute myocardial infarction. Matetzky et al. (2003) sought to evaluate the association of elevated homocysteine levels with an increased risk of recurrent coronary events and mortality in patients with acute myocardial infarction. These researchers determined the homocysteine level of 157 patients within 24 hours of presentation with acute myocardial infarction. These patients were divided into two groups: those with homocysteine levels of 2.7mg/L or more, which was 14% of the sample, and those with homocysteine levels of less than 2.7mg/L, which was composed of 86% of the sample.
Results of this study indicated that diabetic and female patients exhibited lower homocysteine levels than males and non-diabetic patients (P<0.01 and P=0.005, respectively). Also no significant correlation emerged with age or other risk factors. Furthermore, patients with homocysteine levels that were greater than or equal to 2.7 mg/L and less than 2.7mg/L did not differ considerably in regards to the extent of cardiovascular disease as reflected by the occurrence of multivessel disease and their course while in hospital. However, patients in the group exhibiting homocysteine levels that were greater than or equal to 2.7mg/L demonstrated a higher incidence of recurrent coronary events (36% vs. 17%, P=0.04) as well as a higher incidence of death. These researchers maintain that increased levels of homocysteine are associated with a higher risk of coronary events and death, independent of any other risk factors and the extent of cardiovascular disease in patients with acute myocardial infarction. Further research could determine whether these findings extend to other types of cardiovascular dysfunction, such as atherosclerosis.
Along with homocysteine, fibrinogen has also been established as a predictor of cardiovascular events in the general population (Zoccali et al., 2003). Zoccali et al. (2003) conducted a study to investigate the relationship between fibrinogen, mortality and cardiovascular disease in patients with end-stage renal disease, taking into account the risk posed by elevated homocysteine levels. These researchers assessed this relationship between fibrinogen and all-cause mortality and cardiovascular outcomes in a prospective cohort study with 192 patients receiving chronic hemodialysis treatment, and the follow-up time was from 18 to 50 months.
The results of this study indicated that levels of fibrinogen were significantly higher in patients who died during the follow-up than in patients that survived. Also, fibrinogen was higher in patients who had fatal or non-fatal cardiovascular events than in patients that did not experience any of these events. Using multivariate Cox regression analysis, fibrinogen was found to be and independent predictor of survival (P=0.006), as well as a highly significant independent predictive factor for fatal and non-fatal cardiovascular events (P=0.0008). Fibrinogen was determined as an independent risk factor independent of plasma homocysteine levels and traditional risk factors, which points to the possibility that fibrinogen may play a role in cardiovascular disease that is as important as that played by homocysteine.
The possible causal effects that homocysteine levels may have on cardiovascular disease and mortality were explored and expanded upon by El-Khairy et al. (2003). These researchers reported a significant, positive associateion between total homocysteine levels and hospitalizations due to cardiovascular disease, as well as mortality. Furthermore, these researchers aimed to investigate the relationship between plasma total cysteine, not homocysteine, and mortality from all causes and from cardiovascular as well as non-cardiovascular events. They also aimed to assess the association between total cysteine levels and the risk of hospitalization due to cardiovascular disease.
To conduct this study, El-Khairy et al. (2003) measured the levels of plasma cysteine in blood samples from 12,595 men and women aged 40-42 years, as well as from 4766 men and women aged 65-67 years, which were collected as part of a previous homocysteine study that took place in 1992 to 1993. Follow-up data in regards to mortality and hospitalizations due to cardiovascular disease were available in regards to the participants of the study. Results indicated that after a follow-up time of 6.6-7.6 years, there were a total of 610 deaths, which included 243 from cardiovascular disease and 367 non-cardiovascular deaths. The researchers indicated that there was no significant relationship between total cysteine levels and all-cause, cardiovascular or non-cardiovascular mortality. Furthermore, there were also no significant associations between total cysteine levels and the 1275 hospitalizations due to cardiovascular disease that occurred among the sample, except in the case of coronary artery bypass grafting, where a significant association with total cysteine levels was determined. Based on these results the researchers concluded that plasma total cysteine is not significantly associated with mortality or hospitalizations due to cardiovascular disease, and the focus on homocysteine as an independent factor in cardiovascular outcomes and mortality is re-asserted.
Genetic variation and homocysteine
Weisberg et al. (2003) explain how hyperhomocysteinemia, which has been established as a risk factor for cardiovascular disease, can be caused by genetic mutations in enzymes that are involved in homocysteine metabolism. These researchers further explain the process of how homocysteine remethylation to methionine is catalyzed by methionine synthase that is dependent on folate, or by betaine-homocysteine methyltransferase, which utilizes bethaine as a methyl donor. Genetic variants in folate-dependent remethylation have been observed to increase risks for cardiovascular disease, so these researchers therefore screened betaine-homocysteine methyltransferase for sequence changes that might change the level of risk for cardiovascular disease.
Through this study by Weisberg et al. (2003), a variant in exon 6-R239Q was identified, and the frequency of this change was examined in 504 subjects who had received coronary angiography and were divided into controls, which were those with no or little evidence of disease, and cases, which were those showing significant evidence of disease. The researchers discovered that this variant did not affect plasma homocysteine, but the QQ genotype was present in higher frequency in those with no or mild disease, in comparison to those with significant disease (11% vs. 6%). These findings suggest that this QQ genotype may decrease the risk of cardiovascular disease. These researchers conclude that the Q allele of the R239Q mutation may decerase the risk of cardiovascular disease, and that this genetic variant warrants further investigation of its association with the development of cardiovascular disease and other disorders that are dependent upon homocysteine levels.
Further research into the cellular factors involved in the relationship between homocysteine and cardiovascular disease, was conducted by Hossain et al. (2003). These researchers sought to investigate the cellular factors involved in the process by which homocysteine causes endoplasmic reticulum stress and programmed cell death in cultured human vascular cells, and examine how these cellular factors are related to the development and progression of atherosclerosis. Based on their findings, these researchers reported that homocysteine induces the expression of T-cell death associated gene 51 (TDAG51), which is a member of the domain family that is related to pleckstrin homology, in cultured human vascular endothelial cells. Furthermore, transient overexpression of TDAG51 brought about significant changes in cell morphology, decreased adhesion of cells, and promoted detachment-mediated programmed cell death. Moreover, TDAG51 expression was increased and associated with programmed cell death in the atherosclerotic lesions from apolipoprotein E-deficient mice that were fed hyperhomocysteinemic diets, in comparison to mice fed a control diet. Based on these findings, the researchers concluded that TDAG51 is induced by homocysteine, promotes detachment mediated programmed cell death, and plays an important role in the development of atherosclerosis among individuals with elevated levels of homocysteine. This research has both research and clinical implications for the prevention and treatment of atherosclerosis.
Another genetic mechanism involved in the relationship between homocysteine and atherosclerosis was explored by Girelli et al. (2003). These researchers explain how the 677 Cà
T polymorphisms in the 5,10 methylenetetrahydrofolate reductase (MTHFR) gene interacts with folate status in the determination of elevated total plasma homocysteine levels, which, of course, has been established as a risk factor for cardiovascular disease. In this study, the researchers aimed to define the specific threshold values of the 677Cà
T genotype of both RBC folate and plasma, which is associated with hyperhomocysteinemia. They also sought to determine the risk of cardiovascular disease among subjects that exhibited folate levels below the genotyp-specific threshold considered at risk for increased levels of homocysteine. Based on results obtained from the study, a gene-nutrient interaction that defines an increased risk for cardiovascular disease is determined by the presence of folate levels below specific thresholds, which were found to differ depending on the MTHFR 677 Cà
T genotype. These findings provide further direction for research in regards to the relationship between dietary factor and genetic mechanisms in homocysteine processes.
Implications for intervention
Based on the available research, it is evident that there is a direct relationship between homocysteine levels and risk of cardiovascular disease. Furthermore, dietary factors, especially vitamin status with regards to B vitamins and folate, are involved in this possibly causal relationship. This points to several possibilities for preventions and treatments, including genetic-based therapies, vitamin supplementations, and screening methods to indentify those at risk for atherosclerosis and other cardiovascular diseases. Linton et al. (2003) explain how as much as 30% of the adult US population may be in need of aggressive risk reduction therapies, and that the most practical approach for implementation would be to initiate early intervention in people who have familial hypercholesterolemia or a family history of early cardiovascular disease. Furthermore, possibilities for intervention measures may lie in the differences in homocysteine levels between males and females. For instance, research conducted by Spencer et al. (2003) reported that estrogen significantly prevents homocysteine-mediated endothelial dysfunction in porcine coronary arteries, which could extend to possible preventions and treatment measures in humans.
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