Controversial Role of Homocysteine in Atherosclerosis - Term Paper Example

Controversial role of homocysteine in atherosclerosis 1. Brief introduction. 200 w The role of elevated levels of homocysteine in blood plasma has been the subject of intense study and literature reviews for more than 50 years since an association between defects in homocysteine metabolism and thromboembolism was observed (Mudd, Finkelstein, Irreverre, & Laster, 1964). This role was further verified in patients with homocystinuria and abnormalities in vitamin B12 metabolism with general vascular damage and widespread thrombosis (McCully, 1969). Subsequently, the homocysteine theory of arteriosclerosis was formulated (McCully & Wilson, 1975). Since then, the positive association between the risk of cardiovascular disease and homocysteine levels in the general population was validated in many epidemiological studies (Boushey, Beresford, Omenn, & Motulsky, 1995; Verhoef, et al., 1996; Eikelboom, Lonn, Genest, Hankey, & Yusuf, 1999; Humphrey, Fu, Rogers, & Helfand, 2008). Significantly, these studies found that small increases of homocysteine levels in blood increase the risk of coronary heart disease. Another important finding was the role of diet, vitamins and folic acid in lowering homocysteine levels. Some authors have questioned the direct homocysteine-cardiovascular disease link, basing their conclusions on a review of longitudinal, prospective studies (Kaul, Zadeh, & Shah, 2006). Accordingly, the evidence is not strong enough to warrant a causal effect, the mechanisms for how homocysteine causes cardiovascular disease has not been elucidated, and that there has been no proof showing that interventions of decreasing homocysteine levels have modified the risk for atherosclerosis (Kaul, Zadeh, & Shah, 2006). Moreover, homocysteine has been proposed to be a marker, and not a cause of CVD (Wierzbicki, 2007). Recent studies have proposed a mechanism on increased risk of cardiovascular disease (CVD) due to elevated homocysteine. Inhibition of the growth of endothelial cells and promotion of vascular smooth muscle cell proliferation results in damage of vascular cells (Zou, 2007). The abnormalities in the production of endothelial cells was caused by homocysteine’s inhibition of DNA methylation in the promoter region in the gene of an activator of the cell cycle (Jamaluddin, et al., 2007). 2. Amino acid and focus on cysteine. 300 w All living cells contain the biomolecules proteins, carbohydrates, nucleic acids and lipids (Mathews & Van Holde, 1996; McKee & McKee, 2004). These are made up of the repeating sub-units of amino acids, sugars, nucleotides and fatty acids respectively. Cells also contain small organic molecules that are involved in complex biosynthetic and regulatory pathways that are tightly controlled at the molecular level. Amino acids are naturally occurring compounds containing an amino group, a carboxyl group, and a unique side chain or R group (Figure 1). The chemical and functional properties of an amino acid are largely determined by its R group (McKee & McKee, 2004) (Mathews & Van Holde, 1996). Figure 1. General structure of an amino acid (ChemCards , 2010) Although there are hundreds of amino acids, only twenty have been identified to be building blocks of proteins. Among these is cysteine, which contains a sulfhydryl, or thiol (SH) in its R group (–CH2-SH) (Berg, Tymoczko, & Stryer, 2002). The sulfhydryl group is very reactive and could react with another thiol group, forming disulfide bonds or sulfide bridges. These bridges are important in increasing the stability of some proteins. Two sulfhydryl moieties of two cysteine bonds can oxidize forming cystine. This commonly occurs in extracellular fluid like blood and urine. However, the solubility of cystine is very low, and in large amounts, cystine can cause kidney stones (McKee & McKee, 2004).The sulfhydryl moiety also forms weak bonds with nitrogen and oxygen. Another amino acid which contains a sulfhydryl group is methionine. Its side chain is –CH2CH2SCH3. The sulfur in methionine can form bonds with electrophiles (Berg, Tymoczko, & Stryer, 2002). The methyl group (-CH3) can be activated and is involved in many reactions where just one carbon atom is being added to another compound. Cysteine is considered a non-essential amino acid because it can be synthesized de novo in both plants and animals (Figure 2). In animals, cysteine is formed from the cleavage of cystathionine through the action of γ-cystathionase. Cystathionine is produced when serine condenses with homocysteine that is derived from methionine (McKee & McKee, 2004). Methionine, an essential amino acid, cannot be synthesized in humans, and thus, have to be provided in the diet. Its metabolism involves homocysteine. Figure 2. The biosynthesis of cysteine in plants and bacteria (a) and animals (b). Figure adapted from McKee and McKee, 2004. 3. Homocysteine structure and metabolism. 200 w Homocysteine is a non-protein amino acid and thus, it is not utilized in protein synthesis. Its structure is similar to cysteine but it has an additional carbon before the sulfhydryl moiety (R group: –CH2 -CH2 –SH). The tendency of homocysteine to form cyclic compounds may have limited its potential as a protein building block. Homocysteine is not provided in the diet, but is biosynthesized from methionine in a process involving several steps. Methionine is first adenosylated to form S-adenosyl methionine (SAM), and then the methyl group is transferred to an acceptor molecule to form S-adenosyl homocysteine (SAH) in a process called transmethylation. Adenosine is then removed thereby forming homocysteine (Figure 3) (Durand, Prost, Loreau, Lussier-Cacan, & Blache, 2001; Selhub J. , 1999; Marinou, Antoniades, Tousoulis, Pitsavos, Goumas, & Stefanadis, 2005). Transmethylation is the only pathway for producing homocysteine in the body. Homocysteine then undergoes two pathways: remethylation to methionine, and transsulfuration to produce cystathionine, cysteine, pyruvate and taurine. Remethylation requires folate and cobalamin (vitamin B12), and transsulfuration requires pyridoxine (vitamin B6). Homocystinuria results from genetic errors in the metabolic pathways (Kluijtmans, et al., 2003; Klerk, Verhoef, Clarke, Blom, Kok, & Schouten, 2002), but the current focus is on the more common hyperhomocysteinemia that results from genetic variations or nutritional inadequacy (Finkelstein & Martin, 2000). Polymorphisms in the gene methylenetetrahydrofolate reductase (MTHFR) which catalyzes the transfer of a methyl group to homocysteine to re-form methionine have been found in many studies to increase homocysteine levels (Klerk, Verhoef, Clarke, Blom, Kok, & Schouten, 2002). Among the proposed effects of increased homocysteine levels resulting in CVD are oxidation of low density lipoprotein, decreasing the thrombomodulin expression inhibiting the anticoagulant pathway leading to thrombosis, platelet activation and aggregation, and smooth muscle cell proliferation (Eikelboom, Lonn, Genest, Hankey, & Yusuf, 1999; Jamaluddin, et al., 2007). Figure 3. Homocysteine metabolism. ( adapted from(Durand, Prost, Loreau, Lussier-Cacan, & Blache, 2001) 4. Normal Homocysteine levels. 200 w The concentration of circulating total homocysteine (tHcy) is an accurate marker of low folate and vitamin B12. In 1999, 3563 male participants and 4523 female participants were surveyed to determine the normal blood homocysteine levels (Selhub, et al., 1999; Selhub, 1999). The survey found that homocysteine increased with age and was higher in adult males than and females. Screening of babies confirmed that homocysteine levels are higher in baby boys (Refsum, et al., 2004). It was recommended that blood homocysteine levels equal to or greater than 11.4 µmol/L (males) and 10.4 µmol/L (females) are to be considered above normal and was associated with low vitamin concentrations in two-thirds of the subjects surveyed (Selhub, et al., 1999). This verified that an assessment of homocysteinemia requires the knowledge of the health status of the patient. The degree of homocysteinemia is classified based on determination fasting levels of serum homocysteine. Currently, a plasma tHcy level of 15 µmol/L is considered as ‘normal’ (Refsum, et al., 2004). In healthy adults with good folate and B vitamins status, the upper reference limit is 12 µmol/L. Accordingly, homocysteinemia is classified as moderate (15-30 µmol/L), intermediate (30-100 µmol/L) and severe (> 100 µmol/L). This categorization is necessary to come up with a decision regarding the treatment of the condition. 5. Homocysteine and nutrition. 300 w Studies have shown that the enzymes regulating the metabolism of homocysteine are activated by the B vitamins and folate which can be sourced from the diet. The balance of the remethylation and transmethylation pathways are coordinated by nutritional inputs, specifically methionine (Selhub, 1999). Decreasing dietary methionine increased homocysteine remethylation, while increased methionine increased the production of cystathionine through upregulation of the transsulfuration pathway. The molecular mechanisms for the effects of dietary methionine are based on the capacity of S-adenosyl methionine SAM to inhibit methylenetetrahydrofolate reductase (MTFHR) and activate cystathionine synthase (Selhub & Miller, 1992). Thus, when dietary methionine is high, there is a rapid conversion of methionine to SAM. The high levels of SAM promotes the inhibition of MTHFR and subsequently, remethylation of homocysteine is also depressed. The pathway is channeled to the transsulfuration pathway, to produce cysteine. In opposite conditions of low dietary methionine, low SAM levels are not enough to inhibit MTFHR activity, resulting in the remethylation of homocysteine. The role of folate in homocysteine metabolism has been the subject of many research studies (Antoniades, Antonopoulos, Tousoulis, & Stefanadis, 2009; Boushey, Beresford, Omenn, & Motulsky, 1995; Lonn, et al., 2006). A diet that is poor in folate was also found to impair remethylation and synthesis of SAM (Miller, Nadeau, Smith, Smith, & Selhub, 1994). Folate deficiency increased the concentration of tHcy and hepatic SAM concentration. Introducing dietary folate and methionine also decreased tHcy. Since vitamins and trace minerals are lost due to food processing (Schroeder, 1971), fortification is proposed in order to replenish the lost nutrients. Folic fortification of breakfast cereals when consumed daily was recommended to be the most effective means of decreasing serum folate (Riddell, Chisholm, Williams, & Mann, 2000). High-dose folic acid supplementation also decreased tHcy levels in cases where there is vitamin B12 deficiency (Min, 2009). However, a high methionine diet is not recommended since it can decrease body weights and HDL-cholesterol production in mice (Velez-Carrasco, Merkel, & Smith, 2008). Aside from the observed effects of folic acid on reducing tHcy concentrations, folic acid is also thought to be involved in ameliorating endothelial dysfunction through its action of maintaining endothelial nitric oxide synthase in its coupled state which favors the formation of nitric oxide, and not oxygen radicals (Moens, Vrints, Claeys, Timmermans, Champion, & Kass, 2008). 6. Role of Homocysteine and vitamins. 200 w The enzymes involved in metabolism of homocysteine require the participation of vitamin cofactors B6 and B12, and folic acid. The direct association between low folate, vitamin B6 and B12 status and hyperhomocysteinemia have been validated many times Hao, et al; 2007; (Hao, et al., 2007; Boushey, Beresford, Omenn, & Motulsky, 1995; Kluijtmans, et al., 2003; Koehler, Baumgartner, Garry, Allen, Stabler, & Rimm, 2001). Investigations have geared towards the probability of using these vitamins in the prevention and treatment of homocysteinemia in the general population, and in those who already have cardiovascular disease. The use of multivitamins supplementation for several months improved the concentration of the B vitamins and folate acid in plasma, and decreased homocysteine and LDL cholesterol levels in 182 study participants (Earnest, Wood, & Church, 2003). Similar homocysteine-lowering effects were also observed in patients with celiac disease, who have malabsorption problems, who take vitamin supplements (Hadithi, et al., 2009). However, the case for the vitamins is different in patients who already have CVD. Although the treatment of different combinations of vitamins B6, B12 and folic acid of patients with coronary artery disease resulted in a 30% reduction of tHcy one year after receiving vitamin B12 and folic acid, follow-up measurements of homocysteine levels showed that the vitamin supplementation did not significantly affect the total cardiovascular events (Ebbing, et al., 2008). Thus, the findings do not support the use these vitamins for secondary prevention of coronary artery disease. The Norwegian Vitamin Trial (NORVIT) also found that vitamin supplementation did not reduce the risk of cardiovascular disease recurrence after a heart attack (Bønaa, et al., 2006). Moreover, preliminary results of the NORVIT suggested that the B vitamin treatment increased risk of cancer, and that such treatment should not be given. 7. Role of Homocysteine in atherosclerosis. 500w Many studies since the 1960s have validated the relationship between hyperhomocysteinemia and risk of atherosclerosis (Eikelboom, Lonn, Genest, Hankey, & Yusuf, 1999). Severe hyperhomocysteinemia (homocysteine levels greater than 100 µmol/L) can be caused by several inherited genetic disorders. Foremost among the disorders are mutations in the gene encoding for the major transsulfuration enzyme cystathionine β-synthase, remethylation enzymes methylenetetrahydrofolate reductase, methionine synthase in the methionine cycle, or defects in vitamin B12 metabolism (Finkelstein, 1998; Kraus, 1998). These genetic conditions lead to extreme elevations of plasma homocysteine and early atherothrombotic disease, where the typical pathologic features of endothelial injury, vascular smooth muscle cell proliferation, and progressive arterial stenosis are observed (Mudd, Skovby, Levy, Pettigrew, Wilcken, & Pyeritz, 1985). While these genetic errors in metabolism are rare, they gave researchers a model for studying cardiovascular injury that was associated with high homocysteine levels. The clinical and pathologic features observed under the conditions of homocystinuria resulted in the “homocysteine theory of atherosclerosis” that declared high plasma homocysteine concentrations to be responsible for vascular damage (McCully & Wilson, 1975). Results of iindividual studies and meta-analysis have come up with support for the theory of McCully and Wilson (1975). However, there are still controversy over whether homocysteinemia causes CVD or vice versa, or is it that homocysteinemia is just a marker or indicator of CVD. Mounting evidence for the causal effect of homocysteine was backed up by basic and cellular studies which utilized advanced molecular and genetic techniques. Early studies infer that homocysteine damages the endothelial cells lining the blood vessels and increase the production of vascular smooth muscle (Berg, Tymoczko, & Stryer, 2002). From a pathophysiologic point of view, homocysteinemia is associated with increased oxidative stress in the cells, and development of thrombosis (Tyagi, Sedoris, Steed, Ovechkin, Moshal, & Tyagi, 2005), impaired endothelial function (Stuhlinger, Tsao, Her, Kimoto, Balint, & Cooke, 2001) and the activation of inflammatory pathways that are sensitive to changes in cellular redox states (Weiss, Heydrick, Postea, Keller, Keaney, & Loscalzo, 2003). It was originally proposed that homocysteinemia promotes the accumulation of S-adenosyl homocysteine, a potent inhibitor of cellular DNA methylation (Zou, 2007). The hypomethylation of DNA could affect promoter activity resulting in remodelling of chromatin and changes in the transcription of certain genes. However, it was found that In the case of homocysteinemia, increased tHcy levels cause the hypomethylation of DNA promoter region of the cyclin A gene (Jamaluddin, et al., 2007; Zou, 2007). Cyclin A protein is a sub-unit of cyclin-dependent kinases (CDK) that activates CDK during the cell cycle. In the presence of high tHcy concentration, methylation was inhibited at two CpG sites in the cyclin A promoter, resulting in a 6-fold increase in promoter activity, and therefore increased proliferation of endothelial cells. Homocysteine directly inhibited the activity of DNA methyltransferase I (DNMT1) by 30% (Jamaluddin, et al., 2007). Furthermore, homocysteine reduced binding of methyl CpG binding protein 2, while it also increased the binding of cyclin A promoter to histones H3 and H4 which led to chromatin remodelling. 8. Review methods of analysing homocysteine and differences between measurements. 3000 w 9. Checking of Homocysteine Level (Homocysteine test) 300w The results of homocysteine testing express the amount of total homocysteine (tHcy) which is the sum of all the chemical forms of homocysteine in the body. Reduced homocysteine constitutes 1% of tHcy, while the oxidized forms homocysteine-homocysteine or cysteine-homocysteine linked by disulfide bridges each constitutes 5-10%. Mixed disulfides or protein-bound homocysteine comprise the majority of tHcy levels (80-90%) (Jacobsen, 1998). A blood or urine homocysteine test is ordered if a patient is suspected of having homocystinuria, or deficiencies in B12 and folate (Homocysteine, 2010). Homocysteine testing is also recommended in the elderly, malnourished individuals, and drug and alcohol addicts. The test is also ordered if the methionine levels of newborn babies are abnormally high (Homocysteine, 2010). It can also be used to screen for people who have a family history of cardiovascular heart disease despite the absence of known risks. However, the test is still not recommended for routine testing. Prior to taking blood samples for homocysteine analysis, fasting is required for ten to twelve hours. Fasting levels of tHcy are constant for healthy individuals who are not on a special diet (Hortin, 2006). A meal rich in protein can increase tHcy concentration that is still detectable after several hours, thus a more realistic estimate can be achieved with fasting. At room temperature, homocysteine levels may increase by more than 10% within an hour. This is due to the release of homocysteine from the cellular component of blood. Leaving whole blood for 24 hours may increase homocysteine levels by 35-75%. Therefore it is necessary to separate, by centrifugation, the serum or plasma components from the cellular fraction. Ideally, the samples should be processed immediately within 15 minutes after collection or immediately stored on ice (Ueland, Refsum, Stabler, Malinow, Andersson, & Allen, 1993). Homocysteine in frozen serum samples are stable for several years frozen (Hortin, 2006). After processing, total homocysteine levels can then be determined by one of the methods discussed earlier. Some laboratories have developed kits that allow for home sampling of blood for tHcy determination. In this method, a drop of blood is applied to a membrane which stabilizes the plasma portion. The kit can then be sent to the company for analysis (Holford & Braly, 2010). The recommendation for appropriate treatment strategies are based on the results and the indicated degree of homocysteinemia (see previous discussion). 10. Prevention of high homocysteine level. 200 w Hyperhomocysteinemia is caused by a multitude of factors that can be broadly classified as genetic factors, vitamin deficiencies, lifestyle or demographic characteristics, chronic medical conditions, and drug and alcohol use (Fowler, 1997; Eikelboom, Lonn, Genest, Hankey, & Yusuf, 1999). The fortification of the diet by folate is viewed as the most effective means and is the primary recommendation for reducing tHcy in the general population (Riddell, Chisholm, Williams, & Mann, 2000). Grain products in the US are enriched with folic acid, and consumption of these foods significantly reduced tHcy levels in a population of older adults (Jacques, Selhub, Bostom, Wilson, & Rosenberg, 1999). Supplementation with vitamins B6 and B12 also decrease tHcy levels (Earnest, Wood, & Church, 2003; Lonn, et al., 2006). Eating foods rich in folic acid and the B vitamins is recommended to maintain low homocysteine levels in healthy individuals. Green and leafy vegetables and some animal products are rich in folic acid (Kim, 2007). Vitamin B12 can only be found in meat and dairy products, which puts strict vegetarians at risk of deficiency. It is also not synthesized in the body; therefore it has to be provided by nutrition or supplementation. On the other hand, vitamin B6 deficiency is uncommon because it can be sourced from many foods, and is stored in the liver. However, if an individual has liver disease or poor nutritional status, vitamin B6 deficiency is possible, and thus, supplementation is recommended (Maron & Loscalzo, 2009). A Mediterranean diet, which is rich in folate and vitamins, was found to be effective in reducing tHcy (Homocysteine Lowering Trialists’ Collaboration, 2005). 11. Treatment of high homocysteine level. 200 w Homocysteine- lowering treatment should be given even when mild homocysteinemia is present. Moderate homocysteinemia due to poor diet, heterozygosity, drug use, and presence of medical conditions (Refsum, et al., 2004) requires removable of the cause. If the cause is heterozygosity MTHFR gene, then methylene tetrahydrofolate, which is the product of the MTHFR catalysis, should be given orally. This treatment is considered safe (Antoniades, Antonopoulos, Tousoulis, & Stefanadis, 2009). If there is intermediate homocysteinemia, it usually indicates renal failure or moderate to severe folate or vitamin B deficiency (Refsum, et al., 2004). The treatment for moderate homocysteinemia should depend on the assessment of vitamin status. Administration of single or combination treatment of vitamin B12 and folate should be based on this assessment. When 400 mg/day of folic acid is given, plasma tHcy is reduced by 25-30%. Co-administration of vitamin B12 (20 µg – 1 mg/day) was found to decrease tHcy by another 7% (Malinow, Bostom, & Krauss, 1999). Serum tHcy greater than 100 µmol/L, indicates homocystinuria or severe vitamin B12 deficiency. This is associated with increased risk of thrombosis (Yap, 2003), and should be treated daily with 20 µg-1 mg vitamin B12. If homocystinuria was due to the absence of the cystathionine β synthase vitamin B6 (50–250 mg/day) and folic acid (0.4–5 mg/day) with or without vitamin B12 (0.02–1 mg/day) is recommended for patients who respond to vitamin therapy. Non-responders to vitamins are given a diet where methionine is restricted while cystine is supplemented (Yap, 2003). 12. Conclusion. 200 w Most researchers support the theory that increases in circulating homocysteine is an independent risk factor for atherosclerosis. Whereas before all the evidence was circumstantial, now there is compelling proof that increased circulating homocysteine directly affects cardiovascular cell growth by altering the gene expression of cell cycle intermediates. The results of the new studies confirm that increased homocysteine increases the risk for thrombosis and cardiovascular disease in affected individuals. It is fortunate that despite the uncertainty of causality of homocysteine and CVD, researchers continue to study the cellular factors and the pathways where homocysteine is a major intermediate. Genetic studies also paved the way for treating patients with heterozygous genes of the methionine pathway. The most important outcome is the identification of the treatment modalities for increased tHcy. Homocysteinemia is the perfect example of how a complete and proper diet serves to correct errors in metabolism and improve over-all health. However, there is no recommended and accepted therapy for homocysteinemia in adults who have been diagnosed with CVD. Therefore, more studies should be conducted on the cellular events that occur after arteriosclerosis set. 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