Role of Elevated Levels of Homocysteine in Blood Plasma

Role of Elevated Levels of Homocysteine in job PlasmaHomocysteine, a non-protein amino stinging, is an intermediate in the metabolism of methionine and biosynthesis of cysteine. It has gained prominence in the past half-century beca theatrical role its accumulation in the body has been connect to amplificationd risk and occurrence of atherosclerosis and cardiovascular disease. Homocystinuria is oerimputable to errors in metabolism and homocysteinemia is attri thoed to polymorphisms in the genes obscure in methionine metabolism and cysteine biosynthesis, and deficiencies in the nutrients folic acid, and vitamins B12 and B6. deduction of these do- nonhing be put together in the results of genic and feedary studies. The clinical quantification of homocysteine has evolved through the years and turn tests be currently available. Baseline homocysteine intentness for normal big(a)s has been identified to be between 12-15 mol/L. Elevated directs of homocysteine tail assemb ly be shortend with pteroylglutamic acid and B vitamins supplementation, but this intervention does not work in patients who boast suffered stroke or mall attacks. to a greater extent than research studies rush supported the hypothesis that elevated homocysteine causes CVD and is not honourable a marker for the disease. However, despite the amount homocysteine research per numbered, umpteen some other(prenominal) issues quell to be resolved, among which atomic number 18 the elucidation of the molecular mechanism of the direct consummation of homocysteine and the precedentization of techniques for the quantification of homocysteine levels.Brief introduction. cc wThe parting of elevated levels of homocysteine in neckcloth plasm has been the subject of intense conceive and literature go oers for more than 50 years since an connector between defects in homocysteine metabolism and thromboembolism was discover. This character was further confirm in patients with hom ocystinuria and abnormalities in vitamin B12 metabolism with general vascular damage and widespread thrombosis. Subsequently, the homocysteine theory of arteriosclerosis was formulated by McCully and Wilson in 1975. Since and thusly, the positive association between the risk of cardiovascular disease and homocysteine levels in the general population was validated in many epidemiological studies (Boushey, et al., 1995 Verhoef, et al., 1996 Eikelboom et al., 1999 Humphrey et al., cc8). Signifi seattly, these studies found that wee increases of homocysteine levels in ancestry increase the risk of coronary subject matter disease. Another important finding was the role of diet, vitamins and folic acid in impenetrable homocysteine levels.Some authors arouse questioned the direct homocysteine-cardiovascular disease link, basing their conclusions on a review of longitudinal, prospective studies (Kaul, Zadeh and 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 verification showing that interventions of decreasing homocysteine levels have modified the risk for atherosclerosis (Kaul, Zadeh and 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 change magnitude risk of cardiovascular disease (CVD) due to elevated homocysteine. Inhibition of the growth of endothelial jail cells and promotion of vascular thawny muscle cell proliferation results in damage of vascular cells (Zou, 2007). The abnormality in the drudgery of endothelial cells was ca apply by homocysteines inhibition of desoxyribonucleic acid methyl radicalation in the booster station region in the gene of an activator of the cell cycle (Jamaluddin, et al., 2007).Amino acid and focus on cysteine. 300 w each living cells break off the biomole cules proteins, carbohydrates, nucleic acids and lipids (Mathews and Van Holde, 1996 McKee and McKee, 2004). These be made up of the repeating sub-units of amino acids, sugars, nucleotides and juicy acids respectively. Cells similarly contain small organic molecules that atomic number 18 involved in complex biosynthetic and regulatory pathways that ar tightly overtopled at the molecular level.Amino acids argon naturally occurring compounds containing an amino theme, a carboxyl group, and a unique side chain or R group (Figure 1). The chemical substance substance and kick the bucketal properties of an amino acid argon largely determined by its R group (McKee and McKee, 2004 Mathews and Van Holde, 1996).Although there are hundreds of amino acids, exclusively 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 and 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 somewhat proteins. Two sulfhydryl moieties of two cysteine bonds can oxidate forming cystine. This jointly occurs in extracellular fluid same affinity and urine. However, the solvability of cystine is very let out, and in large amounts, cystine can cause kidney stones (McKee and McKee, 2004).The sulfhydryl mediety in addition 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 and 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 anim als, cysteine is formed from the cleavage of cystathionine through the action of -cystathionase. Cystathionine is produced when serine condenses with homocysteine that is derived from methionine (McKee and 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.Homocysteine structure and metabolism. 200 wHomocysteine 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 whitethorn have limited its potential as a protein building block.Homocysteine is not provided in the diet, but is biosynthesized from methionine in a treat involving some(prenominal) steps. Methionine is first adenosylated to form S-adenosyl methionine (SAM), and and so the methyl group is movered to an acc eptor molecule to form S-adenosyl homocysteine (SAH) in a process called transmethylation. Adenosine is accordingly removed thereby forming homocysteine (Figure 3) (Durand et al., 2001 Selhub J. , 1999 Marinou, et al., 2005). Transmethylation is the nevertheless 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, et al., 2002), but the current focus is on the more common hyperhomocysteinemia that results from genetic variations or nutritional inadequacy (Finkelstein and 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, et al., 2002).Among the proposed effects of increase homocysteine levels resulting in CVD are oxidation of depression density lipoprotein, decrease in the thrombomodulin expression inhibiting the anticoagulant pathway maven story to thrombosis, surfacelet activation and aggregation, and smooth muscle cell proliferation (Eikelboom, et al., 1999 Jamaluddin, et al., 2007).Normal Homocysteine levels. 200 wThe concentration of circulating heart and soul homocysteine (tHcy) is an accurate marker of let out folate and vitamin B12. In 1999, 3563 male participants and 4523 female participants were surveyed to determine the normal melody homocysteine levels (Selhub, et al., 1999 Selhub, 1999). The survey found that homocysteine increased with age and was high(prenominal) 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 livestock homocysteine levels equal to or greater than 11.4 mol/L (males) and 10.4 mol/L (females) are to be considered above normal and was associated with down(p) 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 base on ratiocination fasting levels of serum homocysteine. Currently, a subscriber line plasma tHcy level of 15 mol/L is considered as normal (Refsum, et al., 2004). In vigorous adults with good folate and B vitamins status, the upper reference limit is 12 mol/L. Accordingly, homocysteinemia is classified as moderate (15-30 mol/L), intermediate (30-100 mol/L) and severe ( 100 mol/L). This categorization is necessary to come up with a decision regarding the treatment of the condition.Homocysteine and nutrition. 300 wStudies 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, magic spell 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 and Miller, 1992). Thus, when dietary methionine is high, there is a speedy conversion of methionine to SAM. The high levels of SAM promote the inhibition of MTHFR and subsequently, remethylation of homocysteine is as well as 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, et al., 2009 Boushey, et al., 1995 Lonn, et al., 2006). A diet that is poor in folate was to a fault found to corrupt remethylation and synthesis of SAM (Miller, et al., 1994). Folate deficiency increased the concentration of tHcy and liverwort SAM concentration. Introducing dietary folate and methionine as well as decreased tHcy. Since vitamins and trace minerals are lost due to food processing, fortification is proposed in order to refill the lost nutrients. Folic fortification of breakfast cereals when consumed daily was recommended to be the most impelling means of decreasing serum folate (Riddell, et al., 2000). High-dose folic acid supplementation alike decreased tHcy levels in cases where there is vitamin B12 deficien cy (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 and Smith, 2008). forth 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 landed estate which favors the formation of nitric oxide, and not oxygen radicals (Moens, et al., 2008).Role of Homocysteine and vitamins. 200 wThe 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 multiplication Hao, et al 2007 (Hao, et al., 2007 Boushey, et al., 1995 Kluijtmans, et al., 2003 Koehler, et al., 2001). Investigations have geared towards the probability of usi ng these vitamins in the legal community 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 and Church, 2003). interchangeable 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 arterial blood vessel disease resulted in a 30% reduction of tHcy one year after receiving vitamin B12 and folic acid, consume evaluatements of homocysteine levels showed that the vitamin supplementation did not sig nificantly affect the add together 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 (Bnaa, 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.Role of Homocysteine in atherosclerosis. 500wMany studies since the sixties have validated the relationship between hyperhomocysteinemia and risk of atherosclerosis (Eikelboom, et al., 1999). dire hyperhomocysteinemia (homocysteine levels greater than 100 mol/L) can be ca apply by several inherited genetic disorders. Foremost among the disorders are mutations in the gene convert for the major transsulfuration enzyme cystathionine -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. While these genetic errors in metabolism are rare, they gave researchers a work 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.Results of individual studies and meta-analysis have come up with support for the homocysteine theory of atherosclerosis. However, there are still controversy over whether homocystein emia 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 and Stryer, 2002). From a pathophysiologic point of view, homocysteinemia is associated with increased oxidative stress in the cells, and development of thrombosis (Tyagi, et al., 2005), impaired endothelial function (Stuhlinger, et al., 2001) and the activation of inflammatory pathways that are in the raw to changes in cellular oxidation-reduction states (Weiss, et al., 2003).It was originally proposed that homocysteinemia promotes the accumulation of S-adenosyl homocysteine, a potent inhibitor of cellular desoxyribonucleic acid methylation (Zou, 2007). The hypomethyla tion of DNA could affect promoter activity resulting in remodelling of chromatin granule and changes in the recording 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 indeed increased proliferation of endothelial cells. Homocysteine directly inhibited the activity of DNA methyltransferase I (DNMT1) by 30% (Jamaluddin, et al., 2007). Furthermore, homocysteine lessen 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.Review modes of analysing homocyste ine and differences between measurements. 2800 wTotal homocysteine, tHcy, is the sum of all chemical species of homocysteine that is present in the blood (Figure 4) (Ueland, et al., 1993 Refsum, et al., 2004). As mentioned earlier, only a small portion of the tHcy comes from free homocysteine, a large portion comes from mixed disulfides and protein-bound homocysteine. All the checkouts used to quantify tHcy cleave and convert all these forms into a iodin species by adding a reducing mover, therefore what is measured is the reduced form. rake for homocysteine determination is collected in tubes. If plasma homocysteine is to be measured, the tube contains anticoagulants like EDTA, heparin or sodium citrate. If serum homocysteine levels are to be determined, then the samples are allowed to coagulate, which takes thirty minutes and not more, to reduce the risk of increased homocysteine levels coming from the cellular fraction of blood (comprised of red blood cells and platelets). inc rease release of tHcy from cells also makes it necessary to centrifuge and separate blood components inwardly 30 minutes of collection. Centrifugation eon and speed are relatively low (5 min and 300g respectively, for example) and is performed at 4 C. The supernatant (serum/ plasma fraction) that is collected may be analyzed right off, or stored for 2-3 weeks in the refrigerator or fixed for 2-3 months (Homocysteine, 2008). It is possible to store the samples once processed because the homocysteine levels have been shown to be unchanging after separation of plasma/serum from the cellular fraction (Refsum, et al., 2004). there are reports showing that tHcy in serum can remain fixed for several years if stored at -70 C.Scheme for the steps followed in the determination of total homocysteine from blood serum or plasma. (Hcy-SR, Hcy-mixed disulfide EC, electrochemical detection LC, still chromatography CE-LIF, capillary electrophoresis with laser-induced fluorescence detection Ab, antibody EIA, enzyme immunoassay FPIA, fluorescence polarization immunoassay MS-MS, in tandem nap spectrometry). Figure from Refsum et al., 2004.The first step is to generate free homocysteine by chemical reduction of disulfide bonds using dithioethritol, sodium borohydride, n-tributyl phosphine, 2-mercaptoethanol and phosphine tris (2-carboxyethyl) phosphine, which is water-soluble (Jacobsen, 1998). After the reduction, homocysteine is disjunct from the cysteine, glutathione and cysteinylglycine, low molecular weight thiols, by HPLC. Homocysteine can also be derivatized for heavy weapon chromatography determination/ HPLC or subjected to immunoassay (Jacobsen, 1998 Refsum, et al., 2004).The development of the mannerologies for determining total homocysteine started in the 1980s. There are many variants of the regularityologies, but they can be categorized into three general methodologies (1) chromatographic, (2) enzyme immunoassays and, (3) enzyme cycling.Chromatographic methodsThe earliest methods used were the chromatographic methods, of which high pressure liquid chromatography (HPLC) is more used compared to gas chromatography (GC). Variations in the methodology include the combination of liquid chromatography (LC) with mass spectrometry (MS) and GC with MS. The equipment and the skill requirements for these techniques have limited their use mainly in research laboratories.In the HPLC method, the reduced samples are deproteinated and then directly separated on an ion exchange column. The separated homocysteine molecules undergo derivatization with ninhydrin (a chromophore for colorimetric quantification), or fluorescent compounds (e.g. fluorescamine, SBD-F). Alternatively, the reduced homocysteine molecules are first derivatized before separation through reversed-phase HPLC. 6-Aminoquinolyl-n-hydroxylsuccinimidyl carbamate (AQC) is a fluorophore that gives higher sensitivity when used to derivatize cystathionine and homocysteine (Seo, 2005).The derivatized samples are then passed through an ion exchange column. The distance of time that it takes for the sample to go through the column is called the retention time. The samples loss the column pass through a detector, which could be a UV absorbance detector (spectrophotometer), a fluorescence detector or a mass spectrometer which is a powerful detector. The detectors quantify the samples, and send signals to a recorder which produces the chromatogram, where quantities can be seen as full stops.An internal standard of known quantity is used in the determination of the actual quantity of the sample. Normally, the internal standard should have a retention time that is the same as homocysteine. Internal standards are added to the sample tubes and derivatized together with the reduced homocysteine. To quantify homocysteine, the tHcy peak area is compared with the peak area of the standard eluted at the same retention time.Although there are many advancements in the HPLC techn ology, tHcy quantification using this method is relatively slow, time-consuming, requires very studious sample preparation and handling, and skilled technicians. Sample pre-treatment requires 30-60 minutes, while an HPLC run takes 10-30 minutes. Samples are run sequentially, which makes HPLC not suitable for high throughput determinations.Some laboratories use gas chromatography or gas chromatography with mass spectrometry to get more sensitive results. The principles behind GC and HPLC determination are similar, except that in GC, the samples are vaporized and passed through a gas before elution from a column. Similar to HPLC, GC is also more time-consuming compared to other methods.Homocysteine of modify blood samples have been determined using reversed phase HPLC (Bowron, et al., 2005). In this technique, blood samples were collected in tubes that contain potassium EDTA. After mixing gently, the blood was spotted on filter paper cards that are used in standard neonatal screenin g. The cards were air-dried and stored at populate temperature until analysis. Six-mm discs of the cards were punched out, and incubated in tubes with an internal standard and a deproteinization agent. The produced thiols were then derivatized for fluorescent detection, and separated by reversed phase HPLC. The resulting measurements showed that the method was linear up to a homocysteine concentration of 140 mol/L, in comparison with HPLC of plasma homocysteine which showed a linearity up to 200 mol/L. The coefficient of variation inwardly a batch of measurement was 5% and within batches was 8%. HPLC of plasma samples was only 1% within batch and 4% between batches. computer memory of the dried blood sample spots for more than 24 hours in room temperature decreased the homocysteine concentration, probably due to the hemolysis of whole blood during drying. The hemolysis could have been accompanied by the denaturation of enzymes that release homocysteine from blood cells. Overall, although the use of dried blood samples offers flexibility in sampling and storage, the assay had lower precision compared to HPLC. It may not be able to quantify dried blood homocysteine in newborns because its lower limit of detection is above the levels that some healthy neonates have. The method can be used in the detection of homocysteinuria.A more rapid protocol for tHcy measurement is liquid chromatography electrospray ionization-tandem mass spectrometry (LC/MS/MS) (Gempel, et al., 2000). It removes the derivatization step and can perform 400 analyses daily. kind samples are applied to filter cards that are used for neonatal homocysteine screening. The cards are incubated in the presence of a reducing agent and a deuterated internal standard. The tHcy is then extracted in an acetonitrile solution and loaded on a cyano column. Eluates are ionized with an ion spray device before press release through a mass spectrometer. The procedure makes use of an autosampler, which hasten s the analysis. The advantage of the method is the removal of the derivatization step, because the mass spectrum is determined directly. Impurities of the samples are removed by red them through the cyano column. Since the procedure takes only 3 minutes to finish 1 sample, more samples can be processed in less time compared to HPLC (Gempel, et al., 2000).An innovation in a high throughput liquid chromatography-tandem mass spectrometry method is the use of a 96-well plate format that skips precipitation, centrifugation and derivatization. Plasma samples, calibrator and control were mixed well in 96-well plates. From each well, an aliquot was transferred to another plate with sodium hydroxide solution (diluent) and dithioethritol (or another suitable reductant). After organic shaking and a short incubation period, the plate was placed in the autosampler for LC-MS/MS analysis. Comparison of the results with those obtained by HPLC showed that the LC-MS/MS method gave consistently high er homocysteine readings, which was attributed to the use of different standards by the two methods. The biggest advantage of the new method over HPLC was the expense, because LC-MS/MS reduced the costs for material and manpower by more than 50% (Arndt, et al., 2004).Enzyme ImmunoassayThe immunoassay-based tests for homocysteine were developed in the 1990s. Most assays hire a mouse monoclonal antibody antibody against S-adenosylhomocysteine (SAH), which is formed when adenosine and S-adenosylhomocysteine hydrolase are added to reduced total homocysteine of the plasma/serum sample. The earliest report on the procedure involved the use of flouresceinated SAH as tracer. This method is fluorescent polarization immunoassay or FPIA. After SAH is generated, it is mixed with a fluorescein-tagged SAH. The mixture is coated onto a microtiter plate, then made to bind with mouse anti-SAH (Figure 6). When antibody-SAH bind, the changes in fluorescent polarization of the SAH-tracer are detected and serve as basis for the quantification after a standard curve of known homocysteine calibrators is constructed (Shipchandler and Moore, 1995).The newer versions of enzyme immunoassays were performed on microtiter plates and used the formation of colored complexes to measure absorbance which was used as the basis of the quantification (Figure 7) (Frantzen, et al., 1998). After the conversion of reduced tHcy to SAH, another enzyme is added to the mixture to hydrolyze the excess adenosine. After reaction with a monoclonal anti-SAH antibody, a specific anti-mouse-antibody is added, which forms a colored product that can be detected spectrophotometrically for quantification.The immunoassays are simpler to perform compared to chromatography because they do away with the derivatization step and the valuable equipment needed in chromatographic separation. The method has been proven to be rapid and precise. The coefficient of variation between and within assay is 8% and less than 6% res pectively, and the results also correlated well with those obtained using HPLC. The advantage of the immunoassay over HPLC is speed and ease of determination (Frantzen, et al., 1998). Many samples can be run simultaneously on several microtiter plates, which make the immunoassay ideal for routine screening. Furthermore, the technique can be and has been fully change using robotics technology, and is super flexible and can be used on different uninflected platforms.Other similar imunoassays have been developed. In a variant of the assay, recombinant homocysteine lyase, converts homocysteine into -ketoglutarate, with the ontogenesis of ammonia and H2S (Tan, et al., 2000). A second reaction reacts H2S with N,N-Dibutylphenylene diamine (DBPDA) resulting in the formation of form 3,7-bis(dibutyl amino)phenothiazine-5-ium chloride, a highly fluorescent compound. Both highly purified recombinant homocysteine lyase and DBPDA were synthesized in the authors laboratory, which someways limi ts the utility of the procedure because their production requires skill and specialized equipment.Another assay utilizes the recombinant enzyme, methionine -lyase, and the commercially available fluorophore N,N-diethyl-p-phenylenediamine (Chan, et al., 2005). The rest of the procedure is similar to that used in typical immunoassays. The method can be used in an automated analyzer or manually, which makes it ideal for laboratories that cannot afford the expensive automated analyzers. Although the recombinant enzyme was purified only from a crude extract of E. coli, the results compared well with those that were obtained using HPLC. These results showed that the need for tiresome and careful purification procedures can be removed. However, a main drawback was the non-specificity of the enzyme for homocysteine it also reacts, although with less specificity, with cysteine which is present in larger amounts than homocysteine in the plasma. Thus, the use of pure homocysteine as calibrat or is not suitable for the assay. Instead, the recommendation is to use pooled plasma as the calibrator, but this requires the determination of the tHcy by HPLC, which complicates the procedures for small laboratories who do not have access to HPLC equipment. Moreover, the precision of the procedure is also lowered if samples are diluted, because this also reduces the amount of cysteine in the sample relative to its concentration the calibrators. The presence of cysteine is also the cause for the lower sensitivity of this assay, because at lower tHcy levels the background fluorescence due to cysteine is amplified (Chan, et al., 2005).Enzyme cyclingThe most recent method for tHcy determination is the enzyme cycling method which amplifies the detection signal to improve the sensitivity of the detection (Figure 8) (Dou, et al., 2005). Similar to the prior methodologies, all protein-bound and oxidized forms of homocysteine are first reduced to free homocysteine. Next, methionine and S- adenosyl homocysteine (SAH) are formed from the methylation of free tHcy by the action of the enzyme homocysteine methyltransferase with S-adenosyl methionine as methyl sponsor or co-substrate. The SAH so formed comes from SAM and the transmethylation reaction, and not from the free homocysteine molecules. Upon formation, SAH is hydrolyzed immediately to homocysteine and adenosine by SAH hydrolase. The homocysteine originating from SAM enters the homocysteine transmethylation reaction catalyzed by homocysteine methyltransferase to form homocysteine and methionine again, resulting in an enzymatic cycle and an increase in detection signals. The reactions caus