Homocysteine, a non-protein amino acid, is an intermediate in the metabolism of methionine and biosynthesis of cysteine. It has gained prominence in the past half-century because its accumulation in the body has been linked to increased risk and occurrence of atherosclerosis and cardiovascular disease. Homocystinuria is due to errors in metabolism and homocysteinemia is attributed to polymorphisms in the genes involved in methionine metabolism and cysteine biosynthesis, and deficiencies in the nutrients folic acid, and vitamins B12 and B6. Proof of these can be found in the results of genetic and dietary studies. The clinical quantification of homocysteine has evolved through the years and routine tests are currently available. Baseline homocysteine concentration for normal adults has been identified to be between 12-15 Âµmol/L. Elevated levels of homocysteine can be reduced with folate and B vitamins supplementation, but this intervention does not work in patients who have suffered stroke or heart attacks.
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More research studies have supported the hypothesis that elevated homocysteine causes CVD and is not just a marker for the disease. However, despite the amount homocysteine research performed, many issues remain to be resolved, among which are the elucidation of the molecular mechanism of the direct action of homocysteine and the standardization of techniques for the quantification of homocysteine levels.
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. This role was further verified in patients with homocystinuria 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 then, 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., 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 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 proof 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 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 abnormality 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 and Van Holde, 1996; McKee and 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 and McKee, 2004; Mathews and 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 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 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 and McKee, 2004).The sulfhydryl moiety also forms weak bonds with nitrogen and oxygen.
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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 animals, cysteine is formed from the cleavage of cystathionine through the action of Î³-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.
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 et al., 2001; Selhub J. , 1999; Marinou, et al., 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, 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 increased homocysteine levels resulting in CVD are oxidation of low density lipoprotein, decrease in the thrombomodulin expression inhibiting the anticoagulant pathway leading to thrombosis, platelet activation and aggregation, and smooth muscle cell proliferation (Eikelboom, et al., 1999; Jamaluddin, et al., 2007).
Figure 3. Homocysteine metabolism. Figure is adapted from Durand, et al., 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 Âµmol/L (males) and 10.4 Âµmol/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 Âµmol/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 Âµmol/L. Accordingly, homocysteinemia is classified as moderate (15-30 Âµmol/L), intermediate (30-100 Âµmol/L) and severe (> 100 Âµmol/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 and Miller, 1992). Thus, when dietary methionine is high, there is a rapid conversion of methionine to SAM. The high levels of SAM promote 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.
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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 also found to impair remethylation and synthesis of SAM (Miller, et al., 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, 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, et al., 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 and 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, et al., 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, et al., 1995; Kluijtmans, et al., 2003; Koehler, et al., 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 and 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, et al., 1999). Severe hyperhomocysteinemia (homocysteine levels greater than 100 Âµmol/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 Î²-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 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.
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 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 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 sensitive to changes in cellular redox states (Weiss, et al., 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. 2800 w
Total 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 assays used to quantify tHcy cleave and convert all these forms into a single species by adding a reducing agent, therefore what is measured is the reduced form.
Figure 4. Molecular species of homocysteine that are present in plasma. Figure from Refsum et al., 2004.
Blood 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). Increased release of tHcy from cells also makes it necessary to centrifuge and separate blood components within 30 minutes of collection. Centrifugation time and speed are relatively low (5 min and 300g respectively, for example) and is performed at 4Â° C. The supernatant (serum/ plasma fraction) that is collected may be analyzed immediately, or stored for 2-3 weeks in the refrigerator or frozen for 2-3 months (Homocysteine, 2008). It is possible to store the samples once processed because the homocysteine levels have been shown to be stable after separation of plasma/serum from the cellular fraction (Refsum, et al., 2004). There are reports showing that tHcy in serum can remain stable for several years if stored at -70 Â°C.
Figure 5 presents the overlying principle behind all the current assays for tHcy determination.
Figure 5. 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, liquid chromatography; CE-LIF, capillary electrophoresis with laser-induced fluorescence detection; Ab, antibody; EIA, enzyme immunoassay; FPIA, fluorescence polarization immunoassay; MS-MS, tandem mass 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 separated from the cysteine, glutathione and cysteinylglycine, low molecular weight thiols, by HPLC. Homocysteine can also be derivatized for gas chromatography determination/ HPLC or subjected to immunoassay (Jacobsen, 1998; Refsum, et al., 2004).
The development of the methodologies for determining total homocysteine started in the 1980s. There are many variants of the methodologies, but they can be categorized into three general methodologies: (1) chromatographic, (2) enzyme immunoassays and, (3) enzyme cycling.
8.1. Chromatographic methods
The 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 length of time that it takes for the sample to go through the column is called the retention time. The samples leaving 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 peaks.
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 technology, tHcy quantification using this method is relatively slow, time-consuming, requires very careful 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 dried 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 screening. The cards were air-dried and stored at room 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 Âµmol/L, in comparison with HPLC of plasma homocysteine which showed a linearity up to 200 Âµmol/L. The coefficient of variation within a batch of measurement was 5% and within batches was 8%. HPLC of plasma samples was only 1% within batch and 4% between batches. Storage 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. Blood 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 passing through a mass spectrometer. The procedure makes use of an autosampler, which hastens 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 passing 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 thorough 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 higher 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).
8.2. Enzyme Immunoassay
The immunoassay-based tests for homocysteine were developed in the 1990s. Most assays employ a mouse monoclonal 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).
Figure 6. Scheme for determination of tHcy using fluorescence polarization immunoassay (Figure adapted from Diazyme, 2010)..
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 expensive 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% respectively, 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 automated using robotics technology, and is highly flexible and can be used on different analytical platforms.
Figure 7. Steps for the enzyme immunoassay determination of total homocysteine (Frantzen, et al., 1998).
Other similar imunoassays have been developed. In a variant of the assay, recombinant homocysteine lyase, converts homocysteine into Î±-ketoglutarate, with the evolution 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 somehow limits the utility of the procedure because their production requires skill and specialized equipment.
Another assay utilizes the recombinant enzyme, methionine Î³-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 tedious 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 calibrator 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).
8.3. Enzyme cycling
The 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 previous 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 donor 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 cause the accumulation of adenosine, which is converted to inosine and ammonia through the action of adenosine deaminase. NH3, in the presence of NADH and 2-oxoglutarate are converted by glutamate dehydrogenase to glutamate, water and NAD+ . The amount of tHcy is proportional to the concentration of NAD+ in solution, which is determined by its absorbance at 340 nm.
Figure 8. General principles behind the measurement of tHcy (or HCY) concentration by the enzyme cycling assay (Dou, et al., 2005).
The first commercially available HCY assay developed has three homogenous reagents that were developed for analyzers that can handle 3 reagents. If the analyzer can handle only 2 reagents, reagent 1 and 2 are mixed before the run (Diazyme, 2010). Only 20 ÂµL or less of sample volume is necessary. The duration of the entire test is only 10 minutes whether 2 or 3 reagents are used. This method has been tested on many analytical analyzers of different brands. The method is highly sensitive, differences in homocysteine concentrations lower than 1.5 Âµmol/L can be detected. The assay can be used to measure homocysteine concentrations of 3 to 50 Âµmol/L, which is lower than that of other procedures. However, 50 Âµmol/L is beyond the upper limit of most normal adults.
An innovation to the enzyme cycling method described previously utilizes the conversion of pyruvate to lactate and the amount of NAD+ produced (Roberts and Roberts, 2004). Bound and oxidized homocysteine is reduced, and reacted with serine to form cystathionine. Cystathionine Î²-lyase is added to the mixture to catalyze the degradation of cystathionine to homocysteine, pyruvate and ammonia. Pyruvate is reduced to lactate by the action of lactate dehydrogenase, and the concomitant oxidation of NADH to NAD+. The amount of NAD+ produced is directly proportional to the amount of homocysteine in the sample, and can be detected spectrophotometrically at 340 nm. This method can detect up to 0.31 Î¼mol/L of homocysteine. Compared to the earlier enzyme cycling technique, this method was linear from 1 to 100 Î¼mol/L, meaning that it can detect homocysteine concentrations as high as 100 Âµmol/L albeit with a maximum deviation of <10%. For tHcy concentrations from 11.4 to 39.4 Âµmol/L, the imprecision or variation was <5.4% which compares well with the previous enzyme cycling method developed by Dou and co-workers which had a total imprecision of <5% (Dou, et al., 2005).
A comparison of the attributes and the precision of the three general methods discussed will show that the HPLC method is the most precise, but it requires sample pretreatment steps that have to be performed manually, and are thus labor intensive. The additional requirement for specialized and expensive equipment makes it less suitable for clinical laboratories that perform routine homocysteine quantification.
The immunoassay methods are less expensive compared to chromatography, but still, there is a requirement for instruments that can handle four reagents or more, and specialized detectors fluorescence detectors. Moreover, immunoassays have lower precision than HPLC and enzymatic cycling tests.
Among all methods, the enzyme cycling test appears to be the best for routine homocysteine quantification. Fewer reagents are used, and samples do not need laborious pre-treatment. The technique can be used manually, with a spectrophotometer, or performed on any automated chemical analyzer. Enzyme cycling test kits are available, removing the need for manual preparation and handling of reagents that add to errors in the measurement.
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, et al., 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 and Braly, 2010). The recommendation for appropriate treatment strategies are based on the results and the indicated degree of homocysteinemia.
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, et al., 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, et al., 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, et al., 1999). Supplementation with vitamins B6 and B12 also decrease tHcy levels (Earnest, Wood and 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 and 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, et al., 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 Âµg - 1 mg/day) was found to decrease tHcy by another 7% (Malinow, Bostom and Krauss, 1999).
Serum tHcy greater than 100 Âµmol/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 Âµg-1 mg vitamin B12. If homocystinuria was due to the absence of the cystathionine Î² 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 cysteine 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.
Another area than should also be given more focus is the development of more standardized, sensitive and rapid methods of homocysteine analysis. It is also important that a single homocysteine standard should be identified, to make the comparison of results from different laboratories equivalent.
Despite the perceived uncertainty and continuing debate on 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.