Role of Elevated Levels of Homocysteine in Blood Plasma

Role of Elevated Levels of Homocysteine in Blood Plasma 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 Â µmol/L. Elevated levels of homocysteine can be reduced with folate and B vitamins supplementation, but this intervention does not work in patients who have suffere d stroke or heart attacks. 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. 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 h omocysteine 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 homocysteines inhibition of DNA methylation in the promoter region in the gene of an activator of the cell cycle (Jamaluddin, et al., 2007). 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). 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. 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 ÃŽ ³-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 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 fr om 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). 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 Â µmol/L (males) and 10.4 Â 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 Â 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 Â µmol/L. Accordingly, homocysteinemia is classified as moderate (15-30 Â µmol/L), intermediate (30-100 Â µmol/L) and severe (> 100 Â µmol/L). This categorization is necessary to come up with a decision regarding the treatment of the condition. 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. 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 S mith, 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). 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 (BÃ ¸naa, 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. 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 Â 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 ÃŽ ²-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 homocys teine 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. 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. 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. 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. 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 signa ls 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 Â µmol/L, in comparison with HPLC of plasma homocysteine which showed a linearity up to 200 Â µmol/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., 20 00). 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). 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). 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. Other similar imunoassays have been developed. In a variant of the assay, recombinant homocysteine lyase, converts homocysteine into ÃŽ ±-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 ÃŽ ³-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 cali brator, 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 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 reacti ons caus

An Indivisible Peace

It is very inevitable that somewhere in our lives, we have been touched by a special bond called â€Å"friendship†.   That special bond might happen in the most unusual time and place. It might even be connected not just with love, but also with envy and selfishness.A Separate Peace is a timeless novel which depicts a tale of two best friends named Gene and Finny or Phineas who are both away from their homes to attend boarding school during World War II. Since both of them are experiencing the same hardships, pressures and turmoils that come with boarding school, they both create a unique bond and help each other survive tough times. They help each other develop independence and courage while trapped in a boarding school that's full of distressing things.The plot revolves around Devon School which is said to be the most beautiful school in New England because it helps its male students master both scholarly and athletic facets of their lives. Devon School is a boarding schoo l which has an atmosphere of privileged students.The students who are accepted in Devon both possess class and money. It is very usual that a school is just a place for educational achievements and at the same time, personality growth. But Devon isn't just an ordinary   institution for it's a place wherein the boys get the chance to be carefree without worrying about their reputations being ruined.The novel is set on World War II, a very crucial point for the boys because it is the time wherein they'll choose which certain military branch will they enlist in. The boys aren't really forced to enlist in the military but during that time, being involved in the war is truly a great status symbol for it proves the courage of a man. Both Gene and Finny possess contrasting personalities.Gene is a man who excels in his academic and on the other hand, Finny is excellent in sports. The state of being a pacifist also lies in Finny's nature. Although Gene's character isn't that bad, it is Fin ny who   is well-adored by their teachers and schoolmates. He doesn't have enemies and a lot of people look up to him and want to be in his shoes for his extraordinary skills in sports. Gene is the type of man who runs rebellious things inside his head. When they both go to the beach, Finny told Gene that they're now â€Å"best pals† but Gene doesn't respond for a darkness in his heart is already accummulating.He is doubtful of Finny's â€Å"pure† character. He believes that Finny is just showing his sincere side because he is deeply jealous with him and his achievements and that he wants to sabotage him. Since Finny is not doing well academically, Gene doubts that Finny wants to destroy him academically by diverting his attention. With this in mind, Gene decides to get even with Finny. Gene even mentions that â€Å"[Finny] could get away with anything. I couldn't help envying him that a little, which was perfectly normal. There was no harm in envying even your best friend a little.† (Knowles 18).But what Gene thought isn't harmful turns out to be a very critical situation. Gene's grudge towards Finny is unbearable that's why while jumping on a tree during a summer day, he intentionally shook the tree which causes Finny to lose his balance and fall drastically.Although Finny already has a doubt that Gene intentionally did it, he never seriously thinks about it for he believes that his best friend cannot do it. Finny's leg become broken which disabled to him to play sports again. He somehow loses his self-confidence knowing the fact that he cannot engage himself with the one thing he's passionate with which is sports.He returns to Devon in crutches and in a pitiful state, but he never bears a grudge in his heart. Both he and Gene develops their bond more stronger. They willingly share each other's skills. Gene tutors Finny in his academic subjects while Finny teaches Gene about the foundations, tactics and techniques of sports.For a while , they forget about the war in which they are pressured to enlist to. Time comes wherein Gene is put in a mock trial for someone suspects that he's the cause of Finny's injury. Finny then realizes that his best friend actually knocked him off the tree. Fifteen years later, Finny knocked himself again off a marble staircase which causes his recovering leg to become worse again.Gene's conscience undoubtedly bothers him so in the infirmary, he visits Finny and asks for forgiveness for the monstrous crime that he has committed. Finny unconditionally accepts his plea for forgiveness and assures him that nothing will change between them. Finny dies and although Gene is shocked about his death, he still continues to enlist in the Navy. Gene then realizes the misery that he created out of his dark and jealous heart and decides to change his view of the world.The central focus of the heartwarming novel is to depict that each individual in the world is longing for his own separate peace and w ill do anything to attain it. Each individual has his own way of achieving his separate peace. In Gene's case, he attained his separate peace by knocking off Finny in the tree.Gene fears that his bestfriend will sabotage and betray him that's why he chooses to get even with him. Although he doesn't have a concrete evidence that Finny is really set to destroy him, he still continues his plan and thus, satisfying himself with Finny's downfall. Looking at Finny's case, his separate peace is to detach himself from the war and so he uses his excellence in sports to get away from the enlistment. The characters of the novel both have fears that's why they create their own defense mechanisms to hide the truth that is driving their lives.During those times, the boys in Devon also act in conformity. They do things which they think would be acceptable with their friends. Instead of listening to what their hearts really want, they listen to what their friends must say about what is acceptable a nd what is not. Of all the schoolboys, Finny is the only one who enjoys his life using his own standards. His kind-hearted and genuine character sets him apart from his friends including Gene. Finny follows everything that his heart dictates and he always sees that the things that he's doing is a heartwarming reflection of his love for life.A Separate Peace is beautifully woven to show the evils that are lurking inside a human's heart. It depicts that a human has fears and insecurities which cause him to release his â€Å"dark† and â€Å"flawed† side. On the lighter side, it is truly humbling to know that not all humans are indeed evil for there exist some who's love and light for life overpower the evil and greed of human hearts.It's clearly demonstrated in Finny's tale who has always been loving and pure. He forgives his best friend and shows him that it's never too late to change and open up his heart to the world.   Finny's sincere character was proven when he sa id to Gene: â€Å"It was just some kind of blind impulse you had in the tree there . It wasn't anything you really felt against me, it wasn't some kind of hate you've felt all along. It wasn't anything personal.When Finny dies, Gene is left to realize how his conceit killed his best friend. It's a tough time for Gene but he later gives forgiveness to himself and   moves on with his life using a sadder yet wiser perspective. He even recalls his best friend in a heartbeaking way:   â€Å"During the time I was with him, Phineas created an atmosphere in which I continued now to live, a way of sizing up the world with erratic and entirely personal reservations, letting its rocklike facts sift through and be accepted only a little at a time, only as much as he could assimilate without a sense of chaos and loss.No one else I have ever met could do this†¦.When [others] began to feel that there was this overwhelmingly hostile thing in the world with them, then the simplicity and u nity of their characters broke and they were not the same again.† (Knowles 194)A Separate Peace not only chronicles a tale of two best friends but it also has a historical context that lies beneath its story. It is known that the novel was first published in England in 1959, but it doesn't actually mean that the time of the novel is 1959. The novel focuses on the early 1940s wherein United States had proclaimed its participation in World War II. Aside from the country's participation, World War II brought out enormoue valor and patriotism in each citizen's heart.Even though some of them are not actually engaged in combat, each citizen felt When A Separate Peace was first published in the United States in 1960, the Korean War had been over for approximately seven years. Also at that time, the country's participation in Vietnam War had not yet heightened to greater proportions. The United States made it compulsary for men to enroll in the military particularly the draft which is the U.S.'s role in Vietnam in the early 1960s.A small protest happened to diminish the compulsory participation but in the end, the young antiwar protesters were forced to respond by burning their draft notices.It was also in 1960s where the youngest president of United States, John F. Kennedy, was elected. At the age of forty-three, he had defeated Vice President Richard M. Nixon. During Kenndy's term, the population was hugely comprised of adolescents and by the time they have reached college, they greatly became a dominant part of American politics and culture. They have started to doubt and question the authority of their parents' generation. In line with their scrutiny, they have also started to search their own meaning of identity which was also reflected in the novel through Gene's search of his individuality.By the end of 1960s, A Separate Peace   already praised by a wide array of conservative critics. The novel was praised in various ways. Some critics find the book's a ntiwar sentiments very moving, accurate and timely while some deeply admire the book's moral lessons especially the part wherein it showed that a sin's redemption is still possible no matter how great a sin can be.A Separate Peace is not only hailed for its moving historical contexts, but also because itemulates several valuable themes such as war, rivalry, guilt and friendship. On a certain level, the novel emulates the war topic for it concentrates the huge impact World War II has created on the male adolescents.The novel focuses on the dificulty the young men are facing with their decisions about the war. Most of them are in the verge of deciding whether to enlist themselves in the war. Some of them are even preoccupied with the thoughts of their tentative acceptance on their desired military branch.The Word War II caused great implications on Gene's generations especially the fact that most of their young teachers are away to do war-related jobs. Thus, most of the teachers left to instruct them were men between the ages of fifty and seventy. Since a huge age discrepancy lies between them, the teachers cannot directly relate with the students' mind. The students' bond immediately intensified.It is also very evident that the existence of rivalry is very dominant in the novel. All the students in Devon do their best to fit in with the â€Å"society† their friends have created.   If not trying to fit in, some of them are doing implicit things to get even with their friends. It is well portrayed in Gene and Finny's frienship wherein Gene does his best to exceed Finny.Guilt is also a very domineering theme in the novel. In the end, Gene realizes that he's living in his self-contained world which is filled with hatred and disgust for others particulary his best friend Finny.Of the mentioned themes, friendship stands out most for it is the root of everything. It depicts that friendship doesn't just curtain all the good things, but underneath the relations hip lies hidden desires and envy.Gene's experience with Finny depicts to the readers that harbouring false motives towards other people can lead to disastrous things. He also shows that it is impossible for an individual to create complete peace in his heart until he confronts the evil in his heart and let the light shine through it.He realizes that  Ã‚   â€Å"wars were not made by generations and their special stupidities, but that wars were made instead by something ignorant in the human heart.†Ã‚   It's a compelling fact that Gene is able to acknowledge that the real enemy lies within himself. He admits that his false attitude is the cause of the corruption of his entire character .The novel ends with the portrayal that â€Å"separate peace† can only be achieved when an individual learns how to fight his personal evils. What's good in this tale is that it clearly demonstrates that no matter how impure a human's heart is, there's still a chance for him to change a nd put things in its right places.Works CitedBryant, Hallman Bell. A Separate Peace: The War Within. Twayne Publishers: Boston, 1990.Knowles, John. A Separate Peace. Bantam Books: New York, 1959.McDougal-Littell. â€Å"Author Spotlight: John Knowles.† Available online at: http://www.mcdougallittell.com/lit/guest/knowles/

Medical Cannabis Should be Legal Essay - 1657 Words

In society today, many people look for a feeling of freedom, Many people go on vacation and spend money. The most common gateway for people is drug.Our American society is facing a tremendous drug problem. In order to eradicate the drug problem, a public debate is going on to find some solutions to this drug dilemma. It has been highly controversial issue whether drugs such as marijuana should be legalized or not. Some people advocate this issue and believe that legalization is the only solution left for the nation while other oppose because it will increase the numbers of drug users and drug related crimes.Marijuana is a drug that is legal in the United States.This drug is bad and cause some side effect to your brain and body.Scientists†¦show more content†¦However, drug use is a matter of supply and demand. As Bakalar as demand exists, someone is going to supply it, either legally or illegally. Supporters of legalization believe that if government regulates drugs such as cocaine and marijuana by imposing taxes, then the black market will be eliminated (Bakalar). nbsp;nbsp;nbsp;nbsp;nbsp;However, the higher the tax rate, the higher the price of the drugs, and not many users gangs who sell marijuana and cocaine under the market price. In addiction, children and teenagers will be obviously banned from purchasing marijuana and cocaine. Hence, legalization will encourage a growing criminal black market. The main benefit of marijuana legalization can be in medicine and will give patients with severe and dangerous disease a new and effective medicine to help them, Yes, marijuana can help in medicine. â€Å" Marijuana is one of the safest therapeutically active substances known.† One of marijuana’s greatest advantages as a medicine is its remarkable safety. It has little effect on major physiological functions. 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TheRead MoreShould Medical Marijuana Be Legalized?967 Words   |  4 Pagesout whether to legalize or not is medical and recreational marijuana. Many look at cannabis or marijuana as a gateway drug and many look at it as another tobacco product. I personally don’t see marijuana as a bad thing for the states to legalize. People look down on marijuana because of it only being legal in 4 states and the fact of it being a drug. If they look into the facts that s spoken in this essay of the substance it can be positive in many ways. Medical mar ijuana can be positive to our hospitals

Analysis Of The Poem Hero And Claudio Essay - 1357 Words

Hero and Claudio are the traditional romantic lovers; he falls for Hero right after meeting her before even speaking a word to her. His admiration from afar over her beauty echoes the idea of courtly love. Following his entrapment, Claudio immediately asks Benedict â€Å"didst thou note the daughter of Signor / Leonato?† (I.I.132-133). He asks his friend about the lady, before he has even properly met her; he excites himself with the prospect of courting her. He considers Hero to be the ideal woman: â€Å"Can the world buy such a jewel?† (I.I.148). His love is irrational. Claudio is already willing to pour himself into winning her over; he speaks of marriage a few moments after seeing her: â€Å"if Hero would be my wife† (I.I.161). In this way, Claudio actually matches well with Hero, even though her character is not as developed as his is; she has few lines, and her stage time is often spent in silence. Additionally, other than a few quips to Pedro disgu ised as Claudio during the masquerade, she does not express interest in Claudio until the matchmaking scene where she tricks Beatrice. In this scene, Hero describes Benedict as â€Å"the only man of Italy, / Always excepted my dear Claudio† (III.I.92-93). While giving Beatrice a fond description of Benedict, Hero also expresses her interest in her own lover. She refers to him as dear, even though the audience has not seen any interaction between the two characters. Benedict and Beatrice represent the rational love, but the sensualShow MoreRelated Analysis of Much Ado About Nothing Essay3681 Words   |  15 PagesAnalysis of Much Ado About Nothing Much Ado About Nothing illustrates a kind of deliberately puzzling title that seems to have been popular in the late 1590s (ex quot;As You Like Itquot;). Indeed, the play is about nothing; it follows the relationships of Claudio and Hero (which is constantly hampered by plots to disrupt it), and in the end, the play culminates in the two other main characters falling in love (Beatrice and Bena*censored*), which, because it was an event that was quite predictableRead MoreWilliam Shakespeare s Much Ado About Nothing3289 Words   |  14 PagesSection One Title Analysis: As an author, William Shakespeare had titles ranging from the light at heart and ethereal to the rigid and formal. The title, Much Ado About Nothing, is one such title that fits very neatly into his light at heart category. However this doesn t mean that the title doesn t reflect the story as in Shakespeare s other plays. His light at heart stories have titles that are just as reflective as his more serious titles. The title is an obvious indicator of the story that