The metabolism of methionine - Free Essay Example

Sample details Pages: 19 Words: 5833 Downloads: 1 Date added: 2017/06/26 Category Statistics Essay Did you like this example? 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. Don’t waste time! Our writers will create an original "The metabolism of methionine" essay for you Create order 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  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 suffered 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 ÃŽÂ 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  µmol/L (males) and 10.4  µmol/L (females) are to be considered above normal and was associated with low vitamin concentrations in two-thirds of the subjects surveyed (Selhub, et al., 1999). This verified that an assessment of homocysteinemia requires the knowledge of the health status of the patient. The degree of homocysteinemia is classified based on determination fasting levels of serum homocysteine. Currently, a plasma tHcy level of 15  µmol/L is considered as normal (Refsum, et al., 2004). In healthy adults with good folate and B vitamins status, the upper reference limit is 12  µmol/L. Accordingly, homocysteinemia is classified as moderate (15-30  µmol/L), intermediate (30-100  µmol/L) and severe (100  µmol/L). This categorization is necessary to come up with a decision regarding the treatment of the condition. 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 (BÃÆ' ¸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  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 ÃŽÂ 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 hi gh 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. 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 4 ° 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 s table 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  °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  µmol/L, in comparison with HPLC of plasma homocysteine which showed a linearity up to 200  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., 2 000). 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 ÃŽÂ ±-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 ÃŽÂ 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 calibrator, but this requires the determination of the tHcy by HPLC, which complicates the procedures for small laboratories who do not have access to HPLC equipment. Moreover, the precision of the procedure is also lowered if samples are diluted, because this also reduces the amount of cysteine in the sample relative to its concentration the calibrators. The presence of cysteine is also the cause for the lower sensitivity of this assay, because at lower tHcy levels the background fluorescence due to cysteine is amplified (Chan, et al., 2005). Enzyme 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 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. 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  µL 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  µmol/L can be detected. The assay can be used to measure homocysteine concentrations of 3 to 50  µmol/L, which is lower than that of other procedures. However, 50  µmol/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 ÃŽÂ ²-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 ÃŽÂ ¼mol/L of homocysteine. Compared to the earlier enzyme cycling technique, this method was linear from 1 to 100 ÃŽÂ ¼mol/L, meaning that it can detect homocysteine concentrations as high as 100  µmol/L albeit with a maximum deviation of T Polymorphism and Risk of Coronary Heart Disease: A Meta-analysis. JAMA, 288 (16), pp.2023-2031. Kluijtmans, L., Young, I., Boreham, C., Murray, L., McMaster, D., McNulty, H. et al. [2003] Genetic and nutritional factors contributing to hyperhomocysteinemia in young adults. Blood , 101, pp.2483-2488. Koehler, K., Baumgartner, R., Garry, P., Allen, R., Stabler, S. and Rimm, E. [2001] Association of folate intake and serum homocysteine in elderly persons according to vitamin supplementation and alcohol use. American Journal of Clinical Nutrition, 73, pp.628-637. 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Menlo Park, The Benjamin Cummings Publishing Company, Inc. McKee, T. and McKee, J. [2004] Biochemistry: The Molecular Basis of Life Third Edition. Columbus, The McGraw-Hill Companies. Miller, J., Nadeau, M., Smith, J., Smith, D. and Selhub, J. [1994] Folate-deficiency-induced homocysteinaemia in rats: disruption of S-adenosylmethionines co-ordinate regulation of homocysteine metabolism. Biochemistry Journal, 298, pp. 415-419. Min, H. [2009] Effects of dietary supplementation of high-dose folic acid on biomarkers of methylating reaction in vitamin B12-deficient rats. Nutrition Research and Practice, 3 (2), pp.122-127. Moens, A., Vrints, C., Claeys, M., Timmermans, J., Champion, H, and Kass, D. [2008] Mechanisms and potential therapeutic targets for folic acid in cardiovascular disease. American Journal of Physiology- Heart and Circulatory Physiology, 294, pp.H1971-H1977. Refsum, H., Grindflek, A., Ueland, P., Fredriksen, A., Meyer, K., Ulvik, A., et al [2004] Screening for serum total homocysteine in newborn children. Clinical Chemistry, 50, pp.10-25. Refsum, H., Smith, D., Ueland, P. N., Clarke, R., McPartlin, J., Johnston, C., et al. [2004] Facts and recommendations about total homocysteine determinations: an expert opinion. Clinical Chemistry, 50, pp.3-32. Riddell, L., Chisholm, A., Williams, S., and Mann, J. [2000] Dietary strategies for lowering homocysteine concentrations. American Journal of Clinical Nutrition, 71, pp.1448-1454. Roberts, R., and Roberts, W. [2004] Performance characteristics of a recombinant enzymatic cycling assay for quantification of total homocysteine in serum or plasma. Clinica Chimica Acta, 344 (1-2), pp.95-99. Selhub, J. [1999] Homocysteine metabolism. Annual Review of Nutrition, 19, pp.217-246. Selhub, J., and Miller, J. 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Quotes on Funny Thoughts to Make You Smile

Have you ever had a funny thought pop into your brain? Maybe you put a couple of things together and realized it told a truth in a quirky way. Our brains often have a natural comedian deep inside and you might even make yourself laugh. Then when you stop and think about thinking, things start getting a little weird. Famous thinkers and witty people might seem to have more funny thoughts, but we have the benefit of only seeing their really good material. You might be inspired by a humorous remark, or hearing about an incident that causes you to giggle. Share your funny thoughts with others and see how you create a riot of laughter around you. If you need some help, read these funny thoughts of well-known people. Will Rogers Everything is funny, as long as its happening to somebody else.   Robert  Frost The brain a wonderful organ.  It starts working the moment you get up in the morning and does not stop until you get into the office. Charles Schulz Sometimes I lie awake at night, and I ask, Where have I gone wrong? Then a voice says to me, This is going to take more than one night. Friedrich Nietzsche All things are subject to interpretation whichever interpretation prevails at a given time is a function of power and not  truth. Miguel de Cervantes Sanity may be madness but the maddest of all is to see life as it is and not as it should be. Norm Papernick Those who can laugh without cause have either found the true meaning of happiness or have gone stark raving mad. Ethel Barrymore You grow up the day you have your first real laugh — at yourself.   Reba McEntire To succeed in life, you need three things: a wishbone, a backbone, and a funnybone.   Isaac Asimov People who think they know everything are a great annoyance to those of us who do. Abraham Lincoln No man has a good enough memory to be a successful liar. Oscar Wilde The public is wonderfully tolerant. It forgives everything except genius. The public have an insatiable curiosity to know everything, except what is worth knowing.   Victor Borge Santa Claus has the right idea - visit people only once a year. Edward Gibbon Beauty is an outward gift which is seldom despised, except by those to whom it has been refused. Quentin Crisp The trouble with children is that theyre not returnable. George Bernard Shaw Love is a gross exaggeration of the difference between one person and everybody else. Joe Namath Until I was thirteen, I thought my name was shut up. Mark Twain While the rest of the species is descended from apes, redheads are descended from cats. Ogden Nash Middle age is when youve met so many people that every new person you meet reminds you of someone else. The Cable Guy You know what the trouble about real life is? Theres no danger music.

Record Management Free Essays

string(34) " of being complete and unchanged\." Running head: Records Management Name Instructor Date of submission Executive Summary An effective records management program is an integral part of an organization’s effective business operations. Organizations must consider records management requirements when implementing the system management strategies or whenever they design and augment an electronic information system. Organizations are required by law to â€Å"make and preserve records containing adequate and proper documentation of the organization, functions, policies, decisions, procedures, and essential transactions of the organization. We will write a custom essay sample on Record Management or any similar topic only for you Order Now This legal requirement applies to electronic records kept by the organizations as well. Organizations that do not consistently adhere to standard records management practices run the risk of not having records that can be depended upon in the course of subsequent business transactions or activities. This paper focuses on the various methodologies that organizations can implement in order to develop ECM/ERM strategies that are backed with electronic signature systems. It also gives an insight into the various ways through which organizations can implement good IT practices to complement or parallel existing records management practices. In systems implemented in line with ECM/ERM guidelines, developing the most efficient systems will form the core of organizational success. This will be achieved by making electronically signed records the core of organizational IT systems. The organizational IT professionals will come to terms with the fact that signatures are an integral part of the records they keep. If the records need to be preserved, whether for a short duration of time or permanently, then the organization is required to promote integrity of its records by electronically signing them in scheduled series. Additionally, this paper discusses the general principles that govern application of electronic signature technology in organizations. Organizations can accomplish electronic signatures through the use of different technologies such as Personal Identification Number (PIN), smart cards and biometrics. However, some organizations can decide to apply additional technology specific-record management systems. Introduction Records Life Cycle vs. System Development Life Cycle According to Adam (2008), the terms â€Å"records life cycle† and â€Å"system development life cycle† are significant concepts that are often confused in information technology and records management discussions. Records life cycle: The records life cycle refers to the life span of a record from the time it is created or received to its eventual disposition. The process is usually carried out in three main stages: creation, maintenance and use, and eventual disposition (Sampson, 2002). Majorly, this paper focuses on information creation stage since the electronic signature record is created at the initial stage of the records life cycle. The second stage, maintenance and use, is the part in the records life cycle in which the record is maintained at the organizational level while in active use, or is maintained when not in frequent use. The final stage of the records life cycle is disposition, which marks the ultimate fate finish to the record. Most organizational records are categorized as having either a â€Å"temporary† or â€Å"permanent† disposition status (Addey, 2002). Temporary records are held by organizations for stated periods before they are destroyed or deleted. On the other hand, permanent records are initially held by organizations before they are eventually transferred to state and other involved agencies. The eventual disposition of the electronically-signed records is subject to debate between the involved agency and the statutory bodies, in which some organizations may be authorized to dispose some of the records. System development life cycle: The â€Å"system development life cycle† gives a description of the developmental phases that an electronic information system entails. These phases typically include initiation, definition, design, development, deployment, operation, maintenance, enhancement, and retirement. The most important steps in all this are the definition, development, and refinement of the data model, which mostly involves treatment of the records being created or managed (Stephens, 2007). Information systems are developed according to system development methodologies, including those that organizations use to implement the electronic signature as required by the statutory bodies; which govern production and augmentation of existing records. The records life cycle usually exceeds the system development life cycle. When it does the organization involved needs to retain the particular record for a period of time longer than the life of the electronic information system that generated the electronic signature. However, this presents special challenges, such as maintaining the integrity of record in case of system migration. Background Characteristics of Trustworthy Organizational Records Reliability, authenticity, integrity, and usability are the features used to describe trustworthy records from a records management perspective. An organization needs to consider these features when laying implementation plans for ERM programs; such that it can meet its internal business and legal needs, as well as external regulations (Boiko, 2002). The degree of effort that an organization puts into ensuring that these characteristics are attained depends on the organizational business strategies and the structure of the market environment. Transactions that are of great importance to the organization require greater assurance level than those usable with transactions of less criticality to the organization Reliability: A reliable record is one that carries contents that can be trusted as a whole and actual representation of the transactions, activities, or facts to which it refers and can be relied upon in the subsequent transactions Authenticity: An authentic record is one that is proven to be what it purports to be and to have been created or sent by the person who purports to have created and sent it. A record should be created at the point in time of the transaction or incident to which it relates, or soon afterwards, by individuals who have direct knowledge of the facts or by instruments routinely used within the business to conduct the transaction (Wiggins, 2007). To demonstrate the authenticity of records, organizations should implement and document policies and procedures which control the creation, transmission, receipt, and maintenance of records to ensure that records designers are authorized and identified and that records are protected against unauthorized addition, deletion, and alteration. Integrity: The integrity of a record refers to the state of being complete and unchanged. You read "Record Management" in category "Essay examples" It is essential that a record be protected against changes without signed permission. Records management policies and procedures should specify what, if any, additions or annotations may be made to a record after it is created, under what circumstances additions or annotations may be authorized, and the people authorized to make the changes. Any authorized annotation or addition to a record made after it is complete should be explicitly indicated as annotations or additions. Another aspect of integrity is the structural integrity of organizational records. The structure of a record refers to its physical and logical format; as well as the relationship between the data elements contained in the record. Failure to maintain the structural integrity of organizational records can easily impair reliability and authenticity of the record Usability: These are records that can be located, retrieved, presented, and interpreted. In any subsequent retrieval and use, the record needs to be directly connected to the business activity or transaction which produced it. It should be possible to identify a record within the context of broader business activities and functions. The connection between records which document a sequence of activities should be maintained. These contextual linkages of records should carry the information needed for an understanding of the transaction that created and used them. Preserving Trustworthy Records For a record to remain reliable, authentic, with its integrity maintained, and useable for as long as the record is needed, it is necessary that it’s content, context and sometimes structure is maintained. A trustworthy record preserves the actual content of the record itself and information about the record that draws relation to the context in which it was first designed and used. Specific contextual information will vary depending upon the business, legal, and regulatory requirements of the business activity (e. g. , issuing land use permits on Federal lands). It is also necessary to preserve the structure of the record, as well as the content arrangement. Failure to preserve the content structure of the record will affect its structural integrity. That, in turn, undermines the record’s reliability and authenticity; which is of great essence. There are special considerations when dealing with the preservation of the content, context, and structure of records that are made possible by Electronic Record Management through electronic signatures: †¢ Content: The electronic signature or signatures in a record are part of the content. They give a clear indication on who signed a record and whether that person gave approval for the record content. In organizations, multiple signatures are an indication of initial approval and subsequent approvals. It should be understood that in ERM, signatures should accompanied by dates and other identifiers such as organizational titles. All of these peripherals are part of the content of the record and needs to be kept well. Lack of this information seriously impairs the reliability and authenticity of a record †¢ Context: Some electronic signature technologies are centered on individual identifiers which are not embedded in the record content, trust paths, and other means used to create and verify the validity of an electronic signature. This information is not inclusive of the record content but is nevertheless significant. It provides contextual support to the record since it provides evidence that can be used to support the authenticity and reliability of the record. Lack of these contextual records seriously impairs subsequent attempts to verify validity of the organizational records. †¢ Structure: Preserving the structure of a record means that the physical and logical formats of a record are well drawn. In doing this, organizations must ensure that the physical and logical formats of the record elements remain intact physically and logically. An organization may find it necessary to maintain the record structural form through the use of an electronic signature. In that case, the organization is required to preserve both the hardware and software that created the electronic signature, which can either be encryption algorithms or chips. This ensures that the electronic record can be revalidated at a later time when required Advantages and disadvantages of using ECM/ERM systems in organizations The main advantage of organizations using ECM/ERM approaches in organizations is the fact that it offers the platform to verify the validity of records. There are various approaches that organizations can use to achieve trustworthiness of electronically-signed records within their systems over time. This requires that organizations choose an approach that is applicable, fit for their particular line of business; as well as risk assessment The first approach may involve an organization deciding to maintain adequate documentation of its records’ validity. This involves maintaining of adequate documentation of the records such as, trust verification of records gathered at or near the time of record signing. This record keeping approach enables organizations to retain contextual information through an adequate document processes carried out at the time the record was electronically-signed, along with the electronically-signed record itself. The additional contextual information is then retained for as long as the electronically-signed record is retained. Thus the agency preserves the signature’s validity and meets the adequacy of documentation requirements by retaining the contextual information that documented the validity of the electronic signature at the time the record was signed. Maintaining adequate documentation of validity gathered at or near the time of record signing may be preferable for records that have permanent or long-term retentions since it is less dependent on technology and much more easily maintained as technology evolves over time (Rockley, 2003). However, using this approach, the signature name may not remain readable over time because of bit-wise deterioration in the record or as a result of technological obsolescence. Agencies must ensure that for permanent records the printed name of the signer and the date when the signature was executed be included as part of any human readable form (such as electronic display or printout) of the electronic record. Similarly, an organization may opt to maintain the capacity to re-validate digital signatures. The re-validation approach demands that an organization retains the ability to revalidate the digital signature, together with the electronically-signed record itself. The information necessary for revalidation (i. e. , the public key used to validate the signature, the certificate related to that key, and the certificate revocation list from the certificate authority that corresponds to the time of signing) must be retained for as long as the digitally-signed record is retained. Both contextual and structural information of the record must be retained. This is of benefit to the organization since it can review it records over time effectively (Jenkins, 2005). However, this approach of record keeping is potentially burdensome, particularly for records that are digitally signed records with long retention requirements. Conclusions Record keeping is consistently becoming a priority for many organizations with advancement in technology. The challenging part is keeping up with the drastic options that are being launched within short periods. As discussed in this paper, the most efficient method of managing organizational records is through the adoption of Electronic Content Management or Electronic Record Management systems (Halvorson, 2009). This is mostly achieved by electronically signing records depending on their importance and usage in the organization. In doing this, organizations are able protect the reliability, authenticity, integrity, and usability, as well as the confidentiality, and legitimacy of their records. When implementing electronic signature technology, organizations are expected to accord special consideration to the use of electronic signatures in electronic records that preserve organizational legal rights. This is based on the fact that long-term temporary and permanent electronically signed records have greater longevity than typical software obsolescence cycles, it is certain that organizations will be required to migrate those records to updated versions of software to maintain access to the records (Hackos, 2002). The software migration (as opposed to media migration) process may invalidate the digital signature embedded in the record. This may adversely affect an agency’s ability to recognize or enforce the legal rights documented in those records. References Adam, A. (2008). Implementing electronic document and record management systems. Boca Raton: Auerbach Publications. Addey, D. (2002). Content management systems. Birmingham: Glasshaus, cop. Boiko, B. (2005) Content Management Bible. Hoboken: John Wiley Sons. Hackos, T. (2002). Content management for dynamic web delivery. New York: John Wiley Sons. Halvorson, K. (2009). Content strategy for the web. Indianapolis: New Riders. Jenkins, T. (2005). Enterprise content management: what you need to know; [turning content into competitive advantage]. Waterloo: Open Text Corp. Rockley, A. (2003). Managing enterprise content: a unified content strategy. Indianapolis: New Riders. Sampson, K. (2002). Value-added records management: protecting corporate assets, reducing business risks. West port: Quorum Books. Stephens, D. (2007). Records management: making the transition from paper to electronic. Alexa: ARMA. Wiggins, B. (2000). Effective document management: unlocking corporate knowledge. Aldershot: Gower. How to cite Record Management, Essay examples

Human rights in tibet Essay Example For Students

Human rights in tibet Essay Human rights in tibet Essay In 1949, newly communist China sent 35,000 troops to invade Tibet (Tibet Support Group UK 1). The year after that a treaty was made. The treaty acknowledged sovereignty over Tibet, but recognized the Tibetan governments autonomy with respect to internal affairs. The Chinese violated the treaty on many occasions, though. This lead to the National Uprising in 1959, and after that, the exile of the Dalai Lama, spiritual leader of Tibet, and many governmental leaders (Office of Tibet 1). During and after the Chinese invasion of Tibet, there was mass destruction of Tibetan buildings. Over 6,000 monasteries, temples and other cultural and historic buildings were destroyed. The contents of the thousands of buildings destroyed was taken back to China and sold (Office of Tibet 3). The Tibetan people tried to rebuild their country, but the political leader who tried to start the recuperation policy was forced to resign from office shortly after (Office of Tibet 2). During the National Uprising alone 87,000 Tibetans were killed. Another 430,000 died in the fifteen years of guerilla warfare that followed. Sources also say that up to 260,000 have died in prisons and in labour camps (Tibet Support Group UK 3). Also, 200 unarmed civilians were killed during non-violent protests between 1987 and 1989. Overall 1,200,000 Tibetans have died since 1959. That is roughly one fifth of the population of Tibet (Office of Tibet 1). That does not include all of the deaths of Tibetans during the Chinese invasion, and all of those who froze to death trying to flee Tibet. The Tibetan people who survived the killing were denied what most consider primal freedoms. One of which is freedom of religion. Tibetan religious practice was forcibly suppressed until 1979 (Tibet Support Group UK 4). Also, in early 1989, Chinese authorities undertook a campaign to tighten control over religious practice. This campaign intensified the crackdown on the pro-democracy movement (Churchward 1). The campaign affected Catholics, Protestants, Muslims, and Buddhists. Another religious suppression on the part of the Chinese is that they have banned public celebrations of Tibets Great Prayer Festival because China believed that it would lead to nationalist demonstrations (Churchward 2). Now all Tibetan churches, mosques, and temples must be registered, and to do so, they must meet official standards (Churchward 1). Also, the only people permitted to perform religious duties, according to Document #19, are those who after examination are deemed politically reliable, patriotic, and law-abiding (Churchward 3). On May 23, 1951, the 17-point agreement was enacted. It stated that the Chinese would not interfere with Tibets existing system of government and society. China never kept those promises, though, and in 1959 reneged on the treaty altogether (Tibet Support Group UK 1). China renamed two of Tibets three provinces as part of China. The remaining province was named Tibet Autonomous Region (TAR), but there is no evidence to support Chinas claim that TAR is autonomous. All of TARs local legislation is subject to approval of the central government in Beijing, and all local government is subject to the regional party, which in Tibet has never been run by a Tibetan (Tibet Support Group UK 3). The Tibetan people also do not have the right to a fair trial. In Tibet non-violent opposition to the Chinese is met with charges of counter revolution and the offender is classed an enemy of the people. Chinese authorities regard anyone arrested for nationalist activities as undeserving of the protection of the law, because they have lost their right to be considered part of the people (Lawasia and Tibet Information Network 31). The Tibetans suspected of opposing the Peoples Republic of China (PRC) have been held as political prisoners for lengthy periods, decades for some. The Great Gatsby - The Characters Essay The US government presented China with a list of 108 political prisoners in 1993. Nine months later China finally responded. They listed 51 as can not be found and did not even say where the other prisoners were being held. The charges against them are often unknown, some were even sentenced or executed without a trial. Thousands of Tibetans are in custody for political reasons, but accurate figures are impossible to find due to Chinas reluctance to provide any information and their .