Homocysteine and Endothelial Dysfunction - Case Study Example

Homocysteine and Endothelial Dysfunction Name Institution Course Instructor Date HOMOCYSTEINE AND ENDOTHELIAL DYSFUNCTION ABSTRACT Endothelial dysfunction is associated with elevated levels of homocysteine (Hcy) while presence of tocotrienol is associated with vasodilation and smooth function of blood vessels. Vasoconstriction and vasodilation processes were studied following exposure of rabbit aortic rings in an organ bath in which Tocotrienol was added and left for a 30 minute time period. Phenylephrine α-adrenergic receptor enabled vasodilation of the vessels and increased viability. On addition with 3mM Hcy, a time-dependent elevation of Homocysteine causes constriction of the arterial rings. Homocysteine-induced vasoconstriction was however inhibited by the concomitant incubation with Tocotrienol. Results showed that vasodilation of the blood vessels in the aortic rings underwent vasodilation when bathed with Tocotrienol alone. After addition of Hcy, vasoconstriction was noticed in a time-dependent fashion; the higher the Hcy-exposure time, the more intense the vasoconstrictions. Conclusion: Elevated homocysteine levels caused endothelial dysfunction while vasodilation and smooth functioning of vessels was promoted by Tocotrienol. INTRODUCTION This study investigated whether homocysteine causes endothelial dysfunction of blood capillaries and whether tocotrienol can be used to improve endothelial functions by causing vasodilation. Various laboratory studies and human trials have contested the role of homocysteine in endothelial dysfunction, with others citing a significant association while others denying it. Tocotrienol however has been shown to cause vasodilation of the endothelium by its role in the Nitrogen Oxide pathway enabling smooth functioning of blood vessels. Tocotrienol studies revealed benefits in prevention of hypertension, cholesterol formation, tumour formation and inflammation among other important pathogeneses in cardiovascular and oncology research. Presented here are the findings of this experiment, and a discussion about findings from other laboratory and human studies that have discussed the role of homocysteine and benefits of tocotrienol in various conditions. METHODS Prior the experiment, approval was sought from the Animal Care Committee at the University approved of all animal procedures. Rabbits were killed though physical technique in which a trained personnel struck the occipital region, at the base of the head, near the top of the neck. Chemical and inhalational methods of killing rabbits were avoided to ensure that there are no compromises to experimental results. Aorta from the rabbits were cut and stored in ice for preservation. The aorta arteries were then dissected from the surrounding tissue with the use of surgical equipment including scalpel, scissors and tweezers. The arteries were cleaned and cut into small-ring pieces which were then placed in an organ bath machine. Readings were presented on the graph which manifested because of tension forces from the blood vessels. The pieces were prepared for testing constriction and relaxation. The aorta pieces were left to rest in the organ bath for 1 hour and Tocotrienol was added and left to rest for another 30 minutes. 3mM Homocysteine was added to the pieces and left for 1 hour. The aorta pieces were treated with acetylcholine, a muscarinic receptor, used to stimulate the aorta endothelium to cause vasodilation. Viability of the vessels was proved by greater vasodilation. Aorta constrictions were measured using phenylephrine α-adrenergic receptor agonists. RESULTS All data are expressed as mean ±SEM. The relation between the constriction and Hcy constrictions were recorded in the graph as shown below. Relaxation responses are also expressed as a proportion of the appropriate acetylcholine-induced constriction. The results showed that vasoconstriction and endothelial dysfunction were caused by homocysteine. They also showed that vasodilation and improvement of endothelial functions of the vessels was caused by tocotrienol. NB: Error bars showed the standard deviations (SD) for the mean. DISCUSSION The data presented demonstrate that homocysteine caused vasoconstriction and oxidative stress, which led to endothelial dysfunction. Results generated from this study-1) Endothelial dysfunction was caused by homocysteine and 2) Vasodilation and improvement of endothelial function was caused by Tocotrienol, were compared and contrasted with those of other studies. The findings were similar to other studies and contrasts with that of others. Endothelial dysfunction is the impairment of the normal homeostatic characteristics of the vascular endothelium, including blood vessels relaxation that is dependent on the endothelium (Austin et al. 2004). Despite variations on what induces endothelium dysfunction, experimental studies have consistently found that elevated endothelium-mediated vasodilation impairment was triggered by deprivation of the endothelium with nitric oxide, a potent vasodilator (Austin et al. 2004). Nitric oxide is produced by the endothelial isoform of nitric oxide synthase (eNOS) in reaction to physiological impulse, and is a significant endothelium-dependent relaxation mediator (Thambyrajah & Townend 2000). According to Chambers et al (1993) endothelial damage is caused by high homocysteine levels, which affects coagulation factors and platelet function, triggering LDL oxidation. Homocysteine may impart these effects through an action on the endothelium, which is, increased oxidative stress mechanisms (Chambers et al. 1993). Homocysteine linked with Endothelial Dysfunction Lang et al. (2001) also found that elevated homocysteine is linked with endothelial dysfunction but cited that it was due to an unknown mechanism. The experiment used recordings of isometric tension and lucigenic chemiluminescence to evaluate the effects of homocysteine exposure on endothelium-dependent relaxation by isolated rabbit aortic rings and endothelium-independent superoxide anion production by cultured porcine aortic endothelial cells. Results were that homocysteine produced a significant concentration and time-dependent inhibition of endothelium-dependent relaxation. The data suggested that the inhibitory impact of homocysteine on endothelium-dependent relaxation is because of elevation in the endothelial cell intracellular levels of reduced Oxygen, which provided a likely mechanism for the endothelial dysfunction associated with hyperhomocysteinemia. Shukla et al. (2008) used diabetic and non-diabetic rabbits’ aortas as controls to test folic therapy to reduce endothelial dysfunction. The study found that plasma homocysteine increased angiopathy risk in diabetes and only a high dose of folic acid reversed the homocysteine concentration. Zhou et al. (2005) harvested coronary arteries from pigs and used a similar method as this study to investigate molecular changes in arteries treated with Hcy. The study revealed that a ginsenoside Rb1 from ginseng can effectively inhibit Hcy-induced endothelial dysfunction. Stuhlinger et al. (2001) treated aortas of bovine with DL-homocysteine, an equivalent of L-methionine, and ADMA concentration in the introduced into the cell culture medium in increasing doses and time-dependent style. Findings showed that the homocysteine-induced accumulation of ADMA was linked with reduced nitric oxide synthesis by the endothelial cells and segments of the aorta. Thus, after exposure, homocysteine inhibited DDAH enzyme activity causing accumulation of ADMA and inhibition of nitric oxide synthesis, and this explained why homocysteine impaired the endothelium-mediated nitric oxide-dependent vasodilation. Omae et al. (2013) used retinal arterioles from porcine animals to investigate whether homocysteine had an impact on endothelium-dependent nitric oxide-mediated dilation of retinal arterioles, and whether oxidative stress and distinct protein kinase signalling pathways are involved in the mediated effect. The study found that homocysteine inhibited the nitrous oxide dependent vasodilation by producing superoxide from NADP (H) oxidase, which is seemingly linked with p38 kinase. Virdis et al. (2003) evaluated the effect of hyperhomocysteinemia and angiotensin II in the structure and vascular function in mice aorta that had been exposed to methylenetetrahydrofolate reductase (Mthfr +-). The findings revealed that the mice had endothelial dysfunction of the mesenteric vessels mostly attributable to a reduced nitric oxide bioavailability triggered by oxidative excess due to disintegration of the eNOS without vascular structural changes. According to Weiss et al. (2003) oxidative stress contributed to the homocysteine’s effect of damaged vasculatures. Concentrated levels of homocysteine led to increased availability of superoxide by a biochemical mechanism that involved eNOS, and to a lesser extent, by an increased process of chemical oxidation of homocysteine and other amino thiols in the circulatory system. As a result of the increased levels of the superoxide, homocysteine-dependent changes increased in the function of cellular antioxidant enzymes. A direct clinical consequence of increased superoxide levels is the inactivation of the vasorelaxant messenger nitric oxide, which led to the endothelial dysfunction (Weiss et al. 2003). Clinical trials: Human subjects In humans, the condition hyperhomocysteinemia occurred when there was increased homocysteine concentration. Basing on the hypothesis that hyperhomocysteinemia is a major risk factor for vascular disease and venous thrombosis, Chambers et al. (1999) studied 17 healthy human volunteers of which 7 were female and 10 male, with an average age of 33 years. Within 2 hours of oral methionine administration, brachial artery flow-mediated dilation had been impaired; thus the study concluded that the nitric oxide activity on the endothelium may have been impaired leading to acute hyperhomocysteinemia in the subjects. Data’s regression analysis showed an inverse relationship existed between homocysteine concentration and flow-mediated dilation. The findings were that increased homocysteine concentration was linked with acute endothelial dysfunction, but which could be reversed with vitamin C therapy in the healthy individuals. Hassan et al (2004) found that raised homocysteine levels were toxic to endothelium and presented a risk factor to patients with cerebral small vessel disease (SVD). The trial participants included 172 SVD Caucasian patients and 172 community controls that matched them on age and sex. Results showed that average homocysteine levels were higher in patients with SVD than in controls. Using a sample of 93 patients with ischemic heart disease, Torfi et al (2005) investigated the relationship between elevated homocysteine concentration, oxidative stress and inflammation. Findings revealed that the concentration of the inflammatory markers was higher in patients with high plasma homocysteine levels compared to patients with low to normal levels. It was suggested that homocysteine was linked with low grade inflammation. Wotherspoon et al. (2006) tested the hypothesis, and found true that increased cardiovascular risk in patients with Type 1 diabetes and microalbuminuria was partly due to hyperhomocysteinemia-mediated oxidative stress which led to impaired endothelial function. The experiment involved measuring forearm blood flow, total antioxidant status, total plasma homocysteine and whole blood glutathione in 31 diabetes mellitus patients, 16 with microalbuminuria, a control of 15 non-diabetic patients. Abdulle et al (2010) studied the association between homocysteine and endothelial dysfunction markers in stroke disease and used a population of 40 patients with ischaemic stroke, controlled by 42 non-stroke patients. Fasting venous blood was taken within 24 hours, 3 days and 7 days after stroke onset and the total plasma homocysteine was measured. Endothelial dysfunction markers including i-CAM (intracellular adhesion molecule), v-CAM (vascular cell adhesion molecule-1), E-selectin (leukocyte adhesion molecule-1) and C-reactive proteins were compared between the study and control groups. Findings showed that the markers increased significantly in ischemic stroke patients during the study period, but it had no relationship with total plasma homocysteine concentrations. Laboratory studies Jin et al. (2007) explained that elevated concentrations of plasma homocysteine increased oxidative stress and reduced endothelial-dependent relaxation. The study determined whether hyper homocysteine-induced endothelium dysfunction was mediated through cessation of L-arginine cellular transport. Results showed that elevated homocysteine treatment for 6 hours increased L-arginine uptake by 34%, and decreased it by 25% after 24hr exposure. Membrane hyperpolarisation occurred during both periods, which showed that the electron facilitating arginine uptake was maintained. CAT-1’s expression, which stimulated arginine transport, was significantly reduced, whereas eNOS protein levels and basal activity remained the same. Still, nitric oxide formation was significantly reduced. The conclusion given was that reduced arginine supply may have led to eNOS uncoupling and superoxide generated, contributing to hyperhomocysteine-induced oxidative stress. Distrutti et al. (2008) found that endothelial dysfunction increased intrahepatic resistance in cirrhotic livers, and also due to formation of the vasodilators nitric oxide and hydrogen sulphide (H2S). Through studying rats that had been diet-induced with mild hyperhomocysteine, the study demonstrated that in the systemic circulation, hyperhomocysteinemia impaired vasodilation and nitric oxide production from endothelial cells. In another study Dahiya et al. (2010) fed 20 rabbits with fresh leaves of Holy Basil (O. Sanctum), which is known to have strong anti-inflammatory properties, and used 20 others as controls. After 8 days, fasting plasma samples were estimated for homocysteine and lipid profile and revealed that the homocysteine levels were decreased on rabbits supplemented with Holy Basil leaves. In a more or less similar study, Sudarshan et al. (2009) fed rabbits with a diet supplemented with 1% methionine+ 0.1% cholesterol and 5% peanut oil for four weeks. Examination of the endothelial function of the abdominal aorta was done by standard methods and revealed severe endothelial dysfunction of the abdominal aorta. Present too was eNOS, NT and GRP78 which indicated that oxidative mechanisms and inflammatory markers were present. Gurin and Subratty (2003) found that TAME-induced contractions were more prominent when rat aortic strips were pre-incubated in homocysteine. Homocysteine NOT linked with Endothelial Dysfunction Spijkerman et al. (2005) investigated the extent to which homocysteine is associated with endothelium-dependent, flow-mediated vasodilation, and whether the link was strong in individuals with diabetes, and other cardiovascular risk factors. The study population involved 608 elderly people whose brachial arteries were ultrasonically estimated for endothelium-independent nitroglycerin-mediated dilation. The study found that elevated homocysteine was not related with flow-mediated vasodilation, and no interactions between diabetes and cardiovascular risk factors were observed. The study concluded that the effect of homocysteine on endothelial function was small and further clarification of how homocysteine affects endothelial and smooth muscle cell function was needed. Hucks et al. (2004) investigated the role of homocysteine on endothelial dysfunction. The study involved use of 4-hr pre-incubated rat femoral arteries, which were exposed to racemic D, L-homocysteine and placed on a myograph. The arteries were pre-constricted with phenylephrine of which responses to acetylcholine-dependent vasorelaxation were examined. The incubations were then repeated in the presence of indomethacin and other relevant enzymes and markers. Results showed that elevated concentrations of homocysteine had no effect when added directly to the pre-constricted basally-relaxed freshly isolated vessels. The acetylcholine levels also shifted significantly 4 hour pre-incubation, with or without homocysteine treatment. The response to endothelial independent relaxation remained unchanged. Can et al (2008) performed an experiment that tested the association between homocysteine concentration and endothelial dysfunction. The study used the aortas of 30 rats weighing between 200 and 220g. The study found that there was a possible link between tHcy and decreased vascular sensitivity to endothelium-dependent vasodilation, but endothelial dysfunction was not linked with the Hcy metabolism agent. In a contrasting laboratory study, Zivkovic et al. (2013) who investigated the effects of DL-Homocysteine Thiolactone on coronary flow, cardiac contractility, and oxidative stress markers in isolated rat heart found that acute administration of DL-Hcy TLHC and different gasotransmitter inhibiters did not result to pro-oxidant ability. Although there was a little cardiodepressive result, the study suggested that the negative effects of DL-Hcy TLHC on myocardium were not associated with oxidative stress. In testing the link between endothelial dysfunction and homocysteine in 55 renal transplantation patients, Arnadottir et al. (1998) found that tHcy does not correlate directly with glomerular filtration rate, and the rate was approximately three times as high in hemodialysis patients as the control group. Although the tHcy was marked higher in the post-transplant patients than in the control group, it was explained by other factors. Arnadottir et al. (1998) concluded that post-transplant reduction in tHcy was far smaller than expected with respect to renal function. The post-transplant changes in the major tHcy biochemical determinants contributed relatively reliable data to explain the change in tHcy. Basing on the evidence that post-menopausal state was linked with increase plasma homocysteine; Roo et al. (1999) sought to test whether the elevated homocysteine was related to endothelial dysfunction. The population of the study entailed 63 postmenopausal women that were healthy but had undergone hysterectomy. Fasting tHcy was measured as free plus protein-bound homocysteine. Endothelial function was evaluated by measuring plasma concentrations of the endothelins, which are proteins that are endothelium-derived. Other factors in the assessment included the vWF factor, plasminogen activator inhibitor type 1, and the brachial artery, flow endothelium-dependent vasodilation. After adjusting for likely variables, results showed that plasma tHcy was linked to elevated levels of endothelins and increased vWF but there were no statistically significant associations between tHcy and flow endothelium-dependent vasodilation. The study concluded that some aspects of the endothelial function may be impaired by the homocysteine elevated during fasting. Therefore, future research should study whether lowering homocysteine can improve endothelial function and thus morbidity and mortality rates in post-menopausal women with cardiovascular ailments. Hanratty et al. (2001) investigated whether mild hyperhomocysteinemia was associated with endothelial dysfunction in healthy young males. Endothelial function was compared by measuring forearm blood flow in 17 males, treated with mild tHcy >10 micromol/l and 14 controls treated with low tHcy Read More