Injection Of Homocysteine Research Articles

Chronic Hyperhomocysteinemia Increases Inflammatory Markers in Hippocampus and Serum of Rats

This study investigated the effects of chronic homocysteine administration on some parameters of inflammation, such as cytokines (TNF-α, IL-1β and IL-6), chemokine CCL(2) (MCP-1), nitrite and prostaglandin E(2) levels, as well as on immunocontent of NF-κB/p65 subunit in hippocampus and/or serum of rats. Since acetylcholinesterase has been associated with inflammation, we also evaluated the effect of homocysteine on this enzyme activity in hippocampus of rats. Wistar rats received daily subcutaneous injections of homocysteine (0.3-0.6 μmol/g body weight) or saline (control) from the 6th to the 28th days-of-age. One or 12 h after the last injection, rats were euthanized and hippocampus and serum were used. Results showed that chronic hyperhomocysteinemia significantly increased pro-inflammatory cytokines (TNF-α, IL-1β and IL-6), chemokine CCL(2) (MCP-1) and prostaglandin E(2) in hippocampus and serum of rats at 1 and 12 h after the last injection of homocysteine. Nitrite levels increased in hippocampus, but decreased in serum at 1 h after chronic hyperhomocysteinemia. Acetylcholinesterase activity and immunocontent of citoplasmic and nuclear NF-κB/p65 subunit were increased in hippocampus of rats subjected to hyperhomocysteinemia at 1 h, but did not alter at 12 h after the last injection of homocysteine. According to our results, chronic hyperhomocysteinemia increases inflammatory parameters, suggesting that this process might be associated, at least in part, with the cerebrovascular and vascular dysfunctions characteristic of some homocystinuric patients.

Increased Expression of Tumor Necrosis Factor-Alpha in the Rat Hippocampus after Acute Homocysteine Administration

Background and Purpose:This paper evaluated the effect of acute homocysteine administration on inflammatory cytokine tumor necrosis factor-alpha (TNF-α) expression and neuronal apoptosis in the rat hippocampus and investigated the effects of vitamin C treatment on homocysteine-induced inflammation and neuronal death.Methods:Subjects were three-week-old, male Sprague-Dawley rats. Rats for the control group, we injected saline solution into the rats’ abdominal cavities for one week. Rats in the second group received 1 injection of homocysteine (11 mmol/kg) into their abdominal cavities after 1 week of saline solution administration. For the third group, we injected the rats with vitamin C (100 mg/kg) for a week, followed by 1 injection of homocysteine. The hippocampi were stained with an anti-TNF-α antibody, and apoptosis was evaluated using the TUNEL staining method.Results:The homocysteine-injected rats had strong TNF-α expression in every hippocampal region. Vitamin C significantly reduced TNF-α expression in the hippocampus’s CA1 region. Acute homocysteine administration did not cause apoptosis in the hippocampus.Conclusions:The pro-inflammatory cytokine TNF-α may mediate elevated homocysteine levels’ contributions to inflammatory reactions, and vitamin C has some protective effect on inflammatory reactions in the CA1 hippocampal region.

Homocysteine Induces Oxidative–Nitrative Stress in Heart of Rats: Prevention by Folic Acid

Hyperhomocysteinemia is a risk factor for cardiovascular disease, stroke, and thrombosis; however, the mechanisms by which homocysteine triggers these dysfunctions are not fully understood. In the present study, we investigated the effect of chronic hyperhomocysteinemia on some parameters of oxidative stress, namely thiobarbituric acid reactive substances, an index of lipid peroxidation, 2',7'-dichlorofluorescein (H(2)DCF) oxidation, activities of antioxidant enzymes named superoxide dismutase and catalase, as well as nitrite levels in heart of young rats. We also evaluated the effect of folic acid on biochemical alterations elicited by hyperhomocysteinemia. Wistar rats received daily subcutaneous injection of homocysteine (0.3-0.6 μmol/g body weight) and/or folic acid (0.011 μmol/g body weight) from their 6th to the 28th day of life. Controls and treated rats were killed 1 h and/or 12 h after the last injection. Results showed that chronic homocysteine administration increases lipid peroxidation and reactive species production and decreases enzymatic antioxidant defenses and nitrite levels in the heart of young rats killed 1 h, but not 12 h after the last injection of homocysteine. Folic acid concurrent administration prevented homocysteine effects probable by its antioxidant properties. Our data indicate that oxidative stress is elicited by chronic hyperhomocystenemia, a mechanism that may contribute, at least in part, to the cardiovascular alterations characteristic of hyperhomocysteinemic patients. If confirmed in human beings, our results could propose that the supplementation of folic acid can be used as an adjuvant therapy in cardiovascular alterations caused by homocysteine.

Hyperhomocysteinemia reduces glutamate uptake in parietal cortex of rats

In the present study we evaluated the effect of acute and chronic homocysteine administrations on glutamate uptake in parietal cortex of rats. The immunocontent of glial glutamate transporter (GLAST) and sodium-dependent glutamate/aspartate transporter (GLT-1) in the same cerebral structure was also investigated. For acute treatment, neonate or young rats received a single injection of homocysteine or saline (control) and were sacrificed 1, 8, 12h, 7 or 30 days later. For chronic treatment, homocysteine was administered to rats twice a day at 8h interval from their 6th to their 28th days old; controls and treated rats were sacrificed 12h, 1, 7 or 30 days after the last injection. Results show that acute hyperhomocysteinemia caused a reduction on glutamate uptake in parietal cortex of neonate and young rats, and that 12h after homocysteine administration the glutamate uptake returned to normal levels in young rats, but not in neonate. Chronic hyperhomocysteinemia reduced glutamate uptake, and GLAST and GLT-1 immunocontent. According to our results, it seems reasonable to postulate that the reduction on glutamate uptake in cerebral cortex of rats caused by homocysteine may be mediated by the reduction of GLAST and GLT-1 immunocontent, leading to increased extracellular glutamate concentrations, promoting excitotoxicity.

Acute homocysteine administration impairs memory consolidation on inhibitory avoidance task and decreases hippocampal brain-derived neurotrophic factor immunocontent: prevention by folic acid treatment

In the present study, we first investigated the effect of single homocysteine administration on consolidation of short- and long-term memories of inhibitory avoidance task in Wistar rats. We also measured brain-derived neurotrophic factor levels in the hippocampus and parietal cortex of rats. The influence of pretreatment with folic acid on behavioral and biochemical effects elicited by homocysteine was also studied. Wistar rats were subjected to a folic acid or saline pretreatment from their 22nd to 28th day of life; 12 h later they were submitted to a single administration of homocysteine or saline. For motor activity and memory evaluation we performed open-field and inhibitory avoidance tasks. Hippocampus and parietal cortex were obtained for brain-derived neurotrophic factor immunocontent determination. Results showed that homocysteine impaired short- and long-term memories and reduced brain-derived neurotrophic factor levels in the hippocampus. Pretreatment with folic acid prevented both the memory deficit and the reduction in the brain-derived neurotrophic factor immunocontent induced by homocysteine injection. Further studies are required to determine the entire mechanism by which folic acid acts and its potential therapeutic use for memory impairment prevention in homocystinuric patients.

Hyperhomocysteinemia Increases β-Amyloid by Enhancing Expression of γ-Secretase and Phosphorylation of Amyloid Precursor Protein in Rat Brain

Hyperhomocysteinemia and beta-amyloid (Abeta) overproduction are critical etiological and pathological factors in Alzheimer disease, respectively; however, the intrinsic link between them is still missing. Here, we found that Abeta levels increased and amyloid precursor protein (APP) levels simultaneously decreased in hyperhomocysteinemic rats after a 2-week induction by vena caudalis injection of homocysteine. Concurrently, both the mRNA and protein levels of presenilin-1, a component of gamma-secretase, were elevated, whereas the expression levels of beta-secretase and presenilin-2 were not altered. We also observed that levels of phosphorylated APP at threonine-668, a crucial site facilitating the amyloidogenic cleavage of APP, increased in rats with hyperhomocysteinemia, although the phosphorylation per se did not increase the binding capacity of pT668-APP to the secretases. The enhanced phosphorylation of APP in these rats was not relevant to either c-Jun N-terminal kinase or cyclin-dependent kinase-5. A prominent spatial memory deficit was detected in rats with hyperhomocysteinemia. Simultaneous supplementation of folate and vitamin-B12 attenuated the hyperhomocysteinemia-induced abnormal processing of APP and improved memory. Our data revealed that hyperhomocysteinemia could increase Abeta production through the enhanced expression of gamma-secretase and APP phosphorylation, causing memory deficits that could be rescued by folate and vitamin-B12 treatment in these rats. It is suggested that hyperhomocysteinemia may serve as an upstream factor for increased Abeta production as seen in patients with Alzheimer disease.

Concurrent folate treatment prevents Na+,K+-ATPase activity inhibition and memory impairments caused by chronic hyperhomocysteinemia during rat development

We investigated the hypothesis that folate administration would prevent hyperhomocysteinemia-induced memory deficits and Na+,K+-ATPase activity inhibition. Chronic hyperhomocysteinemia was induced from the 6th to the 28th day of life by subcutaneous injection of homocysteine (0.3–0.6μmol/g), twice a day; control Wistar rats received the same volume of saline solution (0.9% NaCl). Half of the homocysteine- and saline-treated groups also received intraperitoneal administration of folate (0.011μmol/g) from the 6th to the 28th day of life. A group of animals was killed 12h after the last injection, plasma and parietal cortex were collected for biochemical analysis. Another group stayed at Central Animal House until 60th day of life, when the rats were submitted to behavioral testing in water maze or were killed for evaluation of cortical Na+,K+-ATPase activity. Results showed that hyperhomocysteinemia impaired reference memory for platform location, as assessed by fewer crossings to the platform place and increased latency for the first crossing, when compared to controls. In the working memory task homocysteine-treated animals also needed more time to find the platform. We also observed that Na+,K+-ATPase activity was reduced in parietal cortex of hyperhomocysteinemic rats sacrificed 12h after the last injection of homocysteine (29-day-old rats). In contrast, this enzyme was not altered when the rats were sacrificed 31 days after the treatment (60-day-old rats). Hyperhomocysteinemic rats treated with folate had all those impairments prevented, an effect probably related to folate antioxidant properties.

Electrochemical determination of homocysteine at a gold nanoparticle-modified electrode

The construction of a colloidal gold-cysteamine-carbon paste electrode, Au coll-Cyst-CPE, for the electrochemical determination of homocysteine is reported. The improved voltammetric behaviour of homocysteine at Au coll-Cyst-CPE with respect to that observed at a gold disk electrode is attributed to an enhanced electron transfer kinetics as a consequence of the array distribution of gold nanoparticles immobilized onto the Cyst SAM. Cyclic voltammtery of homocysteine showed an adsorption-controlled current for scan rates between 500 and 5000 mV s −1. The hydrodynamic voltammogram constructed for homocysteine allowed the selection of a potential value of +600 mV, where the background current is negligible, for the amperometric detection of the analyte at the Au coll-Cyst-CPE. Using a flow rate of 0.8 ml min −1, the R.S.D. value for i p after 25 repetitive injections of homocysteine was of 4.3%, and one single electrode could be used for more than 15 days without any treatment or regeneration procedure of the modified electrode surface. An HPLC method for the separation and quantification of homocysteine and related thiols, using amperometric detection at the modified electrode has been developed. A mobile phase consisting of 2:98% (v/v) acetonitrile:0.05 mol l −1 buffer solution of pH 2.0, and a detection potential of +0.80 V were selected. Separation with baseline resolution and retention times of 3.00, 3.60, 4.52, 5.71 and 7.79 min were obtained for cysteine, homocysteine, glutathion, penicillamine and N-acetyl-cysteine, respectively. Calibration graphs were constructed for all the separated compounds. Detection limits ranged between 20 nM for cysteine and 120 nM for penilcillamine, with a value for homocysteine of 30 nM. These values compare advantageously with those achieved with previously reported HPLC methods using electrochemical, UV, fluorescence and MS detection modes. The developed method was applied to the determination of cysteine and homocysteine serum samples with good results.

Homocysteine reduces cholinesterase activity in rat and human serum

In the present study we investigated the effect of homocysteine administration, the main metabolite accumulating in homocystinuria, on cholinesterase activity in rat and human serum. For the in vivo study, 8-, 15- and 60-day-old rats received one subcutaneous injection of homocysteine (0.3, 0.4 or 0.6μmol/g of body weight, respectively) or saline (control) and were sacrificed 1h later, when serum was collected in order to determine cholinesterase activity. For the in vitro studies, serum of 8-, 15- and 60-day-old untreated rats or 20–25- and 52–60-day-old human beings (healthy volunteers) were incubated with 10–500μM homocysteine. Results showed that acute hyperhomocysteinemia (in vivo study) significantly reduced cholinesterase activity in the serum of rats of all ages tested. We also observed that 500μM homocysteine added to the incubation medium (in vitro study) significantly inhibited cholinesterase activity both in serum of rats and humans. Our findings seem to reinforce the proposed associations of cholinesterase activity with hyperhomocysteinemia.

Hyperhomocysteinemia increases damage on brain slices exposed to in vitro model of oxygen and glucose deprivation: prevention by folic acid

In the present study we evaluate the effects of homocysteine on cellular damage using hippocampal slices from Wistar rats exposed to oxygen and glucose deprivation (OGD, followed by reoxygenation), an in vitro model of hypoxic–ischemic events. For chronic treatment, we induced elevated levels of homocysteine in blood (500μM), comparable to those of human homocystinuria, and in brain (60nmol/g wet tissue) of young rats by subcutaneous injections of homocysteine (0.3–0.6μmol/g of body weight), twice a day with 8h intervals, from the 6th to the 28th postpartum day and controls received saline. Rats were sacrificed 1, 3 or 12h after the last injection. For acute treatment, 29-day-old rats received one single injection of homocysteine (0.6μmol homocysteine/g body weight) or saline and were sacrificed 1h later. In another set of experiments rats were pretreated with Vitamins E (40mg/kg) and C (100mg/kg) or folic acid (5mg/kg) during 1 week; 12h after the last administration they received a single injection of homocysteine or saline and were sacrificed 1h later. Results showed that both chronic (1h after homocysteine administration) and acute hyperhomocysteinemia increased the cellular damage measured by LDH released to de incubation medium, suggesting an increase of tissue damage caused by OGD. Pretreatment with folic acid completely prevented the damage caused by acute hyperhomocysteinemia, whereas Vitamin E just partially prevented such effect. These findings may be relevant to explain, at least in part, the higher susceptibility of hyperhomocysteinemic patients to be susceptible to ischemic events and point to a possible preventive treatment.

Folic acid pretreatment prevents the reduction of Na+,K+-ATPase and butyrylcholinesterase activities in rats subjected to acute hyperhomocysteinemia

The main objective of the present study was to evaluate the effect of folic acid pretreatment on parietal cortex Na+,K+-ATPase and serum butyrylcholinesterase activities in rats subjected to acute hyperhomocysteinemia. Animals were pretreated daily with an intraperitoneal injection of folic acid (5mg/kg) or saline from the 22th to the 28th day of age. Twelve hours after the last injection of folic acid or saline, the rats received a single subcutaneous injection of homocysteine (0.6μmol/g of weight body) or saline and were killed 1h later. Serum was collected and the brain was quickly removed and parietal cortex dissected. Results showed that acute homocysteine administration significantly decreased the activities of Na+,K+-ATPase and butyrylcholinesterase on parietal cortex and serum, respectively. Furthermore, folic acid pretreatment totally prevented these inhibitory effects. We also evaluated the effect of acute homocysteine administration on some parameters of oxidative stress, namely thiobarbituric acid-reactive substances and total thiol content in parietal cortex of rats. No alteration of these parameters were observed in parietal cortex of homocysteinemic animals, indicating that these oxidative stress parameters were probably not responsible for the reduction of Na+,K+-ATPase and butyrylcholinesterase activities. The presented results confirm previous findings that acute hyperhomocysteinemia produces an inhibition of Na+,K+-ATPase and butyrylcholinesterase activities and that pretreatment with folic acid prevents such effects. Assuming that homocysteine might also reduce the activities of these enzymes in human beings, our results support a new potential therapeutic strategy based on folic acid supplementation to prevent the neurological damage found in hyperhomocysteinemia.

Effects of Homocysteine on the Dopaminergic System and Behavior in Rodents

Long-term treatment of levodopa for Parkinson's disease (PD) patients is known to elevate homocysteine level in their plasma. The present study was designed to examine the possible neurotoxic effects of the increased homocysteine level on the dopaminergic system. Homocysteine was administered into Sprague–Dawley male rats intracerebroventricularly or C57BL/6 mice intraperitoneally. Following homocysteine injection the locomotor activities, the levels of dopamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and immunohistochemistry of dopaminergic neurons were examined. The results obtained indicate that homocysteine administration (1 or 2 μmol, i.c.v.) into the rat brains for 5 days significantly decreased the locomotor activities and dopamine as well as its metabolites, DOPAC and HVA, in the rat striatal regions. Two different doses of homocysteine (50 and 100 mg/100 g, i.p. daily) were administered into mice for 36 days to evaluate the effect of systemic treatment of homocysteine on the dopaminergic neurons of the brain. The intraperitoneal injections of two doses of homocysteine significantly increased homocysteine levels in the striatal regions of mouse brains by 21.5 and 39.2%, while reducing dopamine turnover rates in the striatal regions by decreasing (DOPAC + HVA)/DA, 23.7 and 51.6%, respectively. Accordingly, homocysteine decreased locomotor activities significantly by decreasing movement time by 29 and 38%, total distance by 32 and 42%, and numbers of movement by 28 and 41%, respectively. Moreover, homocysteine decreased tyrosine hydroxylase immunoreactivity in substantia nigra of mouse brain. The data obtained indicate that the potential of homocysteine to be toxic to the dopaminergic system. Consequently, long-term levodopa therapy for PD may accelerate the progression of PD, at least in part by elevated homocysteine.

A reduction of endogenous asymmetric dimethylarginine contributes to the effect of captopril on endothelial dysfunction induced by homocysteine in rats

We examined whether captopril exerts beneficial effect on homocysteine-induced endothelial dysfunction in vivo and whether this effect of captopril is associated with a reduction of endogenous inhibitor of nitric oxide synthase (NOS) asymmetric dimethylarginine (ADMA) in rats. Male Sprague–Dawley rats were given intravenous injections of homocysteine (10 mg/kg/day) to induce endothelial dysfunction. Captopril treatment (3 mg/kg/day, i.v.) was taken in some rats after homocysteine administration. Endothelium-dependent relaxation was tested in aortic rings. Serum levels of ADMA, nitrite/nitrate, malondialdehyde (MDA), and creatinine were measured. Furthermore, superoxide dismutase activity in liver and angiotensin converting enzyme activity in serum were also assayed. Administration of homocysteine to rats for 4 weeks significantly impaired endothelium-dependent relaxation compared with control rats. This impairment of endothelium-dependent relaxation was accompanied by elevated serum concentration of ADMA and decreased serum content of nitrite/nitrate. Moreover, serum concentration of MDA was remarkably increased, whereas liver superoxide dismutase activity was decreased in homocysteine-treated group compared with control. Chronic captopril treatment not only improved the impaired endothelium-dependent relaxation, but also prevented the elevation of serum ADMA and MDA levels, as well as reduction of serum nitrite/nitrate contents and liver superoxide dismutase activity. Serum angiotensin converting enzyme activity and creatinine had no significant difference between the three groups. These results suggest that chronic captopril treatment reduces endogenous inhibitor of NOS in rats with homocysteine injection, which may contribute to the beneficial effect of captopril on homocysteine-induced endothelial dysfunction in vivo, and may be secondary to the antioxidative action of captopril.

NTPDase and 5'-nucleotidase activities in synaptosomes of hippocampus and serum of rats subjected to homocysteine administration.

Patients with homocystinuria, an inborn error of metabolism, present neurological dysfunction and commonly experience frequent thromboembolic complications. The nucleoside triphosphate diphosphohydrolase (NTPDase) and 5'-nucleotidase enzymes regulate the nucleotide/nucleoside ratio in the central nervous system and in the circulation and are thought to be involved in these events. Thus, the current study investigated the effect of homocysteine administration on NTPDase and 5'-nucleotidase activities, in the synaptosomal fraction of rat hippocampus, and on nucleotidase activities in rat serum. Twenty-nine-day-old Wistar rats were divided in two groups: group I (control), animals received 0.9% saline; group II (homocysteine-treated), animals received one single subcutaneous injection of homocysteine (0.6 micromol/g). Rats were killed 1 h after the injection. NTPDase and 5'-nucleotidase activities from brain and serum were significantly increased in the homocysteine-treated group. Results show that, in hippocampus, ATP and ADP hydrolysis increased by 20.5% and 20%, respectively, and AMP hydrolysis increased by 48%, when compared to controls. In serum, ATP and ADP hydrolysis increased 136% and 107%, respectively, and AMP hydrolysis increased 95%, in comparison to controls. The current data strongly indicate that in vivo homocysteine administration alters the activities of the enzymes involved in nucleotide hydrolysis, both in the central nervous system and in the serum of adult rats.

Chronic hyperhomocysteinemia provokes a memory deficit in rats in the Morris water maze task

Homocystinuria is an inherited metabolic disease biochemically characterized by tissue accumulation of homocysteine. Affected patients present mental retardation and other neurological symptoms whose mechanisms are still obscure. In the present study, we investigated the effect of chronic hyperhomocysteinemia on rat performance in the Morris water maze task. Chronic treatment was administered from the 6th to the 28th day of life by s.c. injection of homocysteine, twice a day at 8-h intervals; control rats received the same volume of saline solution. Animals were left to recover until the 60th day of life. Morris water maze tasks were then performed, in order to verify any effect of early homocysteine administration on reference and working memory of rats. Results showed that chronic treatment with homocysteine impaired memory of the platform location and that homocysteine treated animals presented fewer crossings to the place where the platform was located in training trials when compared to saline-treated animals (controls). In the working memory task, homocysteine treated animals also needed more time to find the platform. Our findings suggest that chronic experimental hyperhomocysteinemia causes cognitive dysfunction and that might be related to the neurological complications characteristic of homocystinuric patients.

Homocysteine, Vitamin Deficiency and Prevention of Arteriosclerosis

The homocysteine theory of arteriosclerosis was discovered by study of children with homocystinuria caused by different enzymatic disorders of homocysteine metabolism. Deficiency of cystathionine synthase, a pyridoxal phosphate dependent enzyme, causes elevated levels of plasma homocysteine. Deficiency of methyl transferase, a folic acid and cobalamin dependent enzyme, causes elevated levels of plasma homocysteine and cystathionine. Arteriosclerosis was discovered to be associated with homocysteinemia in both of these inherited dirorders. Because of different metabolic consequences of these two diseases, it was possible to conclude that homocysteine causes arteriosclerosis by damaging the endothelial cells and producing hyperplasia of smooth muscle cells and other connective tissue changes. Deficiency of a third enzyme, methylenetetrahydrofolate reductase, also causes homocystinuria and arteriosclerotic plaques, providing independent evidence to support this conclusion. Further evidence to support this conclusion came from studies with animals showing that injection of homocysteine causes arteriosclerotic plaques and thrombosis that closely resemble the vascular changes in children with homocystinuria. The homocysteine theory of arteriosclerosis relates the pathogenesis of the disease in the general population to dietary deficiencies of vitamin B6, folic acid, and vitamin B12. Deficiencies of these vitamins are produced by losses incurred during processing and preservation of basic foodstuffs. Population surveys have concluded that elevated levels of plasma homocysteine are an independent risk factor for arteriosclerosis, heart disease, and stroke. Elevated homocysteine levels have in turn been related to widespread deficiencies of folic acid and vitamin B6 within susceptible populations. The decline in mortality from coronary heart disease in the U.S. during the past three decades is attributed to increased vitamin B6 and folic acid that were introduced into the food supply through voluntary fortification and supplementation. A properly designed intervention trial is needed to show a major reduction in cardiovascular disease risk and complications by a diet and supplementary nutrients that reduce levels of homocysteine in the plasma.

Focal and generalized experimental seizures induced by homocysteine

Electrocorticographic and behavioral effects of parenteral injections of homocysteine in rats are described. Homocysteine activates experimental foci and produces focal seizures in experimental animals with pre-existing lesions. It produces generalized ECoG seizures and generalized convulsions in unlesioned animals in higher doses.