Intranasal vitamin B12 administration in elderly patients: A randomized controlled comparison of two dosage regimens.

AIM: Vitamin B12 deficiency is common in the elderly population. Standard treatment via intramuscular injections, however, has several disadvantages. Safer and more convenient dosage forms such as intranasal are therefore being explored. This study compares the effects of two intranasal vitamin B12 dosage regimens in elderly vitamin B12-deficient patients. METHODS: Sixty patients ≥65 years were randomly assigned to either a loading dose (daily administration for 14 days followed by weekly administration) or a no loading dose (administration every 3 days) regimen for 90 days. Each dose contained 1000 µg cobalamin. Total vitamin B12, holotranscoblamin (holoTC), methylmalonic acid (MMA) and total homocysteine (tHcy) levels in serum were measured on days 0, 7, 14, 30, 60 and 90. RESULTS: Both dosage regimens resulted in a rapid increase of vitamin B12 and holoTC concentrations and normalization of initial high, MMA and tHcy concentrations. The loading dose regimen resulted in the fastest and greatest increase to a median vitamin B12 of 1090 pmol/L (reference 350-650 pmol/L) concentration after 14 days. Following weekly administration, B12 rapidly decreased to a median concentration of 530 pmol/L after 90 days. The no loading dose regimen resulted in a steady increase to a median vitamin B12 of 717 pmol/L after 90 days. CONCLUSIONS: Intranasal vitamin B12 administration is an effective and suitable way to replenish and sustain vitamin B12 levels in elderly patients.

N-Methyl-D-aspartate receptor activation, novel mechanism of homocysteine-induced blood-retinal barrier dysfunction.

Elevated levels of amino acid homocysteine (Hcy) recognized as hyperhomocysteinemia (HHcy) was reported in several human visual disorders, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). Breakdown of blood-retinal barrier (BRB) is concomitant with vision loss in DR and AMD. We previously reported that HHcy alters BRB. Here, we tested the hypothesis that HHcy alters BRB via activation of N-methyl-D-aspartate receptor (NMDAR). Human retinal endothelial cells subjected to high level of Hcy and mouse model of HHcy were used. We injected Hcy intravitreal and used a mouse model of HHcy that lacks cystathionine-ß-synthase (CBS). RT-PCR, western blot, and immunofluorescence showed that retinal endothelial cells (RECs) express NMDAR at the gene and protein levels both in vitro and in vivo and this was increased by HHcy. We assessed BRB function and retinal morphology using fluorescein angiogram and optical coherence tomography (OCT) under HHcy with and without pharmacological inhibition of NMDAR by (MK801) or in mice lacking endothelial NMDAR (NMDARE-/- mouse). Additionally, retinal albumin leakage and tight junction proteins ZO-1 and occludin were assessed by western blotting analysis. Inhibition or elimination of NMDAR was able to improve the altered retinal hyperpermeability and morphology under HHcy as indicated by significant decrease in retinal albumin leakage and restoration of tight junction proteins ZO-1 and occludin. Our findings underscore a potential role for endothelial NMDAR in mediating Hcy-induced breakdown of BRB and subsequently as a potential therapeutic target in retinal diseases associated with HHcy such as DR and AMD. KEY MESSAGES: • Elevated levels of homocysteine (Hcy) are defined as hyperhomocysteinemia (HHcy). • HHcy is implicated in diabetic retinopathy and age-related macular degeneration. • HHcy alters BRB via activation of N-methyl-D-aspartate receptor.

Betaine-rich sugar beet molasses protects from homocysteine-induced reduction of survival in Caenorhabditis elegans.

PURPOSE: Homocysteine (Hcy) in humans represents a blood-borne biomarker which predicts the risk of age-related diseases and mortality. Using the nematode Caenorhabditis elegans, we tested whether feeding betaine-rich sugar beet molasses affects the survival under heat stress in the presence of Hcy, in spite of a gene loss in betaine-homocysteine methyltransferase. METHODS: Knockdown of the genes relevant for remethylation or transsulfuration of Hcy was achieved by RNA interference (RNAi). Survival assay was conducted under heat stress at 37 °C and Hcy levels were determined by enzyme-linked immunosorbent assay. RESULTS: Addition of 500 mg/l betaine-rich sugar beet molasses (SBM) prevented the survival reduction that was caused by exposure to Hcy at 37 °C. Although SBM was no longer capable of reducing Hcy levels under RNAi versus homologues for 5, 10-methylenetetrahydrofolate reductase or cystathionine-ß-synthase, it still enabled the survival extension by SBM under exposure to Hcy. In contrast, RNAi for the small heat shock protein hsp-16.2 or the foxo transcription factor daf-16 both prevented the extension of survival by betaine-rich molasses in the presence of Hcy. CONCLUSIONS: Our studies demonstrate that betaine-rich SBM is able to prevent survival reduction caused by Hcy in C. elegans in dependence on hsp-16.2 and daf-16 but independent of the remethylation pathway.

N-Acyl l-Homocysteine Thiolactones Are Potent and Stable Synthetic Modulators of the RhlR Quorum Sensing Receptor in Pseudomonas aeruginosa.

The RhlR quorum sensing (QS) receptor in the pathogen Pseudomonas aeruginosa plays a prominent role in infection, and both antagonism and agonism of RhlR have been shown to negatively regulate important virulence phenotypes. Non-native lactone ligands are known to modulate RhlR activity, but their utility as chemical probes is relatively limited due to hydrolytic instability. Herein, we report our design and biological evaluation of a suite of hybrid AHL analogs with structures merging (1) features of reported lead RhlR ligands and (2) head groups with improved hydrolytic stabilities. The most promising compounds identified were N-acyl l-homocysteine thiolactones, which displayed enhanced stabilities relative to lactones. Moreover, they were highly selective for RhlR over another key QS receptor in P. aeruginosa, LasR. These compounds are among the most potent RhlR modulators known and represent robust chemical tools to dissect the complex roles of RhlR in the P. aeruginosa QS circuitry.

Vitamin intake is associated with improved visuospatial and verbal semantic memory in middle-aged individuals.

OBJECTIVES: Factors maintaining cognitive health are still largely unknown. In particular, the cognitive benefits associated with vitamin intake and vitamin supplementation are disputed. We investigated self-reported vitamin intake and serum vitamin levels with performance in cognitive factors sensitive to dementia progression in two large middle-aged general population cohorts. METHODS: Survey data were used to assess regular vitamin intake in 4400 NCDS 1958 and 1177 TwinsUK cohort members, and serum homocysteine and B vitamin levels were measured in 675 individuals from the TwinsUK study. Principal component analysis was applied to cognitive test performance from both cohorts resulting in two dementia-sensitive cognitive factors reflecting visuospatial associative memory and verbal semantic memory. RESULTS: In both cohorts, individuals who reported regular intake of vitamins, particularly B vitamins, showed significantly better performance in visuospatial associative memory and verbal semantic memory (P < 0.001). A significant association was also found between homocysteine levels, vitamin serum concentration and visuospatial associative memory performance which indicated that individuals with high B vitamin and homocysteine levels showed better visuospatial associative memory performance than individuals with low vitamin B levels (P < 0.05). DISCUSSION: The findings demonstrate that early dementia-sensitive cognitive changes can be identified in middle-aged asymptomatic individuals and that regular vitamin intake is associated with improved cognitive performance. These findings reinforce the potential cognitive benefits of regular vitamin intake, which should be considered as an economically viable therapeutic strategy for maintaining cognitive health.

Ultra-micronized palmitoylethanolamide rescues the cognitive decline-associated loss of neural plasticity in the neuropathic mouse entorhinal cortex-dentate gyrus pathway.

Chronic pain is associated with cognitive deficits. Palmitoylethanolamide (PEA) has been shown to ameliorate pain and pain-related cognitive impairments by restoring glutamatergic synapses functioning in the spared nerve injury (SNI) of the sciatic nerve in mice. SNI reduced mechanical and thermal threshold, spatial memory and LTP at the lateral entorhinal cortex (LEC)-dentate gyrus (DG) pathway. It decreased also postsynaptic density, volume and dendrite arborization of DG and increased the expression of metabotropic glutamate receptor 1 and 7 (mGluR1 and mGluR7), of the GluR1, GluR1s845 and GluR1s831 subunits of AMPA receptor and the levels of glutamate in the DG. The level of the endocannabinoid 2-arachidonoylglycerol (2-AG) was instead increased in the LEC. Chronic treatment with PEA, starting from when neuropathic pain was fully developed, was able to reverse mechanical allodynia and thermal hyperalgesia, memory deficit and LTP in SNI wild type, but not in PPARα null, mice. PEA also restored the level of glutamate and the expression of phosphorylated GluR1 subunits, postsynaptic density and neurogenesis. Altogether, these results suggest that neuropathic pain negatively affects cognitive behavior and related LTP, glutamatergic synapse and synaptogenesis in the DG. In these conditions PEA treatment alleviates pain and cognitive impairment by restoring LTP and synaptic maladaptative changes in the LEC-DG pathway. These outcomes open new perspectives for the use of the N-acylethanolamines, such as PEA, for the treatment of neuropathic pain and its central behavioural sequelae.

Homocysteine causes dysfunction of chondrocytes and oxidative stress through repression of SIRT1/AMPK pathway: A possible link between hyperhomocysteinemia and osteoarthritis.

Emerging evidence has indicated that the perturbed expression of homocysteine (Hcy) may induce mitochondrial dysfunction and disturb bone metabolism. Sirtuin 1 (SIRT1) and AMP-activated protein kinase (AMPK) are two critical sensors that regulate mitochondrial biogenesis and have been recognized as therapeutic targets in osteoarthritis (OA). This study was designed to test whether Hcy caused pro-osteoarthritic changes through modulation of SIRT1 and AMPK. Our results showed that administration of Hcy reduced the SIRT1/AMPK/PGC-1α signaling in chondrocytes, leading to mitochondrial dysfunction as a result of increased oxidative stress and apoptosis. Moreover, we demonstrated that the expression of NF-κB, COX-2, IL-8, and MMP-13 were elevated subsequent to inhibition of SIRT1/AMPK/PGC-1α/PPAR-γ pathway by homocysteine, thereby causing detrimental effects on chondrocytes. In the animal model of diet-induced hyperhomocysteinemia (HHcy), we observed the similar findings that SIRT1/PGC-1α/PPAR-γ cascades were downregulated with elevated MMP-13 and COX-2. Taken together, data from the current study revealed that the reduced SIRT1 by Hcy may contribute to degradative cartilage process, which provided insight into the etiology of OA.

Metabolomic study of altered energy metabolism during global forebrain ischemia and ischemic precoditioning in blood plasma in homocysteine treated rats.

Elevated homocysteine (Hcy) level is a well known risk factor for cardiovascular and neuropsychiatric diseases. In this study, we investigated metabolic changes in blood plasma in Hcy-treated rats. In combination with Hcy injections to induce hyperhomocysteinemia-like state, we used an animal model of global cerebral ischemia to investigate metabolic changes after 24 h reperfusion in rats. We also focused on the endogenous phenomenon known as ischemic tolerance induced by the preischemic treatment. The experiments were carried out on blood plasma samples as they are easily available and metabolically reflect the overall changes in injured organism. We observed significant changes in plasma metabolite levels of: pyruvate, citrate, acetate implicating alterations in energy metabolism, and increase in triacylglycerols, arginine and lysine, in Hcy-treated rats compared with naive animals. Ischemic insult with 24 reperfusion in Hcy-treated rats led to increase in plasma lactate, and decrease in plasma glucose, pyruvate, citrate and acetate. Complementary, an increase in ketone body 3-hydroxybutyrate was observed. The plasma metabolites: alanine, lactate, valine, glucose, leucine, isoleucine, acetate, citrate and 3-hydroxybutyrate were considered to reflect the response induced by ischemic preconditioning in Hcy rats, where the extent of postischemic damage was not as high as in the non-preconditioned rats. Our results provide evidence that nuclear magnetic resonance (NMR) spectra analysis can identify a specific group of metabolites present in plasma with the capability of discriminating between individual groups of animals. Regarding the effect of elevated Hcy level on plasma metabolome, we showed, that acetate, pyruvate and glucose had the excellent discriminatory power between Hcy-treated and naive rats plasma. Concerning ischemic insult in Hcy-treated animals, we also document the ideal discrimination of ischemic from non-ischemic rats by various groups of metabolites, that can be considered as a potential plasma biomarkers.

Melatonin protects against behavioral deficits, dopamine loss and oxidative stress in homocysteine model of Parkinson's disease.

AIM: Hyperhomocysteinemia and homocysteine (Hcy) mediated dopaminergic neurotoxicity is a matter of concern in the pathophysiology of Parkinson's disease (PD). Our previous study established the involvement of oxidative stress in the substantia nigra (SN) of Hcy rat model of PD; however, the role of antioxidants, such as melatonin, was not tested in this model. MAIN METHODS: Melatonin (10, 20 and 30mg/kg, i.p.) was administered to rats injected with Hcy in right SN (1.0µmol in 2µl saline) to investigate its potency in attenuating the behavioral abnormalities, dopamine depletion and oxidative stress prompted by Hcy. KEY FINDINGS: Treatment of melatonin protected against nigral dopamine loss and replenished the striatal dopamine loss that resulted in amelioration of rotational behavioral bias in Hcy denervated animals. Melatonin administration significantly improved mitochondrial complex-I activity and protected the SN neurons from the toxic insults of oxidative stress induced by Hcy. Amelioration of oxidative stress by melatonin in Hcy-infused SN was bought by dose-dependently scavenging of hydroxyl radicals, restoration of glutathione level and elevation in the activity of antioxidant enzymes. SIGNIFICANCE: The observations bring into light the significant neuroprotective potentials of melatonin in Hcy model of PD which is attributed to the attenuation of oxidative stress in SN.

Both nitric oxide and nitrite prevent homocysteine-induced endoplasmic reticulum stress and subsequent apoptosis via cGMP-dependent pathway in neuronal cells.

Growing evidence indicates that endoplasmic reticulum (ER) stress and/or ER stress-mediated apoptosis may play a role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease. The present study investigated the effects of non-cytotoxic concentrations of nitric oxide (NO) and nitrite, a metabolite of NO, on ER stress and ER stress-mediated apoptosis in Neuro-2a cells exposed to homocysteine (Hcy), an endogenous ER stress inducer. Hcy induced ER stress, as confirmed by inositol-requiring enzyme 1α (IRE1α) phosphorylation and X-box-binding protein-1 (Xbp1) mRNA splicing as well as C/EBP homologous protein (CHOP) expression, and apoptosis, as verified by Annexin V-positive cells. Surprisingly, non-cytotoxic NO (S-nitrosoglutathione) and nitrite markedly reduced Hcy-induced IRE1α phosphorylation, Xbp1 mRNA splicing, CHOP expression, and Annexin V-positive cells, indicating the cytoprotection of NO and nitrite against Hcy-induced ER stress and apoptosis. Moreover, inhibition of sGC/cGMP pathway abolished the cytoprotective effects of NO and nitrite, whereas cellular elevation of cGMP levels mimicked the cytoprotective actions of NO and nitrite. These findings provide the first evidence showing that both NO and nitrite can reduce ER stress and subsequent apoptosis via NO-sGC-cGMP pathway in neuronal cells and suggesting that NO and/or nitrite may have therapeutic value in the treatment of ER stress-associated neurodegenerative diseases.

Role of osteocytes in mediating bone mineralization during hyperhomocysteinemia.

Hyperhomocysteinemia (HHCY) is a risk factor for osteoporosis but whether HHCY affects bone mineralization or not is still ambiguous. Herein we evaluated whether homocysteine affects tissue mineral density (TMD) of cortical bone and if so the role of osteocytes. CD1 mice administered with homocysteine (5 mg/100 g body weight, i.p.) for 7, 15 and 30 days showed temporal changes in TMD and osteocyte lacunar density in femoral cortices. Short-term administration of homocysteine (day 7) increased osteocyte lacunar density and reduced TMD evidenced by microCT50 while prolonged administration of homocysteine (day 30) reinstated TMD and lacunar density to baseline values. Major differences were decreased number of nucleated osteocyte lacunae, increased number of empty lacunae and cleaved caspase 3-positive osteocyte lacunae in day 30 HHCY bone evidenced by H&E staining and immunohistochemistry. Other differences were induction in mineralization genes like Dmp1, Phex and Sost in cortical bone by real-time PCR and increased number of Dmp1- and Sost-positive osteocyte lacunae in day 30 HHCY bone evidenced by immunohistochemistry. Both HHCY day 7 and day 30 samples showed reduced Young's modulus demonstrating that biomechanical property of bone was lost during early HHCY itself, which did not improve with recovery of TMD. Our results thus demonstrate occurrence of two phases in cortical bone upon HHCY: the early phase that involved loss of TMD and increase in osteocyte numbers and a late phase that involved osteocyte reprogramming, apoptosis and mineralization, which reinstated TMD but compromised biomechanical property. To conclude, osteocytes have a potential role in arbitrating bone pathogenesis during HHCY.

Hydrogen sulfide attenuates homocysteine-induced cognitive deficits and neurochemical alterations by improving endogenous hydrogen sulfide levels.

Hyperomocysteinemia (HHcy) has been associated with mild cognitive impairment and dementia. Hydrogen sulfide (H2 S) has been suggested to be an endogenous modulator of neuronal functions. However, the effect and mechanisms involved in beneficial effect of H2 S has not been investigated in homocysteine (Hcy)-induced cognitive deficits. This study has been designed to evaluate the effect of exogenous H2 S on behavioral deficits and neurochemical alterations in HHcy animals. Hcy levels were significantly elevated in plasma, cortex, and hippocampus of Hcy administered animals. A progressive decline in memory functions and increased anxiolytic behavior was observed in HHcy animals. This was accompanied by decrease in endogenous H2 S levels along with decreased activity of cystathionase (CSE) and cystathionine ß-synthase (CBS). However, a significant increase in CSE and CBS mRNAs was observed. In addition, the catecholamine and serotonin levels were reduced and the activity of monoamine oxidase A and B were increased in brain regions of HHcy animals. Haematoxylin and eosin staining revealed higher number of pyknotic cells in brain regions of HHcy animals. H2 S administration was found to lower elevated plasma and brain Hcy levels. The activities of CBS, CSE, and levels of H2 S were restored in HHcy animals administered H2 S. Exogenous H2 S also ameliorated behavioral deficits accompanied by significant increase in catecholamines. Histological analysis revealed normal cell morphology in Hcy-treated animals supplemented with H2 S. These results clearly demonstrate that the protective effect of H2 S on Hcy-induced cognitive deficits is mediated through increased catecholamine and H2 S levels thereby suggesting its beneficial role in preventing HHcy-induced neurodegeneration. © 2016 BioFactors, 43(3):434-450, 2017.

Effects of Folic Acid and Homocysteine on the Morphogenesis of Mouse Cephalic Neural Crest Cells In Vitro.

Folate deficiency and hyperhomocysteinemia have long been associated with developmental anomalies, particularly neural tube defects and neurocristopathies-a group of diverse disorders that result from defective growth, differentiation, and migration of neural crest (NC) cells. However, the exact mechanisms by which homocysteine (Hcys) and/or folate deficiencies disrupt NC development are still poorly understood in mammals. In this work, we employed a well-defined culture system to investigate the effects of Hcys and folic acid (FA) supplementation on the morphogenetic processes of murine NC cells in vitro. We demonstrated that Hcys increases outgrowth and proliferation of cephalic NC cells and impairs their differentiation into smooth muscle cells. In addition, we showed that FA alone does not directly affect the developmental dynamics of the cephalic NC cells but is able to prevent the Hcys-induced effects. Our results, therefore, suggest that elevated Hcys levels per se cause dysmorphogenesis of the cephalic NC and might contribute to neurocristopathies in mammalian embryos.

Upregulation of DAPK contributes to homocysteine-induced endothelial apoptosis via the modulation of Bcl2/Bax and activation of caspase 3.

Hyperhomocysteinemia is characterized by an abnormally high level of homocysteine (Hcy) in the blood and is associated with cardiovascular diseases such as atherosclerosis. Endothelial dysfunction may lead to the pro-atherogenic effects associated with hyperhomocysteinemia. Endothelial dysfunction induced by Hcy has been previously investigated; however, the underlying molecular mechanism remains to be fully elucidated. The present study investigated whether death-associated protein kinase (DAPK) is involved in Hcy­induced apoptosis in human umbilical vein endothelial cells (HUVECs). It was determined that Hcy treatment upregulated the mRNA and protein expression levels of DAPK in HUVECs. Additionally, it was identified that the knockdown of DAPK using small interfering RNA may attenuate the Hcy-induced apoptosis and dissipation of mitochondrial membrane potential. DAPK inhibition may also reverse the effect of Hcy by the upregulation of B cell leukemia/lymphoma 2 (Bcl2) and poly ADP­ribose polymerase, and the downregulation of Bcl2­associated X protein (Bax) and of caspase 3. In conclusion, the present study demonstrated that DAPK contributed to the Hcy­induced endothelial apoptosis via modulation of Bcl2/Bax expression levels and activation of caspase 3.

Berberine activates peroxisome proliferator-activated receptor gamma to increase atherosclerotic plaque stability in Apoe-/- mice with hyperhomocysteinemia.

AIMS/INTRODUCTION: An elevated level of plasma homocysteine has long been suspected as a metabolic risk factor for the development of atherosclerotic vascular diseases in diabetes. Berberine (BBR) has several preventive effects on cardiovascular diseases. The effects of BBR on atherosclerotic plaque stability increased by homocysteine thiolactone (HTL) remain unknown. MATERIALS AND METHODS: The model of atherosclerotic vulnerable plaque was induced by placing a collar around the carotid artery in Apoe-/- mice. Endothelium-dependent relaxation was assayed by organ chamber. RESULTS: Homocysteine thiolactone (50 mg/kg/day, 8 weeks) reduced the atherosclerotic plaque stability in the carotid artery of Apoe-/- mice, which was reversed by BBR administration (1.0 g/kg/day). In vivo and ex vivo experiments showed that HTL dramatically reduced acetylcholine-induced endothelium-dependent relaxation and superoxide dismutase activity, and increased malondialdehyde content, which were inhibited by BBR. Importantly, all effects induced by BBR were abolished by GW9662, an antagonist of peroxisome proliferator-activated receptor-γ. Incubation of cultured endothelial cells with HTL significantly reduced cell viabilities and enhanced production of reactive oxygen species. Pretreatment of cells with BBR dose-dependently reversed HTL-induced detrimental effects, which were GW9662-reversible. CONCLUSIONS: Berberine increases atherosclerotic plaque stability in hyperhomocysteinemia mice, which is related to the activation of peroxisome proliferator-activated receptor-γ and subsequent suppression of oxidative stress in endothelial cells.

Homocysteine upregulates hepcidin expression through BMP6/SMAD signaling pathway in hepatocytes.

Subjects with severe hyperhomocysteinemia have hypoferric anemia and excessive iron deposition in the liver. Hepcidin, the central regulator of iron homeostasis, plays a key role in iron metabolism. However, the regulation of homocysteine (Hcy) on hepcidin is largely unclear. We conducted experiments in HepG2 cells to identify the mechanisms with which Hcy modulates hepcidin expression. We found that treatment with Hcy dose-dependently increased both hepcidin transcript levels and protein levels, as assessed by quantitative real-time reverse-transcriptase polymerase chain reaction and western blotting, respectively. Hcy also activated BMP6 signaling and increased the phosphorylation of SMAD1/5/8 in HepG2 cells. We found that Hcy's effect on hepcidin expression was impaired by the knockdown of BMP6 and its receptors ALK2/3/6 with siRNAs. These results demonstrated that Hcy up-regulated hepcidin expression through the BMP6/SMAD pathway, suggesting a novel mechanism underlying the hyperhomocysteinemia-associated perturbation of iron homeostasis.

Homocysteine induces glyceraldehyde-3-phosphate dehydrogenase acetylation and apoptosis in the neuroblastoma cell line Neuro2a.

High plasma levels of homocysteine (Hcy) promote the progression of neurodegenerative diseases. However, the mechanism by which Hcy mediates neurotoxicity has not been elucidated. We observed that upon incubation with Hcy, the viability of a neuroblastoma cell line Neuro2a declined in a dose-dependent manner, and apoptosis was induced within 48 h. The median effective concentration (EC50) of Hcy was approximately 5 mM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear translocation and acylation has been implicated in the regulation of apoptosis. We found that nuclear translocation and acetylation of GAPDH increased in the presence of 5 mM Hcy and that higher levels of acetyltransferase p300/CBP were detected in Neuro2a cells. These findings implicate the involvement of GAPDH in the mechanism whereby Hcy induces apoptosis in neurons. This study highlights a potentially important pathway in neurodegenerative disorders, and a novel target pathway for neuroprotective therapy.

COMMERCIAL EXTRACT FROM ARONIA AS A MODULATOR OF ADHESIVE PROPERTIES OF FIBRINOGEN TREATED WITH HOMOCYSTEINE AND ITS THIOLACTONE IN VITRO.

Research has confirmed the positive effect of berries of Aivnia melanocarpa on the cardiovascular system. The protective effects of polyphenol-rich extract from berries of A. melanocarpa against changes in biological properties of fibrinogen were studied. In in vino model of hyperhomocysteinemia the capability of fibrinogen to interact with human blood platelets was measured by platelet adhesion in the presence of extract fromA. nelanocapa. We induced hyperhomocystenemia using a reduced form of homocysteine (Hey, at a final concentration of 0.01. 0.1 and 1 µM) and the most reactive form of Hey - its cyclic thioester, homocysteine thiolactone (HTL, at a final concentration of 0.1, 0.5 and I µM). It was observed that Hey or HTL-treated fibrinogen, in comparison with untreated molecule, had a distinct capability to mediate blood platelet adhesion. The experiments also indicate that polyphenol-rich extract from black chokeberries (at final concentrations of 2.5-10 pM/mL) reduced the toxic action of Hey and HTL on the adhesive properties of fibrinogen. The possible protection exerted by black chokeberry extract, through restoring the platelet adhesion of Hey or HTL treated fibrinogen, may be important for vascular diseases.

Homocysteine induces glyceraldehyde-3-phosphate dehydrogenase acetylation and apoptosis in the neuroblastoma cell line Neuro2a

High plasma levels of homocysteine (Hcy) promote the progression of neurodegenerative diseases. However, the mechanism by which Hcy mediates neurotoxicity has not been elucidated. We observed that upon incubation with Hcy, the viability of a neuroblastoma cell line Neuro2a declined in a dose-dependent manner, and apoptosis was induced within 48 h. The median effective concentration (EC50) of Hcy was approximately 5 mM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) nuclear translocation and acylation has been implicated in the regulation of apoptosis. We found that nuclear translocation and acetylation of GAPDH increased in the presence of 5 mM Hcy and that higher levels of acetyltransferase p300/CBP were detected in Neuro2a cells. These findings implicate the involvement of GAPDH in the mechanism whereby Hcy induces apoptosis in neurons. This study highlights a potentially important pathway in neurodegenerative disorders, and a novel target pathway for neuroprotective therapy.

Homocysteine Exposure Impairs Myocardial Resistance to Ischaemia Reperfusion and Oxidative Stress.

BACKGROUND/AIMS: Hyperhomocysteinaemia is recognised as a strong independent risk factor for developing cardiovascular disease. This study investigated how an acute homocysteine dose affected cardiac performance during ischaemia reperfusion and cardiomyocyte contractility and morphology under normal conditions and during oxidative stress. METHODS: Cardiac function was measured in isolated and perfused rat hearts before and after 40 minutes' global normothermic ischaemia. Where used, 0.1 mM L-homocysteine was present prior to, and throughout ischaemia, before wash out after 10 minutes' reperfusion. Calcium transients under normal conditions and changes in contractile synchronicity during oxidative stress (exposure to 0.2 mM H2O2) were measured in freshly isolated rat cardiomyocytes incubated for 60 minutes ± 0.1 mM L-homocysteine. RESULTS: During ischaemia reperfusion 0.1 mM L-homocysteine significantly reduced the rate pressure product during reperfusion (10,038 ± 749 vs. 5955 ± 567 mmHg bpm, p < 0.001), but did not affect time to ischaemic contracture. Incubation of freshly isolated cardiomyocytes with 0.1 mM L-homocysteine significantly decreased the amplitude of the calcium transient and slowed the time to half relaxation. CONCLUSIONS: These findings suggest that homocysteine exposure affected myocardial recovery from ischaemia and contractile homeostasis although the exact mechanisms for these changes remain to be determined.