Supplementation with dairy matrices impacts on homocysteine levels and gut microbiota composition of hyperhomocysteinemic mice.

PURPOSE: Several studies highlighted a correlation between folic acid deficiency and high plasma homocysteine concentration, considered a risk factor for multifactorial diseases. Natural folates represent an emerging alternative strategy to supplementation with synthetic folic acid, whose effects are controversial. The present work was, therefore, performed in hyperhomocysteinemic mice to study the impact of supplementation with dairy matrices containing natural folates on plasma homocysteine levels and faecal microbiota composition. METHODS: Forty mice were divided into six groups, two of which fed control or folic acid deficient (FD) diets for 10 weeks. The remaining four groups were fed FD diet for the first 5 weeks and then shifted to a standard control diet containing synthetic folic acid (R) or a FD diet supplemented with folate-enriched fermented milk (FFM) produced by selected lactic acid bacteria, fermented milk (FM), or milk (M), for additional 5 weeks. RESULTS: Supplementation with dairy matrices restored homocysteine levels in FD mice, although impacting differently on hepatic S-adenosyl-methionine levels. In particular, FFM restored both homocysteine and S-adenosyl-methionine levels to the control conditions, in comparison with FM and M. Next generation sequencing analysis revealed that faecal microbiota of mice supplemented with FFM, FM and M were characterised by a higher richness of bacterial species in comparison with C, FD and R groups. Analysis of beta diversity highlighted that the three dairy matrices determined specific, significant variations of faecal microbiota composition, while hyperhomocysteinemia was not associated with significant changes. CONCLUSIONS: Overall, the results represent a promising starting point for the applicability of food matrices enriched in natural folates to manage hyperhomocysteinemia.

Hyperhomocysteinemia is key for increased susceptibility to PND in aged mice.

BACKGROUND: Postoperative neurocognitive disorder (PND) is a severe postoperative complication with no effective therapy that affects up to 19-52% of senior patients. Age and surgery type have been identified as risk factors. However, what caused the increased risk in the elderly is poorly understood. METHODS: We utilized a PND model in aged mice undergoing experimental laparotomy with general anesthesia to evaluate the causal relationship between hyperhomocysteinemia and increased PND susceptibility. PND was assessed by Novel Object Tasks, Fear Conditioning Tests, and Barnes Maze Tests. Serum homocysteine (Hcy) as well as vitamin B12 and folate acid levels were tested before, immediately after surgery and from day 1 to day 29 after surgery by ELISA. The effectiveness of preventative strategy including diet supplementation of vitamin B12 + folic acid (Vit B12 + FA) and S-adenosylmethionine (SAM) injection targeting hyperhomocysteinemia were also tested. RESULTS: PND in aged mice lasted for at least 2 weeks after experimental laparotomy, which was not observed in young adult mice. Serum Hcy results indicated a significant correlation between postoperative cognitive performance and perioperative Hcy level. Preoperative supplementation with VB12 and folic acid (FA) in the diet or S-adenosylmethionine (SAM) injection reduced perioperative serum Hcy level and inhibited the development of PND in aged mice. CONCLUSIONS: Serum homocysteine accumulation is a fundamental cause for increased susceptibility of PND in aged mice. Preoperative diet supplementation of VitB12 + FA can effectively reduce PND in aged mice, which may be a promising prophylaxis treatment in clinical settings.

The immunomodulatory mechanism of brain injury induced by hyperhomocysteinemia in spontaneously hypertensive rats.

BACKGROUND: Elevated plasma homocysteine (Hcy) concentration is considered as the diagnostic criteria of Hyperhomocysteinemia (HHcy), which is associated with the inflammatory response and blood-brain barrier disruption. Previous studies have proposed that HHcy with hypertension was associated with the brain injury by enhancing the cerebrovascular permeability, however, the immune mechanism remains obscure. The purpose of the study is to explore the immunomodulatory mechanism of brain injury in spontaneously hypertensive rats (SHRs) induced by HHcy. MATERIALS AND METHODS: Sixty SHRs were randomly assigned to three groups: SHR-C (control group), SHR-M (methionine group) and SHR-T (treatment group). Physical examination of body weight, systolic blood pressure (SBP) and plasma Hcy content was measured every 4 weeks. Besides, T-helper cell 17 and regulatory T cells (Treg)-related inflammatory cytokines (interleukin [IL]-6, IL-17, IL-10, and transforming growth factor beta [TGF-ß]) and genes (RORγt and FoxP3) were detected by enzyme-linked immunosorbent assay, quantitative polymerase chain reaction , Western blot, and immunohistochemistry. RESULTS: High methionine diet could cause weight loss, SBP rising, and plasma Hcy content significantly elevated. IL-16 and IL-17A levels in peripheral blood and in brain tissue both lifted, while IL-10 and TGF-ß levels dropped; RORγt expression raised in brain, nevertheless, FoxP3 levels were the opposite. After the intervention with vitamin B6, B12, and folic acid in SHR-T group, these trends would be eased or completely changed. Furthermore, brain tissue slices showed that IL-17-positive cells tended to decrease, and IL-10-positive cells increased in SHR-T group, which was reversed in SHR-M group. CONCLUSIONS: HHcy may promote inflammation that can lead to brain lesions and down-regulate immune response to protect the brain.

Association between malnutrition and hyperhomocysteine in Alzheimer's disease patients and diet intervention of betaine.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disease, which is associated with malnutrition and hyperhomocysteine. The current study aimed to analyze the relationship between malnutrition and hyperhomocysteine in AD patients, and effects of diet intervention with betaine on the disease. METHODS: The nutritional statuses of the AD patients were assessed by short form mini nutritional assessment (MNA-SF). The levels of Hcy, tau hyperphosphorylation, synaptic proteins, blood inflammatory factors were measured by enzymatic cycling assay, Western blot and ELISA. The cognitive function was measured by AD assessment scale (ADAS-cog). RESULTS: There was a significant difference in mental status between normal people and AD patients (P<.05). Overall, malnutrition was reported in a larger proportion of AD patients and high level of Hcy was closely associated with malnutrition. Betaine decreased the levels of phosphorylated tau, elevated PP2Ac activity and inhibited Aß accumulation (P<.05). The levels of IL-lß and TNF-α were significantly higher in the untreatment group while much lower in the intervention group (P<.05). After intervention of betaine treatment, the expression level of Hcy can be restored and betaine can effectively suppress inflammation as well as trigger an increase in memory-related proteins. ADAS-Cog suggested that significant improvement was found after the intervention of betaine. CONCLUSIONS: AD was associated with both malnutrition and higher levels of Hcy. Betaine could restore Hcy expression to normal level in AD patient, which might ameliorate memory deficits.

Effect of lyophilized prune extract on hyperhomocysteinemia in mice.

Altered homocysteine metabolism defined as hyperhomocysteinemia is implicated as pathogenic factor in several cardiovascular diseases and atherosclerosis. The purpose of this study was to investigate the efficacy of prune extract, a good source of phenolic antioxidants, on lowering plasma homocysteine level in male hyperhomocysteinemic mice from average weight of 28 g. The administration of lyophilized prune extract was carried out by intraperitoneal injection one day preceding and one hour before sacrifice of mice. Prune extract decreased significantly plasma homocysteine level, correlated with an increased activity of S-adenosylhomocysteine (SAH) hydrolase and NAD(P)H: quinone oxydoreductase-1 activities. Our results suggest a beneficial effect of prune extract on hyperhomocysteinemia with reduction of homocysteine level by its conversion on to SAH by S-adenosylhomocysteine hydrolase, which is activated by NAD+, a by-product of NAD(P)H: quinone oxydo reductase-1.

EGCG protects against homocysteine-induced human umbilical vein endothelial cells apoptosis by modulating mitochondrial-dependent apoptotic signaling and PI3K/Akt/eNOS signaling pathways.

Homocysteine (Hcy) induced vascular endothelial injury leads to the progression of endothelial dysfunction in atherosclerosis. Epigallocatechin gallate (EGCG), a natural dietary antioxidant, has been applied to protect against atherosclerosis. However, the underlying protective mechanism of EGCG has not been clarified. The present study investigated the mechanism of EGCG protected against Hcy-induced human umbilical vein endothelial cells (HUVECs) apoptosis. Methyl thiazolyl tetrazolium assay (MTT), transmission electron microscope, fluorescent staining, flow cytometry, western blot were used in this study. The study has demonstrated that EGCG suppressed Hcy-induced endothelial cell morphological changes and reactive oxygen species (ROS) generation. Moreover, EGCG dose-dependently prevented Hcy-induced HUVECs cytotoxicity and apoptotic biochemical changes such as reducing mitochondrial membrane potential (MMP), decreasing Bcl-2/Bax protein ratio and activating caspase-9 and 3. In addition, EGCG enhanced the protein ratio of p-Akt/Akt, endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) formation in injured cells. In conclusion, the present study shows that EGCG prevents Hcy-induced HUVECs apoptosis via modulating mitochondrial apoptotic and PI3K/Akt/eNOS signaling pathways. Furthermore, the results indicate that EGCG is likely to represent a potential therapeutic strategy for atherosclerosis associated with Hyperhomocysteinemia (HHcy).

Protective effect of quercetin on homocysteine-induced oxidative stress.

OBJECTIVE: The aim of this study was to evaluate whether quercetin (QUER) treatment could have a protective effect against oxidative stress induced by homocysteinemia in rats. MATERIALS AND METHODS: Thirty-two male Sprague-Dawley rats (adult) were assigned randomly to four groups: the control group was given physiological saline (PS; 1.5 mL/d); the QUER group was given QUER (50 mg/kg body weight [BW] daily) in distilled water and 0.25 mL PS; the homocysteine (HCY) group was given HCY (1 mg/kg BW daily) in distilled water and 1.25 mL PS; and the QUER + HCY group was given QUER 1 h before the administration of HCY. QUER, HCY, and PS were injected intraperitoneally every other day for 30 d. Plasma malondialdehyde (MDA), carbonyl, erythrocyte-reduced glutathione (GSH), plasma sulphydril (-SH) levels, erythrocyte catalase (CAT), and superoxide dismutase (SOD) activities were determined. RESULTS: Plasma CAT levels in the QUER group were found to be significantly higher than in the control group, whereas plasma MDA levels in the QUER group significantly decreased compared with the control group. In the HCY group, plasma MDA and carbonyl levels significantly increased and GSH and SOD levels significantly decreased compared with the control group. Plasma MDA levels significantly decreased and GSH and CAT levels significantly increased in the QUER + HCY group compared with the HCY group. Plasma -SH levels were significantly lower in the HCY group than in the control group. Plasma -SH levels were higher in the QUER + HCY group than in the HCY group, but they were not significant. CONCLUSION: The exposure of rats to HCY leads to oxidative stress reflected in increased MDA and decreased antioxidant enzyme levels. Administration of QUER might attenuate oxidative damage induced by HCY or have a protective effect against it.

CerefolinNAC Therapy of Hyperhomocysteinemia Delays Cortical and White Matter Atrophy in Alzheimer's Disease and Cerebrovascular Disease.

We examined whether using a medical food therapy for hyperhomocysteinemia (HHcy) in patients with Alzheimer's disease (AD) or cognitive impairment due to cerebrovascular disease (CVD) with Cerefolin®/CerefolinNAC® (CFLN: L-methylfolate, methylcobalamin, and N-acetyl-cysteine) slowed regional brain atrophy. Thirty HHcy patients with AD and related disorders (ADRD) received CFLN (HHcy+CFLN: duration [µ ±  σ] = 18.6±16.1 months); a sub-sample of this group did not receive CFLN for varying periods of time (HHcy+NoCFLN: duration [µ ±  σ] = 12.6±5.6 months). Thirty-seven NoHHcy patients with ADRD did not receive CFLN (NoHHcy+NoCFLN: duration [µ ±  σ] = 13.3±17.7 months). No participant took supplemental B vitamins. Regional brain volumes were measured at baseline and end of study, and covariate-adjusted rates of hippocampal, cortical, and forebrain parenchymal (includes white matter) atrophy were predicted. The HHcy+CFLN group's hippocampal and cortical atrophy adjusted rates were 4.25 and 11.2 times slower than those of the NoHHcy+NoCFLN group (p < 0.024). The HHcy+CFLN group's forebrain parenchyma atrophy rate was significantly slower only for CVD; the rate of slowing was proportional to the degree of homocysteine lowering (p < 0.0001). CFLN was associated with significantly slowed hippocampal and cortical atrophy rates in ADRD patients with HHcy, and forebrain parenchymal atrophy rates in CVD patients with HHcy. The present results should be further validated.

Homocysteine, iron and cardiovascular disease: a hypothesis.

Elevated circulating total homocysteine (tHcy) concentrations (hyperhomocysteinemia) have been regarded as an independent risk factor for cardiovascular disease (CVD). However, several large clinical trials to correct hyperhomocysteinemia using B-vitamin supplements (particularly folic acid) have largely failed to reduce the risk of CVD. There is no doubt that a large segment of patients with CVD have hyperhomocysteinemia; therefore, it is reasonable to postulate that circulating tHcy concentrations are in part a surrogate marker for another, yet-to-be-identified risk factor(s) for CVD. We found that iron catalyzes the formation of Hcy from methionine, S-adenosylhomocysteine and cystathionine. Based on these findings, we propose that an elevated amount of non-protein-bound iron (free Fe) increases circulating tHcy. Free Fe catalyzes the formation of oxygen free radicals, and oxidized low-density lipoprotein is a well-established risk factor for vascular damage. In this review, we discuss our findings on iron-catalyzed formation of Hcy from thioethers as well as recent findings by other investigators on this issue. Collectively, these support our hypothesis that circulating tHcy is in part a surrogate marker for free Fe, which is one of the independent risk factors for CVD.

Nutri-epigenetics ameliorates blood-brain barrier damage and neurodegeneration in hyperhomocysteinemia: role of folic acid.

Epigenetic mechanisms underlying nutrition (nutrition epigenetics) are important in understanding human health. Nutritional supplements, for example folic acid, a cofactor in one-carbon metabolism, regulate epigenetic alterations and may play an important role in the maintenance of neuronal integrity. Folic acid also ameliorates hyperhomocysteinemia, which is a consequence of elevated levels of homocysteine. Hyperhomocysteinemia induces oxidative stress that may epigenetically mediate cerebrovascular remodeling and leads to neurodegeneration; however, the mechanisms behind such alterations remain unclear. Therefore, the present study was designed to observe the protective effects of folic acid against hyperhomocysteinemia-induced epigenetic and molecular alterations leading to neurotoxic cascades. To test this hypothesis, we employed 8-weeks-old male wild-type (WT) cystathionine-beta-synthase heterozygote knockout methionine-fed (CBS+/− + Met), WT, and CBS+/− + Met mice supplemented with folic acid (FA) [WT + FA and CBS+/− + Met + FA, respectively, 0.0057-µg g−1 day−1 dose in drinking water/4 weeks]. Hyperhomocysteinemia in CBS+/− + Met mouse brain was accompanied by a decrease in methylenetetrahydrofolate reductase and an increase in S-adenosylhomocysteine hydrolase expression, symptoms of oxidative stress, upregulation of DNA methyltransferases, rise in matrix metalloproteinases, a drop in the tissue inhibitors of metalloproteinases, decreased expression of tight junction proteins, increased permeability of the blood-brain barrier, neurodegeneration, and synaptotoxicity. Supplementation of folic acid to CBS+/− + Met mouse brain led to a decrease in the homocysteine level and rescued pathogenic and epigenetic alterations, showing its protective efficacy against homocysteine-induced neurotoxicity.

The polyphenol-rich extracts from black chokeberry and grape seeds impair changes in the platelet adhesion and aggregation induced by a model of hyperhomocysteinemia.

OBJECTIVE: The mechanism action of the polyphenol-rich extracts from berries of Aronia melanocarpa (black chokeberry) and from grape seeds in the defence against homocysteine (Hcy) and its derivatives action in blood platelets is still unknown. In this study, the influence of the aronia extract and grape seeds extract (GSE) on the platelet adhesion to collagen and fibrinogen and the platelet aggregation during a model of hyperhomocysteinemia was investigated. The aim of our study in vitro was also to investigate superoxide anion radicals (O2⁻•) production after incubation of platelets with Hcy, HTL and the aronia extract and GSE during a model of hyperhomocysteinemia (induced by reduced form of homocysteine at final dose of 100 µM) and the most reactive form of Hcy--its cyclic thioester, homocysteine thiolactone (HTL, 1 µM). Moreover, the additional aim of our study was also to establish and compare the influence of the aronia extract, GSE and resveratrol (3,4',5-trihydroxystilben), a phenolic compound, which has been supposed to be beneficial for the prevention of cardiovascular events, on selected steps of platelet activation. METHODS: The effects of tested extracts on adhesion of blood platelets to collagen and fibrinogen were determined according to Tuszynski and Murphy. The platelet aggregation was determined by turbidimetry method using a Chrono-log Lumi-aggregometer. RESULTS: We have observed that HTL, like its precursor-Hcy stimulated the generation of O2⁻• (measured by the superoxide dismutase-inhibitable reduction of cytochrome c) in platelets and caused an augmentation of the platelet adhesion and aggregation induced by the strong physiological agonist-thrombin. Our present results in vitro also demonstrated that the aronia extract and grape seeds extract reduced the toxicity action of Hcy and HTL on blood platelet adhesion to collagen and fibrinogen, the platelet aggregation and superoxide anion radicals production in platelets, suggesting its potential protective effects on hemostasis during hyperhomocysteinemia. CONCLUSION: In the comparative studies, the aronia extract was found to be more effective antiplatelet factors, than GSE or resveratrol during a model of hyperhomocysteinemia. It gives hopes for development of diet supplements, which may be important during hyperhomocysteinemia.

Effect of catechin/epicatechin dietary intake on endothelial dysfunction biomarkers and proinflammatory cytokines in aorta of hyperhomocysteinemic mice.

PURPOSE: Hyperhomocysteinemia is well recognized as an independent risk factor for the development of premature atherosclerosis. Atherosclerosis, however, may be prevented by polyphenols, potent antioxidant compounds with anti-atherogenic properties. Previously, we used cystathionine beta synthase-deficient mice [Cbs (±)] fed a high-methionine diet-a murine model of hyperhomocysteinemia-to show that daily intake of a red wine polyphenolic extract, mainly comprised of catechin and epicatechin, has a beneficial effect on aortic expression of endothelial dysfunction biomarkers and pro-inflammatory cytokines. The aim of the present study was to understand whether catechin and epicatechin, in purified forms, have anti-atherogenic effects in hyperhomocysteinemia. METHODS: Cbs (±) mice received 50 µg of catechin and/or epicatechin daily in drinking water for 1 month. Plasma homocysteine (Hcy) level and aortic expression of several endothelial dysfunction biomarkers (Vcam-1, Icam-1, E-selectin, and Lox-1) and pro-inflammatory cytokines (Tnf-α, Il-6) were assessed. RESULTS: We found that both catechin and epicatechin had a beneficial effect on plasma homocysteine levels and endothelial dysfunction biomarker expression; however, only catechin had a beneficial effect on pro-inflammatory cytokine expression. Further, when both polyphenols were given, a beneficial effect was observed only on pro-inflammatory cytokine expression. CONCLUSIONS: Catechin seems to be a more potent anti-atherogenic compound than epicatechin in hyperhomocysteinemia and should be considered as a novel therapeutic approach against endothelial dysfunction induced by this condition.

Homocysteine, heat shock proteins, genistein and vitamins in ischemic stroke--pathogenic and therapeutic implications.

Stroke is one of the most devastating neurological conditions, with an approximate worldwide mortality of 5.5 million annually and loss of 44 million disability-adjusted life-years. The etiology of stroke is often unknown; it has been estimated that the etiology and pathophysiology remains unexplained in more than 40% of stroke cases. The conventional stroke risk factors, including hypertension, diabetes mellitus, smoking, and cardiac diseases, do not fully account for the risk of stroke, and stroke victims, especially young subjects, often do not have any of these factors. It is very likely that inflammation, specific genetic predispositions and traditional risk factors interact with each other and may together increase the risk of stroke. Inflammatory and immune responses play important roles in the course of ischemic stroke. Hyperhomocysteinemia (hcy) is considered a modifiable risk factor for stroke, possibly through an atherogenic and prothrombotic mechanism. Both genetic and environmental factors (e.g., dietary intake of folic acid and B vitamins) affect homocysteine level. Identification of the role of hcy as a modifiable risk factor for stroke and of HSPs as regulators of the immune response may lead to more effective prevention and treatment of stroke through dietary and pharmacological intervention. Dietary modification may also include supplementation with novel preventive compounds, such as the antioxidative isoflavones--genistein or daidzein.

Aronia melanocarpa extract suppresses the biotoxicity of homocysteine and its metabolite on the hemostatic activity of fibrinogen and plasma.

OBJECTIVE: Aronia melanocarpa fruits (Rosaceae) are one of the richest plant sources of phenolic substances, and it has been shown to have various biological activities. Berries of A. melanocarpa (chokeberry) have been supposed to be beneficial for the prevention of cardiovascular events. In this study the influence of aronia extract on the clot formation (using human plasma and purified fibrinogen) and the fibrin lysis during the model of hyperhomocysteinemia was investigated. METHODS: Hyperhomocysteinemia was induced using a reduced form of Hcys (at final dose of 0.1mM) and the most reactive form of Hcys - its cyclic thioester, homocysteine thiolactone (HTL, 1 µM). The aim of our study in vitro was also to investigate the modifications of human plasma total proteins and the oxidative stress (by measuring the total antioxidant level - TAS) in plasma after incubation with Hcys, HTL and/or aronia extract. The biological properties of aronia extract were compared with the action of a well characterized antioxidative commercial polyphenol - resveratrol (3,4',5- trihydroxystilbene). RESULTS: The HTL, like its precursor, Hcys stimulated polymerization of fibrinogen. The results also demonstrated that Hcys (0.1mM) and HLT at lower doses than Hcys (1 µM) reduced the fibrin lysis in human plasma. Moreover, Hcys and HTL change the level of thiol and amino groups in plasma total proteins and induce the oxidative stress in plasma. Our results indicate that aronia extract reduced the biotoxicity action of Hcys and HTL on hemostatic properties of fibrinogen or plasma, suggesting its possible protective properties in hyperhomocysteinemia - induced cardiovascular diseases. Moreover, our results showed that the extract from berries of A. melanocarpa due to antioxidant action, significantly reduced the oxidative stress (measured by TAS) in plasma during the model of hyperhomocysteinemia. CONCLUSION: In the comparative studies, the extract from berries of A. melanocarpa and reseveratrol had similar protective properties. It gives hopes for development of diet supplements, which may be preventing thrombosis in pathological states where plasma procoagulant activity and oxidative stress are observed e.g. in hyperhomocysteinemia.

Changes in predictors and status of homocysteine in young male adults after a dietary intervention with vegetables, fruits and bread.

BACKGROUND: Elevated plasma total homocysteine (p-tHcy) is associated with increased risk of cardiovascular disease, and an inverse association has been shown between the dietary intake of B-vitamins, B-vitamin profile and the concentration of p-tHcy. AIM OF THE STUDY: The main objective of this investigation was to study the effect of a dietary intervention focusing on an increased intake of vegetables, fruits and bread. The effect of the dietary intervention was determined by the changes in plasma concentrations of tHcy, cysteine (cys), riboflavin, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) and serum concentrations of folate and vitamin B(12). METHOD: An intervention study with duration of 5 months, including 541 male recruits from the Norwegian National Guard, Vaernes and a control group, including 209 male recruits from the Norwegian Army, Heggelia. RESULTS: The dietary intervention resulted in decreased concentration of p-tHcy (-10%, P = 0.002), p-cys (-6%, P < 0.001) and FMN (-11%, P = 0.310) and increased concentration of riboflavin (+23%, P < 0.001) and FAD (+10%, P = 0.008) in the intervention group compared with the control group. The change in p-tHcy concentration was positively related to the change in the concentration of p-cys (P < 0.001) and FMN (P = 0.035) and inversely related to the change in concentration of folate (P = 0.021). CONCLUSIONS: A dietary intervention program focusing on an increased intake of vegetables, fruits and bread showed a favourable effect on the concentration of p-tHcy and its metabolites. Our findings suggest that the changes in the concentration of p-cys, folate and FMN seem to be predictors of changes in the p-tHcy concentration.

Gestational vitamin B deficiency leads to homocysteine-associated brain apoptosis and alters neurobehavioral development in rats.

Hyperhomocysteinemia has been identified as a risk factor for neurological disorders. To study the influence of early deficiency in nutritional determinants of hyperhomocysteinemia on the developing rat brain, dams were fed a standard diet or a diet lacking methyl groups during gestation and lactation. Homocysteinemia progressively increased in the offspring of the deficient group and at 21 days reached 13.3+/-3.7 micromol/L versus 6.8+/-0.3 micromol/L in controls. Homocysteine accumulated in both neurons and astrocytes of selective brain structures including the hippocampus, the cerebellum, the striatum, and the neurogenic subventricular zone. Most homocysteine-positive cells expressed p53 and displayed fragmented DNA indicative of apoptosis. Righting reflex and negative geotaxis revealed a delay in the onset of integration capacities in the deficient group. Between 19 and 21 days, a poorer success score was recorded in deficient animals in a locomotor coordination test. A switch to normal food after weaning allowed restoration of normal homocysteinemia. Nevertheless, at 80 days of age, the exploratory behavior in the elevated-plus maze and the learning and memory behavior in the eight-arm maze revealed that early vitamin B deprivation is associated with persistent functional disabilities, possibly resulting from the ensuing neurotoxic effects of homocysteine.

Chemical and biomechanical characterization of hyperhomocysteinemic bone disease in an animal model.

BACKGROUND: Classical homocystinuria is an autosomal recessive disorder caused by cystathionine beta-synthase (CBS) deficiency and characterized by distinctive alterations of bone growth and skeletal development. Skeletal changes include a reduction in bone density, making it a potentially attractive model for the study of idiopathic osteoporosis. METHODS: To investigate this aspect of hyperhomocysteinemia, we supplemented developing chicks (n = 8) with 0.6% dl-homocysteine (hCySH) for the first 8 weeks of life in comparison to controls (n = 10), and studied biochemical, biomechanical and morphologic effects of this nutritional intervention. RESULTS: hCySH-fed animals grew faster and had longer tibiae at the end of the study. Plasma levels of hCySH, methionine, cystathionine, and inorganic sulfate were higher, but calcium, phosphate, and other indices of osteoblast metabolism were not different. Radiographs of the lower limbs showed generalized osteopenia and accelerated epiphyseal ossification with distinct metaphyseal and suprametaphyseal lucencies similar to those found in human homocystinurics. Although biomechanical testing of the tibiae, including maximal load to failure and bone stiffness, indicated stronger bone, strength was proportional to the increased length and cortical thickness in the hCySH-supplemented group. Bone ash weights and IR-spectroscopy of cortical bone showed no difference in mineral content, but there were higher Ca2+/PO4(3-) and lower Ca2+/CO3(2-) molar ratios than in controls. Mineral crystallization was unchanged. CONCLUSION: In this chick model, hyperhomocysteinemia causes greater radial and longitudinal bone growth, despite normal indices of bone formation. Although there is also evidence for an abnormal matrix and altered bone composition, our finding of normal biomechanical bone strength, once corrected for altered morphometry, suggests that any increase in the risk of long bone fracture in human hyperhomocysteinemic disease is small. We also conclude that the hCySH-supplemented chick is a promising model for study of the connective tissue abnormalities associated with homocystinuria and an important alternative model to the CBS knock-out mouse.

Vitamin B-12 deficiency and hyperhomocysteinemia are partly ameliorated by cobalt and nickel supplementation in pigs.

Vitamin B-12 deficiency and hyperhomocysteinemia alter the metabolism of trace elements. This study tested the hypothesis that there is a reverse relationship in which diets high in iron, copper, nickel and cobalt would influence vitamin B-12 deficiency outcomes including hyperhomocysteinemia. Piglets (German Landrace x Pietrain) were assigned to six groups of 8 and fed one of the following diets for 166 d: a vitamin B-12-adequate and folate-fortified diet (30 microg/kg vitamin B-12 and 0.5 mg/kg folate) with normal trace element concentrations or one of five vitamin B-12-free, folate nonsupplemented diets (0.36 mg/kg), with either normal trace element concentrations or high concentrations of iron (300 mg/kg), copper (30 mg/kg), cobalt (1 mg/kg) or nickel (6 mg/kg). Feed intake and weight gain did not differ significantly among the groups. Vitamin B-12-deficient pigs developed diminished serum and liver concentrations of vitamin B-12 and folate, an accumulation of iron in the liver and hyperhomocysteinemia. The magnitude of changes differed among vitamin B-12-deficient groups. Vitamin B-12-deficient pigs fed 6 mg/kg nickel had distinctly higher vitamin B-12 concentrations in liver and serum and 45% lower serum concentration of homocysteine than the corresponding deficiency group fed 1 mg/kg nickel; iron concentration in liver was completely normalized. Vitamin B-12-deficient pigs fed 1 mg/kg cobalt had 47% lower homocysteine concentrations in serum than the vitamin B-12-deficient group fed 0.13 mg/kg cobalt, but the vitamin B-12 status was unaffected. Supplementation of iron and copper did not affect these variables. The dietary manipulations had no detrimental effects on variables symptomatic of oxidative stress. The findings indicate a collaborative relationship between vitamin B-12 metabolism and the trace elements nickel and cobalt.

Vascular complications of severe hyperhomocysteinemia in patients with homocystinuria due to cystathionine beta-synthase deficiency: effects of homocysteine-lowering therapy.

Homocystinuria (HCU) due to cystathionine beta-synthase (CBS) deficiency leads to severe hyperhomocysteinemia (HHcy). Vascular events (VE) remain the major cause of morbidity and mortality in the untreated patients with HCU. The study on the natural history of untreated HCU disclosed that, at the time of maximal risk, in other words beyond 10 years old, there was one event per 25 years. Recent studies from Australia (n = 32), The Netherlands (n = 28), and Ireland (n = 24) have documented the effects of long-term treatment on the vascular outcome of a total of 84 patients with 1314 patient-years of treatment for HCU. The mean (range) age was 27.8 (2.5 to 70) years. Five VE were recorded during treatment; one pulmonary embolism, two myocardial infarctions, and two abdominal aneurysms. All five VE occurred in B6-responsive patients at a mean (range) age of 48.8 (30 to 60) years. In 1314 patient-years of treatment, 53 VE would have been expected if they remained untreated; instead only 5 were documented, relative risk = 0.091 (95% confidence interval [CI] 0.043 to 0.190; p < 0.001). Appropriate homocysteine-lowering therapy for severe HHcy significantly reduced the vascular risk in patients with HCU. VE were rare with treatment despite the fact that the post-treatment homocysteine levels were several times higher than the cutoff point for homocysteine in the normal population. The present findings may have relevance to the current concept of "mild HHcy" as a risk factor for vascular disease, with elevated plasma homocysteine levels considerably lower than that of the post-treatment levels in this group of reported patients.