Methotrexate-induced subacute myelopathy: a serious but treatable complication.

Subacute myelopathy is a rare but serious complication of methotrexate (MTX) that may cause paraplegia. Although its underlying mechanisms have not been fully elucidated, homocysteine is thought to play a role in the pathogenesis of this adverse effect. Herein, we report the case of a 34-years old female patient with diffuse large B-cell lymphoma who developed progressive paraplegia accompanied by dysfunctional bladder and bowel movements after treatment with a modified CODOX-M/IVAC regimen, including high-dose intravenous MTX and intrathecal (IT-) MTX. Neurological symptoms gradually improved to almost normal levels within 4.5 months of onset following treatment with a combination of S-adenosylmethionine, methionine, cyanocobalamin, and folate. During chemotherapy, including high-dose MTX and IT-MTX for hematological malignancies, MTX-induced subacute neuronal damage should be carefully evaluated, and appropriate treatment should be initiated as early as possible.

Metoclopramide as a Potential Antipsychotic Against Long-Term Methionine Exposure in Zebrafish.

Methionine (MET) contributes to brain function and is required for proper functioning of the central nervous system. However, exceptionally high levels of MET and its metabolites in plasma have been found to be toxic and can lead to cell alterations. Long-term exposure to MET has been shown to mimic psychotic symptoms in schizophrenic patients and rodents. The present study evaluated behavioral and neurochemical effects of long-term exposure to MET in zebrafish. Five groups of zebrafish were exposed to MET at a concentration of 4.5 mM for 7 days, along with acute exposure to 25 µM of clozapine and 750, 1000, and 1250 µM of metoclopramide. In contrast, the normal group was exposed to only water and dimethyl sulfoxide. After the treatment, social interaction, anxiety, memory, and locomotion of zebrafish and serotonin levels in zebrafish brains were evaluated. Our results showed that metoclopramide was not only beneficial in improving MET-induced cognitive impairment but it also prevented social withdrawal in zebrafish exposed to MET. In addition, metoclopramide reversed anxiety-like behavior, as indicated by significant changes in locomotion activity. Despite slight changes in serotonin levels in the zebrafish brain, an in vitro serotonin assay failed to demonstrate significant differences between the disease control, normal, and two treatment groups. Finally, results from the study showed that repeated administration of MET induced schizophrenia-like symptoms, although metoclopramide ameliorated the MET-mediated negative symptoms and cognitive deficits in zebrafish. Overall, our findings suggest a new perspective to further explore the antipsychotic properties of metoclopramide.

Dietary Methionine via Dose-Dependent Inhibition of Short-Chain Fatty Acid Production Capacity Contributed to a Potential Risk of Cognitive Dysfunction in Mice.

High-methionine diets induce impaired learning and memory function, dementia-like neurodegeneration, and Alzheimer's disease, while low-methionine diets improve learning and memory function. We speculated that variations in intestinal microbiota may mediate these diametrically opposed effects; thus, this study aimed to verify this hypothesis. The ICR mice were fed either a low-methionine diet (LM, 0.17% methionine), normal methionine diet (NM, 0.86% methionine), or high-methionine diet (HM, 2.58% methionine) for 11 weeks. We found that HM diets damaged nonspatial recognition memory, working memory, and hippocampus-dependent spatial memory and induced anxiety-like behaviors in mice. LM diets improved nonspatial recognition memory and hippocampus-dependent spatial memory and ameliorated anxiety-like behavior, but the differences did not reach a significant level. Moreover, HM diets significantly decreased the abundance of putative short-chain fatty acid (SCFA)-producing bacteria (Roseburia, Blautia, Faecalibaculum, and Bifidobacterium) and serotonin-producing bacteria (Turicibacter) and significantly increased the abundance of proinflammatory bacteria Escherichia-Shigella. Of note, LM diets reversed the results. Consequently, the SCFA and serotonin levels were significantly decreased with HM diets and significantly increased with LM diets. Furthermore, HM diets induced hippocampal oxidative stress and inflammation and selectively downregulated the hippocampus-dependent memory-related gene expression, whereas LM diets selectively upregulated the hippocampus-dependent memory-related gene expression. In conclusion, dietary methionine via dose-dependent inhibition of SCFA production capacity contributed to a potential risk of cognitive dysfunction in mice.

A High-Methionine Diet for One-Week Induces a High Accumulation of Methionine in the Cerebrospinal Fluid and Confers Bipolar Disorder-like Behavior in Mice.

Methionine (Met) is considered the most toxic amino acid in mammals. Here, we investigated biochemical and behavioral impacts of ad libitum one-week feeding of high-Met diets on mice. Adult male mice were fed the standard rodent diet that contained 0.44% Met (1×) or a diet containing 16 graded Met doses (1.2×-13×). High-Met diets for one-week induced a dose-dependent decrease in body weight and an increase in serum Met levels with a 2.55 mM peak (versus basal 53 µM) on the 12×Met diet. Total homocysteine (Hcy) levels were also upregulated while concentrations of other amino acids were almost maintained in serum. Similarly, levels of Met and Hcy (but not the other amino acids) were highly elevated in the cerebrospinal fluids of mice on the 10×Met diet; the Met levels were much higher than Hcy and the others. In a series of behavioral tests, mice on the 10×Met diet displayed increased anxiety and decreased traveled distances in an open-field test, increased activity to escape from water soaking and tail hanging, and normal learning/memory activity in a Y-maze test, which were reflections of negative/positive symptoms and normal cognitive function, respectively. These results indicate that high-Met ad libitum feeding even for a week can induce bipolar disorder-like disease models in mice.

Protective Effects of Dendrobium nobile Lindl. Alkaloids on Alzheimer's Disease-like Symptoms Induced by High-methionine Diet.

BACKGROUND: High methionine-diet (HMD) causes Alzheimer's disease (AD)-like symptoms. Previous studies have shown that Dendrobium nobile Lindle. alkaloids (DNLA) have potential benefits for AD Object: The objective of this study has been to explore whether DNLA can improve AD-like symptoms induced by HMD. METHODS: Mice were fed with 2% HMD diet for 11 weeks; the DNLA20 control group (20 mg/kg), DNLA10 group (10 mg/kg), and DNLA20 group (20 mg/kg) were administered DNLA for 3 months. Morris water maze test was used to detect learning and memory ability. Neuron damage was evaluated by HE and Nissl staining. Levels of homocysteine (Hcy), beta-amyloid 1-42 (Aß1-42), S-adenosine methionine (SAM) and S-adenosine homocysteine (SAH) were detected by ELISA. Immunofluorescence and western blotting (WB) were used to determine the expression of proteins. CPG island methylation levels were accessed by Methylation-specific PCR (MSP) and MethylTarget methylation detection. RESULTS: Morris water maze test revealed that DNLA improved learning and memory dysfunction. HE, Nissl, and immunofluorescence staining showed that DNLA alleviated neuron damage and reduced the 5-methylcytosine (5-mC), Aß1-40) and Aß1-42) levels. DNLA also decreased the levels of Hcy and Aß1-42) in the serum, along with decreasing SAM/SAH level in the liver tissue. WB results showed that DNLA down-regulated the expression of amyloid-precursor protein (APP), presenilin-1 (PS1), beta-secretase-1 (BACE1), DNA methyltransferase1 (DNMT1), Aß1-40) and Aß1-42) proteins. DNLA also up-regulated the proteins expression of insulin-degrading enzyme (IDE), neprilysin (NEP), DNMT3a and DNMT3b. Meanwhile, DNLA increased CPG island methylation levels of APP and BACE1 genes. CONCLUSION: DNLA alleviated AD-like symptoms induced by HMD via the DNA methylation pathway.

The Protective Effects of Pioglitazone Against Cognitive Impairment Caused by L-methionine Administration in a Rat Model.

PURPOSE: Accumulating evidence indicates that elevated levels of methionine are associated with cognitive decline, including loss of memory. The exact mechanisms behind this observation are not completely understood but could be related to an increase in oxidative stress markers in hippocampal tissues. The above increase in oxidative stress could be directly caused by an increase in the blood levels of methionine (hypermethioninemia) or one of its metabolites, such as homocysteine. Pioglitazone is a drug primarily used for the treatment of type 2 diabetes mellitus. Several reports showed that using pioglitazone protects against cognitive decline observed in Alzheimer's disease. Pioglitazone has antioxidant properties independent of its hypoglycemic effects. Taken together, we hypothesized that pioglitazone protects against memory loss triggered by elevated levels of methionine through lowering oxidative stress in the hippocampus. METHODS: To test this hypothesis, we used chronic administration of L-methionine in a rat model. Spatial learning and memory were evaluated in the model using a radial arm water maze (RAWM). The levels of several markers related to oxidative stress were measured in hippocampal tissues recovered from experimental rats. RESULTS: Current results showed that administration of L-methionine was associated with a significant loss of short- and long-term memory and an increase in blood homocysteine levels. The above memory changes were associated with an increase in lipid peroxidation and a decrease in the activity of catalase and glutathione peroxidase antioxidant enzymes in the hippocampus. The combined treatment of pioglitazone with L-methionine protected rat model from memory loss. It also prevented changes observed in lipid peroxidation and changes in the activity of catalase and glutathione peroxidase enzymes. CONCLUSION: Current findings indicate that pioglitazone is a viable therapeutic option that protects against cognitive changes observed upon administration of L-methionine.

Ionizing radiations induce shared epigenomic signatures unraveling adaptive mechanisms of cancerous cell lines with or without methionine dependency.

BACKGROUND: Although radiation therapy represents a core cancer treatment modality, its efficacy is hampered by radioresistance. The effect of ionizing radiations (IRs) is well known regarding their ability to induce genetic alterations; however, their impact on the epigenome landscape in cancer, notably at the CpG dinucleotide resolution, remains to be further deciphered. In addition, no evidence is available regarding the effect of IRs on the DNA methylome profile according to the methionine dependency phenotype, which represents a hallmark of metabolic adaptation in cancer. METHODS: We used a case-control study design with a fractionated irradiation regimen on four cancerous cell lines representative of HCC (HepG2), melanoma (MeWo and MeWo-LC1, which exhibit opposed methionine dependency phenotypes), and glioblastoma (U251). We performed high-resolution genome-wide DNA methylome profiling using the MethylationEPIC BeadChip on baseline conditions, irradiated cell lines (cumulative dose of 10 Gy), and non-irradiated counterparts. We performed epigenome-wide association studies to assess the effect of IRs and methionine-dependency-oriented analysis by carrying out epigenome-wide conditional logistic regression. We looked for epigenome signatures at the locus and single-probe (CpG dinucleotide) levels and through enrichment analyses of gene ontologies (GO). The EpiMet project was registered under the ID#AAP-BMS_003_211. RESULTS: EWASs revealed shared GO annotation pathways associated with increased methylation signatures for several biological processes in response to IRs, including blood circulation, plasma membrane-bounded cell projection organization, cell projection organization, multicellular organismal process, developmental process, and animal organ morphogenesis. Epigenome-wide conditional logistic regression analysis on the methionine dependency phenotype highlighted several epigenome signatures related to cell cycle and division and responses to IR and ultraviolet light. CONCLUSIONS: IRs generated a variation in the methylation level of a high number of CpG probes with shared biological pathways, including those associated with cell cycle and division, responses to IRs, sustained angiogenesis, tissue invasion, and metastasis. These results provide insight on shared adaptive mechanisms of the epigenome in cancerous cell lines in response to IR. Future experiments should focus on the tryptic association between IRs, the initiation of a radioresistance phenotype, and their interaction with methionine dependency as a hallmark of metabolic adaptation in cancer.

Dietary Methionine Supplementation Exacerbates Gastrointestinal Toxicity in a Mouse Model of Abdominal Irradiation.

BACKGROUND AND PURPOSE: Identification of appropriate dietary strategies for prevention of weight and muscle loss in cancer patients is crucial for successful treatment and prolonged patient survival. High-protein oral nutritional supplements decrease mortality and improve indices of nutritional status in cancer patients; however, high-protein diets are often rich in methionine, and experimental evidence indicates that a methionine-supplemented diet (MSD) exacerbates gastrointestinal toxicity after total body irradiation. Here, we sought to investigate whether MSD can exacerbate gastrointestinal toxicity after local abdominal irradiation, an exposure regimen more relevant to clinical settings. MATERIALS AND METHODS: Male CBA/CaJ mice fed either a methionine-adequate diet or MSD (6.5 mg methionine/kg diet vs 19.5 mg/kg) received localized abdominal X-irradiation (220 kV, 13 mA) using the Small Animal Radiation Research Platform, and tissues were harvested 4, 7, and 10 days after irradiation. RESULTS: MSD exacerbated gastrointestinal toxicity after local abdominal irradiation with 12.5 Gy. This was evident as impaired nutrient absorption was paralleled by reduced body weight recovery. Mechanistically, significant shifts in the gut ecology, evident as decreased microbiome diversity, and substantially increased bacterial species that belong to the genus Bacteroides triggered proinflammatory responses. The latter were evident as increases in circulating neutrophils with corresponding decreases in lymphocytes and associated molecular alterations, exhibited as increases in mRNA levels of proinflammatory genes Icam1, Casp1, Cd14, and Myd88. Altered expression of the tight junction-related proteins Cldn2, Cldn5, and Cldn6 indicated a possible increase in intestinal permeability and bacterial translocation to the liver. CONCLUSIONS: We report that dietary supplementation with methionine exacerbates gastrointestinal syndrome in locally irradiated mice. This study demonstrates the important roles registered dieticians should play in clinical oncology and further underlines the necessity of preclinical and clinical investigations in the role of diet in the success of cancer therapy.

Proposals for Upper Limits of Safe Intake for Methionine, Histidine, and Lysine in Healthy Humans.

Based on research presented during the 10th Amino Acid Assessment Workshop, no observed adverse effect levels (NOAELs) for supplemental methionine at 46 mg/(kg·d) (∼3.2 g/d), for supplemental histidine at 8.0 g/d, and for supplemental lysine at 6.0 g/d have been proposed. These NOAELs are relevant to healthy adults and are applicable only to high-purity amino acids administered in fortified foods or dietary supplements. Because individuals are exposed to the above supplemental amino acids in the context of complex combinations of essential amino acids or individually in dietary supplements for various physiologic benefits, such as body fat reduction, skin conditioning, mental energy increase, or herpes simplex treatments, the above safety recommendations will make an important contribution to regulatory and nutritional practices.

Metabolic Consequences of Supplemented Methionine in a Clinical Context.

The central position of methionine (Met) in protein metabolism indicates the importance of this essential amino acid for growth and maintenance of lean body mass. Therefore, Met might be a tempting candidate for supplementation. However, because Met is also the precursor of homocysteine (Hcy), a deficient intake of B vitamins or excessive intake of Met may result in hyperhomocysteinemia (HHcy), which is a risk factor for cardiovascular disease. This review discusses the evidence generated in preclinical and clinical studies on the importance and potentially harmful effects of Met supplementation and elaborates on potential clinical applications of supplemental Met with reference to clinical studies performed over the past 20 y. Recently acquired knowledge about the NOAEL (no observed adverse effect level) of 46.3 mg · kg-1 · d-1 and the LOAEL (lowest observed adverse effect level) of 91 mg · kg-1 · d-1 of supplemented Met will guide the design of future studies to further establish the role of Met as a potential (safe) candidate for nutritional supplementation in clinical applications.

Effect of Methionine Diet on Time-Related Metabolic and Histopathological Changes of Rat Hippocampus in the Model of Global Brain Ischemia.

Hyperhomocysteinemia (hHcy) represents a strong risk factor for atherosclerosis-associated diseases, like stroke, dementia or Alzheimer's disease. A methionine (Met)-rich diet leads to an elevated level of homocysteine in plasma and might cause pathological alterations across the brain. The hippocampus is being constantly studied for its selective vulnerability linked with neurodegeneration. This study explores metabolic and histo-morphological changes in the rat hippocampus after global ischemia in the hHcy conditions using a combination of proton magnetic resonance spectroscopy and magnetic resonance-volumetry as well as immunohistochemical analysis. After 4 weeks of a Met-enriched diet at a dose of 2 g/kg of animal weight/day, adult male Wistar rats underwent 4-vessel occlusion lasting for 15 min, followed by a reperfusion period varying from 3 to 7 days. Histo-morphological analyses showed that the subsequent ischemia-reperfusion insult (IRI) aggravates the extent of the sole hHcy-induced degeneration of the hippocampal neurons. Decreased volume in the grey matter, extensive changes in the metabolic ratio, deeper alterations in the number and morphology of neurons, astrocytes and their processes were demonstrated in the hippocampus 7 days post-ischemia in the hHcy animals. Our results suggest that the combination of the two risk factors (hHcy and IRI) endorses and exacerbates the rat hippocampal neurodegenerative processes.

S-Adenosine Methionine (SAMe) and Valproic Acid (VPA) as Epigenetic Modulators: Special Emphasis on their Interactions Affecting Nervous Tissue during Pregnancy.

S-adenosylmethionine (SAMe) is involved in many transmethylation reactions in most living organisms and is also required in the synthesis of several substances such as monoamine neurotransmitters and the N-methyl-D-aspartate (NMDA) receptor. Due to its important role as an epigenetic modulator, we discuss in some length the process of DNA methylation and demethylation and the critical periods of epigenetic modifications in the embryo, fetus, and thereafter. We also discuss the effects of SAMe deficiency and the attempts to use SAMe for therapeutic purposes such as the treatment of major depressive disorder, Alzheimer disease, and other neuropsychiatric disorders. SAMe is an approved food additive and as such is also used during pregnancy. Yet, there seems to scanty data on the possible effects of SAMe on the developing embryo and fetus. Valproic acid (VPA) is a well-tolerated and effective antiepileptic drug that is also used as a mood stabilizer. Due to its high teratogenicity, it is contraindicated in pregnancy. A major mechanism of its action is histone deacetylase inhibition, and therefore, it acts as an epigenetic modulator, mainly on the brain. This prompted clinical trials using VPA for additional indications i.e., treating degenerative brain disease such as Alzheimer disease, dementia, HIV, and even cancer. Therefore, we discuss the possible effects of VPA and SAMe on the conceptus and early postnatally, during periods of susceptibility to epigenetic modifications. VPA is also used as an inducer of autistic-like behavior in rodents and was found by us to modify gene expression when administered during the first postnatal week but not when administered to the pregnant dams on day 12 of gestation. In contrast, SAMe modified gene expression when administered on day 12 of pregnancy but not postnatally. If administered together, VPA prevented the changes in gene expression induced by prenatal SAMe administration, and SAMe prevented the gene expression changes and autistic-like behavior induced by early postnatal VPA. It is concluded that both VPA and SAMe are powerful epigenetic modifiers with antagonistic actions on the brain that will probably be used in the future more extensively for the treatment of a variety of epigenetic diseases of the nervous system.

Sexually Dimorphic Effects of Dietary Methionine Restriction are Dependent on Age when the Diet is Introduced.

OBJECTIVE: Restricting dietary methionine to 0.17% in male mice increases energy expenditure, reduces fat deposition, and improves metabolic health. The goal of this work was to compare each of these responses in postweaning male and female mice and in physically mature male and female mice. METHODS: Methionine-restricted (MR) diets were fed to age-matched cohorts of male and female mice for 8 to 10 weeks beginning at 8 weeks of age or beginning at 4 months of age. The physiological and transcriptional responses to MR were compared in the respective cohorts. RESULTS: Dietary MR produced sexually dimorphic changes in body composition in young growing animals, with males preserving lean at the expense of fat and females preserving fat at the expense of lean. The effects of MR on energy balance were comparable between sexes when the diet was initiated after attainment of physical maturity (4 months), and metabolic and endocrine responses were also comparable between males and females after 8 weeks on the MR diet. CONCLUSIONS: The sexually dimorphic effects of MR are limited to nutrient partitioning between lean and fat tissue deposition in young, growing mice. Introduction of the diet after physical maturity produced comparable effects on growth and metabolic responses in male and female mice.

Effect of Methionine Diet on Metabolic and Histopathological Changes of Rat Hippocampus.

Hyperhomocysteinemia (hHcy) is regarded as an independent and strong risk factor for cerebrovascular diseases, stroke, and dementias. The hippocampus has a crucial role in spatial navigation and memory processes and is being constantly studied for neurodegenerative disorders. We used a moderate methionine (Met) diet at a dose of 2 g/kg of animal weight/day in duration of four weeks to induce mild hHcy in adult male Wistar rats. A novel approach has been used to explore the hippocampal metabolic changes using proton magnetic resonance spectroscopy (1H MRS), involving a 7T MR scanner in combination with histochemical and immunofluorescence analysis. We found alterations in the metabolic profile, as well as remarkable histo-morphological changes such as an increase of hippocampal volume, alterations in number and morphology of astrocytes, neurons, and their processes in the selective vulnerable brain area of animals treated with a Met-enriched diet. Results of both methodologies suggest that the mild hHcy induced by Met-enriched diet alters volume, histo-morphological pattern, and metabolic profile of hippocampal brain area, which might eventually endorse the neurodegenerative processes.

Anti-Ischemic Activity of Fabomotizole Hydrochloride under Conditions of Endothelial Dysfunction.

Anti-ischemic activity of fabomotizole hydrochloride was studied on the model of subendocardial ischemia in rats with endothelial dysfunction. Endothelial dysfunction was modeled by intragastric administration of methionine (3 g/kg, once a day for 7 days). Acute subendocardial ischemia was induced in narcotized rats by intraperitoneal injection of isoproterenol (20 µg/kg/min over 5 min). Fabomotizole hydrochloride (intraperitoneally, 15 mg/kg) significantly reduced isoproterenol-induced ST segment depression in animals with endothelial dysfunction and with intact vasculature.

Hyperhomocysteinemia and myocardial remodeling in the sand rat, Psammomys obesus.

OBJECTIVE: Numerous studies have shown that a methionine-rich diet induces hyperhomocysteinemia (Hhcy), a risk factor for cardiovascular diseases. The objective of the present study was to determine the involvement of Hhcy in cardiac remodeling in the sand rat Psammomys obesus. MATERIALS AND METHODS: An experimental Hhcy was induced, in the sand rat Psammomys obesus, by intraperitoneal injection of 300 mg/kg of body weight/day of methionine for 1 month. The impact of Hhcy on the cellular and matricial structures of the myocardium was analyzed with histological techniques (Masson trichrome and Sirius red staining). Immunohistochemistry allowed us to analyze several factors involved in myocardial remodeling, such as fibrillar collagen I and III, metalloproteases (MMP-2 and -9) and their inhibitors (TIMP-1 and -2), TGF-ß1 and activated caspase 3. RESULTS: Our results show that Hhcy induced by an excess of methionine causes, in the myocardium of Psammomys obesus, a significant accumulation of fibrillar collagens I and III at the interstitial and perivascular scales, indicating the appearance of fibrosis, which is associated with an immuno-expression increase of TGF-ß1, MMP-9 and TIMP-2 and an immuno-expression decrease of MMP-2 and TIMP-1. Also, Hhcy induces apoptosis of some cardiomyocytes and cardiac fibroblasts by increasing of activated caspase 3 expression. These results highlight a remodeling of cardiac tissue in hyperhomocysteinemic Psammomys obesus.

Fórmula metabólica isenta de metionina para homocistinúria clássica / Methionine-free metabolic formula for classical homocystinuria

CONTEXTO: A homocistinúria clássica (HCU) é uma doença rara, do tipo erro inato do metabolismo, de herança autossômica recessiva, cujo defeito metabólico na enzima cistationina ß-sintase (CBS) leva ao acúmulo de metionina, homocisteína (responsável pelos fenômenos tromboembólicos associados) e seus derivados, com deficiência de cistationina e cisteína (responsável pelas alterações em tecido conjuntivo associadas, tais como subluxação de cristalino). A prevalência da HCU é estimada entre 1:200.000 a 1:300.000 recém-nascidos vivos, podendo chegar a 1:60.000 (1). No Brasil, existem pelo menos 80 pacientes diagnosticados, de acordo com a Sociedade de Pacientes representante da doença, e o subdiagnóstico ou diagnóstico mais tardio que a média dos outros países acaba sendo mais frequente, uma vez que a doença não está inserida no Programa Nacional de Triagem Neonatal (PNTN). Sem diagnóstico e tratamento precoces, o paciente com HCU manifesta clinicamente as condições que ocorrem devido ao acúmulo de homocisteína e metionina e consequente deficiência de cisteína, tais como subluxação de cristalino, escoliose, redução da densidade mineral óssea (DMO), tromboembolismo, acidentes vasculares cerebrais, retardo mental, manifestações psiquiátricas, entre outras. Como a piridoxina (vitamina B6) é um cofator da CBS, ela melhora a atividade da enzima nos casos responsivos e, desta forma, acarreta em um quadro menos grave de doença. Entretanto, nem todos os pacientes com HCU são responsivos à piridoxina. Para classificar o paciente de acordo com a responsividade à piridoxina, há a necessidade de realizar o teste de suplementação, que culmina por classificar a HCU em três formas: responsiva à piridoxina, não-responsiva à piridoxina e com resposta intermediária à piridoxina. O tratamento recomendado para pacientes responsivos é piridoxina 10 mg/Kg/dia, evitando-se dose acima de 500 mg/dia. O tratamento dietético (dieta restrita em metionina e suplementada com fórmula isenta em metionina - FMIM) deve ser utilizado em pacientes que não atingiram níveis ideais de homocisteína com a suplementação de piridoxina. TECNOLOGIA: Fórmula metabólica isenta de metionina. EVIDÊNCIAS CIENTÍFICAS: Por ser uma doença bastante rara, há pouca literatura disponível que avalie a condição de HCU. Desta forma, foram considerados quaisquer estudos em seres humanos, independente do delineamento. Não foram identificados Ensaios Clínicos Randomizados ou estudos clínicos com grupo controle (sem intervenção). Foram identificados apenas três estudos observacionais nesta revisão, sendo dois irlandeses (onde sabidamente a incidência é maior que em outros países) e um coreano. Em ambos os países, a triagem para HCU é realizada no período neonatal. Nos três estudos, parte dos pacientes avaliados foi diagnosticada em períodos posteriores ao neonatal e em dois deles os autores realizaram análises comparativas entre os grupos com diagnostico neonatal e com diagnóstico tardio para avaliação dos desfechos. Os desfechos de interesse avalidos pelos estudos foram: níveis plasmáticos de homocisteína (2 estudos), densidade mineral óssea (1 estudo), níveis plasmáticos de metionina (2 estudos), eventos tromboembólicos (2 estudos), subluxação do cristalino (1 estudo) e deficiência intelectual (1 estudo) (2-4). Desfechos de sobrevida, qualidade de vida e convulsão em pacientes com HCU não foram diretamente avaliados pelos estudos, embora a ocorrência de fenômenos tromboembólicos esteja associada à sobrevida (tais eventos são a principal causa de óbito de pacientes com HCU). Os estudos demostraram que a intervenção foi capaz de manter os níveis de homocisteína dentro dos níveis-alvo preconizado pelo tratamento (<100 mmol/L). Houve a manutenção da densidade mineral óssea durante o período de seguimento de 3,4 anos do estudo que avaliou esse desfecho. Para eventos troboembólicos, o estudo de Lim e Lee (2013) identificou uma ocorrência (1/5, 20%), mas Yap e Naughten (1998), que também o avaliou, não identificou caso (0/25, 0%). Yap e Naughten (1998) identificaram a ocorrência de seis eventos de subluxação de cristalino em pacientes que não foram aderentes ao tratamento ou que foram diagnosticados tardiamente, quando comparado a nenhum evento de subluxação de cristalino no grupo de pacientes que foram diagnosticas no período neonatal e aderentes à dieta e FMIM. Não houve relatos de eventos adversos à FMIM nos estudos. IMPACTO ORÇAMENTÁRIO: Foram avaliadas duas FMIM para HCU individualmente em todos os pacientes acima de um ano de idade não responsíveis à piridoxina (HCYSMED B PLUS e XMET MAXAMUM). Dois cenários de impacto orçamentário foram projetados na perpectiva do SUS e com horizonte temporal de 5 anos (2019-2023). No Cenário 1 foi assumida a população oriunda da incidência média de HCU em recém nascidos, sendo o impacto para HCYSMED B PLUS de aproximadamente R$ 43 milhões em 2019 e R$ 221 milhões após os cinco anos, ou, para XMET MAXAMUM, de aproximadamente R$ 36,6 milhões em 2019 e R$ 188 milhões após os cinco anos propostos após a incorporação. No Cenário 2 foi assumida a população estimada de subdiagnóstico no Brasil (17,6%) em relação ao número de pacientes registrados pela Associação Brasileira de Homocistinúria (80 em 2016), sendo o impacto para HCYSMED B PLUS de aproximadamente R$ 7,5 milhões em 2019 e R$ 38,9 milhões após os cinco anos, ou, para XMET MAXAMUM, de aproximadamente R$ 6,5 milhões em 2019 e R$ 33,1 milhões após os cinco anos. A principal limitação é a incerteza quanto à população elegível ao tratamento devido à falta de dados publicados no Brasil. A diferença da dieta recomendada entre pacientes também é uma variável que não foi explorada nessa avaliação, assim como os custos com outros medicamentos. RECOMENDAÇÃO PRELIMINAR: A CONITEC, em sua 67ª reunião ordinária, realizada no dia 14 de junho de 2018, recomendou a não incorporação no SUS da fórmula metabólica isenta de metionina para homocistinúria clássica. Foram considerados vários fatores como a dificuldade no diagnóstico, as fragilidades encontradas nas evidências disponíveis e a ausência de dados para os desfechos considerados clinicamente relevantes. CONSULTA PÚBLICA: A matéria esteve em consulta pública no período de 06 a 25 de julho de 2018 e obteve um total de 746 contribuições, sendo 10 pelo formulário de contribuições técnico-científicas e 736 pelo formulário de contribuições sobre experiência ou opinião. Entre as 736 contribuições sobre experiência ou opinião, 722 eram discordantes (715 totalmente e 7 parcialmente) em relação à recomendação preliminar, e a grande maioria foi de familiares, amigos ou cuidadores do paciente (554). Todas as contribuições técnico-científicas discordaram da recomendação da CONITEC (9 totalmente e 1 parcialmente) e foram em sua maioria de profissionais da saúde (6). A empresa fabricante contribuiu sobre o impacto orçamentário. Outra contribuição sobre a análise econômica apresentada no parecer alegou que a análise foi simplista. Após apreciação das contribuições encaminhadas pela Consulta Pública, a CONITEC entendeu que as evidências disponíveis sobre a utilização da FMIM apontam benefícios na redução de eventos tromboembólicos e, consequentemente, na sobrevida de pacientes com HCU, mesmo quando diagnosticados tardiamente. Além disso, sugere-se a avaliação da inclusão da Triagem Neonatal Expandida (por "tandem") no SUS para o diagnóstico precoce da HCU e de outras doenças metabólicas. RECOMENDAÇÃO FINAL: Os membros da CONITEC presentes na 76ª reunião ordinária, no dia 04 de abril de 2019, deliberaram por unanimidade recomendar a incorporação no SUS da fórmula metabólica isenta de metionina para homocistinúria clássica, conforme Protocolo Clínico e Diretrizes Terapêuticas. Foi assinado o Registro de Deliberação nº 437/2019. DECISÃO: Incorporar a fórmula metabólica isenta de metionina para homocistinúria clássica, conforme Protocolo Clínico e Diretrizes Terapêuticas do Ministério da saúde, no âmbito do Sistema Único de Saúde - SUS. Dada pela Portaria nº 32, publicada no Diário Oficial da União nº 141, seção 1, página 89, em 24 de julho de 2019.

Intestinal Microbiota Protects against MCD Diet-Induced Steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease in western countries, with a continuously rising incidence. Gut-liver communication and microbiota composition have been identified as critical drivers of the NAFLD progression. Hence, it has been shown that microbiota depletion can ameliorate high-fat diet or western-diet induced experimental Non-alcoholic steatohepatitis (NASH). However, its functional implications in the methionine-choline dietary model, remain incompletely understood. Here, we investigated the physiological relevance of gut microbiota in methionine-choline deficient (MCD) diet induced NASH. Experimental liver disease was induced by 8 weeks of MCD feeding in wild-type (WT) mice, either with or without commensal microbiota depletion, by continuous broad-spectrum antibiotic (AB) treatment. MCD diet induced steatohepatitis was accompanied by a reduced gut microbiota diversity, indicating intestinal dysbiosis. MCD treatment prompted macroscopic shortening of the intestine, as well as intestinal villi in histology. However, gut microbiota composition of MCD-treated mice, neither resembled human NASH, nor did it augment the intestinal barrier integrity or intestinal inflammation. In the MCD model, AB treatment resulted in increased steatohepatitis activity, compared to microbiota proficient control mice. This phenotype was driven by pronounced neutrophil infiltration, while AB treatment only slightly increased monocyte-derived macrophages (MoMF) abundance. Our data demonstrated the differential role of gut microbiota, during steatohepatitis development. In the context of MCD induced steatohepatitis, commensal microbiota was found to be hepatoprotective.

The effects of an alternative diet regimen with natural methionine ingredients on white striping breast myopathy in broiler chickens.

Conventional broiler diets include synthetic methionine to optimize fast muscle growth. Recently, a conventional synthetic methionine-rich diet was compared to alternative diet regimens providing natural sources of methionine. Broilers fed diets with natural methionine sources grew at a slightly slower rate. From this study, we hypothesized that the difference in a growth rate would be reflected in features of the breast muscle from broilers fed the alternative diet. We hypothesized that white striping of pectoralis major muscle would be reduced in slower growing broilers fed the alternative diet regimen with natural methionine. We also hypothesized that there would be associated differences in gene expression for cell differentiation and pathology markers. Broilers fed a conventional corn/soy diet regimen with synthetic methionine were compared to those fed roasted cowpea and sunflower seed meal (60% corn/soy, 20% sunflower seed meal, and 20% roasted cowpea) and no synthetic methionine. Overall broiler growth, muscle gene expression, and muscle collagen content data were compared. Expression analyses of combinations of MYOD1, PPARG, COL1A2, TRIM63, SOD1, PTGS2, and CD36 genes were used to examine differentiation and inflammation in the pectoralis muscles. The group fed an alternative diet gained less weight than those fed the control diet in the starter and grower phases but not in the finisher phase. Ultimately, the conventional diet resulted in a greater final weight for the broilers. However, mean white striping scores for the pectoralis major muscles were greater in the conventional control diet regimen. Gene expression results indicated greater expression of PPARG, PTGS2, and CD36 in the muscle of broilers fed the control diet. These data associate white striping with fat deposition and inflammation. Thus, whether due to differences in feed intake, growth rate, or actual compositional differences, the alternative diet with natural methionine sources seemed to curtail amounts of white striping in broiler muscle. More studies are necessary to further discern the effect of growth rate and natural methionine sources on white striping.

Diet-induced hyperhomocysteinemia impairs vasodilation in 5/6-nephrectomized rats.

Plasma homocysteine is elevated in patients with impaired renal function, and markedly so at end-stage renal disease. As chronic kidney disease and hyperhomocysteinemia are also independent risk factors for cardiovascular disease, the latter is hypothesized to accelerate vascular abnormalities following renal failure. This study aimed to investigate the combined effect of impaired renal function and hyperhomocysteinemia on vascular function. We show that in 5/6-nephrectomized rats, a model of chronic kidney disease, a methionine-rich diet for 8 weeks induces moderate hyperhomocysteinemia, exacerbates hypertension, and attenuates the vascular response to acetylcholine, sodium nitroprusside, 8-bromo-cGMP, and isoprenaline. However, plasma nitrate/nitrite and total NOS activity in the thoracic aorta were not affected. Collectively, the data imply that hyperhomocysteinemia and end-stage renal disease synergistically impair endothelium-dependent and endothelium-independent vasodilatation by blocking the cGMP/protein kinase G and/or cAMP/protein kinase A pathways. 5/6-Nephrectomized rat with hyperhomocysteinemia induced by a methionine-rich diet would be a useful model for elucidating the pathogenesis of vascular impairment in patients with end-stage renal disease.