I. Tavares de Almeida

Homocysteine metabolism, hyperhomocysteinaemia and vascular disease: An overview

SummaryHyperhomocysteinaemia has been regarded as a new modifiable risk factor for atherosclerosis and vascular disease. Homocysteine is a branch-point intermediate of methionine metabolism, which can be further metabolised via two alternative pathways: degraded irreversibly through the transsulphuration pathway or remethylated to methionine by the remethylation pathway. Both pathways are B-vitamin-dependent. Plasma homocysteine concentrations are determined by nongenetic and genetic factors. The metabolism of homocysteine, the role of B vitamins and the contribution of nongenetic and genetic determinants of homocysteine concentrations are reviewed. The mechanisms whereby homocysteine causes endothelial damage and vascular disease are not fully understood. Recently, a link has been postulated between homocysteine, or its intermediates, and an alterated DNA methylation pattern. The involvement of epigenetic mechanisms in the context of homocysteine and atherosclerosis, due to inhibition of transmethylation reactions, is briefly overviewed.

Guanidinoacetate methyltransferase deficiency identified in adults and a child with mental retardation

Our study describes the adult clinical and biochemical spectrum of guanidinoacetate methyltransferase (GAMT) deficiency, a recently discovered inborn error of metabolism. The majority of the previous reports dealt with pediatric patients, in contrast to the present study. A total of 180 institutionalized patients with a severe mental handicap were investigated for urine and plasma uric acid and creatinine. Patients with an increased urinary uric acid/creatinine ratio and/or decreased creatinine were subjected to the analysis of guanidinoacetate (GAA). Four patients (three related and one from an unrelated family) were identified with GAMT‐deficiency. A fifth patient had died before a biochemical diagnosis could be made. They all had shown a normal psychomotor development for the first year of life, after which they developed a profound mental retardation. Three out of four had convulsions and all four totally lacked the development of speech. Their GAMT activity in lymphoblasts was impaired and two novel mutations were identified: the 59 G > C and 506 G > A missense mutations. Urinary GAA was increased, but highly variable 347–1,624 mmol/mol creat (Controls <150 mmol/mol creat). In plasma and CSF the GAA levels were fairly constant at 17.3–27.0 μmol/L (Controls 1.33–3.33) and 11.0–12.4 μmol/L, respectively (Controls 0.068–0.114). GAMT deficiency in adults is associated with severe mental retardation and absence or limited speech development. Convulsions may be prominent. The nonspecific nature of the clinical findings as well as the limited availability of GAA assays and/or in vivo magnetic resonance spectroscopy of the brain may mean that many more patients remain undiagnosed in institutions for persons with mental handicaps.

Inhibitory effect of 3-hydroxyacyl-CoAs and other long-chain fatty acid beta-oxidation intermediates on mitochondrial oxidative phosphorylation

Among the abnormal biochemical findings, a mild to severe lactic acidaemia (Treem et al 1994) is frequently described in patients with a long-chain fatty acid β-oxidation disorder, together with a markedly increased plasma long-chain acylcarnitine fraction, abnormal excretion of C 6 -C 10 dicarboxylic acids and an accumulation of medium- and long-chain 3-hydroxydicarboxylic acids (Duran et al 1991). Our aim was to explain the lactic acidosis found in these patients and to associate this with the accumulation of long-chain fatty acid derivatives. Previously (Ventura et al 1995) we have reported that long-chain (C 14 -C 20 ) acyl-CoA esters, both saturated and unsaturated, inhibit the synthesis of ATP in digitonin-permeabilized normal human fibroblasts (Wanders et al 1993). Using the same system we investigated in this work the effects of palmitoyl-CoA, α,β-unsaturated palmitoyl-CoA, 3-hydroxy- and 3-ketopalmitoyl-CoA on the oxidative phosphorylation system.

Valproate inhibits the mitochondrial pyruvate-driven oxidative phosphorylation in vitro

Valproic acid (2-n-propylpentanoic acid; VPA), a medium-chain branched fatty acid, has proved to be an effective antiepileptic drug. It has also been used widely in the symptomatic treatment of children for a variety of types of seizure. Hepatotoxicity is one of the adverse effects. Several groups have studied the in vitro deleterious effects of VPA on cellular intermediary metabolism. However, the exact mechanisms of VPA-associated hepatotoxicity are still unknown. Despite the structural simplicity of valproate, its metabolic profile is quite complex. Glucuronidation and β-oxidation are quantitatively the most important routes of biotransformation, generating a complex pattern of intermediates (Baillie 1992) which could potentially interfere with mitochondrial metabolism at different levels. In the past, several papers have described inhibitory effects of VPA and its metabolites on mitochondrial oxidative phosphorylation, both in vitro (Haas et al 1981; Becker and Harris 1983; Ponchaut et al 1992) and in vivo (Rumbach et al 1983). However, the results are not easily comparable and the reported data are quite contradictory. Although our primary interest was the effect of VPA on β-oxidation, we decided to study in parallel the interference of VPA with mitochondrial substrate oxidations. We therefore investigated the effect of this drug on mitochondrial oxidative phosphorylation, in concentrations similar to those found in man during therapy. The effects of two of its unsaturated metabolites, 2-propyl-2-pentenoic acid (1 mmol/L) (Δ 2(E) -VPA; mitochondrial β-oxidation intermediate) and 2-propyl-4-pentenoic acid (1 mmol/L) (Δ 4 -VPA; microsomal dehydrogenation product), were also studied. Two experimental approaches were used for this study: digitonin-permeabilized rat hepatocytes for the study of the rate of ATP synthesis, and rat liver mitochondria for the study of oxygen consumption by polarography, both in the presence of selected respiratory chain substrates.

Quantitative analysis of urinary acylglycines for the diagnosis of beta-oxidation defects using GC-NCI-MS

The analysis of acylglycines is an important biochemical tool for the diagnosis of inherited disorders of mitochondrial fatty acid beta-oxidation. A stable isotope dilution gas chromatography negative chemical ionisation mass spectrometry method for the quantitative analysis of short- and medium-chain acylglycines as their bis(trifluoromethyl)benzyl (BTFMB) ester derivatives is described. The diagnostic usefulness of the method was demonstrated in nine patients with medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) deficiency, and seven patients with multiple acyl-CoA dehydrogenation defect (MAD). The urinary acylglycine profiles in these patients were compared to those in controls (n = 19), children on a medium-chain triglyceride (MCT) supplemented diet (n = 4), and patients with various other diseases (n = 5).

Lactic acidosis in long-chain fatty acid β-oxidation disorders

Among the many disorders of fatty acid β-oxidation known today, the disorders of long-chain fatty acid oxidation are the most severe and life-threatening. One remarkable abnormality, not observed in, for instance, medium-chain acyl-CoA dehydrogenase deficiency, is the moderate to severe lactic acidaemia in long-chain fatty acid β-oxidation-deficient patients, suggesting that oxidation of pyruvate is also compromised. In order to understand the underlying basis of the lactic acidaemia in these patients, we have studied the formation of L-lactate and pyruvate in cultured skin fibroblasts incubated with D-glucose. All long-chain fatty acid β-oxidation-deficient cell lines studied were found to show a moderate elevation of lactate when compared with control and medium-chain acyl-CoA dehydrogenase-deficient fibroblasts. Interestingly, differences were found between cells deficient in long-chain 3-hydroxyacyl-CoA dehydrogenase and very-long-chain acyl-CoA dehydrogenase, suggesting that saturated acyl-CoA esters and their 3-hydroxyacyl-CoA derivatives affect pyruvate metabolism differently.

3-Hydroxy-3-methylglutaric aciduria combined with 3-methylglutaconic aciduria: A new case

The urinary organic acids of a new case of 3-hydroxy-3-methylglutaryl-CoA lyase deficiency have been analysed by gas chromatography-mass spectrometry. This patients fibroblasts showed a reduced ability to oxidize leucine.

Cellular hypomethylation is associated with impaired nitric oxide production by cultured human endothelial cells

Hyperhomocysteinemia (HHcy) is a risk factor for vascular disease, but the underlying mechanisms remain incompletely defined. Reduced bioavailability of nitric oxide (NO) is a principal manifestation of underlying endothelial dysfunction, which is an initial event in vascular disease. Inhibition of cellular methylation reactions by S-adenosylhomocysteine (AdoHcy), which accumulates during HHcy, has been suggested to contribute to vascular dysfunction. However, thus far, the effect of intracellular AdoHcy accumulation on NO bioavailability has not yet been fully substantiated by experimental evidence. The present study was carried out to evaluate whether disturbances in cellular methylation status affect NO production by cultured human endothelial cells. Here, we show that a hypomethylating environment, induced by the accumulation of AdoHcy, impairs NO production. Consistent with this finding, we observed decreased eNOS expression and activity, but, by contrast, enhanced NOS3 transcription. Taken together, our data support the existence of regulatory post-transcriptional mechanisms modulated by cellular methylation potential leading to impaired NO production by cultured human endothelial cells. As such, our conclusions may have implications for the HHcy-mediated reductions in NO bioavailability and endothelial dysfunction.

Organic acid profiles resembling a beta-oxidation defect in two patients with coeliac disease

Inbom defects of mitochondrial fatty acid oxidation are an important group of inherited diseases. Nearly all of these defects present in early infancy as acute life-threatening episodes of hypoketotic hypoglycaemia induced by fasting. In some patients chronic muscle weakness or cardiomyopathy (Hale and Bennett 1992) have also been described. Because affected patients are usually free of symptoms between episodes, their clinical recognition may be difficult and usually depends on a combination of clinical and biochemical investigations. The first indication of the presence of a β-oxidation defect often arises in the course of routine investigation of urinary organic acids. An inappropriate hypoketotic dicarboxylic aciduria is then one of the best clues. So far all defects of intramitochondrial β-oxidation have been found to be associated with dicarboxylic aciduria (Duran et al 1991). Furthermore, specific dicarboxylic acids found in urine or plasma or both are diagnostically useful in the recognition of inborn errors of fat metabolism (Hale and Bennett 1992). However, elevated levels of dicarboxylic acids have also been detected in normal individuals with certain dietary supplements such as medium-chain triglycerides, drugs (valproic acid) or during fasting. In this report we describe two patients with coeliac disease and prolonged malnourishment whose urinary organic acid profile during a crisis of metabolic decompensation was similar to those frequently observed in long-chain fatty oxidation disorders.

Tyrosinaemia Type I with Normal Levels of Plasma Tyrosine

I. TAVARES DE ALMEIDA l, P. P. LEANDRO 1, M. F. B. SILVA 1, C. SILVEIRA l, A. DA SILVA 2, J. SALAZAR DE SOUSA 2 and M. DURAN 3 1Centro de Metabolismos e Genktica (LN.LC.), Faculdade de Farmdcia, 1600 Lisboa, Portugal, 2 paediatric Gastroenterology Unit, Sta. Maria Hospital, Faculdade de Medecina, Lisboa, Portugal; 3 University Childrens Hospital, Het Wilhelmina Kinderziekenhuis, Nieuwe Gracht 137, NL-3512 LK Utrecht, The Netherlands