Ochratoxin A in domestic and imported beers in Belgium: occurrence and exposure assessment.

Determination of ochratoxin A (OTA) concentration was performed in commercial beer in Belgium using immunoaffinity column (OchraTest) clean-up and liquid chromatography. The procedure was validated and fulfilled the European Committee for Standardization's criteria. It offered a detection limit of 3 ng l(-1) and a quantification limit of 10 ng l(-1). Recovery experiments carried out with the spiked samples in the range 50-200 ng OTA l(-1) showed an overall average recovery rate of 97% (RSD = 2.8%). The validated method was applied to the analysis of 62 Belgian beers and 20 commercial beers imported from Denmark, France, Germany, Ireland, Mexico, The Netherlands and Scotland. None of these beers exceeded the previously suggested EU limit of 200 ng l(-1). However, OTA was detected in 60 Belgian beers and in all imported beers. The average levels of contamination were 33 ng l(-1) (RSD = 112%) and 32 ng l(-1) (RSD = 81%), respectively. The highest level found was 185 ng l(-1). On the basis of the established tolerable daily intake (TDI) of 5 ng kg(-1) body weight, accepted by the scientific committee on food of the EU, this study indicates that beer consumption in Belgium is not likely to contribute to more than a few per cent of the TDI based on the average consumption. This study also shows variability of the OTA contamination in beer with time. Thus, there is a potential risk of having highly contaminated batches from time to time. We therefore recommend to control further the OTA contamination in brewery products and to take precautionary measures during harvest, transport and storage of the raw materials to maintain the OTA intake at the lowest achievable level.

Transfer of ochratoxin A during lactation: exposure of suckling via the milk of rabbit does fed a naturally-contaminated feed.

The transfer of ochratoxin A from the blood to the milk of lactating rabbit does and subsequently the exposure of their sucklings to the mycotoxin were investigated. An effective transfer of ochratoxin A from blood to milk was shown in lactating rabbit does fed a naturally-contaminated diet (10-20 g/kg of body weight/day) throughout a lactation period of 19 days. The ochratoxin A concentrations in plasma and in milk did not significantly change throughout the lactation period with a mean milk/plasma concentration ratio of 0.015. These variables were however significantly correlated (p < 0.05), as were the ingested amounts and milk concentrations. At slaughter, the highest concentration of ochratoxin A accumulated in the body of the rabbit does were found in kidney (1.2 g/kg) followed by liver (158 ng/kg), mammary gland (105 ng/kg) and muscle (38 ng/kg). A linear relationship was found between the ochratoxin A concentrations in milk and in the plasma of the sucklings, indicating an effective transfer of the toxin to the sucklings. If the same is true in humans, the exposure of the breast-fed infant to the toxin, which has been largely reported in the literature, should be a major matter of concern for human health.

Inhibition of myocardial glucose uptake by cGMP.

Guanosine 3',5'-cyclic monophosphate (cGMP), a second messenger of nitric oxide (NO), regulates myocardial contractility. It is not known whether this effect is accompanied by a change in heart metabolism. We report here the effects of 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), a cGMP analog, on regulatory steps of glucose metabolism in isolated working rat hearts perfused with glucose as the substrate. When glucose uptake was stimulated by increasing the workload, addition of the cGMP analog totally suppressed this stimulation and accelerated net glycogen breakdown. 8-BrcGMP did not affect pyruvate dehydrogenase activity but activated acetyl-CoA carboxylase, the enzyme that produces malonyl-CoA, an inhibitor of long-chain fatty acid oxidation. To test whether glucose metabolism could also be affected by altering the intracellular concentration of cGMP, we perfused hearts with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, or with S-nitroso-N-acetylpenicillamine (SNAP), a NO donor. Perfusion with L-NAME decreased cGMP and increased glucose uptake by 30%, whereas perfusion with SNAP resulted in opposite effects. None of these conditions affected adenosine 3',5'-cyclic monophosphate concentration. Limitation of glucose uptake by SNAP or 8-BrcGMP decreased heart work, and this was reversed by adding alternative oxidizable substrates (pyruvate, beta-hydroxybutyrate) together with glucose. Therefore, increased NO production decreases myocardial glucose utilization and limits heart work. This effect is mediated by cGMP, which is thus endowed with both physiological and metabolic properties.

Cyclic AMP suppresses the inhibition of glycolysis by alternative oxidizable substrates in the heart.

In normoxic conditions, myocardial glucose utilization is inhibited when alternative oxidizable substrates are available. In this work we show that this inhibition is relieved in the presence of cAMP, and we studied the mechanism of this effect. Working rat hearts were perfused with 5.5 mM glucose alone (controls) or together with 5 mM lactate, 5 mM beta-hydroxybutyrate, or 1 mM palmitate. The effects of 0.1 mM chlorophenylthio-cAMP (CPT-cAMP), a cAMP analogue, were studied in each group. Glucose uptake, flux through 6-phosphofructo-1-kinase, and pyruvate dehydrogenase activity were inhibited in hearts perfused with alternative substrates, and addition of CPT-cAMP completely relieved the inhibition. The mechanism by which CPT-cAMP induced a preferential utilization of glucose was related to an increased glucose uptake and glycolysis, and to an activation of phosphorylase, pyruvate dehydrogenase, and 6-phosphofructo-2-kinase, the enzyme responsible for the synthesis of fructose 2,6-bisphosphate, the well-known stimulator of 6-phosphofructo-1-kinase. In vitro phosphorylation of 6-phosphofructo-2-kinase by cAMP-dependent protein kinase increased the Vmax of the enzyme and decreased its sensitivity to the inhibitor citrate. Therefore, in hearts perfused with various oxidizable substrates, cAMP induces a preferential utilization of glucose by a concerted stimulation of glucose transport, glycolysis, glycogen breakdown, and glucose oxidation.

In vitro effects of valproate and valproate metabolites on mitochondrial oxidations. Relevance of CoA sequestration to the observed inhibitions.

The inhibitory effects of valproate (VPA) and nine of its metabolites on mitochondrial oxidations have been investigated. Valproate, 4-ene-VPE, 2,4-diene-VPA and 2-propylglutaric acid inhibited the rate of oxygen consumption by rat liver mitochondrial fractions with long- and medium-chain fatty acids, glutamate (+/- malate), succinate, alpha-ketoglutarate (+ malate) and pyruvate (+ malate) as substrates. Sequestration of intramitochondrial free CoA by valproate and these three metabolites has been demonstrated and quantified. However, CoA trapping could not account for all the inhibitions observed. 2-ene-VPA and 3-oxo-VPA, metabolites formed during the beta-oxidation of valproate, were not capable of trapping intramitochondrial CoA although they were inhibitors of the beta-oxidation of decanoate, probably by inhibition of the medium-chain acyl-CoA synthetase.

Cytochrome aa3 depletion is the cause of the deficient mitochondrial respiration induced by chronic valproate administration.

Liver mitochondria from rats fed 1% (w/w) valproic acid for 75 days displayed an approximate 30% decrease in coupled respiration rates with substrates entering the respiratory chain at complexes I and II. Uncoupling the respiration from proton-pumping, or measuring the respiration without complex IV removed this inhibition. The treatment induced a loss of activity of cytochrome oxidase consistent with a decrease in the mitochondrial content of cytochrome aa3. The inhibition induced by long lasting administration of valproate is apparently located at the site of the proton-pumping activity of complex IV. Furthermore, the capacity of electron transport through complex IV, being far in excess of that required for normal functioning in coupled mitochondria, seems to be controlled by the coupling to proton-pumping in which cytochrome aa3 appears to play a major role.

Influence of chronic administration of valproate on ultrastructure and enzyme content of peroxisomes in rat liver and kidney. Oxidation of valproate by liver peroxisomes.

Chronic administration to rats of the anticonvulsant drug, valproate, induced proliferation of liver peroxisomes and selectively increased the activity of the enzymes involved in beta-oxidation in these organelles. In kidney cortex, only a moderate increase in enzyme activity could be recorded. Valproate (1% w/w in the diet for 25 to 100 days) caused the appearance on electron micrographs of unusual tubular inclusions in the matrix of liver peroxisomes. SDS-PAGE analysis of purified peroxisomal fractions from treated rats demonstrated an increase in the content of five polypeptides; four of which most likely correspond to enzymes of the peroxisomal beta-oxidation. It is suggested that the peroxisomal inclusions correspond to the accumulation of these polypeptides in the matrix of the organelle. An in vivo evaluation of the peroxisomal hydrogen peroxide production suggested that valproate itself or one of its metabolites is substrate for peroxisomal beta-oxidation. This was confirmed by in vitro studies. Activation of valproate or its metabolites by liver acyl-CoA synthetase could be demonstrated, although it was 50 times slower than that of octanoate. This reaction further led to a small, but significant production of H2O2 by the action of peroxisomal acyl-CoA oxidase.