Curcumin protects against tartrazine-mediated oxidative stress and hepatotoxicity in male rats.

OBJECTIVE: Synthetic dyes have been reported to exert detrimental effects on the health of humans. This study evaluated the effects of a diet containing tartrazine (Tz) on rats which included: i) biochemical parameters including hepatic enzymes, kidney functions and profiles of lipids; ii) markers of oxidative stress in cells by measuring concentrations of malondialdehyde (MDA) and glutathione (GSH); iii) activities of selected, key hepatic antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx); iv) pathologies of liver. Also, protective effects of three doses of curcumin (CUR), a natural food coloring agent, on these parameters in rats that had been co-exposed to Tz. MATERIALS AND METHODS: Fifty Wistar male albino rats were randomly divided into five groups: Group I, control, where rats were fed a normal diet; Group II, rats were fed normal diets containing 7.5 mg Tz/kg diet, dry mass (dm); In Groups III, IV and V, rats were fed diets containing Tz plus 1.0, 2.0 or 4.0 g CUR/kg diet, dm, respectively. Whole blood was collected after 90 d of exposure, homogenates of liver were prepared and the above analyses were conducted. RESULTS: Exposure to Tz in the diet caused statistically significant (p<0.05) greater concentrations of lipids, hepatic enzymes, and kidney function parameters as well as the indicator of oxidative stress MDA. Alternatively, activities of several antioxidant enzymes (i.e. CAT, SOD and GPx) and concentration of the substrate GSH, an indicator of non-enzymatic antioxidant capability, were significantly (p<0.05) less than those in control rats not exposed to Tz. Tz caused various histopathological changes in livers of rats, which were characterized by hemorrhage and dilatation of the central vein and sinusoids, hepatocyte necrosis, intracellular vacuolization. Co-administration of 2.0 (Group IV) or 4.0 g CUR/kg diet (Group V) with Tz significantly mitigated effects on functions of liver and kidney and the profile of relative concentrations of lipids. CUR significantly (p<0.05), and almost completely, reversed effects on enzymatic and non-enzymatic antioxidant and indicators of oxidative stress about rats fed Tz (Group II) to values in control rats. However, co-administration of 1.0 g CUR with Tz (Group III) exhibited a negligible effect on those parameters. The results of this study suggest benefits of the use of CUR, as a promising natural food additive to counteract oxidative stress caused by dietary exposure to the synthetic dye Tz due to potent protective antioxidant activity. CONCLUSIONS: Blending some natural food additives, such as CUR with diets containing synthetic dyes, could moderate potential effects of these artificial dyes. Decreasing or removing toxins in food is an essential step for the amelioration of human health status and decreasing risk of onset or progression of degenerative diseases.

The kinetics of the effect of manganese supplementation on SOD2 activity in senescent human fibroblasts.

OBJECTIVE: To investigate the effect of increasing Mn+2 concentrations on superoxide dismutase 2 (SOD2) activity in pre-senescent and senescent cultured fibroblasts, and to determine the Km Mn+2 values required to achieve maximal SOD2 activities in such cells. MATERIALS AND METHODS: SOD2 activities, and superoxide anion (SOA) generation rates, were assayed in mitochondrial sonicates of young passage 5 fibroblasts sub-cultured in routine growth medium (MEM 1), and in an accurately identified senescent passage 20, 25 and 30 subcultures incubated with media containing supplemental Mn+2 increments equal to 60, 90, 120, 150 and 180 nM (MEM 2, 3, 4, 5 and 6 respectively). RESULTS: Whereas SOD2 activity did not significantly change in any of the cells sub-cultured in MEM 1, the enzyme underwent progressive significant increases in early senescent passage 20 cells and senescent passage 25 and 30 cells. Such increases were relative to Mn+2 concentration and peaked in value in the senescent cells incubated with MEM 5 and MEM 6. Furthermore, whereas SOA generation rates underwent significant progressive increases in MEM 1-incubated senescent passage 20-30 cells, peaking in value at passage 30, the rates were gradually and significantly lowered in the cells incubated with MEM 2-MEM 6, and reached lowest values in those incubated with MEM 6 (p<0.001 for all comparisons). The computed Km values of Mn+2 with respect to SOD2 in senescent passage 20, 25 and 30 cells equalled 19.2, 39.6 and 54.4 nM respectively with corresponding SOD2 Vmax values of 37.6, 55.9 and 71.4 µmol/min/mg protein. CONCLUSIONS: Senescent cells near the end of their replicative life span utilise more Mn+2 and achieve maximal SOD2 activities suggesting that the use of supplementary Mn+2 can help in combating oxidative stress.

Changes in adenosine deaminase activity in ageing cultured human cells and the role of zinc.

The level of adenosine deaminase (ADA; EC 3.5.4.4) was estimated at different passages in six confluent fibroblast cultures established from forearm skin biopsies of healthy adult normal volunteers. After determination of the zinc concentration in standard growth medium, ADA activity was estimated at different passages of subculture in media with different zinc concentrations. The results indicated that the specific activity of ADA in control confluent skin fibroblast cultures (passage 2) cultivated in standard growth medium containing 15.4 microM zinc (similar to that present in normal human plasma) was equal to 226.6+/-19.64 micromol min(-1) mg(-1) protein. The results showed that there were no significant changes in ADA specific activity in any of the control cultures as the zinc concentration of the medium was increased. To characterize the passage of subculture at which fibroblasts enter the ageing phase, three marker enzymes were assayed namely, phosphofructokinase, lactate dehydrogenase and glycogen phosphorylase. The result showed that the cells enter the ageing phase at passage 20 and beyond. Further investigation showed that ADA activity of serially subcultured confluent cultures cultivated in standard growth medium significantly dropped at passages 20, 25 and 30. ADA activity however was not significantly altered in cells at passage 2, 10 and 15 cultivated in standard growth medium and in the presence of higher zinc levels (23.1, 34.6, 53.8 and 73.1 microM). Furthermore there was significant lowering of ADA activities in cells at passages 20, 25 and 30 when cells were cultured in the presence of 15.4, 23.1 and 34.6 microM zinc. Such lowered activities of ADA were restored to normal when the cells were cultured in the presence of higher zinc concentration equal to 53.8 and 73.1 microM. From the results we concluded that it is possible to restore ADA activity in aged skin fibroblasts to normal levels by raising the zinc concentration in the culture medium to four or five times the control normal plasma zinc level.

Separation and purification of Echis coloratus venom and some biological and biochemical effects of the proteins.

Crude venom of Echis coloratus was separated into seven protein fractions using 7% preparative native polyacrylamide gel electrophoresis. The effect of crude venom and seven venom protein fractions (F1-F7) from Echis coloratus on key metabolic activities of fibroblast cultures was investigated. Confluent cultures were incubated with the venom proteins for 3 h at 37 degrees C. The specific activity of phosphofructokinase, was significantly lowered upon incubation with the crude venom and with fractions 2, 3, 4 and 6. Citrate synthase activity was significantly lowered by the crude venom and by fractions 2 and 3. Glycogen phosphorylase activity was significantly increased by the crude venom and by fractions 2, 3, 4 and 6 leading to a significant concurrent drop in glycogen content. Creatine kinase activity was significantly increased by the crude venom and by fractions 3, 4, 5 and 6. Cellular ATP levels rose significantly upon incubation with the crude venom and with fractions 3, 4, 5 and 6. Incubation of cell sonicates with all the venom proteins did not significantly alter the activity or content of any of the studied parameters.

Establishment of a normal reference range for adenosine deaminase in plasma of healthy Saudis using the continuous spectrophotometric method.

The level of adenosine deaminase (EC. 3.5.4.4), was estimated in plasma of 389 healthy males and 493 healthy females in order to establish a normal reference range for Saudis. Using the continuous spectrophotometric method, the reference ranges were calculated in two ways using the mean +/- 2 SD and the 2.5th - 97.5th percentile value methods. In both methods of calculation, a slightly higher range was observed for children as compared to adults. The method of 2.5th - 97.5th percentile values brought almost all of our subjects within the recommended range of 11.5 - 25 U/l. In the current study, the normal range for adenosine deaminase totalled 15.0 - 23.2, 14.8 - 23.6, 15.0 - 23.0 and 16.7 - 24.6 U/l for the overall population, all males, females, and children, respectively. The ranges are discussed in the light of significantly different results obtained by the two calculation methods and recommendation of an appropriate method for healthy Saudis, namely the 2.5th - 97.5th percentile values. The choice of the Ellis and Goldberg kinetic continous monitoring method for the estimation of plasma ADA levels in the current investigation is also hereby justified.

The effect of crude Cerastes cerastes gasperetti venom on the activity of key metabolic enzymes in cultured human fibroblasts.

Cultured human fibroblasts were used to study the effect of a crude extract of Cerastes cerastes gasperetti venom on the activity of a profile of key enzymes of metabolism. A single concentration of the crude venom was incubated with confluent fibroblasts established from six normal subjects for a period of three hours. A dramatic reduction in the specific activities of glucose and glycogen degradative enzymes was observed (23.7 +/- 3.9%, 36.3 +/- 8.7% and 71.1 +/- 5.7% of control for citrate synthase, glucose-6-phosphate and phosphofructokinase respectively). Furthermore, the specific activity of creatine kinase was doubled. No significant change in activity of three transaminases was noticed. Incubation of the same concentration of venom for the same period of time with serum did not result in any change in the activity of the enzymes studied. It is suggested that the cells mobilize stored phosphocreatine for the production of adenosine triphosphate (ATP) to compensate for the reduced rate of sugar catabolism. Furthermore, it is hereby suggested that the effects noticed on the enzyme activities are not directed at the enzyme protein itself, but are of mediated nature.

Changes in the activity of glutamate related enzymes in cerebral cortex, during insulin-induced seizures.

The activity of glutamate related enzymes and the concentration of glutamine, glutamate and gamma-amino n-butyric acid (GABA) were investigated in the cerebral cortex of rats, in different stages of insulin-induced hypoglycemia. Hypoglycemia was produced by intraperitoneal injection of insulin 0.05-100 units per kg body weight. The minimum required dose to produce irreversible severe hypoglycemia was 0.5 units/kg. In 85% of the cases an insulin induced hypoglycemic convulsion, was achieved 130-150 minutes after injection. Blood glucose levels during insulin induced seizures ranged between 8-15 mg%. In the range of 0.5-100 u insulin/kg the degree of hypoglycemia and the onset of convulsions were identical. The concentration of glutamine was significantly reduced during convulsive and postconvulsive stages. Glutamate and GABA concentrations were reduced significantly in all stages of insulin-induced hypoglycemia. The decrease in glutamine concentration was concurrent with an increase in the activity of its degradative enzyme, glutaminase. This was apparent at the preconvulsive, convulsive and postconvulsive stages. The activity of other enzymes related to energy production such as glutamate dehydrogenase (GDH), glutamate transaminase (GPT) and aspartate aminotransferase (AAT) were also increased. The activity of glutamine synthase (GS) was unaffected by hypoglycemia. Insulin induced changes in glutamine, glutamate and their related enzymes could not be attributed to convulsion since a similar pattern of changes was observed in the preconvulsive and postconvulsive stages, and no changes were detected following picrotoxin-induced seizures.

Enzyme studies in biotin-responsive disorders.

There appear to be at least two underlying aetiologies for combined carboxylase deficiency; firstly, a failure of biotinylation of apocarboxylases due to a mutation of holocarboxylase synthetase (EC 6.3.4.10) which results in an enzyme with a high Km with respect to biotin and secondly, a failure of biotinylation due to a lowered availability of biotin due to biotinidase deficiency (EC 3.5.1.12). In both these disorders secondary defects of all four biotin-dependent carboxylases result which in turn causes the excretion of the metabolites characteristic of the isolated carboxylase deficiencies. In addition, both disorders respond biochemically and clinically to the administration of large amounts of biotin.

Pyruvate carboxylase deficiency.

The causes of congenital lactic acidaemia are outlined. Isolated pyruvate carboxylase deficiency is reviewed in detail with a report of a recent case and a discussion of the biochemical consequences. Other causes of defective pyruvate carboxylation are described, particularly the combined carboxylase defects.

Isolated biotin-resistant 3-methylcrotonyl-CoA carboxylase deficiency in two sibs.

Two Vietnamese siblings with an isolated deficiency of 3-methylcrotonyl coenzyme A carboxylase in leucocytes and cultured fibroblasts are described. Both children excreted massive amounts of 3-methylcrotonylglycine and 3-hydroxyisovaleric acid. There was no in vivo or in vitro biochemical response to biotin. Apart from an attack of vomiting leading to subcoma in the elder sib four weeks after arrival in the Netherlands, the children were in good health. There were no signs of delayed mental development.

Evaluation of 3,4-dinitrophenyl tetra-N-acetyl-beta-chitotetraoside as a substrate for the measurement of lysozyme in normal and pathological sera.

Lysozyme was measured using the synthetic substrate 3',4-dinitrophenyl tetra-N-acetyl-beta-chitotetraoside and the LKB Reaction Rate Analyser. This method has been evaluated by comparing levels obtained with serum samples from healthy individuals and patients with either cancer or inflammatory bowel disease with those obtained from the same specimens using a turbidimetric method. In terms of standard egg-white lysozyme, the colorimetric method gave much higher levels for all samples than the turbidimetric method; however, similar group differences were maintained. For individual serum specimens significant correlation between the two methods was found to occur only in the healthy group. Assay precision for the two methods was similar but the turbidimetric method could detect levels of lysozyme activity which were 10 times lower than those detected by the colorimetric method.