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A thin-layer chromatography-2,2-diphenyl-1-picrylhdrazyl (TLC-DPPH) method has been developed for the quantification of withanolides and simultaneous estimation of antioxidant potential of each constituent present in a complex mixture. High-performance thin-layer chromatography (HPTLC) of withanolide-rich fraction of Withania somnifera root was performed on Si 60F254 HPTLC plates with dichloromethane-toluene-methanol-acetone-diethyl ether (6.5:7:4:1.5:1, v/v) as a mobile phase for the separation of withanolides and on the basis of RF values 0.07, 0.55, 0.61, 0.63, 0.66, and 0.77 for withanoside V, withaferine A, 1,2-deoxywithastramonolide, withanone, withanolide A, and withanolide B, respectively. The concentrations of withanoside V, withaferine A, 1,2-deoxywithastramonolide, withanone, withanolide A, and withanolide B were 0.86, 12.9, 1.92, 1.52, 5.24, and 4.52 mg g-1 respectively. A TLC-DPPH rapid test was used to assess the free-radical scavenging activity of constituents in withanolide-rich fraction for the first time. In withanolides, withanoside V and withanolide B show the highest antioxidant potential. In unknown compounds, peak No. 17 at RF 0.92 has the highest antioxidant potential and peak No. 16 at RF 0.87 has recorded the lowest antioxidant potential. It is also found that some of the unidentified components had significant antioxidant potential. This technique has several advantages; therefore, this method will be useful for rapidly validating the Ayurvedic products containing W. somnifera. [ABSTRACT FROM AUTHOR]

Effects of treatment with a single intraperitoneal injection of cadmium (Cd) on oxidative energy metabolism and lipid/phospholipid profiles of rat liver mitochondria were examined at the end of 1 week and 1 month. Following Cd treatment the body weight increased only in the 1 month group, whereas the liver weight increased in both groups. State 3 and 4 respiration rates in general decreased significantly, with the maximum effect being seen with succinate. The 1 week Cd group showed decreased respiratory activity with glutamate, pyruvate + malate, and succinate as the substrates. In the 1 month Cd-treated group respiration rates recovered with glutamate and pyruvate + malate but not with succinate. All cytochrome contents decreased in the 1 week Cd-treated group but recovered in the 1 month group. ATPase activity registered an increase in both Cd-treated groups. Dehydrogenase activities increased in the 1 week group but decreased in the 1 month Cd-treated group. The mitochondrial cholesterol content increased in the 1 week Cd-treated group. In the 1 week Cd-treated group the lysophospholipid (Lyso), sphingomyelin (SPM), and diphosphatidylglycerol (DPG) components increased. By contrast, the phosphatidylethanolamine (PE) component decreased. In the 1 month Cd-treated group the phosphatidylinositol, phosphatidylserine, and DPG components increased, whereas the Lyso, SPM, and phosphatidylcholine components decreased. The results demonstrate that single-dose Cd treatment can have adverse effects on liver mitochondrial oxidative energy metabolism and lipid/phosphopholipid profiles, which in turn can affect membrane structure-function relationships. [ABSTRACT FROM AUTHOR]

Effects of ageing on the lipid/phospholipid profile of brain and liver mitochondria from rats were examined. In the brain mitochondria the contents of total phospholipid (TPL) and cholesterol (CHL) increased with simultaneous increase in the TPL/CHL (mole:mole) ratio. The proportion and contents of lysophospholipid (Lyso), sphingomyelin (SPM), phosphatidylinositol (PI), phosphatidylserine (PS) and diphosphatidylglycerol (DPG) components increased, with maximal increases seen for PS and PI; phosphatidylcholine (PC) and phosphatidylethanolamine (PE) components registered decrease. In the liver mitochondria contents of TPL and CHL increased. However, the TPL/CHL (mole:mole) ratio was not altered. Lyso, PI and PS increased. However, the magnitude of increase was competitively lower; PE and DPG decreased. SPM and PC did not change as a consequence of ageing. These changes altered the contents of individual phospholipids in the two membrane systems. Respiration with glutamate, pyruvate + malate, succinate and ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine was significantly impaired in brain mitochondria from old animals. For liver mitochondria the respiratory activity declined with glutamate and succinate. Correlation studies by regression analysis revealed that the lipid/phospholipid classes regulate respiratory function differently in the mitochondria from the two tissues. The respiration-related parameters in the brain mitochondria were dependent on multiple lipid/phospholipid components, and the process of regulation was complex compared to the liver mitochondrial functions. [ABSTRACT FROM AUTHOR]

The effects of thyroidectomy (Tx) and subsequent treatment with 3,5,3′-triiodothyronine (T3) or combined replacement therapy (TR) with T3 and thyroxine (T4) on the substrate and temperature kinetics properties of Na+,K+-ATPase and lipid/phospholipid makeup of rat kidney microsomes were examined. Enzyme activity was somewhat high in the hypothyroid (Tx) animals and increased significantly following T3 treatment, while TR treatment caused a decrease. In the Tx and T3 groups enzyme activity resolved in two kinetic components, while in the TR group the enzyme showed allosteric behavior up to 0.5 m m ATP concentration. The K m and V max values of both the components decreased in Tx animals without affecting the catalytic efficiency. T3 treatment caused a significant increase in the V max of both the components, with a significant increase in the catalytic efficiency, while the K m values were not upregulated. The TR regimen lowered the K m and V max of component II but improved the catalytic efficiency. Thyroid status-dependent changes were also noted in the temperature kinetics of the enzyme. Regression analysis revealed that changes in the substrate and temperature kinetics parameters correlated with specific phospholipid components. [ABSTRACT FROM AUTHOR]

Effect of thyroidectomy (Tx) and subsequent treatment with 3,5,3′-triiodo- l-thyronine (T3) or replacement therapy (TR) with T3 + l-thyroxine (T4) on the temperature kinetics properties of FoF1 adenosine triphosphatase (ATPase, ATP synthase, H+-translocating ATP synthase EC 3.6.3.14) and succinate oxidase (SO) and on the lipid/phospholipid makeup of rat kidney mitochondria were examined. Tx lowered ATPase activity, which T3 treatment restored. SO activity was unchanged in Tx but decreased further by T3 treatment. TR restored both activities. The energies of ATPase activation in the high and low temperature ranges ( E H and E L) increased in the Tx and T3 animals with decrease in phase transition temperature (Tt). TR restored E H and E L but not Tt to euthyroid levels. E H and E L of SO decreased in Tx animals. T3 and TR restored E H whereas E L was restored only in the TR group; Tt increased in both groups. Total phospholipid and cholesterol contents decreased significantly in Tx and T3-treated animals. In Tx animals, sphingomyelin (SPM) and phosphatidylcholine (PC) components decreased, while phosphatidylserine (PS) and diphosphatidylglycerol components increased. T3 and TR treatments caused decreases in SPM, phosphatidylinositol and PS. PC and phosphatidylethanolamine (PE) increased in the T3 group. TR resulted in increased lysophospolipids and PE. Changes in kinetic parameters of the two enzymes were differently correlated with specific phospholipid components. Both T3 and TR regimens were unable to restore normal membrane structure-function relationships. [ABSTRACT FROM AUTHOR]

Effects of treatment with DHEA (0.2 or 1.0 mg/kg body weight for 7 days) on oxidative energy metabolism of rat liver mitochondria from old (18-24 month old) and young (8-10 weeks old) male albino rats belonging to Charles-Foster strain were examined. Treatment with 1.0 mg DHEA resulted in increased body weights of the young rats without change in the liver weight. In the old animals the liver weight increased progressively with increasing dose of DHEA without affecting body weight. The state 3 respiration rates in liver mitochondria from old animals were, in general, lower than those in the young rats. The state 3 and state 4 respiration rates increased following DHEA treatment in dose-dependent manner bringing them close to values for young animals or beyond that with the effect being more pronounced at 1.0 mg dose. Treatment with DHEA also stimulated state 3 and state 4 respiration rates in young rats in dose-dependent manner, Contents of cytochrome aa3, b and c+c1 increased significantly in old animals in dose-dependent manner. In the young rats the lower dose (0.2 mg) of DHEA was more effective in bringing about a maximum increase in the contents of the cytochromes: the effect declined at the higher dose (1.0 mg). DHEA treatment also stimulated the mitochondrial ATPase activity in the old as well as in the young rats, The dehydrogenases activities were considerably low in the old rats compared to the values for the young animals. Treatment with DHEA stimulated dehydrogenases activities in old rats in dose-dependent manner bringing them close to values for the young animals or beyond. Treatment with lower dose (0.2 mg) of DHEA maximally stimulated dehydrogenases activities in young animals. [ABSTRACT FROM AUTHOR]

Early and late effects of alloxan diabetes and subsequent treatment with insulin on the temperature kinetics properties of succinate oxidase (SO) activity in rat kidney mitochondria were examined. In diabetic animals SO activity increased significantly and the increase was more pronounced at the late stage. Insulin treatment partially restored SO activity. However, the effect was temperature-dependent. In diabetic animals the energy of activation in the low temperature range (E(L)) increased significantly while that in the high temperature range (E(H)) decreased. The latter seems to be responsible for improving catalytic efficiency in the diabetic state. Insulin treatment normalized E(H) only in the 1-month diabetic group. The phase transition temperature (Tt), decreased in diabetic animals. Insulin treatment caused an increase beyond the control value in Tt in 1-month diabetic animals. The results suggest that insulin status-dependent modulation of SO activity is a complex process. [ABSTRACT FROM AUTHOR]