Quantification of parthenolide in Tanacetum species by LC-UV/LC-MS and microscopic comparison of Mexican/US feverfew samples.

LC-UV and LC-MS methods have been developed which permit the analysis of parthenolide in different Mexican/US feverfew samples and commercial products. The study was undertaken to confirm the presence of parthenolide in Mexican plant samples and its comparison with US feverfew samples. The best results were obtained with a Phenomenex Luna C18 (2) column using gradient mobile phase of water and acetonitrile:methanol (9 : 1). Elution was run at a flow rate of 1.0 mL per min and ultraviolet detection at 210 nm. The results obtained using LC-UV were comparable to those obtained using LC-MS. Parthenolide was detected in all the samples analyzed and is the major chemical constituent of feverfew. The samples collected in Oaxaca, Mexico (0.28%) and Puebla, Mexico (0.25%) showed the highest content of parthenolide. All Parthenium samples were also examined under light and fluorescent microscopy.

Colchicine inhibition of insulin induction of stearoyl-CoA desaturase and fatty acid synthetase in cultured avian liver explants.

Chicken embryo liver explants cultured in chemically defined medium in the absence of serum provide a unique system to probe into the mechanism of insulin induction of lipogenic enzymes. Colchicine at concentrations of 0.2 and 1 microM in the culture medium caused inhibition of insulin induction of stearoyl-CoA desaturase and fatty acid synthetase by 50 and 90%, respectively. As measured by immunochemical techniques, the inhibition of the induction of these two enzyme systems resulted from the decreased content of the delta 9-terminal desaturase component of the stearoyl-CoA desaturase and the fatty acid synthetase. Colchicine, however, had no effect on the general protein synthesis, nor did it affect the malic enzyme, which is induced by triiodothyronine but not by insulin. Also, colchicine had no influence on the binding of 125I-insulin to isolated plasma membrane. Pretreatment of liver explants with insulin for 0.5-1 h and subsequent incubation in insulin-free media for 48 h resulted in induction of the desaturase and fatty acid synthetase. However, inclusion of colchicine in the media for 3 h subsequent to the treatment with insulin completely abolished the inductive effect of insulin, suggesting that colchicine affects events occurring subsequent to insulin binding to the cell surface membranes. Since lumicolchicine, an inactive isomer of colchicine, had no effect on insulin action, it is suggested that the inhibition of insulin induction of the desaturase and synthetase is related to the depolymerizing action of colchicine. Therefore, in eliciting long-term responses to insulin, microtubular integrity of the cell may be required for the transfer of a putative from cell surface insulin receptor to intracellular sites.

Stearoyl-coenzyme A desaturase activity in the mammary gland and liver of lactating rats.

Stearoyl-CoA desaturase activity in microsomes from lactating rat mammary gland is very low (0.05-0.15 nmol/min/mg of protein) regardless of lactating time. In such microsomes, reductase activities and content of cytochrome b5 are several-fold lower than in normal rat liver microsomes. Preincubation of the mammary microsomes with purified terminal desaturase gives a 55-fold stimulation of stearoyl-CoA desaturase activity, whereas preincubation with cytochrome b5 has no effect. However, preincubation of mammary microsomes with both cytochrome b5 and terminal desaturase results in a 200-fold stimulation of overall desaturation. These observations suggest that negligible stearoyl-CoA desaturase activity in lactating rat mammary microsomes is due to a cytochrome b5 content and the absence of terminal enzyme. The hepatic stearoyl-CoA desaturase activity increases 9-fold during lactation. There is little or no change in the NADH-cytochrome c reductase activity or in the concentrations of cytochrome b5 during this period, but the activity of the terminal desaturase increases with the increase of overall desaturation. These results suggest that liver is one of the more important sources of oleic acid for milk triglycerides.

Inhibition of the microsomal stearoyl coenyme A desaturation by divalent copper and its chelates.

Divalent copper and copper complexes of tyrosine, histidine and lysine inhibited at low concentrations the stearoyl-CoA desaturation reaction in both chicken liver microsomes and in a purified system consisting of chicken liver delta 9 terminal desaturase, cytochrome b5, ascorbate and liposome. Although the copper chelates lowered the steady-state level of ferrocytochrome b5 by 20%, and partially inhibited the NADH-ferricyanide reductase activity, the availability of the ferrocytochrome b5 during the time course of desaturation was not affected, indicating that the site of inhibition of desaturation was at the terminal step, i.e., on the delta 9 terminal desaturase. The presence of chalates during catalysis was essential for the observed inhibition. Based on the observation that O2 is involved in the desaturation and that there is an initial electron reduction of desaturase iron, it is plausible that the copper chelates are inhibiting by acting as superoxide scavengers.

Interaction of the fluorescent analogue stearoyl-(1,N6)-etheno coenzyme A with chicken liver acetyl coenzyme A carboxylase.

The interaction of stearoyl-(1,N6)-etheno coenzyme A (stearoyl-epsilon-CoA) with acetyl coenzyme A carboxylase was investigated by using fluorescence spectroscopy. The fluorescence emission of stearoyl-epsilon-CoA was partially quenched by acetyl coenzyme A carboxylase. Analysis of the data for dissociation constant (KD) and the stoichiometry of the interaction (n) gave values of 5.06 nM and 1.2, respectively, at pH 7.6 in 50 mM Tris-HCl and 25 degrees C. The KD value is comparable to the inhibition constant (Ki) obtained previously by others for the inhibition of rat liver acetyl coenzyme A carboxylase by long chain fatty acyl-CoAs. Citrate (which is known to polymerize and thus activate carboxylase) caused a partial quenching of the protein fluorescence of carboxylase, presumably due to polymerization of the enzyme. The quenching of the stearoyl-epsilon-CoA fluorescence caused by carboxylase as well as the inhibition of carboxylase activity by stearoyl-epsilon-CoA was reversed by citrate, but only in the presence of 6-O-methylglucose polysaccharide which forms a stable complex with fatty acyl-CoA. This shows that the stearoyl-epsilon-CoA bound to the enzyme is displaced by citrate only in the presence of an acceptor of fatty acyl-CoA. These results support the reciprocal relationship of citrate and fatty acyl-CoA in the regulation of acetyl coenzyme A carboxylase.

Stearoyl-coenzyme A desaturase activity in Novikoff hepatoma.

The activities of microsomal stearoyl-CoA desaturation, NADH-cytochrome b5 reductase, NADH-cytochrome c reductase, and the content of cytochrome b5 were similar in livers of normal and host rats. On the other hand, stearoyl-CoA desaturation activity was absent in Novikoff hepatoma. The activities of NADH-cytochrome b5 and NADH-cytochrome c reductases in the hepatoma microsomes were 4.8% and 2.2%, respectively, of those in normal liver. Furthermore, in hepatoma microsomes, cytochrome b5 was absent. An active stearoyl-CoA desaturation was reconstituted only on addition of both cytochrome b5 and the terminal desaturase enzyme to the hepatoma microsomes. These results indicated that a complete absence of cytochrome b5 and terminal desaturase is responsible for the lack of stearoyl-CoA desaturation in Novikoff hepatoma microsomes.

Regulation of rat hepatic stearoyl coenzyme A desaturase. The roles of insulin and carbohydrate.

The rat hepatic stearoyl-CoA desaturation decreased by 3.7-fold in streptozotocin-induced diabetes. Insulin treatment of diabetic rats increased the enzyme activity by 7-fold. In marked contrast to glucose administration, fructose feeding in diabetic rats resulted in 20-fold stimulation of stearoyl-CoA desaturation, although both carbohydrates stimulated stearoyl-CoA desaturation in normal rats. Measurement of the microsomal electron transfer components showed no significant changes in the NADH-cytochrome b5 reductase activity or in the concentration of cytochrome b5. However, the activity of the terminal desaturase changed in a parallel fashion as the amount of terminal desaturase reflect changes in the overall desaturation. Supplementation of various microsomes with the saturating amount of purified terminal desaturase resulted in the formation of similar amounts of catalytically active complex and increased the stearoyl-CoA desaturation to the same level suggesting that the changes in the amount of terminal desaturase reflect changes in the overall desaturation. The results support the suggestion that both insulin and the intermediates of carbohydrate metabolism are involved in the regulation of terminal desaturase.