A method for the determination of the activity of steroid 5α-reductase in rat ventral prostate has been newly developed. It includes the enzyme reaction with cortexolone as a substrate, and the process of derivatization of androgens with fluorescent agent and their separation by high-performance liquid chromatography with fluorescence detection: After the enzyme reaction at 35°C and pH 6.6 in the presence of 100μM NADPH, cortexolone and reduction products (5α-pregnane-17α, 21-diol-3, 20-dione and 5α-pregnane-3α, 17α, 21-triol-20-one) were derivatized by treatment with 1-anthroyl nitrile. The resulting fluorescent esters of the three compounds were separated on an ODS column using 84% (v/v) methanol in water as a mobile phase, and the enzyme activity was estimated from the amounts of the products. The detection limit was 0.2pmol as an injection amount of each derivative. The applicability of this method was successfully demonstrated in the measurement of the inhibition of the 5α-reductase activity by certain steroids.
Male Sprague-Dawley rats were fed 5% and 25% levels of coconut fiber to examine the effect of fiber on cholesterol metabolism. Rats fed fiber showed a significant lowering of serum total cholesterol and (LDL+VLDL) cholesterol, and an increase in HDL cholesterol. A hypocholesterolemic effect was observed in the aorta and other tissues. There was increased cholesterogenesis in the liver, as was evident from increased incorporation of radioactive acetate into cholesterol and increased activity of β-hydroxy-β-methylglutaryl-coenzyme A reductase (HMG-CoA reductase). Rats fed fiber showed an increased concentration of hepatic bile acids, increased the fecal excretion of neutral sterols and bile acids, and lower release of lipoproteins into the circulation. Increased incorporation of radioactive acetate into hepatic bile acids was also observed in these groups. Activities of plasma lecithin:cholesterol acyl transferase (LCAT) and lipoprotein lipase (LPL) were increased, while glucose-6-phosphate dehydrogenase and malic enzyme showed a decrease, in rats fed the coconut fiber.
The extract of Phyllanthus amarus (Syn. P. niruri) was found to be a potent inhibitor of the hepatocarcinogenesis induced by N-nitrosodiethylamine (NDEA). None of the P. amarus extract-treated animals developed any tumors even 32 weeks after the NDEA administration, whereas all of the animals died due to tumor burden in the control group. Liver weight was significantly increased in the NDEA-treated group, whereas it was not altered after treatment with NDEA and P. amarus. Liver γ-glutamyl transpeptidase (GGT), glutamyl-S-transferase (GST), reduced glutathione (GSH) and aniline-4-hydroxylase P450 enzyme were elevated in NDEA-treated animals, whereas they were almost normal in animals treated with the carcinogen plus P. amarus extract. The serum parameters indicative of liver injury such as bilirubin, lipid peroxides, alkaline phosphatase, and glutamate-pyruvate transaminase, which were elevated by NDEA treatment, were also normal in the NDEA and P. amarus-treated group. Even though the exact mechanism of action is not known at present, the observed antitumor activity may be due to the inhibition of P450 enzyme activity and subsequent inhibition of the production of the ultimate carcinogen as well as scavenging of oxygen free radicals during promotion of the transformed cell.
To estimate the chondroprotective effect of Cynodon dactylon, an indigenous drug, we measured the activities of lysosomal enzymes, β-glucuronidase, N-acetyl-β-D-glucosaminidase, and cathepsin D in cartilage tissues of guinea pigs with experimentally induced osteoarthritis (OA). Induction of OA was achieved by intra-articular injections of papain into the knee joints of guinea pigs. OA-induced animals were treated with either Cynodon dactylon, an indigenous drug, or tenoxicam, a known chondroprotective allopathy drug. The result showed that the activities of the lysosomal enzymes were increased in the cartilage tissue of OA-induced animals and that these values were reversed to normal by both of the interventional agents. The reason for this normalcy may be attributed to the anti-inflammatory effect of the drugs, probably due to stabilization of the lysosomal membrane.
Enteroglucagon has been reported to have trophic effects on the intestinal mucosa, and is associated with proliferation and repair after various injuries of the small intestine. Glicentin is an active enteroglucagon, and is present in the largest amount in the intestine. We established a method for quantifying human plasma levels of glicentin, and measured these levels in patients with Crohn's disease. Twenty-three patients with Crohn's disease (14 males and 9 females) and 8 healthy adults were enrolled in this study. A sandwich ELISA method was used to quantify immunoreactive glicentin in plasma sampled from subjects fasting in the early morning and after the oral administration of 75g glucose (Trelan G®). The plasma glicentin levels in fasting subjects were significantly higher in patients with active-stage Crohn's disease, and were similar between the remission-stage Crohn's disease and normal controls. These levels after glucose administration were significantly higher in patients with active-stage Crohn's disease than in those with the disease in remission or in healthy controls. However, the pattern of the changes in the plasma glicentin levels after the glucose administration was similar among the three groups. These data suggest that plasma glicentin levels might reflect the pathological conditions and activity in Crohn's disease.
Activities of various enzymes including elastase were determined in sera from healthy control and from patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), IgA nephropathy (IgA NP), and other renal failures. In IgA NP, the activity of elastase increased significantly in parallel with the disease stage. It correlated well with the increase in the amount of polymorphonuclear leukocyte-elastase (PMN-elastase)·α1-antitrypsin complex assayed by ELISA, with the increase in myeloperoxidase (MPO) activity, and also with the increase in proteinuria. Studies on both substrate and inhibitor specificities of the elastase showed that the enzyme is indeed from PMN.