Metabolism of norethisterone and norethisterone derivatives in rat uterus, vagina, and aorta.

The 19-nor-progestogen norethisterone is used as a progestogen component in contraceptives and in continuous- and sequential combined hormone replacement therapy (HRT) in postmenopausal women. Metabolism of norethisterone in HRT target tissues may play a role in its biological response. The aim of this study was to investigate which steroid-metabolizing enzymes are present in rat uterus, vagina, and aorta, three HRT target tissues. Next, the ability of the tissues to metabolize norethisterone was assessed. Furthermore, to investigate the effect of substituents at the 7- and 11-position, the metabolism of Org OM38 (7alpha-methyl-norethisterone), Org 4060 (11beta-ethyl-norethisterone), and Org 34694 (7alpha-methyl,11-ethylidene-norethisterone) was studied. Using radiolabeled progesterone, the presence of 20alpha-hydroxysteroid dehydrogenase, 5alpha-reductase, and 3alpha-hydroxysteroid dehydrogenase activity could be demonstrated in uterus, vagina, and to a lesser extent in aorta. The combined action of the latter two enzyme activities resulted in 3alpha-OH,5alpha-H-norethisterone as the major metabolite of radiolabeled norethisterone in uterus (26.9%), vagina (37.1%), and aorta (1.4%). The norethisterone derivatives, however, were metabolized to a much lesser extent (1.0-7.6%). No formation of 5alpha-reduced forms of Org 4060, Org OM38, or Org 34694 was found, while formation of minor amounts of 3alpha-OH-Org 4060 and 3alpha-OH-Org OM38 could be demonstrated in both uterus, vagina, and aorta. These findings confirm the role of 5alpha-reductase as a rate-limiting step in the metabolism of norethisterone derivatives and show important inhibitory effects of substituents at the 7alpha- and 11-position of the steroid skeleton on 5alpha-reduction.

Metabolism of estradiol, ethynylestradiol, and moxestrol in rat uterus, vagina, and aorta: influence of sex steroid treatment.

Estrogen replacement therapy for postmenopausal women consists of an estrogenic and a progestagenic compound. The treatment has a positive estrogenic effect on bone, the cardiovascular system, and vagina but is dependent of the estrogen-progestagen balance in uterus to prevent unwanted proliferation. We were interested in the influence of estrogens and progestagens on estrogen metabolism in target tissues of estrogen replacement therapy. Therefore, we studied the metabolism of estradiol, 17alpha-ethynylestradiol, and moxestrol (11beta-methoxy-17alpha-ethynylestradiol) in rat uterus, vagina, and aorta. In uterus and vagina, estradiol was converted to estrone, estradiol-3-glucuronide, and estrone-3-glucuronide. These metabolites demonstrate the presence of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) and UDP-glucuronosyl transferase (UDP-GT) in uterus and vagina. We found that the conversion of estradiol by 17beta-HSD in uterus was increased in animals treated with estradiol or with a combination of estradiol and progesterone. The conversion of estradiol in uterus by UDP-GT was estradiol-induced and in contrast, progesterone-suppressed. In the vagina, steroid hormone treatment had no effect on estradiol conversion by 17beta-HSD or UDP-GT. Ethynylestradiol was glucuronidated only, and this was not affected by steroid treatment. Moxestrol was not converted in any of the three organs that were studied, indicating that the 11beta-methoxy substituent renders it a poor substrate for glucuronidation. Overall, the estrogen metabolism, and its regulation by sex steroids, in rat uterus is different compared with human uterus. Therefore, the rat may not be the best-suited model to investigate uterine effects of estradiol-progestagen combined treatment.

The influence of a fish oil-enriched diet on the phospholipid fatty acid turnover in the rabbit red cell membrane in vivo.

The influence of the polyunsaturated fatty acid (PUFA) composition of the diet on the rate of fatty acid turnover of individual phospholipids in the erythrocyte membrane in vivo was studied. Following modification of the fatty acid composition of the membrane phospholipids by the use of a fish oil or a linoleic acid enriched diet, phospholipids--labelled in the unsaturated fatty acid at the 2-position of the glycerol moiety--were introduced into the membrane of freshly isolated rabbit erythrocytes. Thereafter, the labelled erythrocytes were reinjected into the bloodstream of the animal. It appears that, with the exception of 1-palmitoyl,2-linoleoyl phosphatidylcholine, all other phosphatidylcholines disappear faster from the erythrocytes of fish oil-fed rabbits than from the red cells of linoleic acid-fed rabbits. Another parameter, which possibly influences the turnover rates of PUFA containing phospholipids, can be peroxidation. An attempt was made to measure peroxidative damage of lipids in vivo by the introduction of 1-palmitoyl,2-cis-parinaroyl phosphatidylcholine (PnPC)--a probe to measure oxidative stress--into the membrane of freshly isolated erythrocytes, in the same way as is described for the radioactive phospholipids. The data demonstrate that the fluorescent signal from the PnPC decreases at a fast rate which is independent of the dietary conditions.

Comparison of the effects of dietary vitamin E on in vivo and in vitro parameters of lipid peroxidation in the rabbit.

This study has investigated the effect of dietary vitamin E on markers of antioxidant status. Four groups of rabbits received diets containing 30 energy percent (en%) total fat (7.8 en% contributed by linoleic acid) for 12 weeks. D,1-alpha tocopheryl acetate was added to the diets to obtain a range of vitamin E concentrations (49, 114, 179, or 775 tocopherol equivalents per kg diet). Increased vitamin E concentrations were demonstrated in plasma lipoproteins and erythrocyte membranes following supplementation, and dietary effects on lipid peroxidation were investigated by (i) monitoring a fluorescent parinaric acid probe incorporated into erythrocyte membranes in vivo, (ii) determination of malondialdehyde and oxysterols in plasma, and (iii) investigation of the susceptibility of low density lipoprotein (LDL) to copper-induced conjugated diene formation in vitro. No effects of vitamin E were observed on parinaric acid oxidation in vivo or on the accumulation of lipid peroxidation products in plasma, but the resistance of LDL to oxidation in vitro increased significantly as vitamin E was supplemented to the diets. Our results demonstrate that under these dietary conditions (7.8 en% linoleic acid) increasing the vitamin E content of plasma and erythrocytes approximately two-fold does not reduce the level of lipid peroxidation in vivo, indicating sufficient antioxidant capacity on the lowest vitamin E diet. In contrast, LDL became more resistant to an extreme oxidative stress applied in vitro. The relevance of these assays to currently proposed mechanisms of atherosclerosis is discussed.

In vivo turnover of phospholipids in rabbit erythrocytes.

The rate of phospholipid turnover in erythrocyte membranes in vivo has been studied using a recently developed procedure (Kuypers, F.A., Easton, E.W., van den Hoven, R., Wensing, T., Roelofsen, B., Op den Kamp, J.A.F. and van Deenen, L.L.M. (1985) Biochim. Biophys. Acta 819, 170-178). The technique is based on the application of phospholipid transfer proteins in order to introduce trace amounts of radiolabelled phospholipids in the membrane of isolated erythrocytes, followed by re-injection of the erythrocytes into the bloodstream of the animal. The most abundant species of the phosphatidylcholine (PC) class, 1-palmitoyl,2-linoleoyl PC, has, on the basis of loss of the radioactivity in its fatty acyl part, a relatively high turnover with a half-time value of 1.5 days. Other PC species studied exhibit more moderate turnover rates of about 5 days for 1-palmitoyl,2-oleoyl PC and 1-stearoyl,2-arachidonoyl PC. Dipalmitoyl PC, labelled in the polar headgroup, turns over at a slow rate with a half-time value of 9 days. From these data and the relative abundance of the various species, it can be calculated that, on a daily basis in vivo, about one third of the total PC pool in rabbit erythrocyte membranes is replaced and/or modified by de-/reacylation. The only phosphatidylethanolamine (PE) species studied so far, 1-palmitoyl,2-arachidonoyl PE, appeared to be renewed at a relatively low rate with a half-time value of 12 days. The data demonstrate that the in vivo turnover values of phospholipids in the erythrocyte membrane may depend on their polar head group structure, their localization in the membrane and, to a large extent, on their fatty acid composition.