12-Lipoxygenase activity in human uterine cervix.

Homogenates prepared from sections of human uterine cervix or endometrium were incubated with [1-14C]arachidonate and the products examined by radio-TLC. A major product chromatographing with 12-hydroxyeicosatetraenoic acid (12-HETE), and a number of more polar metabolites which were unaffected by 50 microM indomethacin but decreased to the same extent as 12-HETE by 50 microM nordihydroguaiaretic acid, were demonstrated by this technique. The addition of glutathione to the incubation mixture increased the production of 12-HETE, with a proportional decrease in the polar products. There was a large variation in 12-lipoxygenase activity measured in different cervix samples. The levels of the enzyme in the cervix were similar in two groups of uterine samples classified by the histological appearance of the endometrium as proliferative and secretory. However activity was significantly lower in samples taken from post-menopausal patients compared with pre-menopausal patients.

Biochemical studies on a 12-lipoxygenase in human uterine cervix.

Human uterine cervix possesses a high 12-lipoxygenase activity; this enzyme has been isolated in a purified form from the squamous epithelial region of human cervix and its major properties have been investigated. Enzyme activity was present in all subcellular fractions obtained by centrifugation; the highest specific activity was associated with the microsome fraction (160,000 X g pellet). Purification of the enzyme was achieved by acetone precipitation, ion exchange chromatography on CM-cellulose and affinity chromatography on linoleyl-aminoethyl-Sepharose. The product from the incubation of sodium [1-14C]arachidonate with crude enzyme extracts co-chromatographed with authentic 12-hydroxyeicosatetraenoic acid, but the purified enzyme gave a product that behaved like the 12-hydroperoxy derivative. The enzyme had optimum activity at pH 6.5, a Km of 15 microM for arachidonic acid and was stimulated by ATP and Ca2+. Enzyme activity was inhibited by esculetin, nordihydroguaiaretic acid, eicosatetraynoic acid, detergents at concentrations greater than 0.1% (w/v) and preincubation of substrate with GSH and GSH peroxidase. The occurrence of a high 12-lipoxygenase activity is discussed in relation to the specific physiological functions of this tissue.

The effect of acetylsalicylate on aggregation and arachidonate metabolism by human platelets suspended in plasma or buffer.

Acetylsalicylate inhibits prostaglandin and thromboxane production by human platelets suspended in plasma or buffer. Acetylsalicylate inhibits arachidonate-induced aggregation of human platelets suspended in plasma, but the effect of acetylsalicylate on arachidonate-induced aggregation of human washed platelets in buffer has not been reported. We have therefore studied this in relation to arachidonate metabolism in human platelets suspended in plasma or buffer. Platelets suspended in plasma and in buffer were both prepared from each donor, who had not taken acetylsalicylate or like-acting drugs for at least 7 days. Acetylsalicylate was 1500 times less potent in inhibiting arachidonate-induced aggregation in buffer (IC50 = 27.3 +/- 7.5 (s.e.m.)mM) than it was in plasma (IC50 = 18.3 +/- 6.0 microM); whereas it was only 4 times less potent in inhibiting thromboxane production in buffer (IC50 = 110 +/- 51.0 microM) than in plasma (IC50 = 25.3 +/- 8.9 microM). The acetylsalicylate concentration required to inhibit aggregation in buffer was sufficient to inhibit 12-hydroxyeicosatetraenoic acid production whereas the concentration that inhibited thromboxane production in buffer was not. These results indicate that arachidonate-induced aggregation of platelets in buffer may depend on product(s) of lipoxygenase rather than of cyclooxygenase, and is hence insensitive to inhibition by acetylsalicylate compared with arachidonate-induced aggregation of platelets in plasma.

A comparison of gas-liquid chromatography and a titration method for the determination of plasma non-esterified fatty add levels during pregnancy.

The level of plasma non-esterified fatty acids (NEFA) was measured by gas-liquid chromatography (GLC) and a titration method in 194 samples collected during pregnancy and from four days to 24 weeks post partum. Both techniques indicated a similar pattern of changes in plasma NEFA associated with pregnancy. The titration estimates of NEFA level were usually greater than those measured by GLC, and there was some suggestion that the disparity between the methods was increased at the end of pregnancy and was reduced at six weeks after delivery.

Changes in plasma non esterified fatty acids and serum glycerol in pregnancy.

Changes in plasma nonesterified fatty acids (NEFA) and serum glycerol in pregnancy were examined in a semi-serial study of 85 women. A preliminary study showed that it was almost impossible to achieve standardized conditions for sampling, and the compromise of taking a single venous sample without stasis, after 30 minutes rest and about 12 hours fasting, was adopted. Although there were large individual variations the patterns of change were, in general, the same for NEFA and glycereo although the two were not closely correlated. There was no +convincing change before 30 weeks of pregnancy but both increased in the last ten weeks, fell sharply in the first week post partum, and then rose again to late pregnancy levels by 6 weeks post partum before falling to non-pregnant levels by between 3 and 6 months post partum. Those patterns of change are in broad accordin with changes of fat storage and lipolysis associated with the reporoductive cycle. Changes in the patterms of NEFA were triviax the only significant alteration was a small rise in the proportion of C16:0 (palmitic) in late pregnancy.

Inhibition of prostaglandin biosynthesis by 7-oxa- and 5-oxa-prostaglandin analogues.

Some oxaprostaglandin derivatives have been shown to inhibit prostaglandin biosynthesis from arachidonate by a particulate prostaglandin synthetase preparation. The most potent inhibitor was 5-oxaprost-13-trans-enoate, and inhibition by this compound appeared to be competitive. Certain structure-activity relationships were ascertained.

Reversible activation of succinate dehydrogenase.

1. Treatment of particulate respiratory chain preparations in ways expected to raise or lower the concentration of endogenous soluble low-molecular-weight compounds respectively increased and diminished the capacity of succinate dehydrogenase to become activated reversibly and ;spontaneously' when preparations were diluted in tris acetate buffer and incubated at 37 degrees . 2. Addition of critically low concentrations of recognized activators to preparations that failed to undergo reversible ;spontaneous' activation when incubated at 1mg. of protein/ml. conferred on them the capacity to do so. 3. Preparations with a diminished tendency to undergo reversible ;spontaneous' activation had an increased tendency to become irreversibly inactivated on prolonged incubation at 1mg. of protein/ml. in tris acetate. 4. Extraction procedures designed to demonstrate the presence of possible endogenous activators in enzyme preparations failed to reveal a single substance to which such a role could be conclusively attributed. A mixture of compounds was found, however, including certain amino acids that have been shown to act as activators. It is questionable whether these compounds would be present at sufficiently high concentrations to act as activators when enzyme preparations are diluted to 1mg. of protein/ml. 5. Despite the failure to demonstrate conclusively the presence of endogenous activators, the balance of evidence appears to favour the hypothesis that reversible ;spontaneous' activation of these preparations can best be explained by the presence of such substances, and a scheme describing the mechanism of activation and deactivation of succinate dehydrogenase is discussed in relation to these and other observations.