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Urinary estriol and serum unconjugated estradiol were measured in normal pregnant patients given ampicillin for three days. When comparing the first two control days with the third day of drug administration, it was noted that six out of 18 patients sustained a decrease in urinary estriol greater than 35 per cent. This over-all decrease, however, was not as consistent as has been earlier reported. The administration of ampicillin had no noticeable effect on serum unconjugated estradiol. The etiology and current status of this problem were presented.

A radioimmunoassay for 17-OH-pregnenolone was developed and applied to the study of this steroid in human plasma. Steroids that cross-react with the antiserum could be readily separated from 17-OH-pregnenolone; therefore the assay was highly specific. In normal subjects plasma 17-OH-pregnenolone is mainly of adrenal origin. It was largely suppressible with dexamethasone, readily responsive to ACTH and followed a diurnal rhythm similar to that of plasma fluorogenic corticosteroids with which there was a significant correlation (p < 0.001). Testicular secretion of 17-OH-pregnenolone could be inferred from the observation that while dexamethasone suppressed plasma concentrations incompletely combined treatments with dexamethasone and methyltestosterone caused the steroid to disappear completely. In contrast, women receiving dexamethasone alone had similar levels of 17-OH-pregnenolone to women receiving dexamethasone plus estrogen. Ovarian secretion of 17-OH-pregnenolone was suggested by the finding ...

1. Glucose phosphorylation rates of about 1 mumole/g./min. have been measured at room temperature in homogenates of human placental chorionic villi, and these rates are relatively constant throughout gestation. 2. This reaction has an apparent K(m) for glucose of 3x10(-5)m both in early and term placenta. 3. Human foetal membranes, the amnion and chorion, also phosphorylate glucose at a rate about equal to that of the placenta. 4. On incubation of intact bits of villus tissue from 8-12-week or full-term placenta with labelled pyruvate, followed by paper chromatography of the tissue extract, the following distribution of label was observed: residual pyruvate, 40-60%; lactate, 30-50%; glucose, 6%; fructose, 7%; sorbitol, 0.6%. 5. The concept of the placenta acting as a foetal liver during early pregnancy is inconsistent with the observation that glucose production by this organ persists up to term.

SUMMARY A liver extract from which glucose 6-phosphatase activity has been removed by ultracentrifugation has been used as a source of enzyme for assay of hepatic glucokinase activity. The method involves the coupling of glucokinase with an exogenous source of glucose 6-phosphate dehydrogenase, and measurement of TPN+ reduction in the Beckman spectrophotometer at 340 rnp. The specific activity of this soluble glucokinase is sufficiently high to account for previous estimates of glucose phosphorylation in liver slices and for the high rate of glycogen synthesis in the in- tact rat. Fasting or alloxan diabetes caused a lowering of he- patic glucokinase activity on a per g liver basis to about 400/, of the value for normal, fed rats. REFERENCES 1. RENOLD, A. E., HASTINGS, A. B., NESBETT, F. B., AND ASH- MORE, J., J. Biol. Chem., 213, 135 (1955). 2. CAHILL, G. F., JR., ASHMORE, J., RENOLD, A. E.

Apparent Michaelis constants for glucose and fructose of white adipose tissue hexokinase have been estimated by determining the rate of hexose C-1 oxidation through the hexose monophosphate shunt with added glucose-6- P dehydrogenase and NADP + . The values obtained are: fructose, 3 mM; glucose, 0.07 mM. It is likely that a single enzyme catalyzes the phosphorylation of glucose and fructose since a summation of reaction rates is not observed in the presence of both sugars. Glucose competitively inhibits fructose phosphorylation although the presence of fructose has little or no effect on glucose phosphorylation. This observation is in keeping with the relative affinities of the enzyme for the two hexoses. The enzyme displays a specific requirement for ATP and is activated both by magnesium and manganese ions. Under the experimental conditions described, glucose phosphorylation rates of between 30 and 60 μmoles/min/g of adipose tissue nitrogen were observed.

1. Three chromatographically and enzymatically distinct forms of acid phosphatase have been separated from extracts of human placental villus tissue by gel filtration. 2. These enzymes, termed I, II, and III, have molecular weights of g200,000, 105,000, and 35,000, respectively; and pH optima for p-nitrophenyl phosphate hydrolysis of 5.5, 4, and 5.5. 3. Enzyme II, which is probably of lysosomal origin, is 90% inhibited by 20 mm l-(+)tartrate. Enzyme III is insensitive to tartrate but is completely inhibited by 10 µm p-chloromercuribenzoate, and I displays an intermediate sensitivity to tartrate. Both I and II, but not III, are also inhibited by molybdate and fluoride. 4. Whereas enzymes I and II appear to be typical nonspecific acid phosphohydrolases, enzyme III is atypical in that most of the common phosphate esters are poor substrates. It is further distinguished by a stimulatory response to purines and an apparent allosteric type inhibition by pyridoxine 5-phosphate.

With the aid of C14-labeling, various glycolytic and respiratory properties of rat brain preparations were studied. Glucose oxidation as well as glycolysis occurred readily in isotonic whole brain homogenates, fortified with ATP and DPN. Under optimal conditions glucose oxidation accounted for about 60 per cent of the total respiration. Added TPN markedly enhanced the oxidation of glucose carbon-1 but had no effect on the oxidation of other glucose carbons. In short-term experiments conducted over a wide glucose concentration range, glucose oxidation in homogenates was almost maximal at 0.0002 M, whereas in slices, maximal oxidation was reached at 0.001 M. Fructose-U-C14 was readily oxidized by brain slices, but its oxidation was almost completely inhibited in the presence of glucose. Comparison of the radioactivity of lactic acid formed in brain slices and homogenates from glucose-1-C14 and glucose-U-C14 indicated the essential exclusive occurrence of the Embden-Meyerhof process. Although some glucose oxidation was observed with isolated brain mitochondria, it appeared this was due to contaminating cytoplasm, since glucose oxidation was greatly enhanced by the addition of the supernatant fraction.

17-β-Estradiol 3-phosphate has been found to be readily hydrolyzed by one of the acid phosphatases in human placenta (enzyme III). The reaction is characterized by a relatively low apparent Michaelis constant for the steroid phosphate, which is contrasted with substantially higher Michaelis constants and lower maximal hydrolysis rates for the structurally related compounds, l-tyrosine O-phosphate and p-nitrophenyl phosphate. Among those phosphate esters which are not hydrolyzed by this enzyme are 17β-estradiol 17-phosphate, androsterone 3-phosphate, and cyclohexyl phosphate. Dehydroepiandrosterone 3-phosphate is tightly bound by the enzyme but its maximum hydrolysis rate is only about one-third that of estradiol 3-phosphate. Cortisol 21-phosphate is fairly well hydrolyzed at high concentrations, but has a relatively high apparent Km. 17β-Estradiol 3-phosphate is not a favored substrate for potato acid phosphatase, wheat germ acid phosphatase, or placental acid phosphatase enzyme II. The possibility is considered that steroid phosphates may be intermediates in placental metabolism.

A rapid, direct radioimmunoassay for estriol-16α-(β-D-glucuronide) in pregnancy urine has been developed by use of immunogens in which the estriol glucuronide hapten is linked through positions 2 or 4 of ring Δ to bovine serum albumin or agarose gel. The resultant antibodies cross react less than 1 per cent with other estriol conjugates, estradiol conjugates, or unconjugated estrogens. Pregnanediol-3-glucuronide cross reacts less than 0.5 per cent. This high degree of specificity of the antibodies allows for a 10,000-fold dilution of pregnancy urine to be assayed directly without interference from other steroids or urinary constituents. Good linear correlation was obtained for urinary estriol glucuronide equivalents measured in the same subjects by our routine colorimetric method which separates and measures free estriol after hydrolysis. With an accelerated antibody-antigen equilibration of 20 minutes at 40°, 10 samples can be easily processed in less than 90 minutes. The method also allows for the evaluation of variations in the excretion pattern of one estrogen conjugate in normal and high-risk pregnancies.

1. The dissimilation of a number of externally added hexose phosphates and 5'-nucleotides by the perfused rat heart is described, and non-specific esterase and 5'-nucleotidase activity associated with the superficial cell membrane or vascular system has been demonstrated. 2. The rate of production of (14)CO(2) from [U-(14)C]glucose 6-phosphate suggests that oxidation occurred after hydrolysis to glucose. The incorporation of isotope from [U-(14)C]glucose 6-phosphate into glycogen was small, and similar to that obtained with [U-(14)C]glucose as substrate. 3. Glucose 6-phosphate was also partially isomerized to fructose 6-phosphate. Similarly, fructose 6-phosphate was converted mainly into glucose 6-phosphate, but also into glucose and inorganic phosphate. When fructose 1,6-diphosphate was added to the perfusate, a mixture of glucose 6-phosphate, fructose 6-phosphate and triose phosphates accumulated in the medium approximately in the equilibrium proportions of the phosphohexose-isomerase and triose phosphate-isomerase reactions, together with inorganic phosphate and some glucose. Glucose 1-phosphate was hydrolysed to glucose, but was not converted into glucose 6-phosphate. Leakage of enzymes out into the perfusion fluid did not occur. 4. This demonstration that phosphohexose isomerase, triose phosphate isomerase and aldolase may react with extracellular substrates at an appreciable rate suggests that these enzymes are attached to the cell membrane.

The effects of the cis and trans isomers of clomiphene citrate on the uterine hexokinase activity of immature ovariectomized rats were studied in long- and short-term experiments. At a dose level of 600 μg/kg, both isomers caused a similar increase in total uterine hexokinase activity observable in 18 hr. When enzyme activity was expressed per g of uterus (specific activity), significant increases were noted 18 hr after a single injection of transclomiphene, or after 4 daily injections of either isomer. Treatment of these animals with 170-estradiol (5 μg/kg) caused an elevation of both the specific and total hexokinase activities within 18 hr. The antiestrogenic effect of cisclomiphene was demonstrated by its ability to partially suppress the elevation of total uterine hexokinase induced by treatment with estradiol for 3 days. This suppression of total enzyme activity by cisclomiphene was largely a result of a partial inhibition of the normal uterine growth elicited by estradiol stimulation. Transclomiphe...

An electron microscopic study of acid p-nitrophenyl phosphatase activity in placental tissue has revealed two distinct localizations for this enzyme in the syncytiotrophoblast: within lysosome-like bodies and on the microvillous surface. The surface enzyme is distributed in an apparently random patchwork pattern over the chorionic villi. Evidence is presented which suggests that this nonlysosomal form may be phosphatase III, an enzyme which has previously been partially purified and characterized in vitro. Acid phosphatase activity was not observed in the cytotrophoblastic cells of Langhans' layer, and it was not present on microvilli of epithelial cells from uterine glands. These studies were carried out with a modified Gomori-type incubation medium containing a substantially reduced lead ion concentration together with p-nitrophenyl phosphate.

Human placental acid phosohatase III has been purified 5000-fold from the crude tissue homogenate by a combination of gel filtration and ion-exchange chromatography. The resultant material had a specific activity of 10 international units p-nitrophenyl phosphate phosphatase activity per mg protein at 37°, and a pH optimum of 5.2–5.3 in sodium citrate. In sodium acetate buffer the pH curve was flattened and its optimum shifted to pH 4.8–5.0. Addition of 6-ethylmercaptopurine to the reaction mixture greatly stimulated the hydrolysis of p-nitrophenyl phosphate without further changing the pH curve. Purified enzyme III is moderately sensitive to gluteraldehyde and extremely sensitive to inactivation by heavy metal ions. These and other physico chemical properties of this enzyme are discussed in relation to difficulties in its histochemical visualization, possible uniqueness in human tissue, and similarities to an enzyme found in rabbit heterophil leukocytes.