The coenzyme A-synthesizing protein complex and its proposed role in CoA biosynthesis in bakers' yeast.

An improved procedure is described for the recovery and purification of the coenzyme A-synthesizing protein complex (CoA-SPC) of Saccharomyces cerevisiae (bakers' yeast). The molecular mass of the CoA-SPC, determined prior to and following its purification, is estimated by Sephacryl S-300 size exclusion chromatography to be between 375,000-400,000. Two previously unreported catalytic activities attributed to CoA-SPC have been identified. One of these is CoA-hydrolase activity which catalyzes the hydrolysis of CoA to form 3',5'-ADP and 4'-phosphopantetheine, and the other is dephospho-CoA-pyrophosphorylase activity which catalyzes a reaction between 4'-phosphopantetheine and ATP to form dephospho-CoA. The dephospho-CoA then reacts with ATP, catalyzed by the dephospho-CoA-kinase, to reform CoA. This sequence of reactions, referred to as the CoA/4'-phosphopantetheine cycle, provides a mechanism by which the 4'-phosphopantetheine can be recycled to form CoA. Each turn of the cycle utilizes two mol of ATP and produces one mol of ADP, one mol of PPi, and one mol of 3',5'-ADP. Other than the hydrolysis of CoA by CoA-SPC, the 4'-phosphopantetheine for the cycle apparently could be supplied by alternate sources. One alternate source may be the conventional pathway of CoA biosynthesis. Intact CoA-SPC has been separated into two segments. One segment is designated apo-CoA-SPC and the other segment segment is referred to as the 10,000-15,000 M(r) subunit. The 5'-ADP-4'-pantothenic acid-synthetase, 5'-ADP-4'-pantothenylcysteine-synthetase, 5'-ADP-4'-pantothenylcysteine-decarboxylase, and CoA-hydrolase activities reside in the apo-CoA-SPC segment of CoA-SPC. Whereas the dephospho-CoA-kinase and the dephospho-CoA-pyrophosphorylase activities reside in the 10,000-15,000 M(r) subunit. Thus, the 10,000-15,000 M(r) subunit mimics the bifunctional enzyme complex that catalyzes the final two steps in the conventional pathway of CoA biosynthesis.

Urinary endothelin-1: not a useful marker for preeclampsia.

Urinary endothelin-1 levels were assayed from 50 gravidas with preeclampsia and 11 with normotension in the third trimester of pregnancy. Urinary endothelin-1 levels (mean +/- SEM) were similar between gravidas with normotension and preeclampsia (62.7 +/- 7.5 vs 79.8 +/- 9.3 fmol/mg urinary creatinine, respectively). Urinary endothelin-1 excretion is not significantly increased in patients with preeclampsia and therefore not a good marker for preeclampsia.

Magnesium sulfate therapy in preeclampsia is associated with increased urinary cyclic guanosine monophosphate excretion.

OBJECTIVE: Our objective was to determine if maternal urinary cyclic guanosine monophosphate levels are altered in preeclampsia. STUDY DESIGN: Aliquots from 24-hour urine samples collected from 57 women with preeclampsia and 14 normotensive pregnant women in the third trimester of pregnancy were assayed for urinary cyclic guanosine monophosphate. Urinary cyclic guanosine monophosphate values were expressed per milligram of urinary creatinine to standardize for renal function. RESULTS: There was no difference in gestational age at time of urine collection between the two groups. Urinary cyclic guanosine monophosphate levels (mean +/- SD) were similar between normotensive and preeclamptic pregnant women (751 +/- 498 vs 632 +/- 363 pmol/mg urinary creatinine, respectively, p = 0.12). Preeclamptic women receiving magnesium sulfate had significantly higher levels of urinary cyclic guanosine monophosphate than those not receiving magnesium sulfate (786 +/- 360 vs 555 +/- 344 pmol/mg urinary creatinine, respectively, p = 0.02). CONCLUSIONS: These preliminary results indicated that cyclic guanosine monophosphate excretion increases in patients with preeclampsia during magnesium sulfate infusion. The vascular smooth muscle relaxation effects of magnesium sulfate may be mediated by directly increasing cyclic guanosine monophosphate production or indirectly through endothelium-derived relaxing factor.

Oral nifedipine pharmacokinetics in pregnancy-induced hypertension.

The pharmacokinetics of oral nifedipine were studied in 15 women with pregnancy-induced hypertension in the third trimester of pregnancy to determine if the drug's disposition was different from that in nonpregnant patients. Peak serum concentrations of 38.6 +/- 18 ng/ml occurred at approximately 40 minutes after ingestion of nifedipine 10 mg. The terminal elimination half-life (mean 1.3 +/- 0.5 hrs) was shorter than that reported for normotensive volunteers and nonpregnant hypertensives after oral dosing. Mean +/- SD apparent elimination clearance of 2.0 +/- 0.8 L/hr/kg was more rapid than that in healthy volunteers (mean 0.49 +/- 0.09 L/hr/kg). Random serum concentrations were progressively higher in patients receiving larger daily doses. Nifedipine was detected in samples of fetal cord blood and amniotic fluid at concentrations approximately 93% and 53% those of simultaneous maternal vein samples, respectively. The findings indicate that nifedipine may achieve greater antihypertensive efficacy in pregnant women if administered at shorter intervals.

Nifedipine pharmacokinetics and pharmacodynamics during the immediate postpartum period in patients with preeclampsia.

Pharmacokinetic and pharmacodynamic parameters of oral nifedipine were studied in the immediate postpartum period in eight women with preeclampsia. Peak serum concentrations of 18 +/- 2.1 micrograms/L occurred 40 minutes after ingestion of nifedipine (10 mg). The terminal elimination half-life (mean = 1.35 +/- 0.3 hours) was found to be shorter than that reported for normotensive volunteers or nonpregnant hypertensive women (mean, 3.4 +/- 0.4 hours). A mean apparent oral elimination clearance of 3.3 +/- 1.3 L/hr/kg was more rapid than that found in normal volunteers (mean, 0.49 +/- 0.09 L/hr/kg) or in women with pregnancy-induced hypertension in the third trimester (mean, 2.0 +/- 0.8 L/hr/kg). Initial nadirs in mean arterial pressure were noted at 50 minutes after ingestion of nifedipine, with an average reduction in mean arterial pressure of 13.8 mm Hg. A dosing interval of every 3 to 4 hours is suggested when rapid-release nifedipine is used in the postpartum patient with preeclampsia.

Labetalol pharmacokinetics in pregnancy-induced hypertension.

Pharmacokinetic parameters of oral labetalol were studied in eight women with pregnancy-induced hypertension in the third trimester of pregnancy. Labetalol exhibited rapid absorption; peak serum concentrations of 881 +/- 219 ng/ml occurred at 20 minutes after labetalol ingestion. The terminal elimination half-life (mean, 1.7 +/- 0.27 hours) was found to be shorter than that reported for normotensive volunteers or nonpregnant hypertensive patients (mean, 6 to 8 hours). A mean apparent oral elimination clearance of 21.8 ml/min/kg compared favorably with that seen in other pregnant and nonpregnant populations. Food delayed the time to peak serum concentration to approximately 60 minutes. Labetalol was detected in fetal cord samples and amniotic fluid samples at concentrations approximately 50% and 16% that of simultaneous maternal vein samples, respectively.

Evaluation of the B-protein assay in cancer management.

Previous data have suggested that the B-protein assay might prove to be useful in the assessment of patients with cancer after various therapeutic modalities. The assay's effectiveness was evaluated by prospective study of 133 patients with cervical, uterine, or ovarian cancer. After therapy, B-protein levels remained elevated in 17 nonresponding patients who eventually died of the disease. In contrast, 88 patients experienced a significant reduction in B-protein levels measured 90 days after treatment. Among this group, 25 patients demonstrated elevated B-protein levels during the 2-year follow-up period and all were confirmed to have persistent or recurrent disease. These data indicate that monitoring serum B-protein levels can be beneficial in the posttherapeutic management of gynecologic malignancies.

Purification and characterization of B-protein from human serum.

B-Protein, present in the serum of individuals with cancer, has been purified to electrophoretic homogeneity. The purification procedure consisted of chromatography on Sephacryl S-200, Affi-Gel Blue, Con A--Sepharose 4B, wheat germ lectin--Sepharose and preparative polyacrylamide gel electrophoresis. The molecular weight of B-Protein is estimated to be 100 000 to 120 000. It is a glycoprotein which appears to be composed of two subunits, each with a molecular weight of approximately 52 000. Analytical polyacrylamide gel electrophoresis and analytical ultracentrifugation data indicate that purified B-Protein is homogeneous. Isoelectric focusing studies also show the purified B-Protein to be homogeneous in composition consisting of a single band of pI = 4.8. Amino acid analysis is consistent with this acidic isoelectric point. Other analyses indicate that B-Protein contains 7% carbohydrate and 7% lipid in the form of triglycerides.

Comparison of the Lumadex-foam stability index test, lecithin: sphingomyelin ratio, and simple shake test for fetal lung maturity.

Amniotic fluid was obtained from 269 patients within 72 hours of delivery. The sensitivity, specificity, and predictive value of the Lumadex-foam stability index test, lecithin: sphingomyelin (L:S) ratio, and simple shake test were assessed in the diagnosis of respiratory distress syndrome (RDS), as well as the accuracy in identification of functional lung maturity in the growth-retarded fetus. The Lumadex-foam stability index test was found to be an accurate, quantitative, simple, and rapid measure of fetal lung maturity in the normal as well as abnormal pregnancy. The L:S ratio was accurate in the normal pregnancy, but became inaccurate in the small growth-retarded fetus. The nonquantitative simple shake test was accurate when it indicated fetal lung maturity, but an immature result was meaningless because of the large false-negative rate.

Accelerated pulmonary maturity as measured by the Lumadex-foam stability index test.

The Lumadex-foam stability index test and lecithin:sphingomyelin (L:S) ratio were compared in the small preterm fetus. Amniotic fluid was obtained within 72 hours of birth from 70 high-risk patients whose infants were at 34 weeks' gestation or less and/or weighed less than 2000 g. Twenty-nine infants were small for gestational age. Eleven of the 41 appropriate for gestational age babies developed respiratory distress syndrome. Conclusions of the study were as follows: 1. The Lumadex-foam stability index test is an accurate, quantitative, and rapid measure of fetal lung maturity. 2. Growth retardation accelerates functional lung maturity in the preterm fetus. 3. The foam stability index value can discriminate between the small for gestational age and appropriate for gestational age fetus at 32 weeks' gestation or less and/or less than 1500 g birth weight. 4. The L:S ratio cannot discriminate between the small for gestational age and appropriate for gestational age fetus because the false-negative rate increases as fetal weight decreases.

Placental transfer of 14C-hexoprenaline.

The placental transfer of a single intravenous injection of 14C-hexoprenaline was studied in eight pregnant New Zealand white rabbits. Maternal and fetal blood was sampled intermittently for 60 minutes after the injection. An initial rapid decrease in the levels of 14C-hexoprenaline in maternal blood was followed by a second slower phase, whereas fetal levels remained insignificant. The conclusion, therefore, is that the rapid improvement in fetal heart rate after the administration of a single maternal intravenous injection of hexoprenaline in the treatment of fetal distress is due to the action on the uterus and/or on maternal cardiovascular function, and not to direct stimulation of the fetus.

Hexoprenaline pharmacokinetics in pregnant and nonpregnant sheep.

Hexoprenaline, a beta 2-sympathomimetic agent, is used to suppress uterine contractions in the treatment of premature labor. However, little is known regarding the potential of this drug to undergo placental transfer or whether the pregnant state alters any of the pharmacokinetic parameters. Using sheep as a model, intravenous doses of 14C-hexoprenaline were administered to pregnant and non-pregnant animals. Measurable levels of radioactivity did not appear in fetal blood samples. After intravenous bolus administration, blood concentrations in the ewe could be fitted by a triexponential curve characteristic of a three-compartment pharmacokinetic model. Following intravenous infusion, a biexponential curve described the decline in blood concentrations. Mean terminal half lives for total radioactivity ranged from 2.5 to 4.2 hours. The pregnant animals tended to exhibit smaller apparent volumes of distribution and lower values for total body clearance, normalized to body weight, compared to non-pregnant sheep.

Effects of hexoprenaline on the lecithin/sphingomyelin ratio and pressure-volume relationships in fetal rabbits.

A placebo-controlled, double-blind trial was carried out on 74 New Zealand White rabbit fetuses from 15 does to assess the effect of a fetal injection of hexoprenaline on surfactant release. After the uterus was exposed, half the fetuses received 0.1 ml (0.25 microgram) of hexoprenaline injected intraperitoneally through the intact uterine wall; the other half received an equivalent volume of placebo. After 3 hours, the abdomen was reopened, and the fetuses were surgically delivered and killed before breathing. The lecithin/sphingomyelin (L/S) ratios, obtained from lung washings, revealed a mean of 1.59:1 for the placebo group and 1.92:1 for the hexoprenaline group (p less than 0.001). Pressure/volume curves were generated from the lungs of 24 fetuses from 10 does, and the volume of air in the lungs for each pressure was analyzed in four ways: total volume, volume per gram of fetal body weight, volume per gram of dry lung weight, and as a percentage of total lung capacity at a pressure of 40 cm H2O. A first and second inflation-deflation curve was obtained for each experiment. The lungs from the hexoprenaline-treated group retained significantly more air than those from the placebo group. The most significant comparison was obtained when lung volume was expressed per gram of dry lung weight. The possibility of administering a beta 2-sympathomimetic drug to the mother in advanced preterm labor, specifically to release surfactant in the fetal lung, is suggested.

Effect of corticosteroids and fetomaternal disorders on the L:S ratio.

Hydrocortisone or placebo was administered to 126 women at risk for premature delivery who had immature lecithin:sphingomyelin (L:S) ratios in order to induce early surfactant synthesis. In 70 subjects (37 steroid-treated patients, 33 controls), L:S ratio was determined a second time between 9 hours and 7 days after therapy had been initiated. The treatment group showed a significant increase in the L:S ratio when compared to those who received the placebo. Moreover, patients who had fetomaternal disorders that accelerated or delayed lecithin production were also found to have increased L:S ratios after treatment. Fewer newborns developed respiratory distress syndrome (RDS) in the treatment group than in the control group and those in the former category who were affected by RDS had a milder clinical course.

Prophylactic transfusions in pregnant patients with sickle hemoglobinopathies: benefit versus risk.

Pregnancy in patients with sickle cell disease has been a significant threat to maternal survival and good reproductive outcome. In the past several years, statistics for both maternal and perinatal outcome have improved. There is controversy, however, as to whether this improvement has resulted from the use of maternal transfusions or the aggressive and intense medical management afforded these patients in recent years. This study details the reproductive experience of 80 pregnant patients with significant hemoglobinopathies, 75 of whom were treated with partial prophylactic exchange transfusions during gestation. Each of the 75 patients who completed the protocol received 2 transfusions using buffy coat-poor washed packed red cells. The results show that there was no maternal mortality and a significant improvement in maternal morbidity compared to previous studies. There was also a significant improvement in fetal salvage, with a perinatal mortality rate of 26 per 1000. In addition, there were fewer premature and low birth weight infants as compared to other studies in the literature. Although these results were favorable, only a randomized multicentered study in the future will detail advantages and disadvantages of such therapy in the gravid sickle cell patient compared to intensive medical treatment without transfusion.

Lecithin-sphingomyelin ratio and RDS in patients with diabetes mellitus: possible mechanisms.

Controversy exists concerning the positive correlation of maternal diabetes mellitus and the propensity for the progeny to develop respiratory distress syndrome (RDS). The increased incidence of RDS in these offspring seems to occur despite the various physical and biochemical assessments which indicate a mature lung profile. Recent data seem to incriminate insulin antagonism of normal fetal physiologic processes as the principal culprit. Strict control of maternal glucose metabolism in the pregnant diabetic may be essential in reducing RDS in the infant of a diabetic mother.

Coenzyme A-synthesizing protein complex of Saccharomyces cerevisiae.

The coenzyme A-synthesizing protein complex (CoA-SPC) is a multienzyme complex of Saccharomyces cerevisiae (Bakers' yeast), which has a molecular weight in excess of 200,000 as determined by Sephadex G-200 column chromatography. This multienzyme complex, which is insoluble in the crude yeast cell lysate, has been purified 229-fold. A cellular component of the yeast cell lysate, referred to as t-Factor, with a molecular weight of 400-1000 and chloride ion are involved in the solubilization of CoA-SPC. The CoA-SPC requires L-cysteine, D-pantothenic acid and ATP as substrates. The terminal CoA-SPC-bound intermediate is dephospho-CoA, which is subsequently phosphorylated and released from the complex as CoA. The sequence of reactions for the synthesis of CoA by the CoA-SPC differs significantly from those previously proposed for other systems. It could be that the reaction sequence is unique for the yeast cell.

Alternate procedure for the preparation of the coenzyme A-synthesizing protein complex of Bakers' yeast.

The coenzymes A-synthesizing protein complex (CoA-SPC) of Bakers' yeast synthesizes coenzyme A in an in vitro system from the precursors ATP, D-pantothenic acid and L-cysteine. CoA-SPC has been produced on a small scale by freezing Bakers' yeast cells in a mixture of diethyl ether and solid CO2, followed by a thawing period, and subsequent removal of the diethyl ether by vacuum. The resulting yeast lysate was then stirred for 18 h in the presence of t-Factor to solubilize CoA-SPC. When a greater quantity of CoA-SPC was needed, the danger associated with the use of a large volume of diethyl ether was apparent. Therefore, the freezing step involving diethyl ether and solid CO2 has been replaced by a process of slowly drying fresh Bakers' yeast until approximately 34% of the initial weight of the yeast remained as dry solids. The yeast solids were ground to further disrupt the cell wall and membrane structure. The grinding step was followed by rehydration of the dry yeast solids with deionized H2O and stirring the rehydrated yeast for 18 h to solubilize CoA-SPC.