The P4 study: Subsequent pregnancy maternal physiology after hypertensive and normotensive pregnancies.

OBJECTIVES: Hypertensive disorders of pregnancy are associated with subsequent increased risk of cardiometabolic disease. Adverse cardiometabolic measures are noted soon after hypertensive versus normotensive pregnancy (NP); to what degree these persist into a subsequent pregnancy (SP) is unknown. This study aimed to assess women's physiology early in SP after hypertensive pregnancy (HP: preeclampsia or gestational hypertension) or NP and compare SP to 6 months postpartum findings from the index pregnancy. STUDY DESIGN: Prospective sub-study of the P4 (Postpartum, Physiology, Psychology and Paediatric) observational cohort. Measurements six months after NP versus HP, and the SP at 11-13 weeks gestation. MAIN OUTCOME MEASURES: Blood pressure (BP), blood and urine tests (urine ACR, HOMA-IR, LDL cholesterol), body composition, and contribution of maternal characteristics and inter-pregnancy factors to BP and body fat (FM%) in SP. RESULTS: 49 women (34 NP, 15 HP). In the SP, post-HP women had higher BP (112/70 mmHg HP vs 102/64 mmHg NP; p < .001), with no significant drop from six months postpartum to early SP. On regression analysis, systolic and diastolic BP at 6-months were the major predictors for SP systolic (p < 0.001) and diastolic (p = 0.009) BP respectively in the SP. Longer interpregnancy interval and increased FM% 6-months postpartum were associated with higher SP FM% (p < 0.001). CONCLUSIONS: BP and body fat six months postpartum were similar early in the SP for HP group, and postpartum BP and FM% were major predictors of their corresponding SP measurements. Postpartum/inter-pregnancy intervention programs to improve these cardiometabolic risk markers might help improve women's long-term health and require investigation.

Indications for delivery in pre-eclampsia.

OBJECTIVE: Examine the frequency with which the most accepted indicators for delivery in pre-eclampsia are used in a population with predominantly late-onset (birth > 32 weeks) pre-eclampsia (PE). METHODS: Retrospective cohort study using the St George Public Hospital (SGH) Hypertension in Pregnancy database. Demographic, pregnancy, and outcome details were extracted and verified by comparison with data collection sheets. RESULTS: From 2001 to 2013, 908 women (970 babies) with PE were included, of which a subgroup of 303 women (33%) had clearly delineated delivery triggers available. This subgroup of women had similar demographic and outcome characteristics to the total PE population. In this group, the most common maternal trigger for delivery apart from gestational age 37+ weeks was difficult to control/severe hypertension (114 cases, 38%) and the most common fetal trigger intrauterine growth restriction (IUGR: 14 cases, 4%). 78 (35%) of term women had no specific delivery trigger other than gestation. A primary maternal trigger and/or associated complication was slightly more common in those delivering <37 weeks vs 37+ weeks (52 vs 38%, p = .03), while a fetal or combined maternal/fetal complication was over four times more common in preterm women (25 vs 6%, p < .001). CONCLUSION: In our population of predominantly late-onset PE, maternal triggers for delivery (predominantly severe hypertension) far outweigh fetal triggers (predominantly IUGR). Fetal and mixed indicators for delivery were relatively more common in women delivering preterm, possibly reflecting the severity of placental dysfunction in this subgroup.

Pre-eclampsia causes adverse maternal outcomes across the gestational spectrum.

OBJECTIVE: To determine if women with early onset pre-eclampsia (EOP) have worse maternal outcomes than those who present later. Specifically, we aimed to determine whether term preeclamptic women and their infants have better outcomes than either their late pre-term or early onset counterparts. STUDY DESIGN: Between 1991 and 2011, 4657 pregnancies complicated by hypertension were recorded in our database; 2148 (45%) had pre-eclampsia (PE). Six hundred ninety six cases (32%) that had accurate data for the gestation at which PE developed were analysed. Pre-eclampsia was defined as per the International Society for the Study of Hypertension in Pregnancy guidelines. Maternal outcomes included (1) episodes of severe hypertension, (2) proteinuria, (3) acute kidney injury, (4) abnormal liver function, (5) thrombocytopenia and (6) neurological complications. Perinatal outcomes were also analysed. RESULTS: Eighty seven (13%) of 696 cases had EOP; 226 (32%) had late pre-term PE and 383 (55%) term PE. Maternal age was similar amongst the three groups. Women with late pre-term and term PE had similar rates of maternal and foetal outcomes. Compared with term PE, women with EOP had similar rates of adverse maternal outcomes, however their babies had significantly increased rates of morbidity and mortality. CONCLUSION: Pre-eclampsia causes significant maternal organ involvement regardless of gestation at onset. Outcomes for babies of women with EOP are significantly worse than for those who present later. Overall, women presenting with PE after 34 weeks have generally good maternal and foetal outcomes in a unit equipped to manage such cases.

Protein surface roughness and small molecular binding sites.

Pharmaceutical design is usually directed at developing small molecules that can specifically bind and alter the activity of a target protein. Here, we show that high-affinity binding of small molecules requires a rough patch on a protein surface. Drug design strategies should therefore be targeted to rough areas on a protein. Our results indicate that the roughness of small functional sites may reflect the complex local shapes needed to fit specific interactions into small areas.

Glycerol-3-phosphate dehydrogenase activity in human lymphocytes: effects of insulin, obesity and weight loss.

Insulin exposure stimulates an increase in glycerol-3-phosphate dehydrogenase (G3PDH) activity in isolated human lymphocytes that correlates to an increase in G3PDH mRNA and requires new protein synthesis. Synthetic diacylglycerol or phorbol ester can mimic the effect of insulin on G3PDH activity, suggesting that protein kinase C may be involved in regulation of G3PDH levels. In addition, lithium chloride, an inositol phosphate phosphatase inhibitor, and calcium uptake inhibitors can abolish insulin stimulation of G3PDH activity. For obese subjects in whom insulin resistance in vitro can be demonstrated, the extent of insulin stimulation of G3PDH activity is decreased compared to normal weight individuals, and treatment by a very low calorie diet restores insulin stimulation of G3PDH activity. Thus, insulin stimulation of G3PDH activity is dependent upon the metabolic state of the subject from whom the cells are obtained.

Evidence for sulfite as an essential metabolite for human peripheral lymphocytes.

Sulfite has been identified as an essential metabolite by means of growth studies using a chemically-defined, protein-free medium for culture of human peripheral lymphocytes. Sulfite reduced the amount of cysteine required for optimum growth by at least four-fold. In some subjects, sulfite stimulated growth even in the presence of optimal amounts of cysteine indicating that lymphocytes of some individuals are unable to convert cysteine to sulfite in adequate amounts.

The effect of asparagine and adenine on the glutamine requirement for growth of human peripheral lymphocytes.

Quantitative growth responses of lymphocytes directly isolated from individual subjects in a newly developed chemically-defined, protein-free medium are used to demonstrate that supplements of both L-asparagine and a purine source, but neither alone, significantly reduce the quantitative requirement for L-glutamine for growth. This system is useful for exploring individual differences in quantitative glutamine requirements and adequacy of asparagine and purine biosynthesis.

Purification and properties of pyruvate dehydrogenase kinase from bovine kidney.

Pyruvate dehydrogenase kinase was purified about 2,700-fold to apparent homogeneity from extracts of bovine kidney mitochondria. The kinase consists of two subunits (alpha beta) with molecular weights of 48,000 (alpha) and 45,000 (beta) as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinase activity resides in the alpha subunit. The alpha subunit is sensitive to proteolysis by chymotrypsin, whereas the beta subunit is selectively modified by trypsin. These observations, together with the results of peptide mapping, indicate that the two subunits are distinctly different proteins. It is proposed that the beta subunit is a regulatory subunit.

Purification and properties of pyruvate dehydrogenase phosphatase from bovine heart and kidney.

Pyruvate dehydrogenase phosphatase was purified to apparent homogeneity from bovine heart and kidney mitochondria. The phosphatase has a sedimentation coefficient (S20,w) of about 7.4 S and a molecular weight (Mr) of about 150 000 as determined by sedimentation equilibrium and by gel-permeation chromatography. The phosphatase consists of two subunits with molecular weights of about 97 000 and 50 000 as estimated by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Phosphatase activity resides in the Mr 50 000 subunit, which is sensitive to proteolysis. The phosphatase contains approximately 1 mol of flavin adenine dinucleotide (FAD) per mol of protein of Mr 150 000. FAD is apparently associated with the Mr 97 000 subunit. The function of this subunit remains to be established. The phosphatase binds 1 mol of Ca2+ per mol of enzyme of Mr 150 000 at pH 7.0, with a dissociation constant (Kd) of about 35 microM as determined by flow dialysis. Use of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate (EGTA) at pH 7.6 in conjunction with flow dialysis gave a Kd value for Ca2+ of about 8 microM. In the presence of both the phosphatase and the dihydrolipoyl transacetylase (E2) core of the pyruvate dehydrogenase complex, two equivalent and apparently non-interacting CA2+-binding sites were detected per unit of Mr 150 000, with a Kd value of about 24 microM in the absence and about 5 microM in the presence of EGTA. In the presence of 0.2 M KCl, which inhibits phosphatase activity about 95%, the phosphatase exhibited only one Ca2+-binding site, even in the presence of E2. The phosphatase apparently possesses an "intrinsic" Ca2+-binding site, and a second Ca2+-binding site is produced in the presence of E2. The second site is apparently altered by increasing the ionic strength. It is proposed that the second site may be at the interface between the phosphatase and E2, with Ca2+ acting as a bridging ligand for specific attachment of the phosphatase to E2.

Regulation of pyruvate dehydrogenase kinase activity by protein thiol-disulfide exchange.

Endogenous kinase activity of highly purified pyruvate dehydrogenase complex from bovine kidney is markedly inhibited by N-ethylmaleimide and by certain disulfides. Inhibition by disulfides is highly specific and is reversed by thiols. 5,5'-Dithiobis(2-nitrobenzoate) is the most potent inhibitor, showing significant inhibition at a concentration as low as 1 microM. Cystamine, oxidized glutathione, pantethine, lipoic acid, lipoamide, ergothionine, insulin, oxytocin, and vasopressin were ineffective. Hydrogen peroxide and t-butyl hydroperoxide were inactive. The data indicate pyruvate dehydrogenase kinase (EC 2.7.1.99) contains a thiol group (or groups) that is involved in maintaining a conformation of the enzyme that facilitates phosphorylation and inactivation of its protein substrate, pyruvate dehydrogenase (EC 1.2.4.1). These findings suggest that modulation of pyruvate dehydrogenase kinase activity by thiol-disulfide exchange may be an important physiological mechanism for regulation of kinase activity and, hence, activity of the pyruvate dehydrogenase complex.

Use of trypsin and lipoamidase to study the role of lipoic acid moieties in the pyruvate and alpha-ketoglutarate dehydrogenase complexes of Escherichia coli.

The relationships between release of (3)H-labeled lipoyl moieties by trypsin and lipoamidase and accompanying loss of overall enzymatic activity of the Escherichia coli pyruvate and alpha-ketoglutarate dehydrogenase complexes were studied. Trypsin releases lipoyl domains together with their covalently attached lipoyl moieties from the "inner" core of the dihydrolipoyl transacetylase and the dihydrolipoyl transsuccinylase whereas lipoamidase releases only the lipoyl moieties. The results show that release of lipoyl domains by trypsin and release of lipoyl moieties by lipoamidase proceeded at faster rates than the accompanying loss of overall activity of the two complexes. Trypsin released about half of the lipoyl domains in the pyruvate dehydrogenase complex without significant effect on the overall activity. A model is presented to explain these and other observations on active-site coupling via lipoyl moieties.