Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome (HHH) presenting with acute fulminant hepatic failure.

We report on two Aboriginal patients with the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome. Both presented with acute hepatic failure with severe hypertransaminasemia and coagulopathy, prompting evaluation for emergent liver transplantation. The diagnosis of HHH syndrome was based on the presence of typical metabolic abnormalities. A protein-restricted diet and L-arginine or L-citrulline supplementation were immediately started, with rapid normalization of liver function test results and other biochemical abnormalities. Molecular analysis of the SLC25A15 gene showed that the two patients were homozygous for the common French Canadian mutation (F188Delta). The diagnosis of HHH syndrome should be considered in patients with unexplained fulminant hepatic failure. There does not appear to be a genotype-phenotype correlation for this presentation, inasmuch as the only other reported patient presenting with this picture had two different point mutations. Early identification and prompt treatment of these patients is crucial to avoid liver transplantation and can be life saving.

Reduced insulin-stimulated GLUT4 bioavailability in stroke-prone spontaneously hypertensive rats.

AIMS/HYPOTHESIS: Insulin-stimulated glucose transport is impaired in a genetic model of hypertension, the stroke-prone spontaneously hypertensive rat (SHRSP), yet the molecular mechanisms that underlie this defect in the animals remain unclear. METHODS: We examined the effects of insulin on the trafficking of the insulin-responsive glucose transporter GLUT4 to the plasma membrane in isolated adipocytes from SHRSP and normotensive control Wistar-Kyoto (WKY) rats. RESULTS: Treatment of isolated adipocytes with insulin resulted in trafficking of GLUT4 to the plasma membrane. There was no significant difference in the magnitude of insulin-stimulated GLUT4 trafficking from intracellular membranes to the plasma membrane between strains. In contrast, we demonstrated that there is a significant reduction in GLUT4 accessible to the glucose photolabel Bio-LC-ATB-BGPA at the plasma membrane of SHRSP adipocytes compared with control rats. CONCLUSIONS/INTERPRETATION: We propose that a large proportion of GLUT4 translocated to the plasma membrane in response to insulin is not able to bind substrate and catalyse transport in the SHRSP. Therefore, there is a reduction in bioavailable GLUT4 in SHRSP animals that is likely to account, at least in part, for the reduced insulin-stimulated glucose uptake.

Improved methods for the extraction and analysis of isoflavones from soy-containing foods and nutritional supplements by reversed-phase high-performance liquid chromatography and liquid chromatography-mass spectrometry.

An improved method for extraction and analysis of isoflavones from soy protein, soy foods and nutritional supplements is presented. The method uses acetonitrile extraction without acidification, with apigenin as internal standard. Samples extracted in acetonitrile-water are diluted to 50% acetonitrile and directly injected for gradient HPLC separation on a C18 reversed-phase column. This method saves significant time during sample preparation and improves accuracy and precision. Conventional and rapid HPLC analysis methods compatible with the extraction scheme were developed. During development of the methods, unexpected minor forms of malonyl and acetyl isoflavones were discovered in extracts of soy proteins and in pure isoflavone standard preparations. By LC-triple MS, these peaks have identical composition to the respective 6''-O-malonyl- and 6''-O-acetyl-isoflavones from which they form. These minor forms are believed to be malonyl and acetyl isoflavones where the site of attachment is a hydroxyl other than the 6'-OH of the glucose. These compounds can represent significant minor isoflavone components of foods, which contain high concentrations of malonyl or acetyl isoflavones.

Cd36 and molecular mechanisms of insulin resistance in the stroke-prone spontaneously hypertensive rat.

Insulin resistance is of pathogenic importance in several common human disorders including type 2 diabetes, hypertension, obesity and hyperlipidemia, but the underlying mechanisms are unknown. The spontaneously hypertensive rat (SHR) is a model of these human insulin resistance syndromes. Quantitative trait loci (QTLs) for SHR defects in glucose and fatty acid metabolism, hypertriglyceridemia, and hypertension map to a single region on rat chromosome 4. Genetic analysis of an SHR derived from a National Institutes of Health colony led to the identification of a causative mutation in the SHR Cd36. We have investigated glucose and fatty acid metabolism in the stroke-prone SHR (SHRSP). We demonstrate defects in insulin action on 2-deoxy-D-glucose transport (SHRSP 3.3 +/- 1.5 vs. 21.0 +/- 7.4 pmol x min(-1) x [20 microl packed cells](-1), SHRSP vs. WKY, respectively, P = 0.01) and inhibition of catecholamine-stimulated lipolysis (P < 0.05 at all concentrations of insulin) in adipocytes isolated from SHRSP. In contrast, basal levels of catecholamine-stimulated nonesterified free fatty acid (NEFA) release and plasma levels of NEFA are similar in SHRSP and WKY. These results are in agreement with the data on the SHR.4 congenic strain, which suggested that the QTL containing Cd36 mutations accounted for the entire defect in basal catecholamine action but only for approximately 40% of the SHR defect in insulin action. In the SHR, both abnormalities appear consequent of defective Cd36 expression. Because Cd36 sequence and expression are apparently normal in SHRSP, it is likely that the molecular mechanism for defective insulin action in this strain is caused by a gene(s) different than Cd36.

Sex hormones induce insulin resistance in 3T3-L1 adipocytes by reducing cellular content of IRS proteins.

AIM/HYPOTHESIS: Numerous studies have suggested a relation between sex hormones and insulin sensitivity but the ability of sex hormones to directly influence insulin action in peripheral tissues has not been investigated. METHODS: We have examined the effects of estriol, estradiol and estrone on insulin action in cultured 3T3-L1 adipocytes, a useful model of adipocytes. RESULTS: Treatment of these cells with each of these sex hormones resulted in a statistically significant reduction in the ability of insulin to stimulate glucose transport independently of a reduction in total cellular GLUT-4 content. This diminished ability of insulin to stimulate glucose transport was accompanied by a reduction in the total cellular content of insulin receptor substrates -1 and -2 and the p85alpha subunit of phosphatidylinositol 3'-kinase. By contrast, cellular content of protein kinase B was unchanged by hormone treatment but the magnitude of insulin-stimulated kinase activity was statistically significantly reduced after incubation with each of the sex hormones tested. We have further shown that treatment of 3T3-L1 adipocytes with these hormones alters the subcellular distribution of insulin receptor substrate proteins such that the particulate and soluble pools of these proteins were differentially affected by hormone treatment. CONCLUSION/INTERPRETATION: These data show that sex hormones can directly induce a state of insulin resistance in 3T3-L1 adipocytes in culture. The mechanism of this defect seems to be at least in part due to decreased cellular content and altered subcellular distribution of insulin receptor substrate proteins which in turn results in a reduction in proximal insulin-stimulated signalling cascades.

Analytical characterization of electrochemical biosensor test strips for measurement of glucose in low-volume interstitial fluid samples.

BACKGROUND: Minimally invasive interstitial fluid (ISF) sampling and glucose measurement technologies were integrated into a hand-held device for diabetic glucose monitoring investigations. METHODS: Conventional electrochemical test strip technology (Bayer Glucometer Elite) was adapted to measure glucose in small (0.5-2.0 microL) samples of ISF. Test strip glucose measurements were performed on a commercial potentiostat and were compared to various reference glucose methodologies (YSI 2300 analyzer, microhexokinase procedure, Bayer Glucometer Elite). Characterizations of the integrated ISF sampling-glucose test strip design included accuracy and precision in various sample media (saline, ISF surrogates, diabetic ISF samples), sample volume dependence, test strip sterilization studies (electron beam, gamma irradiation), and diabetic ISF sampling and glucose measurements. RESULTS: Glucose measurements were free from significant media effects. Sample volume variations (0.6-3.2 microL) revealed only modest dependence of glucose measurement bias on sample volume (-1.5% per microliter). Sterilization treatments had only a minor impact on glucose response and test strip aging and no significant impact on interferent responses of the glucose test strips. Diabetic subject testing under minimum fasting conditions of at least 2 h with integrated ISF sampling and glucose measurement gave low ISF glucose measurement imprecision (CV, 4%) and mean glucose results that were indistinguishable from reference (microhexokinase) ISF glucose measurements and from capillary blood glucose measurements (Glucometer Elite). CONCLUSIONS: Conventional single-use, electrochemical glucose test strip and ISF collection technologies can be readily integrated to provide real-time ISF sampling and glucose measurements for diabetic monitoring applications.

An initial assessment of the risk approach to antenatal management in Malaysia.

This study was the first assessment of a nationwide risk approach system to antenatal management introduced to Malaysia in 1989. Three rapid, record-based surveys on three different study groups were conducted to determine risk factor prevalence, accuracy of risk assignment, action after risk assignment and the relationship of risk level and place of delivery. The most frequent risk factors were short birth interval, high parity and first pregnancy. Accuracy of risk assignment was highest at the lowest levels of risk and poorest at the highest levels. Women at the lowest levels of risk were more likely to be seen by a doctor than women at highest risk. These was a trend to deliver in hospital, rather than at home, as level of risk increased; but many women at high risk still delivered at home. Recommendations are made on modifications to the system prior to future evaluation.

PIP: In 1990 in Malaysia, 3 distinct surveys were conducted as part of an assessment of a nationwide risk approach system to prenatal management introduced in 1989. After history-taking and examination, a health worker completed a risk checklist, then determined what risk category the pregnant woman belonged to and assigned her chart a color code. Red means a life-threatening condition and immediate labor ward admission. Yellow means risk factors requiring antenatal monitoring and treatment by a physician. White means no risk, so the midwife or the community nurse can monitor the pregnant women. Green means that complications may occur and a senior nurse should monitor the woman's progress. The first survey included all pregnant women attending prenatal clinics in Pasir Mas District, Kelantan State, for the first time. The second survey was a retrospective check of the prenatal cards of all pregnant women attending these clinics within the same area over a 1-week period in January 1990. The third retrospective, record-based survey included all women who delivered in the study area during February 1990. Both the clinic staff and the study team classified 75% of the women as high risk. Only 44.4% of antenatal cards had a completed risk checklist, more than 33% of which were completed after the clinic. The most common risk factors were short birth interval (47.5%) and high parity (i.e., gravida 6 or above) (27.2%). Almost 50% of women coded green delivered at home, when they should have delivered at the hospital, suggesting that they disregarded clinic advice. Sensitivity of the code approach was better at a lower level of risk than at higher levels (71.2% for green vs. 43.2% for yellow and 0 for red). Misunderstanding of risk criteria or coding to own clinical judgment accounted for the incorrect coding by health staff. Overall, midwives were knowledgeable and competent. They knew their communities and clients well. Based on these findings, the researchers suggest replacing the risk checklist with at least 1 laminated reference checklist displayed prominently in the clinic. Further evaluation is needed.

Simplified dual-lumen catheter design for simultaneous potentiometric monitoring of carbon dioxide and pH.

A novel dual-lumen catheter electrode design suitable for the simultaneous measurement of PCO2 (partial pressure of carbon dioxide) and pH in flowing blood is described. The probe is fabricated from a single segment of dual-lumen silicone rubber or polyurethane tubing that is impregnated with the proton ionophore tridodecylamine. The impregnation step imparts H+ permselectivity to both inner and outer walls of the tubing. By filling each lumen with a suitable buffer/electrolyte solution and Ag/AgCl reference electrode wire, simultaneous potentiometric detection of both PCO2 and pH is achieved. Careful optimization of incorporated proton carrier (tridodecylamine), plasticizer (o-nitrophenyl octyl ether), and lipophilic counteranion sites (tetrakis[3,5-bis(trifluoromethyl)phenyl]borate) within the tubing walls yields catheter electrodes with resistance values of 10-20 M omega and relatively high stability in flowing blood. Results from continuous measurements of PCO2 and pH during long-term 30-65-h blood loop experiments demonstrate that, after an initial conditioning period, the catheter exhibits low drift rates (PCO2, 4.7 +/- 1.7 mV/30 h; pH, 1.4 +/- 0.5 mV/30 h) and yields continuously measured values in good agreement with those obtained on discrete samples with a commercial blood gas analyzer (PCO2, r2 = 0.997; pH, r2 = 0.915). In vivo evaluation of the catheter sensors, performed by implanting silicone rubber dual-lumen probes in the arteries of anesthetized dogs, indicates that the proposed catheter design can closely follow PCO2/pH changes induced in the animals during 6-13 h of continuous monitoring.

Current and future directions in the technology relating to bedside testing of critically ill patients.

Significant progress has been made recently in the measurement methods and instrumental approaches applicable to bedside testing of critically ill patients. While the "ideal" technology would involve the ability to obtain accurate stat profile values on a continuous basis via noninvasive methods, given the present state of noninvasive sensing technologies, this capability is unlikely to be achieved in the foreseeable future. In principle, invasive and on-line techniques offer more hope for future success in continuous bedside monitoring of all the key critical care analyses. However, success in these directions will come only when issues regarding sensor stability and sampling device/sensor biocompatibility are completely solved. Until then, it appears that the user-friendly point of care type stat analyzers that can provide accurate values for all the key analytes, used in conjunction with existing noninvasive trend monitors (eg, pulse oximetry), will offer the most attractive approach for the effective treatment of critically ill patients.

Potentiometric combination ion/carbon dioxide sensors for in vitro and in vivo blood measurements.

The development and analytical performance of a novel potentiometric combination ion/pCO2 sensor design for in vitro and in vivo measurements are reported. The design is based on incorporating an appropriate ionophore within the outer silicone gas permeable membranes of both conventional macro and new catheter-type pCO2 sensors. Simultaneous measurement of the potentials across the ion-selective/gas permeable membrane and the inner glass or polymer pH sensitive membrane provides the basis for continuous monitoring of both ionic and pCO2 levels with the same device. A macro-sized K+/pCO2 embodiment of the sensor is constructed from a commercial Severinghaus CO2 sensor and is used to demonstrate the principles and capabilities of the proposed design. A flexible, miniaturized (outer diameter = 1.2 mm) combination K+/pCO2 catheter sensor is also described. The catheter-type sensor is fabricated by inserting a tubular polymer membrane pH electrode into an outer silicone rubber tube doped with valinomycin. Continuous measurements of K+ and pCO2 during 6-h blood pump studies using both the macro and catheter-type combination sensors correlate well with those of conventional bench-top analyzers. In addition, continuous (4 h) intravascular measurements with the combination catheter sensor in dogs show good agreement with those of commercial blood analyzers (R = 0.984 and 0.962 for pCO2 and K+, respectively.

S-thiolation of cytoplasmic cardiac creatine kinase in heart cells treated with diamide.

Two methods for quantitation of protein S-thiolation, by isoelectric focusing or by enzyme activity, were used for studying S-thiolation of cytoplasmic cardiac creatine kinase. With these methods, creatine kinase was identified as a major S-thiolated protein in both bovine and rat heart. In rat heart cell cultures, creatine kinase became 10% S-thiolated during a 10 min incubation with 0.2 mM diamide. This enzyme became S-thiolated more slowly than other heart cell proteins and it also dethiolated more slowly. Two sequential additions of diamide at a 25 min interval caused twice as much S-thiolation after the second addition as compared to the first. This increased sensitivity to the second diamide treatment may have resulted from glutathione loss during the first addition which produced a higher GSSG-to-GSH ratio after the second treatment. The GSSG-to-GSH ratio was highest prior to the maximum S-thiolation of creatine kinase, but, in general, the time course of glutathione was similar to the S-thiolation of creatine kinase. This study demonstrates that cytoplasmic creatine kinase is S-thiolated and, therefore, inhibited during a diamide-induced oxidative stress in heart cells. Implications for regulation of cardiac metabolism during oxidative stress are discussed.

A comparison of protein S-thiolation (protein mixed-disulfide formation) in heart cells treated with t-butyl hydroperoxide or diamide.

Beating neonatal heart cell cultures were treated with diamide or t-butyl hydroperoxide, and changes in glutathione oxidation, cell beating, and protein S-thiolation (protein mixed-disulfide formation) were examined. Both compounds caused extensive oxidation of glutathione. Cells treated with diamide stopped beating within 2 min, and beating returned to normal after 30-45 min. Cells stopped beating 25 min after the addition of t-butyl hydroperoxide, and beating did not resume. t-Butyl hydroperoxide caused S-thiolation of a variety of proteins, but only one protein, of molecular mass 23 kDa, was extensively modified. Diamide caused extensive modification of proteins with molecular masses of 97, 42 and 23 kDa as well as three proteins of about 35 kDa. Though the GSSG content of cell cultures returned to normal by 15 min after diamide treatment. S-thiolation of several proteins persisted. These studies show that S-thiolation of proteins is an important metabolic response in cells exposed to an oxidative challenge by t-butyl hydroperoxide or diamide, and that the specificity of the response depends on the agent used.

Protein mixed-disulfides in cardiac cells. S-thiolation of soluble proteins in response to diamide.

Protein mixed-disulfides in cultured rat heart cells were analyzed by gel electrophoresis under conditions that eliminated artifactual formation of these protein derivatives. Protein S-thiolation (protein mixed-disulfide formation) was detectable under normal culture conditions. Diamide oxidized intracellular glutathione in these cells and produced extensive protein S-thiolation. The specificity of this protein modification indicates a role in the regulation of cardiac metabolism.