Multiplate and TEG platelet mapping in a population of severely injured trauma patients.

OBJECTIVES: The objectives of this study were to compare thromboelastography platelet mapping (TEG PM) with impedance aggregometry (Multiplate, MP) in a single trauma population and relate their results clinically. BACKGROUND: Platelet function as measured by thromboelastography and impedance aggregometry demonstrates significant reductions that persist for days following traumatic injury. However, no study compares these devices and the correlation between them is not known. METHODS: In level 1 trauma patients, TEG PM and MP were conducted at their initial presentation to the emergency department. Within-device repeatability and between-device association were determined using correlation analyses. Demographic variables, Injury Severity Score, blood product transfusion, laboratory test results and mortality rate were recorded. RESULTS: Ninety-two patients were enrolled. Within-device repeatability was high for TEG PM and MP for arachidonic acid (AA) and adenosine diphosphate (ADP) activation pathways. When comparing TEG PM with MP, results correlated poorly in the ADP pathway (Spearman's rho = 0·11, P = 0·44) and moderately in the AA pathway (Spearman's rho = 0·56, P < 0·0001). TEG PM was predictive of blood product transfusion and correlated with increased base deficit, whereas MP was only predictive of mortality. CONCLUSIONS: Intra-device variability was low for TEG PM and MP, but the two point-of-care devices measuring platelet function correlate poorly with each other in injured trauma patients. Each device also had different clinical associations.

Reliability of 24-h void frequency as an index of hydration status when euhydrated and hypohydrated.

BACKGROUND/OBJECTIVES: Void frequency (VF) is significantly correlated to hydration status, but it is unknown whether VF is reliable when an individual is repeatedly euhydrated (EU) or hypohydrated (HY). The purpose of this study was to test the reliability of VF when individuals were EU or HY on multiple occasions. SUBJECTS/METHODS: Fourteen males (age 22±2 years, mass 79.1±12.8 kg) completed three EU trials achieved with 24-h ad libitum fluid intake, and 14 males (age 22±2 years, mass 78.6±10.4 kg) completed three HY trials achieved with 24-h fluid restriction. Twenty-four hour urine was collected and analyzed for specific gravity (USG) and VF. Subjects voided at a 'normal urgency' (rated a '2' on a 0-4 perceptual scale) throughout each 24-h period. RESULTS: Twenty-four hour USG was greater and VF was lower when HY (1.026±0.003 and 5±2, respectively) versus EU (1.014±0.003 and 7±2; both P<0.05). Intra-class correlations for VF between the three trials at each hydration status were deemed acceptable (0.863 and 0.849 for EU and HY, respectively). Within-subject coefficients of variation for VF were 15±9 and 21±14% for the EU and HY trials. CONCLUSIONS: VF is a reliable index of 24-h hydration status when healthy young males are EU or HY and voiding at a consistent 'urgency'.

Effect of passive heat stress on arterial stiffness in smokers versus non-smokers.

In non-smokers, passive heat stress increases shear stress and vasodilation, decreasing arterial stiffness. Smokers, who reportedly have arterial dysfunction, may have similar improvements in arterial stiffness with passive heat stress. Therefore, we examined the effects of an acute bout of whole-body passive heat stress on arterial stiffness in smokers vs. non-smokers. Thirteen smokers (8.8 ± 5.5 [median = 6] cigarettes per day for > 4 years) and 13 non-smokers matched for age, mass, height, and exercise habits (27 ± 8 years; 78.8 ± 15.4 kg; 177.6 ± 6.7 cm) were passively heated to 1.5 °C core temperature (T C) increase. At baseline and each 0.5 °C T C increase, peripheral (pPWV) and central pulse wave velocity (cPWV) were measured via Doppler ultrasound. No differences existed between smokers and non-smokers for any variables (all p > .05), except cPWV slightly increased from baseline (526.7 ± 81.7 cm · s(-1)) to 1.5 °C ΔT C (579.7 ± 69.8 cm · s(-1); p < 0.005), suggesting heat stress acutely increased central arterial stiffness. pPWV did not change with heating (grand mean: baseline = 691.9 ± 92.9 cm · s(-1); 1.5 °C ΔT C = 691.9 ± 79.5 cm · s(-1); p > 0.05). Changes in cPWV and pPWV during heating correlated (p < 0.05) with baseline PWV in smokers (cPWV: r = -0.59; pPWV: r = -0.62) and non-smokers (cPWV: r = -0.45; pPWV: r = -0.77). Independent of smoking status, baseline stiffness appears to mediate the magnitude of heating-induced changes in arterial stiffness.

24-h Void number as an indicator of hydration status.

BACKGROUND/OBJECTIVES: Few user-friendly hydration assessment techniques exist for the general population to use on a daily basis. The present study evaluated void number over 24 h as a potential hydration assessment tool. SUBJECTS/METHODS: Male and female subjects collected urine for 24 h while adequately hydrated (n=44; 22 ± 4 years, 168 ± 16 cm, 73 ± 15 kg) or fluid restricted (n=43; 22 ± 3 years, 175 ± 10 cm, 81 ± 24 kg). As a control, participants were asked to void when feeling the 'first urge to void' on a commonly used urge scale and noted the volume of each void. For each sample, 24-h urine volume, osmolality (U(OSM)), specific gravity (U(SG)) and color were measured in the laboratory. RESULTS: As designed, the level of urge upon voiding was consistent throughout the study (2 ± 0; 'first urge to void'). Samples were classified by U(SG) as either euhydrated (U(SG)<1.020) or hypohydrated (U(SG) ⩾ 1.020). Grouping by U(OSM) did not change results. Euhydrated versus hypohydrated individuals had greater 24-h urine volume (1933 ± 864 versus 967 ± 306 ml, respectively) and lower urine color (2 ± 1 versus 5 ± 1), U(SG) (1.012 ± 0.004 versus 1.025 ± 0.004) and UOSM (457 ± 180 versus 874 ± 175 mOsm/kg H2O; all P<0.001). Euhydrated individuals voided more than hypohydrated individuals over the 24-h period (5 ± 2 versus 3 ± 1 voids; P<0.001). Additionally, void number inversely correlated with hydration status as identified by U(SG) (r=-0.50; P<0.05) and U(OSM) (r=-0.56; P<0.05). CONCLUSIONS: In conclusion, over 24 h, individuals with a higher void number were euhydrated (that is, had less concentrated hydration biomarkers) than those with a lower void number. Based on these data, void number might be utilized as a simple and feasible hydration assessment for the general public, as it utilizes no equipment or technical expertise.

Diverse roles for protein kinase C delta and protein kinase C epsilon in the generation of high-fat-diet-induced glucose intolerance in mice: regulation of lipogenesis by protein kinase C delta.

AIMS/HYPOTHESIS: This study aimed to determine whether protein kinase C (PKC) delta plays a role in the glucose intolerance caused by a high-fat diet, and whether it could compensate for loss of PKCepsilon in the generation of insulin resistance in skeletal muscle. METHODS: Prkcd (-/-), Prkce (-/-) and wild-type mice were fed high-fat diets and subjected to glucose tolerance tests. Blood glucose levels and insulin responses were determined during the tests. Insulin signalling in liver and muscle was assessed after acute in vivo insulin stimulation by immunoblotting with phospho-specific antibodies. Activation of PKC isoforms in muscle from Prkce (-/-) mice was assessed by determining intracellular distribution. Tissues and plasma were assayed for triacylglycerol accumulation, and hepatic production of lipogenic enzymes was determined by immunoblotting. RESULTS: Both Prkcd (-/-) and Prkce (-/-) mice were protected against high-fat-diet-induced glucose intolerance. In Prkce (-/-) mice this was mediated through enhanced insulin availability, while in Prkcd (-/-) mice the reversal occurred in the absence of elevated insulin. Neither the high-fat diet nor Prkcd deletion affected maximal insulin signalling. The activation of PKCdelta in muscle from fat-fed mice was enhanced by Prkce deletion. PKCdelta-deficient mice exhibited reduced liver triacylglycerol accumulation and diminished production of lipogenic enzymes. CONCLUSIONS/INTERPRETATION: Deletion of genes encoding isoforms of PKC can improve glucose intolerance, either by enhancing insulin availability in the case of Prkce, or by reducing lipid accumulation in the case of Prkcd. The absence of PKCepsilon in muscle may be compensated by increased activation of PKCdelta in fat-fed mice, suggesting that an additional role for PKCepsilon in this tissue is masked.

Dilinoleoyl-phosphatidic acid mediates reduced IRS-1 tyrosine phosphorylation in rat skeletal muscle cells and mouse muscle.

AIMS/HYPOTHESIS: Insulin resistance in skeletal muscle is strongly associated with lipid oversupply, but the intracellular metabolites and underlying mechanisms are unclear. We therefore sought to identify the lipid intermediates through which the common unsaturated fatty acid linoleate causes defects in IRS-1 signalling in L6 myotubes and mouse skeletal muscle. MATERIALS AND METHODS: Cells were pre-treated with 1 mmol/l linoleate for 24 h. Subsequent insulin-stimulated IRS-1 tyrosine phosphorylation and its association with the p85 subunit of phosphatidylinositol 3-kinase were determined by immunoblotting. Intracellular lipid species and protein kinase C activation were modulated by overexpression of diacylglycerol kinase epsilon, which preferentially converts unsaturated diacylglycerol into phosphatidic acid, or by inhibition of lysophosphatidic acid acyl transferase with lisofylline, which reduces phosphatidic acid synthesis. Phosphatidic acid species in linoleate-treated cells or muscle from insulin-resistant mice fed a safflower oil-based high-fat diet that was rich in linoleate were analysed by mass spectrometry. RESULTS: Linoleate pretreatment reduced IRS-1 tyrosine phosphorylation and p85 association. Overexpression of diacylglycerol kinase epsilon reversed the activation of protein kinase C isoforms by linoleate, but paradoxically further diminished IRS-1 tyrosine phosphorylation. Conversely, lisofylline treatment restored IRS-1 phosphorylation. Mass spectrometry indicated that the dilinoleoyl-phosphatidic acid content increased from undetectable levels to almost 20% of total phosphatidic acid in L6 cells and to 8% of total in the muscle of mice fed a high-fat diet. Micelles containing dilinoleoyl-phosphatidic acid specifically inhibited IRS-1 tyrosine phosphorylation and glycogen synthesis in L6 cells. CONCLUSIONS/INTERPRETATION: These data indicate that linoleate-derived phosphatidic acid is a novel lipid species that contributes independently of protein kinase C to IRS-1 signalling defects in muscle cells in response to lipid oversupply.

Dysfunctional regulation of endothelial nitric oxide synthase (eNOS) expression in response to exercise in mice lacking one eNOS gene.

BACKGROUND: Previous data suggest that 1 endothelial NO synthase (eNOS) gene is sufficient to allow normal expression and function of eNOS under basal conditions. We hypothesized that this might not hold true for conditions known to increase eNOS gene expression, such as exercise. METHODS AND RESULTS: Male mice heterozygous for a disruption of the eNOS gene (eNOS(+/)(-)) and normal C56Bl/6J mice (eNOS(+/+)), 3 to 4 months of age, underwent exercise training for 3 weeks. Nontrained mice were exposed to the exercise environment (noise and vibration of the treadmill) without exercise for an identical period. In eNOS(+/+) mice (n=7), exercise increased aortic eNOS protein expression by 3.4+/-0.4-fold (P<0.002). This was associated with a greater vascular cGMP accumulation on stimulation with acetylcholine (P<0.05). Furthermore, exercise training increased eNOS mRNA (1.78+/-0.4-fold) and protein (1.76+/-0.17-fold) in left ventricular tissue, as determined by competitive reverse transcription-polymerase chain reaction and Western analysis (P<0.05 for both). In striking contrast, exercise had no effect on aortic eNOS expression and cGMP accumulation in eNOS(+/)(-) mice (P>0.05). Thus, although eNOS expression appears to be normal in eNOS(+/)(-) mice under basal conditions, these mice are unable to increase eNOS expression during exercise. CONCLUSIONS: These findings show that regulation of eNOS expression during exercise requires the presence of both alleles of the gene and may have implications for conditions in which polymorphisms of eNOS are present in only 1 allele in humans. These individuals may have a normal vascular reactivity under basal conditions but may be unable to adapt their vascular reactivity in response to exercise training.

Evidence for a NADH/NADPH oxidase in human umbilical vein endothelial cells using electron spin resonance.

A growing body of evidence has suggested that a membrane-bound NADH/NADPH oxidase is the predominant source of reactive oxygen species (ROS) in vascular cells. Prior studies have used indirect assessments of superoxide including lucigenin-enhanced chemiluminescence, cytochrome c, and fluorescent dye techniques. The present study was performed to determine if NADH/NADPH oxidase function could be detected human endothelial cells using electron spin resonance. Human umbilical vein endothelial cells (HUVEC) were homogenized and fractionated into cytosolic and membrane components. Cell fractions were incubated in buffer containing either NADH or NADPH (100 microM for each) and the spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO). EPR signals were obtained in a Bruker EMX spectrometer. Cytoplasmic fractions were devoid of activity. In contrast, incubation of membrane fractions with NADH produced a signal with a total peak intensity of 1,038 +/- 64, which was significantly greater than that observed with NADPH (540 +/- 101). The signal was completely inhibited by either manganese superoxide dismutase (MnSOD, 100 U/ml) or the flavoprotein inhibitor diphenylene iodinium (DPI, 100 microM). Rotenone (100 microM) did not significantly alter the signal intensity, (833 +/- 88). These data demonstrate direct evidence for a functional NADH/NADPH oxidase in human endothelial cells and show that electron spin resonance is a useful tool for study of this enzyme system.

An anti-smoking project instituted by senior nursing students in a rural community.

The Health Council of Hawkins County, Tennessee, received a State Tobacco Prevention grant to institute an anti-smoking program in the county schools. The program chosen was called TarWars and encompasses a prohealth tobacco-free program curriculum. The goal was to discourage tobacco use among the nation's youth by focusing on tobacco's short-term ill effects. Because the program encourages community involvement, nursing students from East Tennessee State University, Johnson City, identified volunteers from the Hawkins County Retired Teachers Association to develop a teaching program for use in the first year of the program and as well as in subsequent years of the TarWars curriculum. A pretest and posttest were given to the students to assess the program's success. The objectives of the grant were that 80% of the students would be able to name three negative effects associated with tobacco use and three positive effects of not using tobacco. The goal was met when 91% could identify negative effects and 89% could identify three positive effects of not using tobacco.

Protein expression, vascular reactivity and soluble guanylate cyclase activity in mice lacking the endothelial cell nitric oxide synthase: contributions of NOS isoforms to blood pressure and heart rate control.

OBJECTIVE: Both disruption of the endothelial nitric oxide synthase (eNOS) gene and pharmacological inhibition of the NOS produce modest hypertension. It is unclear if and to what extent NOS isoforms other than eNOS contribute to this effect and how loss of one copy of the eNOS gene might impact on vascular reactivity or eNOS protein expression. METHODS: We examined protein expression, vascular reactivity, activity of soluble guanylate cyclase, blood pressure and heart rate in mice completely lacking the eNOS gene (eNOS-/-), wild-type mice (eNOS+/+) and mice heterozygotic for the eNOS gene (eNOS+/-). RESULTS: While eNOS-/- mice had mild hypertension and bradycardia, eNOS+/- mice were normotensive. In control mice, oral administration of L-NAME (approximately 100 mg/kg/day x 21 days) increased blood pressure to levels observed in eNOS-/- mice. In eNOS-/- mice, chronic oral administration of L-NAME had no effect on blood pressure, suggesting that inhibition of other NOS isoforms unlikely contribute to hypertension. L-NAME treatment induced bradycardia in both control and eNOS-/- mice, suggesting that both eNOS and other isoforms of NOS might be involved in heart rate control. Studies of aortic rings from eNOS-/- mice revealed a complete lack of endothelium-dependent vascular relaxation in response to acetylcholine and the calcium ionophore A23187 and an increase in sensitivity to phenylephrine, serotonin and nitroglycerin. Aortic rings from eNOS+/- mice demonstrated only minor alterations of responses to nitroglycerin and a normal relaxation to either acetylcholine or A23187 compared to vessels from eNOS-/+. Western analysis demonstrated that eNOS expression was virtually identical between eNOS+/+ and eNOS+/- mice and was absent in eNOS-/- mice. The activity of lung-isolated soluble guanylate cyclase was identical in the three strains of mice. CONCLUSIONS: We conclude that loss of one copy of the eNOS gene, as observed in heterozygotic animals, has no effect on vascular reactivity, blood pressure or eNOS protein expression. Isoforms of NOS, other than eNOS are unlikely involved in blood pressure regulation but may participate in heart rate control.

Primary valve malfunction of the Krupin eye valve with disk.

PURPOSE: To examine the management and possible causes of primary valve malfunction of the Krupin eye valve with disk. METHODS: The authors reviewed the results of 113 patients undergoing implantation of the Krupin eye valve with disk and identified eight patients with primary valve malfunction requiring surgical revision. RESULTS: Valve revision involved manipulation (n = 1 case), explantation of the malfunctioning valve and implantation of a new valve (n = 2), and amputation of the valve (n = 5). Six of eight patients had final intraocular pressures of < 21 mmHg on one or no medications at a mean interval of 15.9 months (range 5-36) after surgical revision. Transient postoperative hypotony was noted in three patients and chronic hypotony with loss of light perception in one patient. One explanted valve was examined and found to have partially fused leaflets. CONCLUSIONS: Surgical revision in cases of primary valve malfunction of the Krupin eye valve with disk may be accomplished relatively safely with an acceptable level of postoperative complications. The etiology of primary valve malfunction may be related to the sterilization process and prolonged storage before implantation.

Cat model for intraocular pressure reduction after transscleral Nd:YAG cyclophotocoagulation.

Transscleral neodymium:yttrium-aluminum-garnet (Nd:YAG) laser cyclophotocoagulation (TSNYC) is used to lower intraocular pressure (IOP) in glaucoma patients refractory to conventional medical and surgical therapy. Our study investigates the ability of TSNYC to lower IOP in normal cats. One eye of 13 cats was treated with non-contact TSNYC. Mean pretreatment IOP was 25 mm Hg (vs. 25.3 mm Hg in contralateral control eyes). Eyes received 80 laser applications over 360 degrees delivered at least 3 mm posterior to the limbus with maximum power (8 to 9 joules) and maximum retrofocus (3.6 mm). Eyes were retreated if IOP was not reduced below baseline after 2 weeks. By 4 weeks, IOP was decreased in all treated eyes by a mean of 29.2% and was maintained as long as 20 weeks (mean decrease 14.8%). IOP in 10 eyes was lowered after a single treatment session. Of these, 2 eyes had IOP spikes > 10 mm Hg prior to IOP reduction. Three cats required retreatment to maintain IOP reduction. All eyes developed transient (< 4 weeks) postoperative uveitis and 3 eyes developed rubeosis iridis which resolved with topical corticosteroids. Histologic examination (6 weeks post-treatment) showed focal disruption of the pigment epithelium and to a lesser degree the nonpigmented epithelium at the base of the ciliary body and in the valleys of the pars ciliaris. The epithelium at the apices of the ciliary processes appeared intact. Vascular engorgement was variably present. This study demonstrates that TSNYC lowers IOP in cats. This animal model will be useful for investigating mechanism(s) responsible for TSNYC-induced IOP reduction.

A neutral water-soluble broadening agent for spin-label studies.

Cr(maltolate)3 is proposed as a neutral water-soluble reagent for the broadening of accessible nitroxyl spin labels or spin probes in biological experiments. For situations in which the molecular charge is important, it supplements Cr(oxalate)3(3-), which is somewhat more effective on a molar basis. The interaction of the two reagents with spin-labeled creatine kinase is an example of a case in which the charge of the broadening agent is important.

Adaptation of feeding to a low fat yield in breast milk.

The effect of a low milk fat yield was assessed in a blinded prospective study of healthy term infants and mothers encouraged to breast-feed. Fat yield index was calculated as milk volume collected by Egnell pump multiplied by the "creamatocrit." Two weeks after delivery mothers who had a relatively low fat yield index (less than or equal to 30th percentile) were matched with mothers with a higher fat yield index and with formula-feeding mothers. Between 2 and 6 weeks the low fat yield group had a marginally lower weight gain but similar growth in length and head circumference to that of the higher fat yield group. The low fat yield group spent more time per feeding and had more complete breast emptying, resulting in a fat yield index comparable with the higher fat yield group for the mean milk volume ingested by the infant at 6 weeks. A low fat yield had no adverse effect on maternal satisfaction or maternal-infant interaction during feeding. Formula feeders spent the least time in feeding and en face gazing. Adaptation of breast-feeding to a low fat yield sustains infant intake and growth, maternal-infant interaction, and maternal satisfaction. However, there is little reserve if milk production diminishes. Further study is needed to define the limits of adaptation and effective interventions if fat yield is inadequate.

Detecting early glaucomatous field defects with the size I stimulus and Statpac.

The influence of stimulus size and normal database on the detection of visual field defects in automated static threshold perimetry (Humphrey Field Analyzer) was investigated in 82 eyes having a diagnosis of normal, glaucoma suspect, or early glaucoma. Using a mathematically derived 'normal' database, which assumes constantly decreasing threshold sensitivities with increasing eccentricity, the size I stimulus showed significantly greater sensitivity than the size III stimulus for detecting small, shallow scotomata in the central visual field. The use of Statpac, which contains an empirically derived, age-related normal database, increased the sensitivity significantly over that of the size III stimulus (with its mathematical model), and to a degree similar to that of the size I stimulus. The results obtained with the size I stimulus were reproducible and independent of the patient's age. This study suggests a potential role for the size I stimulus in evaluating eyes having or at risk of developing early glaucomatous field loss.