Measurement Of Dehydrogenase Activity Research Articles

Effects of Cr 3+, Cr 6+ and tannery sludge on C and N mineralization and microbial activity in semi-arid soils

The aim of this study was to evaluate the effect of two Cr species (Cr 3+ and Cr 6+) on N and C mineralization and dehydrogenase activity in semi-arid soils. The Cr species (250 mg kg −1 soil) were either added alone or mixed with tannery sludge (0.0125 g g −1) to three soils: cultivated soils, and outside and under the canopy of mesquite trees were then incubated for 180 days at 25 °C. Sole Cr 6+ addition had a higher inhibition of CO 2 production rate in cultivated soil (58–73%) than in soils under the canopy and outside the canopy. Soil outside the canopy amended with Cr 6+ showed the highest inhibition of dehydrogenase activity (40–100%) followed by cultivated and under the canopy soils. However, Cr 6+ added alone increased the inhibition of nitrification in soil outside the canopy (68–84%, from 30 to 120 days), followed by under the canopy and cultivated soils. The addition of tannery sludge to Cr 6+ significantly reduce the CO 2 production rate and dehydrogenase activity in all three soils, and increased the inhibition of nitrification in the following order: outside the canopy, cultivated and under the canopy soils. The addition of Cr 3+ or Cr 3+ plus tannery sludge either stimulated or inhibited CO 2 production rate, dehydrogenate activity and ammonification in the three soils in no clearly defined order. Measurement of dehydrogenase activity was the best tool for assessing the harmful effect of Cr 6+ on soil microbial activity in semi-arid soils exposed for an extended period.

Investigation of the Origin of Extracellular RNA in Human Cell Culture

We have used the human ECV 304 cell line to study the origin and fate of extracellular RNA (exRNA) in cell culture. Quantification of different extracellular RNA species using reverse transcription followed by quantitative PCR revealed a prevalent fraction of ribosomal RNAs. Comparison of intracellular and extracellular ribosomal RNA copy numbers allowed the calculation of the number of destroyed cells that would result in the corresponding number of extracellular rRNAs. Interestingly, this number was comparable to the amount of destroyed cells as determined by the measurement of extracellular lactate dehydrogenase activity.

Optimum Conditions for Measuring Dehydrogenase Activity of Aspergillus niger using TTC

The suitability of the triphenyl tetrazolium chloride (TTC) for dehydrogenase activity measurement of the vegetative cells of the fungal species Aspergillus niger was investigated. The triphenyl formazan (TF) yield increased with the increases in TTC concentration, pH, temperature and incubation time within the studied ranges. The effects of individual parameters, as well as the combined effects, on the TF yield were found to follow exponential expressions. The sensitivity analysis showed that the TF yield is more sensitive to changes in temperature followed by pH, TTC concentration and incubation time. Although the rate of increase in the enzyme activity decreased gradually with the increase in temperature, no enzyme denaturation was observer below 55 oC. The optimum TTC-test conditions for measuring the dehydrogenase activity of A. niger vegetative cells are a TTC concentration of 15 g/L, a pH of 9, a temperature of 55 oC and an incubation time of 4 h. Although the procedure has been successfully tested with mycelia of different stages of growth, it would be interesting to test the effects of different stages of growth on TTC reduction under both aerobic and anaerobic conditions.

Resveratrol Reduces Renal and Lung Injury Caused by Sepsis in Rats

Resveratrol (3,5,4'-trans-trihydroxystilbene), a natural phytoalexin, has various pharmacological effects, including anti-inflammatory properties via inhibition of oxidation, leukocyte priming, and expression of inflammatory mediators. The present study was aimed to investigate the possible beneficial activities of resveratrol on lung and kidney damage in a rat model of sepsis. Sepsis was induced to Sprague-Dawley rats of both sexes (200-250 g) by cecal ligation and perforation. The rats were treated with resveratrol (30 mg/kg; i.p.) or saline after induction of sepsis and at 16 h. Twenty-four hours after the sepsis-induction, all rats were decapitated. Blood was collected for the measurement of tumor necrosis factor-alpha level and lactate dehydrogenase activity. Lung and kidney samples were taken for histological assessment and for the measurement of malondialdehyde, glutathione level, myeloperoxidase activity, and collagen content. Sepsis caused a significant increase in malondialdehyde levels, myeloperoxidase activity, and collagen content of the lung and kidney tissues with a concomitant reduction in glutathione levels. Microscopic examination revealed severe destruction of regular morphology in both lung and kidney tissues. Serum tumor necrosis factor-alpha and lactate dehydrogenase levels also were higher in rats with sepsis compared to those of the sham group. Resveratrol treatment reversed these biochemical parameters and preserved tissue morphology as evidenced by histological evaluation. Resveratrol, a phenolic compound, reduces sepsis-induced remote organ injury, at least in part, through its ability to balance oxidant-antioxidant status, to inhibit neutrophil infiltration and to regulate the release of inflammatory mediators.

Clinical, biochemical, and molecular findings in three patients with 3-hydroxyisobutyric aciduria

3-Hydroxyisobutyric aciduria is a rare entity and affected individuals display a range of clinical manifestations including dysmorphic features and neurodevelopmental problems in the majority of patients. Here, we present two novel patients with 3-hydroxyisobutyric aciduria. To our knowledge, these are the 11th and 12th cases of 3-hydroxyisobutyic aciduria reported. It is believed that a deficiency in 3-hydroxyisobutyrate dehydrogenase is the most likely cause of this disorder. Measurement of 3-hydroxyisobutyrate dehydrogenase activity in fibroblasts homogenates of the two newly identified patients and a previously reported patient, however, revealed similar activities as in control fibroblasts. Since other enzymes with overlapping substrate specificity could conceal abnormal 3-hydroxyisobutyrate dehydrogenase activity, we cloned a candidate human cDNA for 3-hydroxyisobutyrate dehydrogenase ( HIBADH). By heterologous expression in Escherichia coli, we showed that the product of the HIBADH gene indeed displays 3-hydroxyisobutyrate dehydrogenase activity. Mutation analysis of the corresponding gene in the patients suffering from 3-hydroxyisobutyric aciduria revealed no mutations. We conclude that HIBADH is not the causative gene in 3-hydroxyisobutyric aciduria.

Effects of exposure to sublethal copper concentration on glyoxalase system enzymes in specimens of Scapharca inaequivalvis

The present investigation relates the changes in the activity of glyoxalase enzymes (glyoxalase I, G I, and glyoxalase II, G II) in certain tissues (foot, gills and digestive gland) of the bivalve mollusc Scapharca inaequivalvis after a two-week exposure to a sublethal concentration (1.6 μM, 0.1 ppm) of Cu++ and untreated control specimens in marine water. The trials also included determination of Cu content in the tissues and measurements of lactate dehydrogenase (LDH) activity, to estimate either possible Cu accumulation or alterations in glycolytic activity. Copper accumulation was present in all the studied organs, being 45-fold increased in the gills. G I, G II and LDH activities were unchanged in the digestive gland after Cu exposure. G I activity in foot and gills of Cu-exposed animals decreased, likely due to a drop in the glycolytic rate, as pointed out by the parallel decrease of LDH activity. After Cu exposure, G II activity was unchanged in the gills, maybe to secure enough levels of GSH; however, in the foot G II activity decreased, likely giving an increased intracellular amount of S- d-lactoylglutathione as energy supplier.

Overexpression of Apolipoprotein A-IV Enhances Lipid Secretion in IPEC-1 Cells by Increasing Chylomicron Size

Intestinal apolipoprotein A-IV expression is highly regulated by dietary lipid in newborn swine, suggesting a role in lipid absorption. Constitutive overexpression of apoA-IV in newborn swine enterocytes enhances basolateral secretion of triacylglycerol (TG) in TG-rich lipoproteins 4.9-fold (Lu, S., Yao, Y., Meng, S., Cheng, X., and Black, D. D. (2002) J. Biol. Chem. 277, 31929-31937). To investigate the mechanism of this enhancement, IPEC-1 cells were transfected with a tetracycline-regulatable expression system (Tet-On). In cells incubated with oleic acid, a dose response relationship was observed between medium doxycycline concentration and basolateral apoA-IV and TG secretion. Similarly regulated expression of apoA-I did not enhance lipid secretion. The mean diameter of TG-rich lipoproteins secreted from doxycycline-treated cells was larger than from untreated cells (87.0 nm versus 53.4 nm). Basolateral apoB secretion decreased. Using the same expression system, full-length human apoA-IV (376 amino acids); a "pig-like" human apoA-IV, lacking the C-terminal EQQQ repeats (361 amino acids); and a "chicken-like" apoA-IV, further truncated to 343 amino acids, were expressed in IPEC-1 cells. With increasing protein secretion, cells expressing the full-length human apoA-IV displayed a 2-fold increase in TG secretion; in sharp contrast, cells expressing the pig-like human apoA-IV displayed a 25-fold increase in TG secretion and a 27-fold increase in lipoprotein diameter. When human apoA-IV was further truncated to yield a chicken-like protein, TG secretion was inhibited. We conclude that overexpression of swine apoA-IV enhances basolateral TG secretion in a dose-dependent manner by increasing the size of secreted lipoproteins. These data suggest that the region in the human apoA-IV protein from residues 344 to 354 is critical to its ability to enhance lipid secretion, perhaps by enabling the packaging of additional core TG into chylomicron particles. The EQQQ-rich region may play an inhibitory or modulatory role in chylomicron packaging in humans.

Commentary on the British Thoracic Society guidelines for the investigation of unilateral pleural effusion in adults

The publication of guidelines for the investigation of unilateral pleural effusion in adults by the British Thoracic Society has focused attention on this subject which, although comprising only a small proportion of laboratory workload, is a fairly common clinical problem. We critically reviewed the guidance applicable to clinical biochemistry laboratories and found a number of deficiencies. In particular, the need for anaerobic sample collection for pH measurement and preservation of samples for glucose assay is not mentioned and health and safety issues related to the handling of potentially infected fluids are also not considered. There are discrepancies between recommendations in the text and in the accompanying diagnostic algorithm, which require clarification. Measurement of total protein is an essential first step in the analysis of pleural fluid and will usually distinguish transudates from exudates. Measurement of lactate dehydrogenase activity is only required when total protein results are equivocal. There are practical difficulties with measurement of fluid pH as recommended in the guidelines and there is little evidence that such measurements are valuable. Similarly, there is little evidence to support the recommendation for measurement of complement in suspected rheumatoid effusions, and the recommendation for amylase isoenzyme studies if acute pancreatitis is a possibility is not practical. The different nature of pleural fluid demands a good understanding of the handling of these samples, the limitations of the analytical methods and the subsequent result interpretation by laboratory staff. We propose a modified diagnostic algorithm reflecting our criticisms of the original. Dialogue between the laboratory and local clinicians, possibly with the production of local guidelines, informed by these recommendations, should help optimize diagnostic management of patients with pleural effusion.

Suppression of Hyperglycemia-Induced Superoxide Formation and Endothelin-1 Gene Expression by Carvedilol

Carvedilol (CV) is a beta-blocker with favorable effects on cardiovascular disease. To determine whether CV can prevent increases in superoxide (SO) production due to hyperglycemia, human umbilical vein endothelial cells (HUVEC) were treated with either 100 or 500 mg/dL dextrose in the presence or absence of 0.1, 1.0, and 10 micromol/L CV. Superoxide levels were measured using the hydroethidine (HE) fluorescence method. Generation of SO was linear from time 0 to 60 minutes. At 60 minutes, the HE fluorescence in cells treated with 500 mg/dL dextrose (123.3+/-4.9 units) was significantly higher than that in control cells treated with 100 mg/dL dextrose (84.0+/-3.5 units) (P<0.002). Addition of 0.1, 1.0, and 10 micromol/L CV to cells treated with 500 mg/dL dextrose decreased SO generated to 113.3+/-1.8, 98.7+/-8.3, and 70.0+/-1.0 units, respectively (P<0.13, P<0.05, and P<0.004, respectively). Cellular endothelin-1 mRNA and endothelin-1 protein secreted in culture media were significantly increased in the presence of 500 mg/dL dextrose. The addition of 10 micromol/L CV significantly decreased both endothelin-1 (1-21) mRNA and protein levels. Measurements of media lactate dehydrogenase activity indicated that CV inhibited cytotoxicity caused by 500 mg/dL dextrose. These findings suggest that CV not only prevents dextrose-induced SO generation in endothelial cells but may also have favorable effects on gene expression and cell survival.

Mitochondrial Malate Dehydrogenase 활성을 이용한 냉장우육과 냉동우육의 판별법 개발

본 연구는 냉장우육과 냉동우육의 판별법을 개발하기 위하여, 한우 육을 부위별(사태, 등심, 우둔, 양지)로 구입하고 냉장(4±1℃), 냉동(-4, -18, -77℃)상태로 15일 저장하면서 압착육즙채취기를 이용하여 근육 세포내 mitochondria 막에 존재하는 mitochondrial malate dehydrogenase 활성 특성을 비교 연구한 결과 다음과 같은 결과를 얻었다. 본 실험을 위하여 제작된 압착육즙채취기를 이용하여 정량적인 압착 육즙을 얻을 수 있었고, 1.5 g의 우육을 이용하였을 때 0.15 mL의 압착육즙을 얻었다. 냉장우육과 냉동우육 간의 mitochondral malate dehydrogenase의 각 부위별 활성은 사태의 경우 냉장우육은 23.63 Unit/mL의 활성을 나타내었고, -4℃는 20.91 Unit/mL, -18℃는 26.43 Unit/mL 그리고 -77℃는 25.90 Unit/mL의 활성을 나타내었다. 동일한 부위에서 저장온도에 따른 효소의 활성도는 냉장우육보다 -4℃를 제외한 냉동 저장 온도(-18, -77℃)에서 모두 유의적으로 높은 효소의 활성을 나타내었다(p＜0.05). 동일한 저장 온도에서 각 부위 간에 활성의 차이점은 -77℃에서 냉동 저장한 우육이 유의적 차이 없이 가장 높은 효소 활성을 나타내었고, -77℃ 저장온도를 제외하고는 모두 유의성 있는 변화량을 나타내었다(p＜0.05). 실험군의 압착육즙 내 mitochondrial malate dehydrogenase의 활성은 10분간 흡광도 변화량을 조사함으로써 계산되었다. 등심을 제외한 모든 부위에서 효소반응개시 3분 후에 -4℃에서 동결우육을 제외하고 동결우육의 효소 활성이 떨어지는 것을 확인할 수 있었고, 5분 반응 후에는 그 효소의 활성을 발견할 수 없었다. 냉장우육은 12분까지 활성을 측정할 수 있었다. 15일간 냉장우육 및 냉동우육을 저장 온도에 따라 저장하면서 그 활성을 측정하였는데, 저장 기간이 길어져도 그 효소 활성의 차이는 유의적으로 변화하지 않고, 모든 실험군(사태, 등심, 양지, 우둔 부위)에서 유의적으로 유사한 활성을 유지하였다(p<0.05). 따라서 본 연구결과로부터 mitochondrial malate dehydrogenase의 활성을 이용하여 냉장우육과 냉동우육 유무를 판별하는데 유효한 지표로 사용 가능한 것으로 사료되었다.

Hydralazine is a powerful inhibitor of peroxynitrite formation as a possible explanation for its beneficial effects on prognosis in patients with congestive heart failure

The hemodynamic and anti-ischemic effects of nitroglycerin (GTN) are rapidly blunted as a result of the development of nitrate tolerance. Hydralazine has been shown to prevent tolerance in experimental and clinical studies, all of which may be at least in part secondary to antioxidant properties of this compound. The antioxidant effects of hydralazine were tested in cell free systems, cultured smooth muscle cells, isolated mitochondria, and isolated vessels. Inhibitory effects on the formation of superoxide and/or peroxynitrite formation were tested using lucigenin and L-012 enhanced chemiluminescence as well as DHE-fluorescence. The peroxynitrite scavenging properties were also assessed by inhibition of nitration of phenol. Prevention of impairment of NO downstream signaling and GTN bioactivation was determined by measurement of P-VASP (surrogate parameter for the activity of the cGMP-dependent kinase-I, cGK-I) and mitochondrial aldehyde dehydrogenase (ALDH-2) activity. Hydralazine dose-dependently decreased the chemiluminescence signal induced by peroxynitrite from SIN-1 and by superoxide from HX/XO in a cell free system, by superoxide in smooth muscle cells and mitochondria acutely challenged with GTN. Moreover, hydralazine inhibited the peroxynitrite-mediated nitration of phenols as well as proteins in smooth muscle cells in a dose-dependent fashion. Finally, hydralazine normalized impaired cGK-I activity as well as impaired vascular ALDH-2 activity. Our results indicate that hydralazine is a highly potent radical scavenger. Thus, the combination with isosorbide dinitrate (ISDN) will favorably influence the nitroso-redox balance in the cardiovascular system in patients with congestive heart failure and may explain at least in part the improvement of prognosis in patients with chronic congestive heart failure.

Development of a microtitre plate method for determination of phenol utilization, biofilm formation and respiratory activity by environmental bacterial isolates

Abstract Twenty-five aerobic phenol-degrading bacteria, isolated from different environmental samples on phenol agar after several subcultures in phenol broth, utilized phenol (0.2 g l −1 ) within 24 h, but removal of phenol was more rapid when other carbon sources were also present. A microtitre plate method was developed to determine growth rate, biofilm formation and respiratory activity of the strains isolated. Pseudomonas putida strain s C5 and D6 showed maximum growth (as O.D. at 600 nm), P. putida D6 and unidentified bacterial strain M1 were more stable at high concentrations of phenol (0.8 g l −1 ), and P. putida C5 formed the greatest amount of biofilm in 0.5 g phenol l −1 medium. Measurement of dehydrogenase activity as reduction of triphenyl tetrazolium chloride supported data on growth rate and biofilm formation. The microtitre plate method provided a selective method for detection of the best phenol degrading and biofilm-forming microorganisms, and was also a rapid, convenient means of studying the effect of phenol concentration on growth rate and biofilm formation.

Lysis of starters in UF cheeses: Behaviour of mesophilic lactococci and thermophilic lactobacilli

Abstract The objective of this work was to study the autolytic behaviour of strains of mesophilic (Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris) and thermophilic lactic acid bacteria (Lactobacillus helveticus, Lb. delbrueckii subsp. lactis and Streptococcus thermophilus) in UF cheese. Cheeses were made from a UF-retentate (milk concentrated by a factor of 6) of microfiltered milk (0.8 μm pore size membrane) using the following starter systems: (1) single inocula of autolytic strains of L. lactis (US3, AM2 or AM1), non-autolytic strains of L. lactis (AM2-C or CNRZ-144), (2) a co-inocula of strains of Lb. helveticus (ITG-LH1, CNRZ-32 or CNRZ-303), Lb. delbrueckii subsp. lactis (ITG-LL14 or ITG-LL51) with the same strain of S. thermophilus CNRZ-1358. Cell viability was monitored over a 28 day ripening period by enumeration on selective media. Degree of lysis was determined by the measurement of the intracellular marker lactate dehydrogenase (LDH) activity, and also by immunodetection of intracellular proteins with species specific antibodies. In UF cheeses, lysis of autolytic strains of L. lactis was significantly delayed, showing release of intracellular components after 21 days of ripening. No lysis was observed for non-autolytic L. lactis strains or for S. thermophilus. Lysis of thermophilic lactobacilli (Lb. helveticus, Lb. delbrueckii), was observed from the start of ripening, but the onset and the level of lysis observed was strain and species dependent.

Mitochondrial Mediated Thimerosal-Induced Apoptosis in a Human Neuroblastoma Cell Line (SK-N-SH)

Environmental exposure to mercurials continues to be a public health issue due to their deleterious effects on immune, renal and neurological function. Recently the safety of thimerosal, an ethyl mercury-containing preservative used in vaccines, has been questioned due to exposure of infants during immunization. Mercurials have been reported to cause apoptosis in cultured neurons; however, the signaling pathways resulting in cell death have not been well characterized. Therefore, the objective of this study was to identify the mode of cell death in an in vitro model of thimerosal-induced neurotoxicity, and more specifically, to elucidate signaling pathways which might serve as pharmacological targets. Within 2 h of thimerosal exposure (5 μM) to the human neuroblastoma cell line, SK-N-SH, morphological changes, including membrane alterations and cell shrinkage, were observed. Cell viability, assessed by measurement of lactate dehydrogenase (LDH) activity in the medium, as well as the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, showed a time- and concentration-dependent decrease in cell survival upon thimerosal exposure. In cells treated for 24 h with thimerosal, fluorescence microscopy indicated cells undergoing both apoptosis and oncosis/necrosis. To identify the apoptotic pathway associated with thimerosal-mediated cell death, we first evaluated the mitochondrial cascade, as both inorganic and organic mercurials have been reported to accumulate in the organelle. Cytochrome c was shown to leak from the mitochondria, followed by caspase 9 cleavage within 8 h of treatment. In addition, poly(ADP-ribose) polymerase (PARP) was cleaved to form a 85 kDa fragment following maximal caspase 3 activation at 24 h. Taken together these findings suggest deleterious effects on the cytoarchitecture by thimerosal and initiation of mitochondrial-mediated apoptosis.

A phytol-enriched diet induces changes in fatty acid metabolism in mice both via PPARα-dependent and -independent pathways

Branched-chain fatty acids (such as phytanic and pristanic acid) are ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha) in vitro. To investigate the effects of these physiological compounds in vivo, wild-type and PPARalpha-deficient (PPARalpha-/-) mice were fed a phytol-enriched diet. This resulted in increased plasma and liver levels of the phytol metabolites phytanic and pristanic acid. In wild-type mice, plasma fatty acid levels decreased after phytol feeding, whereas in PPARalpha-/- mice, the already elevated fatty acid levels increased. In addition, PPARalpha-/- mice were found to be carnitine deficient in both plasma and liver. Dietary phytol increased liver free carnitine in wild-type animals but not in PPARalpha-/- mice. Investigation of carnitine biosynthesis revealed that PPARalpha is likely involved in the regulation of carnitine homeostasis. Furthermore, phytol feeding resulted in a PPARalpha-dependent induction of various peroxisomal and mitochondrial beta-oxidation enzymes. In addition, a PPARalpha-independent induction of catalase, phytanoyl-CoA hydroxylase, carnitine octanoyltransferase, peroxisomal 3-ketoacyl-CoA thiolase, and straight-chain acyl-CoA oxidase was observed. In conclusion, branched-chain fatty acids are physiologically relevant ligands of PPARalpha in mice. These findings are especially relevant for disorders in which branched-chain fatty acids accumulate, such as Refsum disease and peroxisome biogenesis disorders.

Taurine treatment protects against chronic nicotine‐induced oxidative changes

Experiments have shown that chronic nicotine administration caused oxidative damage in various organs by increasing lipid peroxidation products and decreasing the activity of endogenous antioxidants. The aim of this study was to investigate the effects of taurine treatment on nicotine-induced oxidative changes in rat thoracic aorta and heart and to explore the possible mechanisms of action. Male Wistar albino rats (200-250 g) were injected with nicotine hydrogen bitartrate (0.6 mg/kg; i.p.) or saline for 21 days. Taurine was administered (50 mg/kg; i.p.) alone or along with nicotine injections. After decapitation, the thoracic aorta and heart tissues were excised. The aorta was used for in vitro contractility studies or stored along with the heart samples for the measurement of malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity and collagen content. Tissue samples were also examined histologically. Serum samples were stored for the measurement of MDA, GSH and lactate dehydrogenase (LDH) activity. Chronic nicotine treatment impaired both the contraction and relaxation responses of the aortic rings to phenylephrine and acetylcholine, respectively. It increased lipid peroxidation, MPO levels and tissue collagen content of both aorta and heart samples. Taurine supplementation to nicotine-treated animals reversed the contractile dysfunction and restored the endogenous GSH levels and decreased high lipid peroxidation and MPO activities in both tissues. These data suggest that taurine supplementation effectively attenuates the oxidative damage because of chronic nicotine administration possibly by its antioxidant effects.

Improved Reliability of Measurement of Lactate Dehydrogenase by IFCC Method in Heparin Plasma

Our laboratory recently reported (1) an excessively high frequency of duplicate errors in measurements of lactate dehydrogenase (LD; l-lactate:NAD+ oxidoreductase; EC 1.1.1.27) activity with the Roche IFCC-recommended method (2). This unacceptably high frequency of duplicate errors was found with various Hitachi analyzers (Roche GmbH) with primary lithium-heparin tubes with plasma separator from Becton Dickinson but not with serum or EDTA plasma. Such duplicate errors were not seen with the German- and French-recommended LD methods (3)(4), nor were such duplicate errors seen when nonevacuated heparin tubes from Sarstedt were used (5). In addition to the effects of methods, we investigated the effects of sample preparation, method modifications, mixing procedures, and the use of different lithium-heparin-containing tubes with and without plasma separator, lithium-heparin glass and plastic tubes, different lithium-heparin concentrations, and subsampling into secondary tubes. The results of all of these experiments are shown in Table 1 of the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol51/issue1/. Previously, we showed that the high frequency of duplicate errors was not caused by carryover from reagents for other tests and was not restricted to one specific type of Hitachi analyzer (1). In the present study, we demonstrate that the high frequency of duplicate errors is not restricted to the use of Roche reagents, confirming that the duplicate errors are caused by the combination of evacuated lithium-heparin tubes from Becton Dickinson …

PGE1 abolishes the mitochondrial‐independent cell death pathway induced by D‐galactosamine in primary culture of rat hepatocytes

PGE1 reduces in vivo and in vitro D-galactosamine (D-GalN)-induced cell death in hepatocytes. The present study was undertaken to elucidate the intracellular pathway by which D-GalN induces cell death in cultured hepatocytes. In addition, we evaluated if PGE1 was able to modulate different parameters related to D-GalN-induced apoptosis in cultured rat hepatocytes. Hepatocytes were isolated from male Wistar rats (225-275 g) by the classical collagenase procedure. PGE1 (1 microM) was administered 2 h before D-GalN (5 mM) in primary culture of rat hepatocytes. Apoptosis was determined by DNA fragmentation and caspase-3, -6, -8 and -9 activation in hepatocytes. Caspase activation was evaluated by the detection of the related cleaved product and its associated activity. Cell necrosis was determined by the measurement of lactate dehydrogenase (LDH) activity in culture medium. To elucidate the role of mitochondria, we measured neutral (nSMase) and acid (aSMase) sphingomyelinase, as well as the expression of cytochrome c in mitochondria and cytoplasm fractions from D-GalN treated hepatocytes. D-GalN induced caspase-3 activation and DNA fragmentation in hepatocytes. This apoptotic response was not associated with the activation of caspase-6, -8 or -9. The use of specific inhibitors confirmed that only caspase-3 was involved in D-GalN-induced apoptosis. D-GalN did not modify nSMase and aSMase activities, nor mitochondrial cytochrome c release in hepatocytes. D-GalN induced apoptosis through caspase-3 activation but without modification of the activity of caspase-6, -8, -9, SMases or cytochrome c release. PGE1 appears to prevent D-GalN-induced apoptosis by a mitochondria-independent mechanism.

In vitro and in vivo hemocompatibility evaluation of graphite coated polyester vascular grafts.

Attempts have been made in this study to prepare a homogeneous and stable coating of graphite on polyester vascular grafts (GPVG) using an electrophoresis method to evaluate thromboresistant and blood compatibility of GPVG in comparison to non-coated PVG and InterGard (collagen sealed PVG) as control. Lactate dehydrogenase (LDH) activity measurement was carried out on all PVG types to evaluate platelet adhesion. To examine tissue reaction GPVG and non-coated sheets of knitted polyester fabric were implanted simultaneously in the dorsal flank of rats subcutaneously. The GPVG, non-coated and control were implanted in descending aorta as end-to-end or end-to-side implantation substitution in 25 sheep for 4-60 weeks. Results showed that the graphite coating on polyester vascular grafts reduced the number of adherent platelets and prevent platelet activation and spreading on the surface in comparison with non-coated and control. Pathological investigation showed inflammatory reactions were totally resolved after 12 weeks and there was no difference in the tissue reaction between graphite coated, non-coated and control patches. All grafts remained patent and there was no significant difference in patency rate between these three types of PVG. We found that GPVG has no need for pre-clotting and it showed lower platelet aggregation, thinner capsule formation and lower calcification after 15 months. However, suturing of GPVG was more difficult in comparison with the other types.

In vitro and in vivo hemocompatibility evaluation of graphite coated polyester vascular grafts

Attempts have been made in this study to prepare a homogeneous and stable coating of graphite on polyester vascular grafts (GPVG) using an electrophoresis method to evaluate thromboresistant and blood compatibility of GPVG in comparison to non-coated PVG and InterGard (collagen sealed PVG) as control. Lactate dehydrogenase (LDH) activity measurement was carried out on all PVG types to evaluate platelet adhesion. To examine tissue reaction GPVG and non-coated sheets of knitted polyester fabric were implanted simultaneously in the dorsal flank of rats subcutaneously. The GPVG, non-coated and control were implanted in descending aorta as end-to-end or end-to-side implantation substitution in 25 sheep for 4–60 weeks. Results showed that the graphite coating on polyester vascular grafts reduced the number of adherent platelets and prevent platelet activation and spreading on the surface in comparison with non-coated and control. Pathological investigation showed inflammatory reactions were totally resolved after 12 weeks and there was no difference in the tissue reaction between graphite coated, non-coated and control patches. All grafts remained patent and there was no significant difference in patency rate between these three types of PVG. We found that GPVG has no need for pre-clotting and it showed lower platelet aggregation, thinner capsule formation and lower calcification after 15 months. However, suturing of GPVG was more difficult in comparison with the other types.