Novel system for real-time ex vivo lactate monitoring in human whole blood

The objective of the study was to evaluate the performance of an amperometric enzyme based lactate sensor and to investigate the possibility of replacing a double lumen catheter based blood withdrawal system with a heparin coated single lumen system. The inner lumen of a double lumen catheter which was placed in a peripheral vein was perfused with heparin solution. The outer lumen was used to collect heparinized blood samples at a defined flow rate. The single lumen system was attached to a heparinized catheter which was also placed in a peripheral vein. The undiluted blood samples were collected at a specified flow rate. A sensor flow chamber incorporating an amperometric thin-film lactate microbiosensor was placed in the sampling line for real-time lactate monitoring. Plasma lactate concentrations were measured during frequently performed hyperlactatemia bicycle ergometer experiments in six healthy volunteers (age 25.8±2.8 years, BMI 22.7±1 kg/m²). Additionally, plasma lactate was measured in real-time using the lactate sensors. The first three experiments were performed with a double lumen based catheter system whereas the following three experiments were performed with a heparin coated catheter system. The correlation coefficients of sensor readings and laboratory analyzer results in all six experiments were between 0.93 and 0.99, respectively (P<0.001). The miniaturized lactate sensors showed a linear range up to 25 mmol/l lactate concentration and 95% response times <30 s in undiluted serum. During the experiments maximum lactate concentrations of 14 mmol/l were achieved. Improvements of system performance using heparin coated catheter systems could be shown. The overall SD of the sensor readings compared to laboratory results using three double lumen catheter based systems was 0.91 mmol/l whereas the SD using three heparin coated systems was 0.65 mmol/l. In summary, real-time monitoring of lactate in human whole blood is feasible with such a device and can be improved by using heparin coated catheter systems.     

Purification and properties of a low molecular weight DNA polymerase from Neurospora crassa

A third DNA polymerase 'C' with low molecular weight was isolated and purified 3700-fold from ground hyphae of Neurospora crassa WT 74 A, which shows similarities to beta- and gamma-polymerases from higher eukaryotes: preference for poly(rA)(dT) as a template/primer, inhibition by p-chloromercuribenzoate, resistance against N-ethylmaleimide up to 10 mmol/l, and molecular weight of about 40000. This polymerase elutes as a distinct peak from DEAE-cellulose at 0.60 mol/l KCl and has an optimum for K+ at 2-20 mmol/l, for Mn2+ at 0.8 mmol/l, for Mg2+ at 4.0 mmol/l, the pH optimum is 8.0. Its Km is 1.5 mumol/l using dTTP as substrate. The enzyme activity described here is free of endonuclease but contains detectable amounts of exonuclease.     

Isolation and characterization of salinity resistant mutant of a nitrogen-fixing cyanobacterium, Anabaena doliolum

D.V. SINGH. A.K. TRIPATHI AND H.D. KUMAR. 1991. Sodium chloride, up to 20 mmol/l concentration, had a positive effect on acetylene reducing activity and photosynthetic oxygen evolution of a paddy field cyanobacterium, Anabaena doliolum. Beyond 20 mmol/l level of salinity adverse effects appeared. A mutant resistant to 200 mmol/l NaCl was isolated by nitrosoguanidine mutagenesis. The mutant, NaCl-R₂₀₀, showed about 20–25% more nitrogenase activity and photosynthetic oxygen evolution than the parent. Better capacity of nitrogen fixation and photosynthesis possibly could help the mutant in synthesis of osmotic stabilizer to resist the salinity stress.     

Effect of free and conjugated bile salts on alpha-amylase activity.

The effect of individual bile salts on alpha-amylase hydrolysis of Cibachron Blue starch was studied at pH 6.0. With sodium cholate, taurocholate and taurodeoxycholate, enzyme activity was increased to 150-160 percent of the control value, at a concentration of similar to 1 mmol/l bile salt. The increased activity extended up to 4 mmol/l. The bile salts sodium deoxycholate and taurochenodeoxycholate exerted activation and inhibition depending on the concentration. With deoxycholate (0.75 mmol/l), activation (150 percent) was evident, while inhibition was apparent above 2.5 mmol/l. With taurochenodeoxycholate maximum activity (135 percent) was observed at 0.25 mmol/l, while inhibition was evident above 1.5 mmol/l. Chenodeoxycholate and lithocholate exerted marked inhibition at concentrations as low as 0.5 mmol/l. Inhibition of alpha-amylase by chenodeoxycholate was competitive with both soluble and insoluble starch substrates. Since the pH of the jejunum is in the region of 6.0 the phenomenon of activation and inhibition of alpha-amylase by bile salts at this pH could be of physiological significance.     

A portable, pre-programmable insulin infusion system in diabetes mellitus.

A miniaturized portable pump has been developed in order to study the effects of pre-programmed insulin infusion patterns on the blood glucose levels in juvenile-onset, insulin-requiring diabetics. Six diabetics undergoing 7 insulin infusion periods of up to 4 days were studied. The mean blood glucose was 7.0 +/- 2.3 (SD) mmol/l and the mean amplitude of glycemic excursions was 5.0 +/- 1.4 mmol/l. Success in achieving normoglycemia will depend on choosing the correct dose of insulin for infusion.     

Oxalate modulates thiobarbituric acid reactive species (TBARS) production in supernatants of homogenates from rat brain, liver and kidney: effect of diphenyl diselenide and diphenyl ditelluride

The aim of this paper was to investigate the mechanism(s) involved in the sodium oxalate pro-oxidative activity in vitro and the potential protection by diphenyl diselenide ((PhSe)(2)) and diphenyl ditelluride ((PhTe)(2)) using supernatants of homogenates from brain, liver and kidney. Oxalate causes a significant increase in the TBARS (thiobarbituric acid reactive species) production up to 4mmol/l and it had antioxidant activity from 8 to 16mmol/l in the brain and liver. Oxalate had no effect in kidney homogenates. The difference among tissues may be related to the formation of insoluble crystal of oxalate in kidney, but not in liver and brain homogenates. (PhSe)(2) and (PhTe)(2) reduced both basal and oxalate-induced TBARS in rat brain homogenates, whereas in liver homogenates they were antioxidant only on oxalate-induced TBARS production. (PhSe)(2) showed a modest effect on renal TBARS production, whereas (PhTe)(2) did not modulate TBARS in kidney preparations. Oxalate at 2mmol/l did not change deoxyribose degradation induced by Fe(2+) plus H(2)O(2), whereas at 20mmol/l it significantly prevents its degradation. Oxalate (up to 4mmol/l) did not alter iron (10micromol/l)-induced TBARS production in the brain preparations, whereas at 8mmol/l onwards it prevents iron effect. In liver preparations, oxalate amplifies iron pro-oxidant activity up to 4mmol/l, preventing iron-induced TBARS production at 16mmol/l onwards. These results support the antioxidant effect of organochalcogens against oxalate-induced TBARS production. In addition, our results suggest that oxalate pro- and antioxidant activity in vitro could be related to its interactions with iron ions.     

Influence of Culture Parameters on Biological Hydrogen Production by Clostridium saccharoperbutylacetonicum ATCC 27021

Summary Various medium components (carbon and nitrogen sources, iron, inoculum size) and environmental factors (initial pH and the agitation speed) were evaluated for their effects on the rate and the yield of hydrogen production by Clostridium saccharoperbutylacetonicum. Among the carbon sources assessed, cells grown on disaccharides (lactose, sucrose and maltose) produced on the average more than twice (2.81 mol-H2/mol sugar) as much hydrogen as monosaccharides (1.29 mol-H2/mol sugar), but there was no correlation between the carbon source and the production rate. The highest yield (2.83 mol/mol) was obtained in lactose and sucrose but the highest production rate (1.75 mmol/h) in sucrose. Using glucose as carbon source, yeast extract was the best nitrogen source. A parallel increase between the production rate and the yield was obtained by increasing glucose concentration up to 40 g/l (1.76 mol-H2/mol, 3.39 mmol/h), total nitrogen as yeast extract up to 0.1% (1.41 mol/mol, 1.91 mmol/h) and agitation up to 100 rev/min (1.66 mol-H2/mol, 1.86 mmol/h). On the other hand, higher production rates were favoured in preference to the yield at a neutral initial pH 7 (2.27 mmol/h), 1000 mg iron/l or more (1.99 mmol/h), and a larger inoculum size, 10%, (2.36 mmol/h) whereas an initial alkaline pH of 8.5 (1.72 mol/mol), a lower iron concentration of 25 mg/l (1.74 mol/mol) and smaller inoculum size, 1%, (1.85 mol/mol) promoted higher yield over production rate.     

Purification and properties of a low molecular weight DNA polymerase from Neurospora crassa

A third DNA polymerase ‘C’ with low molecular weight was isolated and purified 3700-fold from ground hyphae of Neurospora crassa WT 74 A, which shows similarities to β- and γ-polymerases from higher eukaryotes: preference for poly(rA)(dT) as a template/primer, inhibition by p-chloromercuribenzoate, resistance against N-ethylmaleimide up to 10 mmol/l, and molecular weight of about 40 000. This polymerase elutes as a distinct peak from DEAE-cellulose at 0.60 mol/l KCl and has an optimum for K⁺ at 2–20 mmol/l, for Mn²⁺ at 0.8 mmol/l, for Mg²⁺ at 4.0 mmol/l, the pH optimum is 8.0. Its K_m is 1.5 μmol/l using dTTP as substrate. The enzyme activity described here is free of endonuclease but contains detectable amounts of exonuclease.     

Effects of different saccharose and electrolyte concentrations on preserved erythrocytes

Preservation solutions for buffy coat-free red cell concentrates with sucrose concentrations from 234 decreasing up to 15 mmol per 1 solution were tested. The hemolysis rate increased from 0.5 up to 1.9% by decreasing the sucrose concentration. The red cell volume was unchanged at low sucrose concentrations. No differences were noticed in ATP content and morphological changes. A considerable extracellular pH shift at high sucrose concentration exists only at the beginning of storage. A sucrose concentration of 30-50 mmol/l solution (3-5 mmol per unit red cell concentrate) at an ionic strength of 0.16 proves to be most suitable.     

Hexose-specific inhibition in vitro of human red cell Ca(2+)-ATPase activity.

In a concentration-dependent manner (5.5-27.5 mmol/l), D-glucose incubated in vitro with human erythrocyte membranes at 37 degrees C for 1 h inhibited membrane Ca(2+)-ATPase activity by up to 75%. The IC50 was 11 mmol/l. L-Glucose was ineffective, as were 3-O-methylglucose, 2-deoxyglucose, sorbitol and myo-inositol. In contrast, D-fructose decreased Ca(2+)-ATPase activity nearly as effectively as D-glucose and mannose and galactose at 11 mmol/l were less than 50% as effective as D-glucose. Tunicamycin (12 pmol/l), but not 10 mmol/l aminoguanidine, progressively antagonized in vitro the D-glucose effect on the enzyme. Erythrocyte membrane Ca(2+)-ATPase activity may be regulated by glycosylation, rather than nonenzymatic glycation.     

Determination of iron, copper, zinc, magnesium and selenium in plasma and erythrocytes in neurosurgical patients.

A direct method for determination of Fe, Cu, Zn, Mg and Se in erythrocytes was developed. The aim of the present study was to establish a method for examining perioperative levels of the above mentioned elements simultaneously in erythrocytes and plasma by atomic absorption spectrophotometry in 11 patients undergoing neurosurgery for acute spinal nerve compressions because of intervertebral disk prolapses. Reference values for erythrocytes were 11.49 +/- 3.48 mmol/mmol Hb; 0.82 +/- 0.087 mmol/mmol Hb; 9.01 +/- 2.20 mmol/mmol Hb; 0.104 +/- 0.032 mmol/mmol Hb; 0.07 +/- 0.050 mmol/mmol Hb for iron, copper, zinc, magnesium, and selenium, respectively. Postoperative erythrocyte concentrations did not differ significantly compared to those obtained preoperatively and remained within the reference ranges perioperatively. For plasma the following reference values were used: 19.0 +/- 8.0 mmol/l (Fe); 20.1 +/- 8.2 mmol/l (Cu); 15.4 +/- 4.6 mmol/l (Zn); 0.9 +/- 0.15 mmol/l (Mg); 1.02 +/- 0.3 mmol/l (Se). There was a significant decrease in the concentration of copper in plasma (13.41 +/- 3.46 mmol/l, p < 0.1) and zinc (10.73 +/- 2.73 mmol/l, p < 0.1) immediately postoperative, iron (10.56 +/- 3.91 mmol/l, p < 0.1) and zinc on day 1 (11.28 +/- 1.88 mmol/l, p < 0.10), and a significant postoperative increase of copper on day 5 (18.81 +/- 3.97 mmol/l, p < 0.1), postoperatively. The mean plasma concentrations of iron, copper, zinc magnesium and selenium remained within the reference ranges during the entire period.     

Preservation of resuspended red cell concentrate. Red cell volume and hemolysis

Sucrose in a concentration of 30 to 50 mmol/l preservation solution (10-20 mmol/l red cell concentrate (RCC) and 3-5 mmol per unit RCC) and an ionic strength of about 0.16 avoid changes of red cell volume during 6 weeks of storage. Increasing sucrose concentrations up to 80 mmol/l RCC decrease the hemolysis. But a sucrose concentration of only 10 mmol/l RCC causes an acceptable low hemolysis rate of 0.25% after 35 days of storage in PCV FENWAL plastic bags. Sucrose can be replaced by mannitol or sorbitol at the same final concentration. Changes in red cell metabolism and viability will not be expected.     

Glucose as a lipolytic agent: studies on isolated rat adipocytes

In order to elucidate the direct effect of glucose on lipolysis in isolated rat adipocytes, cells were incubated in a buffer with different concentrations of this sugar: 2, 8 or 16 mmol/l. The increase in glucose concentration from 2 mmol/l to 8 or 16 mmol/l enhanced basal lipolysis by 30% and 47%, respectively. Epinephrine-induced lipolysis (1 micromol/l) was also increased by 31% and 32%, when glucose concentration was increased from 2 mmol/l to 8 or 16 mmol/l, respectively. The rise in lipolysis caused by glucose was restricted by H-89 (an inhibitor of protein kinase A, 30 micromol/l), but insulin (1 nmol/l) had no inhibitory action. The augmentation of lipolysis by glucose did not require its metabolism (as demonstrated using 2-deoxyglucose) and was due to the action of this sugar on the final steps of the lipolytic cascade, particularly on protein kinase A. However, short-term exposure of adipocytes to higher glucose concentrations did not restrict the inhibitory action of insulin on lipolysis induced by epinephrine.     

Does plasma cholesterol concentration predict mortality from coronary heart disease in elderly people? 18 year follow up in Whitehall study.

OBJECTIVE--To explore the extent to which the relation between plasma cholesterol concentration and risk of death from coronary heart disease in men persists into old age. DESIGN--18 year follow up of male Whitehall civil servants. Plasma cholesterol concentrations and other risk factors were determined at first examination in 1967-9 when they were aged 40-69. Death of men up to 31 January 1987 was recorded. SUBJECTS--18,296 male civil servants, 4155 of whom died during follow up. MAIN OUTCOME MEASURES--Cause and age of death. Cholesterol concentration in 1967-9 and number of years elapsed between testing and death. RESULTS--1676 men died of coronary heart disease. The mean cholesterol concentration in these men was 0.32 mmol/l higher than that in all other men (95% confidence interval 0.26 to 0.37 mmol/l). This difference in cholesterol concentrations fell 0.15 mmol/l with every 10 years'' increase in age at screening. The risk of raised cholesterol concentration fell with age at death. Compared with other men cholesterol concentration in those who died of coronary heart disease was 0.44 mmol/l higher in those who died aged less than 60 and 0.26 mmol/l higher in those aged 60-79 (p = 0.03). For a given age at death the longer the gap between cholesterol measurement and death the more predictive the cholesterol concentration, both for coronary heart disease and all cause mortality (trend test p = 0.06 and 0.03 respectively). CONCLUSION--Reducing plasma cholesterol concentrations in middle age may influence the risk of death from coronary heart disease in old age.     

Effectiveness and hazards of case finding for a high cholesterol concentration.

OBJECTIVE--To see whether adults with high blood cholesterol concentrations (greater than 6.5 mmol/l) detected by case finding return to their doctor, receive appropriate advice, and report changes in their diet and whether adults found not to have high cholesterol concentrations on case finding are discouraged from changing their diet. DESIGN--Follow up study. SETTING--Seven general practices in the lower Hunter region of Australia. PARTICIPANTS--600 Men and women aged 25-65 who had their blood cholesterol concentrations measured three to four months earlier while attending their general practitioner. Analysis conducted on 552 (92%) who returned follow up postal questionnaires. MEASUREMENTS AND MAIN RESULTS--Of the 125 subjects found to have blood cholesterol concentrations greater than 6.5 mmol/l, 102 (82%) returned to their doctor and 83 of these recalled receiving dietary advice. 93 (75%) Subjects with high concentrations reported changing their diet compared with 72 (46%) subjects with concentrations of greater than 5.5-6.5 mmol/l and 44 (17%) subjects with lower concentrations. Overall 333 (61%) of all respondents who answered reported not changing their diet, the most common reason (nearly half the sample; n = 266) being that their cholesterol concentration was "all right." Adults without high cholesterol concentrations who returned to their doctor were also significantly more likely to recall their doctor saying that they did not need to lower their concentrations (less than or equal to 5.5 mmol/l = 61%; greater than 5.5-6.5 mmol/l = 12%; greater than 6.5 mmol/l = 5%) and significantly less likely to recall receiving dietary advice (less than or equal to 5.5 mmol/l = 13%; greater than 5.5-6.5 mmol/l = 60%; greater than 6.5 mmol/l = 82%). CONCLUSIONS--High risk strategies which require everyone to be tested for high cholesterol concentrations may interfere with population strategies designed to reduce everyone''s dietary intake of fat.     

Impact of human growth hormone on plasma lipoprotein concentrations

To evaluate whether the moderately elevated human growth hormone concentration, seen in insulin dependent diabetic patients, has any impact on lipoproteins, human growth hormone was given to nondiabetic persons in doses which would bring their plasma human growth hormone concentration up in the same level as seen in insulin dependent diabetic patients. After one week of treatment with human growth hormone we found total plasma triglyceride to be significantly raised (0.98 mmol/l +/- 0.28 mmol/l (mean +/- SD) before versus 1.27 mmol/l +/- 0.38 mmol/l (mean +/- SD) after treatment). Very low density lipoprotein (VLDL) was separated into two fractions (VLDL-1 and VLDL-2) of which VLDL-2 is regarded as a VLDL-remnant which is suggested to be of importance for development of atherosclerosis. After one week of human growth hormone treatment there were no changes in VLDL-1 concentrations whereas a significant raise in VLDL-2 triglyceride and VLDL-2 cholesterol was seen.     

Effects of 3-isobutyl-1-methylxanthine on secretory response, cAMP accumulation and DNA synthesis of islets from postnatal and adult Wistar rats

A possible role for cyclic adenosine-3'-5'-monophosphate (cAMP) in islet cell replication was examined in collagenase-isolated pancreatic islets from Wistar rats of different age and different metabolic state (non-pregnant, pregnant, days 15.5-17.5). Islets obtained from pregnant rats released significantly more insulin in response to 10 mmol/l glucose (culture for 24 h) and their DNA synthesis (incorporation of [3H]thymidine into islet DNA) was doubled compared to islets from non-pregnant controls. Islets obtained from 4-6 days old rats showed a maximal stimulation of DNA synthesis after exposure to 0.1 mmol/l IBMX (3-isobutyl-1-methylxanthine) whereas the cAMP accumulation and the insulin biosynthesis measured in a subsequent short-term incubation were dose-dependent stimulated up to 1.0 mmol/l IBMX. In islets of 12 days old rats or 3 months old rats, however, IBMX did not stimulate DNA synthesis or insulin release measured during culture, although the cAMP content per islet was significantly enhanced after culture in the presence of IBMX.     

A complicated pregnancy in a patient with lipodystrophic diabetes attributable to a peroxisome proliferator-activated receptor gamma (PPARG) mutation

Diabet. Med. 29, e398-e401 (2012) ABSTRACT: Background We describe an unplanned pregnancy in a 19-year-old with lipodystrophic diabetes caused by a mutation in the peroxisome proliferator-activated receptor gamma (PPARG) gene. The pregnancy was complicated by poor compliance with treatment, severe hypertriglyceridaemia and pancreatitis. Case report The patient presented at 6?weeks' gestation with an HbA(1c) of 140?mmol/mol (15%), cholesterol 8.1?mmol/l and triglycerides 20.1?mmol/l. She wished to continue the pregnancy so lipid-lowering therapy was discontinued. She was severely insulin resistant and poorly compliant with diet and medication. A continuous subcutaneous insulin infusion was required for efficient delivery of large doses of basal insulin, alongside injected mealtime boluses, (up to 300?units insulin per day). At 17?weeks' gestation she developed acute pancreatitis secondary to hypertriglyceridaemia (triglycerides >?100?mmol/l) and required plasmapheresis. Lipid-lowering therapy was reinstated in the third trimester and plasmapheresis was required repeatedly to maintain triglycerides 相似文献   

Effect of glucose on human fetal pancreatic tissue in vitro

For investigating insulin secretion in vitro human fetal pancreatic slices prepared from women with a normal carbohydrate tolerance at a mean gestational age of 12 +/- 1 weeks were incubated in the presence of various secretagogues. Glucose concentration up to 20 mmol/l failed to enhance insulin secretion, whereas an increase of glucose up to 40 mmol/l, the addition of the phosphodiesterase inhibitor IBMX or a priming period of 30 min in the presence of 20 mmol/l glucose resulted in an enhancement of hormone release, calculated per microgram dry weight. For the first time the results demonstrates an intact secretory machinery of the B-cells under controlled conditions in an early stage of gestational development.     

Glucose induces and leptin decreases expression of uncoupling protein-2 mRNA in human islets

Elevated islet uncoupling protein-2 (UCP-2) impairs beta-cell function and UCP-2 may be increased in clinical obesity and diabetes. We investigated the effects of glucose and leptin on UCP-2 expression in isolated human islets. Human islets were incubated for 24 h with glucose (5.5-22 mmol/l)+/-leptin (0-10 nmol/l). Some islet batches were incubated at high (22 mmol/l), and subsequently lower (5.5 mmol/l), glucose to assess reversibility of effects. Leptin effects on insulin release were also measured. Glucose dose-dependently increased UCP-2 expression in all islet batches, maximally by three-fold. This was not fully reversed by subsequently reduced glucose levels. Leptin decreased UCP-2 expression by up to 75%, and maximally inhibited insulin release by 47%, at 22 mmol/l glucose. This is the first report of UCP-2 expression in human islets and provides novel evidence of its role in the loss of beta-cell function in diabetes.