Comparison of local and systemic effects of insulin on myocardial glucose extraction in ischemic heart disease

Physiological increases in circulating insulin level significantly increase myocardial glucose uptake in vivo. To what extent this represents a direct insulin action on the heart or results indirectly from reduction in circulating concentrations of free fatty acids (FFA) is uncertain. To examine this, we measured myocardial glucose, lactate, and FFA extraction in 10 fasting men (ages 49-76 yr) with stable coronary artery disease during sequential intracoronary (10 mU/min, coronary plasma insulin = 140 +/- 20 microU/ml) and intravenous (100 mU/min, systemic plasma insulin = 168 +/- 26 microU/ml) insulin infusion. Basally, hearts extracted 2 +/- 2% of arterial glucose and extracted 27 +/- 6% of FFA. Coronary insulin infusion increased glucose extraction to 5 +/- 3% (P < 0.01 vs. basal) without changing plasma FFA or heart FFA extraction. Conversion to intravenous infusion lowered plasma FFA by approximately 50% and heart FFA extraction by approximately 75%, increasing heart glucose extraction still further to 8 +/- 3% (P < 0. 01 vs. intracoronary). This suggests the increase in myocardial glucose extraction observed in response to an increment in systemic insulin concentration is mediated equally by a reduction in circulating FFA and by direct insulin action on the heart itself. Coronary insulin infusion increased myocardial lactate extraction as well (from 20 +/- 10% to 29 +/- 9%, P < 0.05), suggesting the local action may include stimulation of a metabolic step distal to glucose transport and glycolysis.     

Effects of pregnancy on bone matrix proteins in Wistar rats

Using an EDTA extraction procedure, bones from pregnant Wistar rats were analyzed for their content of collagen and non-collagenous components (sialoprotein, proteoglycan and carbohydrate). The bone matrix size was found to be smaller in pregnant rats than in normal rats (19.5% vs 17.5% of the dry weight bone). The EDTA extractability of the bone protein from pregnant rats was higher than that from controls (2.6% vs 1.9% dry weight bone). EDTA extracts from pregnant rats contained higher amounts of soluble collagen (1.6% vs 0.5% of dry weight tissue) and lower amounts of non-collagenous components (1.65% vs 2.23% for hexoses, 2.38% vs 3.95% for sialic acid and 1.24% vs 1.73% for uronic acid). In bone matrix, collagen content was lower in the pregnant rats (9.45% vs 10.6%). Similarly, the amounts of non-collagenous components were slightly decreased in the bone matrix from the pregnant rats. The respective values were: 0.91% vs 0.93% for hexoses, 0.45% vs 0.52% for sialic acid and 0.39% vs 0.50% for uronic acid. These results suggest that in pregnancy collagen and non-collagenous protein content in bone is decreased while the total mineral content is increased.     

Blood sampling by puncture in the cavernous sinus from hunted wild boar

A new method of sampling based on the extraction of blood from the cavernous sinus of the dura mater has been assessed in hunted wild boar. Blood from 139 animals was obtained by two different extraction methods: the harvesting from thoracic cavity (TC) and intracavernous venipuncture (IV). Sera obtained by the IV method had higher volume (mean 2.85 vs 1.85 ml), were less hemolytic (mean absorbance at 450 nm: 1.01 vs 2.41 nm). A higher number of samples and a higher proportion of sera collected by IV (90.6 %) compared to those obtained using the TC method (78.4 %), could be analyzed against Aujeszky’s disease using blocking ELISA. No statistically significant differences in seroprevalences between samples obtained using both extraction methods were observed. The results obtained indicate that the IV is an easy, fast, reliable, clean, and safe method to collect blood samples from hunted wild boar, proving a real alternative to the traditional collection method.     

Factors affecting emulsion stability and quality of oil recovered from enzyme-assisted aqueous extraction of soybeans

The objectives of the present study were to assess how the stability of the emulsion recovered from aqueous extraction processing of soybeans was affected by characteristics of the starting material and extraction and demulsification conditions. Adding endopeptidase Protex 6L during enzyme-assisted aqueous extraction processing (EAEP) of extruded soybean flakes was vital to obtaining emulsions that were easily demulsified with enzymes. Adding salt (up to 1.5 mM NaCl or MgCl₂) during extraction and storing extruded flakes before extraction at 4 and 30 °C for up to 3 months did not affect the stabilities of emulsions recovered from EAEP of soy flour, flakes and extruded flakes. After demulsification, highest free oil yield was obtained with EAEP of extruded flakes, followed by flour and then flakes. The same protease used for the extraction step was used to demulsify the EAEP cream emulsion from extruded full-fat soy flakes at concentrations ranging from 0.03% to 2.50% w/w, incubation times ranging from 2 to 90 min, and temperatures of 25, 50 or 65 °C. Highest free oil recoveries were achieved at high enzyme concentrations, mild temperatures, and short incubation times. Both the nature of enzyme (i.e., protease and phospholipase), added alone or as a cocktail, concentration of enzymes (0.5% vs. 2.5%) and incubation time (1 vs. 3 h), use during the extraction step, and nature of enzyme added for demulsifying affected free oil yield. The free oil recovered from EAEP of extruded flakes contained less phosphorus compared with conventional hexane-extracted oil. The present study identified conditions rendering the emulsion less stable, which is critical to increasing free oil yield recovered during EAEP of soybeans, an environmentally friendly alternative processing method to hexane extraction.     

Extraction of short-chain-length poly-[(R)-hydroxyalkanoates] (scl-PHA) by the “anti-solvent” acetone under elevated temperature and pressure

A novel method was developed for extraction of short-chain-length poly(hydroxyalkanoates) (scl-PHA) from microbial biomass by the well-known “scl-PHA anti-solvent” acetone at elevated temperature and pressure in a closed system combining components for extraction, filtration, and product work-up. Recovery of scl-PHA using this new approach was compared with established methods using chloroform at ambient pressure. The new method performs similar regarding product purity (98.4 vs. 97.7 %) and extraction yield (96.8 % by both methods), and is by far faster than established chloroform extraction (20 min vs. 12 h). Separation of the polymer from acetone is simply achieved by cooling down the acetone solution of scl-PHA, thus allows for a nearly quantitative recovery of the solvent that conveniently can be reused. Characterization of scl-PHA extracted by both methods does not reveal any significant difference in terms of molar mass and thermo analytical parameters.     

Impaired cardiac reserve and severely diminished skeletal muscle O₂ utilization mediate exercise intolerance in Barth syndrome

Barth syndrome (BTHS) is a mitochondrial myopathy characterized by reports of exercise intolerance. We sought to determine if 1) BTHS leads to abnormalities of skeletal muscle O(2) extraction/utilization and 2) exercise intolerance in BTHS is related to impaired O(2) extraction/utilization, impaired cardiac function, or both. Participants with BTHS (age: 17 ± 5 yr, n = 15) and control participants (age: 13 ± 4 yr, n = 9) underwent graded exercise testing on a cycle ergometer with continuous ECG and metabolic measurements. Echocardiography was performed at rest and at peak exercise. Near-infrared spectroscopy of the vastus lateralis muscle was continuously recorded for measurements of skeletal muscle O(2) extraction. Adjusting for age, peak O(2) consumption (16.5 ± 4.0 vs. 39.5 ± 12.3 ml·kg(-1)·min(-1), P < 0.001) and peak work rate (58 ± 19 vs. 166 ± 60 W, P < 0.001) were significantly lower in BTHS than control participants. The percent increase from rest to peak exercise in ejection fraction (BTHS: 3 ± 10 vs. control: 19 ± 4%, P < 0.01) was blunted in BTHS compared with control participants. The muscle tissue O(2) saturation change from rest to peak exercise was paradoxically opposite (BTHS: 8 ± 16 vs. control: -5 ± 9, P < 0.01), and the deoxyhemoglobin change was blunted (BTHS: 0 ± 12 vs. control: 10 ± 8, P < 0.09) in BTHS compared with control participants, indicating impaired skeletal muscle extraction in BTHS. In conclusion, severe exercise intolerance in BTHS is due to both cardiac and skeletal muscle impairments that are consistent with cardiac and skeletal mitochondrial myopathy. These findings provide further insight to the pathophysiology of BTHS.     

Ruminal phosphorus availability from several feedstuffs measured by the nylon bag technique

The present study was aimed at determining rumen phosphorus availability of some feedstuffs assessed with the nylon bag technique: forage (alfalfa), cereals (control-C wheat, formaldehyde treated-FT wheat, barley, corn), cereal by-products (wheat bran, wheat distillers, corn distillers) and meals (C and FT soya bean meals, rapeseed meals and sunflower meals). Rumen phosphorus availability was not uniform amongst the feedstuffs, varying from 33.1% (FT rapeseed meal) to 84.7% (C wheat). Alfalfa phosphorus release kinetics showed high bacterial phosphorus contamination. Technological treatments affected phosphorus content of by-products by either increasing (wheat bran and distillers) or decreasing (corn distillers) after germ extraction from the seed. Formaldehyde treatment decreasing rumen phosphorus availability (from 77.2% vs. 89.4% for wheat to 33.1% vs. 64.4% for the rapeseed meal) may depreciate the phosphorus nutritional value of FT meals.     

Hemodynamics and O2 uptake during maximal knee extensor exercise in untrained and trained human quadriceps muscle: effects of hyperoxia.

To elucidate the potential limitations on maximal human quadriceps O2 capacity, six subjects trained (T) one quadriceps on the single-legged knee extensor ergometer (1 h/day at 70% maximum workload for 5 days/wk), while their contralateral quadriceps remained untrained (UT). Following 5 wk of training, subjects underwent incremental knee extensor tests under normoxic (inspired O2 fraction = 21%) and hyperoxic (inspired O2 fraction = 60%) conditions with the T and UT quadriceps. Training increased quadriceps muscle mass (2.9 +/- 0.2 to 3.1 +/- 0.2 kg), but did not change fiber-type composition or capillary density. The T quadriceps performed at a greater peak power output than UT, under both normoxia (101 +/- 10 vs. 80 +/- 7 W; P < 0.05) and hyperoxia (97 +/- 11 vs. 81 +/- 7 W; P < 0.05) without further increases with hyperoxia. Similarly, thigh peak O2 consumption, blood flow, vascular conductance, and O2 delivery were greater in the T vs. the UT thigh (1.4 +/- 0.2 vs. 1.1 +/- 0.1 l/min, 8.4 +/- 0.8 vs. 7.2 +/- 0.8 l/min, 42 +/- 6 vs. 35 +/- 4 ml x min(-1) x mmHg(-1), 1.71 +/- 0.18 vs. 1.51 +/- 0.15 l/min, respectively) but were not enhanced with hyperoxia. Oxygen extraction was elevated in the T vs. the UT thigh, whereas arteriovenous O2 difference tended to be higher (78 +/- 2 vs. 72 +/- 4%, P < 0.05; 160 +/- 8 vs. 154 +/- 11 ml/l, respectively; P = 0.098) but again were unaltered with hyperoxia. In conclusion, the present results demonstrate that the increase in quadriceps muscle O2 uptake with training is largely associated with increases in blood flow and O2 delivery, with smaller contribution from increases in O2 extraction. Furthermore, the elevation in peak muscle blood flow and vascular conductance with endurance training seems to be related to an enhanced vasodilatory capacity of the vasculature perfusing the quadriceps muscle that is unaltered by moderate hyperoxia.     

The association of gene polymorphisms with the muscle fiber type composition

Muscle fiber composition of m. vastus lateralis has significant individual variability mainly depending on genetic factors. Present study shows analysis of association between polymorphisms of three muscle performance-related genes and muscle fiber type composition in 48 young healthy men. DNA was obtained from mouthwash samples by alkaline extraction. Polymorphism determination of PPARalpha, ACE and ACTN3 genes was performed using polymerase chain raction. Muscle fiber typing from m. vastus lateralis was performed using immunohistochemistry method. We found an association of increased frequency of intron 7 G allele of PPARalpha gene (93.9% vs 60.0%) and D allele of ACE gene (68.8% vs 34.4%) in the group with the highest proportion of slow-twitch fibers (56-70%) compared to the group with the lowest proportion (25-43%). Thus, PPARalpha and ACE genes can be considered as potential candidate genes for muscle fiber type determination.     

Blood glucose extraction as a mediator of perceived exertion during prolonged exercise

The effect of blood glucose extraction on the perception of exertion was examined during prolonged arm exercise. Eight male subjects consumed in counter-balanced order a standard daily diet containing either (1) 75 g dihydroxyacetone and 25 g sodium pyruvate (DHAP) or (2) an isocaloric amount of placebo, to manipulate blood glucose extraction. Following each 7-day diet, subjects exercised to exhaustion at 60% of peak arm oxygen consumption. Ratings of perceived exertion (Borg, CR-10 scale) were obtained for the arms (RPE-A), legs (RPE-L), chest (RPE-C) and overall body (RPE-O) every 10 min of exercise. After 60 min of continuous exercise, blood samples were drawn from the radial artery and axillary vein. Ratings of perceived exertion did not differ between trials during the first 50 min of exercise. At the 60-min time point, perceived exertion was lower (P less than 0.01) in the DHAP than placebo trials for the arms (RPE-A: 4.25 vs 5.50) and overall body (RPE-O: 3.25 vs 4.00). These differences persisted throughout exercise. RPE-L and RPE-C did not differ between trials. Whole-arm arterial-venous glucose difference was higher (P less than 0.05) in the DHAP (1.00 mmol.l-1) than placebo (0.36 mmol.l-1) trials, as was fractional extraction of glucose (22.5 vs 9.0%). Respiratory exchange ratio was the same between trials. Triceps muscle glycogen was (1) higher in the DHAP than placebo trial at pre-exercise (P less than 0.05), (2) decreased during exercise and (3) did not differ between trials at exercise termination.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of stable isotope dilution assays for ochratoxin A in blood samples

Two new stable isotope dilution assays were developed for the quantification of ochratoxin A in human blood samples for exposure studies. The methods based on two different sample extraction and cleanup procedures including liquid–liquid extraction with following immunoaffinity chromatography (IA) as well as a dispersive solid-phase extraction (DSPE) method. For detection, LC–MS/MS was applied. For the first time, exact quantitation of the reference compound ochratoxin A was performed by quantitative NMR spectroscopy (qNMR). Additionally, a comparison of different blood-drawing procedures revealed no differences for heparin plasma and serum whereas citrate plasma gave significantly lower results for the mycotoxin. Limits of detection (LOD: 0.02 ng/g (IA) vs 0.03 ng/g (DSPE)), limits of quantification (LOQ: 0.07 ng/g (IA) vs 0.08 ng/g (DSPE)), relative recovery (?94%), precision, and linearity indicated excellent performance of the developed methods.     

Stroma-free hemoglobin: its presence in plasma does not improve oxygen supply to the resting hindlimb vascular bed of hemodiluted dogs.

In hemodilution, red cell spacing in the microcirculation is increased, flow distribution may become more heterogeneous, and, as a result, oxygen supply to tissues may suffer. We tested the hypothesis that oxygen extraction from diluted blood may be enhanced by the presence of hemoglobin in the plasma phase in relatively low concentrations. In anesthetized dogs, the hindlimb vascular bed was isolated and perfused with the animal's own blood by a roller pump. One group of dogs (n = 6) was hemodiluted (hematocrit = 15.0 +/- 1.0%) with a 6% solution of dextran. A second group of dogs (n = 6) was similarly hemodiluted (hematocrit = 16.0 +/- 0.4%) with dextran containing stroma-free hemoglobin solution whereby plasma-phase hemoglobin concentration was raised to 1.1 +/- 0.1 g.dL-1. Systemic hemodynamic observations were made repeatedly over the subsequent 2.5 h, while blood flow to the hindlimb was progressively reduced in stepwise decrements. The hemoglobin-hemodiluted group showed increased systemic arterial blood pressure and total peripheral resistance when compared with the control (dextran diluted) group. The isolated hindlimb also showed evidence of increased vascular resistance in the hemoglobin-treated group. In each individual animal, critical oxygen delivery and extraction were determined by finding the intercept of the supply-independent and supply-dependent portions of the oxygen uptake/oxygen delivery relationship. Neither the critical oxygen delivery rates (5.75 +/- 0.83 vs. 6.41 +/- 0.53 mL.kg-1.min-1) nor critical oxygen extraction ratios (0.75 +/- 0.03 vs. 0.76 +/- 0.04) were found to be significantly different in the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional impairment of skeletal muscle oxidative metabolism during knee extension exercise after bed rest

A functional evaluation of skeletal muscle oxidative metabolism during dynamic knee extension (KE) incremental exercises was carried out following a 35-day bed rest (BR) (Valdoltra 2008 BR campaign). Nine young male volunteers (age: 23.5 ± 2.2 yr; mean ± SD) were evaluated. Pulmonary gas exchange, heart rate and cardiac output (by impedance cardiography), skeletal muscle (vastus lateralis) fractional O(2) extraction, and brain (frontal cortex) oxygenation (by near-infrared spectroscopy) were determined during incremental KE. Values at exhaustion were considered "peak". Peak heart rate (147 ± 18 beats/min before vs. 146 ± 17 beats/min after BR) and peak cardiac output (17.8 ± 3.3 l/min before vs. 16.1 ± 1.8 l/min after BR) were unaffected by BR. As expected, brain oxygenation did not decrease during KE. Peak O(2) uptake was lower after vs. before BR, both when expressed as liters per minute (0.99 ± 0.17 vs. 1.26 ± 0.27) and when normalized per unit of quadriceps muscle mass (46.5 ± 6.4 vs. 56.9 ± 11.0 ml·min(-1)·100 g(-1)). Skeletal muscle peak fractional O(2) extraction, expressed as a percentage of the maximal values obtained during a transient limb ischemia, was lower after (46.3 ± 12.1%) vs. before BR (66.5 ± 11.2%). After elimination, by the adopted exercise protocol, of constraints related to cardiovascular O(2) delivery, a decrease in peak O(2) uptake and muscle peak capacity of fractional O(2) extraction was found after 35 days of BR. These findings suggest a substantial impairment of oxidative function at the muscle level, "downstream" with respect to bulk blood flow to the exercising muscles, that is possibly at the level of blood flow distribution/O(2) utilization inside the muscle, peripheral O(2) diffusion, and intracellular oxidative metabolism.     

Sample clean-up methods,immunoaffinity chromatography and solid phase extraction,for determination of deoxynivalenol and deepoxy deoxynivalenol in swine serum

Concentrations of deoxynivalenol (DON) and deepoxy deoxynivalenol (DOM-1) in animal blood are important parameters for studies in toxicology and biological detoxification of DON. Clean-up methods, using either immunoaffinity chromatography (IAC) or solid phase extraction (SPE), were compared in order to determine the free form of DON or DOM-1 and the sum amount (free form plus glucuronide conjugated form of DON or DOM-1), respectively, in swine serum. Detection was achieved by high performance liquid chromatography with ultraviolet detection (HPLC-UV). Compared with the SPE-HPLC method, the IAC-HPLC method provided lower quantitation limit (DON: 18 vs 42 ng/ml; DOM-1: 21 vs 30 ng/ml) and higher recoveries (DON: 93.4–102.7% vs 63.7–85.3%; DOM-1: 85.5–91.1% vs 68.0–82.6%). Compared with previously published methods, the developed IAC-HPLC method removed analytical interferences from swine serum in one quick and easy step, and eliminated steps of extraction with organic solvent and/or pre-purification using SPE cartridges. This IAC-HPLC method was used to analyze swine serum samples collected from pigs that were evaluated in a feeding trial of a microbiological detoxification of DON. No DON or DOM-1 were detected in serum samples from pigs given a toxin-free diet or a microbial control diet. In serum samples from pigs given a DON diet (5 mg/kg of DON), free form DON and sum free DON + conjugated DON were 38.8 ± 13.7 and 49.8 ± 14.1 ng/ml, respectively. In serum samples from those given a detoxified-DON diet (DON was transformed to DOM-1), free form DOM-1 was detected but not quantified, and the sum DOM-1 was found as 47.5 ± 6.3 ng/ml.     

Supercritical fluid extraction of selected pharmaceuticals from water and serum

Selected drugs from benzodiazepine, anabolic agent and non-steroidal anti-inflammatory drug (NSAID) therapeutic classes were extracted from water and serum using a supercritical CO₂ mobile phase. The samples were extracted at a pump pressure of 329 MPa, an extraction chamber temperature of 45°C, and a restrictor temperature of 60°C. The static extraction time for all samples was 2.5 min and the dynamic extraction time ranged from 5 to 20 min. The analytes were collected in appropriate solvent traps and assayed by modified literature HPLC procedures. Analyte recoveries were calculated based on peak height measurements of extracted vs. unextracted analyte. The recovery of the benzodiazepines ranged from 80 to 98% in water and from 75 to 94% in serum. Anabolic drug recoveries from water and serum ranged from 67 to 100% and 70 to 100%, respectively. The NSAIDs were recovered from water in the 76 to 97% range and in the 76 to 100% range from serum. Accuracy, precision and endogenous peak interference, if any, were determined for blank and spiked serum extractions and compared with classical sample preparation techniques of liquid-liquid and solid-phase extraction reported in the literature. For the benzodiazepines, accuracy and precision for supercritical fluid extraction (SFE) ranged from 1.95 to 3.31 and 0.57 to 1.25%, respectively (n=3). The SFE accuracy and precision data for the anabolic agents ranged from 4.03 to 7.84 and 0.66 to 2.78%, respectively (n=3). The accuracy and precision data reported for the SFE of the NSAIDs ranged from 2.79 to 3.79 and 0.33 to 1.27%, respectively (n=3). The precision of the SFE method from serum was shown to be comparable to the precision obtained with other classical preparation techniques.     

Oxygen release from arterioles with normal flow and no-flow conditions.

The rate of oxygen release from arterioles ( approximately 55 microm diameter) was measured in the hamster window chamber model during flow and no-flow conditions. Flow was stopped by microvascular transcutaneous occlusion using a glass pipette held by a manipulator. The reduction of the intra-arteriolar oxygen tension (Po2) was measured by the phosphorescence quenching of preinfused Pd-porphyrin, 100 microm downstream from the occlusion. Oxygen release from arterioles was found to be 53% greater during flow than no-flow conditions (2.6 vs. 1.7 x 10(-5) ml O2.cm(-2).s(-1), P < 0.05). Acute hemodilution with dextran 70 was used to reduce vessel oxygen content, significantly increase wall shear stress (14%, P < 0.05), reduce Hct to 28.4% (SD 1.0) [vs. 48.8% (SD 1.8) at baseline], lower oxygen supply by the arterioles (10%, P < 0.05), and increase oxygen release from the arterioles (39%, P < 0.05). Hemodilution also increased microcirculation oxygen extraction (33% greater than nonhemodilution, P < 0.05) and oxygen consumption by the vessel wall, as shown by an increase in vessel wall oxygen gradient [difference in Po2 between the blood and the tissue side of the arteriolar wall, nonhemodiluted 16.2 Torr (SD 1.0) vs. hemodiluted 18.3 Torr (SD 1.4), P < 0.05]. Oxygen released by the arterioles during flow vs. nonflow was increased significantly after hemodilution (3.6 vs. 1.8 x 10(-5) ml O2.cm(-2).s(-1), P < 0.05). The oxygen cost induced by wall shear stress, suggested by our findings, may be >15% of the total oxygen delivery to tissues by arterioles during flow in this preparation.     

Effect of cryoprotectant concentration, equilibration time and thawing procedure on survival and development of rapid frozen-thawed mature mouse oocytes

Experiments were conducted to develop a simple rapid-freezing protocol for mature mouse oocytes that would yield a high proportion of oocytes with developmental potential. The effects of concentration (3.5, 4.5 and 6.0 M dimethyl sulfoxide (DMSO) all with 0.5 M sucrose) and the duration of exposure (2.5 min vs 45 sec) of oocytes to the cryoprotectant and its extraction after thawing in 2, 3 or 4 steps of descending sucrose concentration were studied. The most effective of the rapid-freezing and thawing protocols (4.5 M DMSO; 45 sec exposure and 3-step thawing) was compared to slow freezing protocols using 1.5 M DMSO and 1.0 M 1,2 propanediol as cryoprotectants. The DMSO concentrations had an effect on survival, fertilization and embryo development using short (45 sec) but not long (2.5 min) exposure. The rate of morphological oocyte survival was significantly higher using 4.5 M DMSO than 3.5 or 6.0 M (92% vs 82 and 73%, respectively). The development of fertilized embryos to blastocysts was also significantly higher at 4.5 M than at 3.5 or 6.0 M (68% vs 42 and 53%, respectively). The extraction of cryoprotectant in 3 or 4 steps of descending sucrose concentration resulted in higher survival (P < 0.01) and fertilization than in 2 steps. The best survival, fertilization and development was achieved with the 3-step procedure. Optimal combinations of conditions were 4.5 M DMSO at 45 sec prefreeze exposure and 3-step extraction of the cryoprotectant. Oocytes frozen by conventional methods had a survival, fertilization and development to blastocyst rate significantly lower than those frozen under the optimal rapid conditions. Thus rapid freezing of mature mouse oocytes with 4.5 M DMSO + 0.5 M sucrose and short prefreeze exposure is effective and has the additional advantage of being less time-consuming than slow freezing methods.     

Validation of ⁹⁹mTechnetium-labeled mebrofenin hepatic extraction method to quantify meal-induced splanchnic blood flow responses using a porcine model

The aim of this study was to evaluate the measurement of the total splanchnic blood flow (SBF) using a clinical diagnostic method based on Fick's principle and hepatic extraction of 99mTc-mebrofenin (99mTc-MBF) compared with a paraaminohippuric acid (pAH) dilution method in a porcine model. Another aim was to investigate whether enterohepatic cycling of 99mTc-MBF affected the SBF measurement. Five indwelling catheters were placed in each pig (n = 15) in the portal, mesenteric, and hepatic veins, as well as in the aorta and the vena cava. The SBF was measured using both methods. The portal blood flow; the intestinal and hepatic oxygen uptake; the net fluxes of oxygen, lactate, and glucose; and the extraction fraction (EF) of 99mTc-MBF were measured before and for 70 min after feeding. The mean baseline SBF was 2,961 ml/min vs. 2,762 ml/min measured by pAH and 99mTc-MBF, respectively, and increased significantly to 3,977 ml/min and 3,981 ml/min postprandially. The hepatic EF of 99mTc-MBF decreased from 40% at the start of the investigation to 16% 70 min after feeding. The arterial-portal difference in 99mTc-MBF concentration was 0.21% (P = 0.48), indicating no intestinal extraction or metabolism. The clinical method for measuring the SBF based on hepatic 99mTc-MBF extraction is robust compared with the indicator dilution method, despite the decrease seen in hepatic extraction of 99mTc-MBF. Because there was no difference in the content of 99mTc-MBF between the arterial and portal vein plasma, the SBF can be calculated from an arterial and a hepatic vein sample.     

Substrate utilization and enzymes in skeletal muscle of extremely endurance-trained men

Substrate utilization during exercise at 65% of maximal O2 uptake (VO2 max) and biochemical characteristics of vastus lateralis were compared between five endurance-trained (T) and five untrained subjects (U). The oxidative enzyme activities were 100% greater in T than in U, and VO2 max was 50% higher. A greater proportion of ATP regeneration occurred through oxidative processes in T than in U (smaller leg lactate release and smaller muscle lactate accumulation). The respiratory exchange ratio together with the local leg respiratory quotient indicated a greater contribution of fat to oxidative metabolism in T than U (53 vs. 33%). No difference, however, in the ratio of plasma free fatty acid extraction to O2 extraction by the working legs was found between T and U. Thus it could be calculated that a greater fraction of fat oxidation would have been covered by intramuscular triglycerides in T than in U (34 vs. 15%, P less than 0.05). T in comparison to U were further characterized by a smaller glycogen breakdown and a smaller glucose uptake, which may have been one contributing factor that prevented the blood glucose level from falling in T. The greater leg muscle citrate concentration in T could have been one factor mediating a lower carbohydrate utilization as a response to an increase in the relative proportion of fat oxidation.     

Rapid and reliable high-throughput methods of DNA extraction for use in barcoding and molecular systematics of mushrooms

We present two methods for DNA extraction from fresh and dried mushrooms that are adaptable to high-throughput sequencing initiatives, such as DNA barcoding. Our results show that these protocols yield ∼85% sequencing success from recently collected materials. Tests with both recent (<2 year) and older (>100 years) specimens reveal that older collections have low success rates and may be an inefficient resource for populating a barcode database. However, our method of extracting DNA from herbarium samples using small amount of tissue is reliable and could be used for important historical specimens. The application of these protocols greatly reduces time, and therefore cost, of generating DNA sequences from mushrooms and other fungi vs. traditional extraction methods. The efficiency of these methods illustrates that standardization and streamlining of sample processing should be shifted from the laboratory to the field.