Acetate Infusion Research Articles

Recurrent Antecedent Hypoglycemia Alters Neuronal Oxidative Metabolism In Vivo

OBJECTIVEThe objective of this study was to characterize the changes in brain metabolism caused by antecedent recurrent hypoglycemia under euglycemic and hypoglycemic conditions in a rat model and to test the hypothesis that recurrent hypoglycemia changes the brain's capacity to utilize different energy substrates.RESEARCH DESIGN AND METHODSRats exposed to recurrent insulin-induced hypoglycemia for 3 days (3dRH rats) and untreated controls were subject to the following protocols: [2-13C]acetate infusion under euglycemic conditions (n = 8), [1-13C]glucose and unlabeled acetate coinfusion under euglycemic conditions (n = 8), and [2-13C]acetate infusion during a hyperinsulinemic-hypoglycemic clamp (n = 8). In vivo nuclear magnetic resonance spectroscopy was used to monitor the rise of13C-labeling in brain metabolites for the calculation of brain metabolic fluxes using a neuron-astrocyte model.RESULTSAt euglycemia, antecedent recurrent hypoglycemia increased whole-brain glucose metabolism by 43 ± 4% (P < 0.01 vs. controls), largely due to higher glucose utilization in neurons. Although acetate metabolism remained the same, control and 3dRH animals showed a distinctly different response to acute hypoglycemia: controls decreased pyruvate dehydrogenase (PDH) flux in astrocytes by 64 ± 20% (P = 0.01), whereas it increased by 37 ± 3% in neurons (P = 0.01). The 3dRH animals decreased PDH flux in both compartments (−75 ± 20% in astrocytes, P < 0.001, and −36 ± 4% in neurons, P = 0.005). Thus, acute hypoglycemia reduced total brain tricarboxylic acid cycle activity in 3dRH animals (−37 ± 4%, P = 0.001), but not in controls.CONCLUSIONSOur findings suggest that after antecedent hypoglycemia, glucose utilization is increased at euglycemia and decreased after acute hypoglycemia, which was not the case in controls. These findings may help to identify better methods of preserving brain function and reducing injury during acute hypoglycemia.

DIFFERENTIAL NEURONAL AND GLIAL METABOLIC RESPONSE DURING HYPOTHERMIA IN AN EXPERIMENTAL ANIMAL MODEL

To study the effect of hypothermia on metabolic compartmentalization in an animal model. [1-(13)C] glucose, [2-(13)C] glucose, [3-(13)C] lactate, and [2-(13)C] acetate were infused into male Sprague-Dawley rats. The (13)C label was detected using (13)C-edited H magnetic resonance spectroscopy or (13)C magnetic resonance spectroscopy to determine the isotopic enrichment of both glutamate and glutamine. The infusion was carried out at either normothermia (37 degrees C) or hypothermia (31 degrees C). The [1-(13)C] glucose infusion during hypothermia resulted in decreased labeling of glutamate and glutamine consistent with decreased metabolism or the shunting of glucose through the pentose phosphate pathway. Unexpectedly, [2-(13)C] glucose infusion during hypothermia resulted in decreased labeling of glutamate but not glutamine, implying decreased neuronal but unaltered glial metabolism. The lactate and acetate infusion showed no temperature effect on labeling, indicating that the dampened neuronal metabolism occurred during glycolysis. The results may explain the mechanism of action of hypothermia by differentially preserving the protective metabolism in glia while selectively dampening neuronal metabolism.

A pilot study of the effects of pioglitazone and rosiglitazone on de novo lipogenesis in type 2 diabetes

Treatment of type 2 diabetes mellitus (T2DM) patients with pioglitazone results in a more favorable lipid profile, and perhaps more favorable cardiac outcomes, than treatment with rosiglitazone. Pioglitazone treatment increases VLDL-triacylglycerol clearance, but the role of de novo lipogenesis (DNL) has not been explored, and no direct comparison has been made between the thiazolidinediones (TZDs). Twelve subjects with T2DM and hypertriacylglyceridemia were randomized to either rosiglitazone or pioglitazone treatment. Stable isotope infusion studies were performed at baseline and after 20 weeks of treatment. Both treatments reduced glucose and HbA(1c) concentrations equally. Pioglitazone treatment resulted in a 40% reduction in hepatic DNL (P < 0.01) and in a 25% reduction in hepatic glucose production (P < 0.05), while rosiglitazone did not significantly change either parameter, although comparisons of changes between treatments were not significantly different. These pilot results indicate that pioglitazone reduces hepatic DNL while rosiglitazone does not. Larger follow-up studies are required to confirm differential effects of these agents definitively. The reduction in DNL may underlie altered assembly or atherogenicity of lipoprotein particles and may reflect PPARalpha or other non-PPARgamma actions on the liver by pioglitazone. These differences might help explain previously reported differences in lipid profiles and cardiovascular disease outcomes for rosiglitazone and pioglitazone.

The effect of short-term hyperammonaemia on milk synthesis in dairy cows

To test the hypothesis that ammonia detoxification in ruminants consumes amino acids to the detriment of milk protein production, we infused four lactating dairy cows with ammonium acetate or sodium acetate in switchback experiments. Plasma ammonia concentrations increased to 411 microm within 1 h of the start of infusion of ammonium acetate at 567 mmol/h. The rate constant for ammonia clearance from plasma was 0 x 054/min and the half-life was 12 x 9 min. Infusion at 567 mmol/h for 1 h followed by 1 h without infusion, repeated four times between am- and pm-milking, caused a decrease in feed intake. Compared with sodium acetate, continuous infusion of ammonium acetate at 360 mmol/h throughout an entire 10-h milking interval increased plasma ammonia concentrations to 193 microm and caused a 20% decrease in milk, protein and lactose production with no effect on percentage composition of milk or the yield of milk fat. Arterial concentrations of glucose and non-esterified fatty acids tended to increase; there was no effect on arterial acetate, beta-hydroxybutyrate or triacylglcerol, and branched-chain amino acids, Lys and Thr decreased. Mammary plasma flow, estimated by assuming 100% uptake/output of Phe+Tyr, was significantly correlated with milk yield. Mammary uptakes of acetate tended to be reduced by hyperammonaemia, but uptakes of other energy metabolites and amino acids were not affected. Thus, while an increase in amino acid consumption during hyperammonaemia was apparent from the drop in circulating concentrations of Leu, Ile, Val, Lys and Thr, there was no evidence to support the hypothesis that milk yield is affected by the lower concentrations. An ammonia-induced depression in feed intake may have caused the decrease in milk synthesis.

Limiting amino acids for growing lambs fed a diet low in ruminally undegradable protein1

Ruminally cannulated Rambouillet wether lambs were used in three 6 x 6 Latin square experiments (n = 6/experiment) to determine which essential AA limit N retention. Lambs (BW = 36.9 +/- 1.9 kg for Exp. 1, 35.1 +/- 1.4 kg for Exp. 2, and 46.0 +/- 1.3 kg for Exp. 3) were housed in metabolism crates and limit-fed (DMI = approx. 1.8% of BW daily) twice daily a soybean hull-based diet low in ruminally undegradable protein. Treatments for Exp. 1 were continuous abomasal infusions of a solution (500 mL/d) containing 1) no AA (CON), 2) a mixture of 10 essential AA and 2 nonessential AA (10EAA), 3) 10EAA with Met removed, 4) 10EAA with Lys removed, 5) 10EAA with His removed, and 6) 10EAA with Thr removed. Treatments for Exp. 2 were abomasal infusions of 1) CON, 2) 10EAA, 3) 10EAA with Leu, Ile, and Val removed (-BCAA), 4) 10EAA with Arg removed, 5) 10EAA with Phe removed, and 6) 10EAA with Trp removed. Treatments for Exp. 3 were abomasal infusions of 1) CON, 2) 10EAA, 3) -BCAA, 4) 10EAA with Leu removed, 5) 10EAA with Ile removed, and 6) 10EAA with Val removed. All lambs received continuous infusions of acetate and propionate into the rumen and dextrose into the abomasum to supply additional energy. Periods were 7 d: 3 d for adaptation to abomasally infused treatments and 4 d for fecal and urinary collections. Blood samples were collected 3 h after feeding on d 7. In all 3 experiments, N retention was greater (P < 0.10) for lambs receiving 10EAA vs. CON, demonstrating that the basal AA supply from CON was limiting. Removal of each of the essential AA from 10EAA decreased (P < 0.10) their concentrations in plasma (except for Trp), indicating that 10EAA supplied these AA in excess of the animal's requirement. In Exp. 1, N retention (g/d) decreased (P < 0.10) in response to the removal of Met and Thr, but was not affected by removal of Lys and His from 10EAA. In Exp. 2, N retention decreased (P < 0.10) in response to removal of all 3 branched-chain AA, Arg, and Trp, whereas the removal of Phe from 10EAA did not affect N retention. In Exp. 3, N retention decreased (P < 0.10) in response to removal of branched-chain AA and Val, but was not affected by the omission of Leu and Ile from 10EAA. The results of this research demonstrated that Met, Thr, Arg, Trp, and Val limited N retention of lambs fed a diet low in ruminally undegradable protein.

Milk Synthetic Response of the Bovine Mammary Gland to an Increase in the Local Concentration of Amino Acids and Acetate

Rates of secretion of components into milk are a function of precursor concentrations and parameters that describe expression of the milk synthetic enzymes and their sensitivity to precursor concentrations. To establish the enzymatic sensitivities of milk fat yield and mammary acetate utilization to circulating acetate concentration, lactating cows were infused for 10h with 0 or 40g of acetate/h in an external iliac artery supplying one udder half. In addition, to investigate the possibility that energy supply influences the milk protein response to an elevated amino acid (AA) concentration, 2 different AA profiles were infused with and without acetate. Six cows, fed a total mixed ration of 21% crude protein ad libitum, were infused with AA at 0g/h, 30g/h in the profile of rumen microbes, or 30g/h in the profile of milk proteins, in a 3×2 factorial arrangement with the 2 acetate treatments of 0 and 40g/h, all in a 6×6 Latin square. Amino acid infusion caused a 60% increase, on average, in plasma concentration of AA entering the infused udder half. From the microbial AA profile, 49% of infused AA were taken up by the udder half, 42% of which occurred during the first pass. From the milk AA profile, 44% of infused AA were taken up by the udder half, 50% of which occurred during the first pass. There was an 8% increase in yield of milk protein with AA infusion, representing 7% capture, but no effect of the infused profile. Acetate infusion caused a decrease in the yields of milk protein and lactose when AA were infused, but not when AA were absent. Milk fat yields were not affected, although acetate concentrations in plasma entering the infused udder half increased by 123% and mammary uptakes increased by 128%. Mammary uptakes of long-chain fatty acids and β-hydroxybutyrate were not affected by acetate infusion, whereas glucose uptakes tended to increase. It was suggested that excess acetate may have been sequestered in adipose tissue in the udder. Yields of both protein and fat in milk showed a low sensitivity to the concentration of their precursors in circulation. It was concluded that the Km in Michaelis-Menten–type equations describing milk synthesis should be assigned a low value, and that the Vmax is regulated to bring about changes in milk yield and composition.

Access to cerebrospinal fluid absorption sites by infusion into vascular channels of the skull diploë

Object The purpose of this human cadaver study was to determine whether or not an intraosseous skull infusion would access the superior sagittal sinus (SSS) via intradural venous channels. The diploic space of the skull bone contains a sinusoidal vascular network that communicates with the underlying dura mater. Diploic veins in the parasagittal area connect with endothelium-lined intradural channels in the subjacent dura and ultimately with the dural venous sinuses. A significant proportion of cerebrospinal fluid (CSF) absorption is thought to occur via arachnoid granulations in the region of the SSS and especially along the parasagittal dura where arachnoid granulations are surrounded by intradural venous channels (lateral lacunae). The CSF is likely to be conducted from the subarachnoid space into the venous system via the fine intradural channels making up the lateral lacunae. Methods Infusion of vinyl acetate casting material into the diploic space of the human cadaveric skull resulted in complete filling of the lateral lacunae and SSS. Corrosion casting techniques and examination under magnification were used to characterize the anatomical connections between diploic spaces and dural venous sinuses. Results Corrosion casting, performed on five formalin-fixed cadavers, clearly showed the anatomical connections between the diploic infusion site and the venous sinuses in the underlying parasagittal dura where some of the CSF is thought to be absorbed. Conclusions The diploic vascular channels of the human skull may represent an indirect pathway into the dural venous sinuses. Intraosseous skull infusion may represent another possible strategy for diversion of CSF into the vascular system in the treatment of hydrocephalus.

Flavour preference conditioned by postabsorptive propionate and acetate in wethers

Previous studies have indicated that intraruminal infusions of acetate and propionate condition flavour preference in sheep. Three experiments were conducted to evaluate and compare the preference-reinforcing effects of postabsorptive acetate and propionate. Eight wethers were used in Experiment 1, and a different eight wethers were used in Experiment 2 and 3. In Experiment 1, wethers were divided into two groups, and a half of each group ( n = 2) was given clove-flavoured straw and the other half was given cardamom-flavoured straw on odd-numbered days during a 12-d conditioning period. During straw ingestion, wethers in one group received intramesenteric infusion of sodium propionate (0.56 mmol/kg) and the other group received saline infusion. On even-numbered days, flavours and infusate were switched, so that each group received a new flavour and a new infusate. When wethers were offered a choice between the two flavoured straw, they preferred ( P < 0.01) the flavoured straw that had been paired with propionate infusion. In Experiment 2 and 3, two trials were conducted using four animals in each trial. In Experiment 2, wethers were given clove-flavoured ( n = 2) or nutmeg-flavoured ( n = 2) straw paired with intramesenteric infusion of sodium acetate (1.69 and 0.85 mmol/kg in Trial 1 and 2, respectively) on odd-numbered days and the other flavoured straw paired with saline infusion on even-numbered days during a 10-d conditioning period. In subsequent two-choice tests, wethers in Trial 1, but not in Trial 2, showed a preference ( P < 0.05) for the flavoured straw paired with the acetate infusion. Wethers used in Trial 2 were then given 10 additional days of conditioning with intrajugular infusion of 1.69 mmol/kg of sodium acetate and saline, but preferences for the flavour paired with acetate infusion were not developed. In Experiment 3, wethers were given fennel-flavoured ( n = 2) or nutmeg-flavoured ( n = 2) straw paired with sodium propionate infusion (1.69 and 0.96 mmol/kg in Trial 1 and 2, respectively) into the ruminal vein on odd-numbered days and the other flavoured straw paired with saline infusion on even-numbered days during a 10-d conditioning period. In subsequent two-choice tests, wethers in Trial 1 tended to prefer ( P < 0.10) the flavoured straw paired with propionate infusion to that with acetate infusion. In Trial 2, wethers avoided nutmeg-flavoured straw more than fennel-flavoured straw, but the intake of each flavoured straw tended to be higher ( P < 0.10) when it was paired with propionate. These results suggest that acetate and propionate generate preference-reinforcing signals in the portal-hepatic area, but the signal generated by acetate is weaker than that by propionate.

Single breath CO measurements normalized to 5% CO2 in Coombs' test positive neonates

A portable, prototype instrument that measures peak CO, CO2 and H2 concentrations of breath samples was evaluated on 58 antibody positive, blood group incompatible infants. The reproducibility of 108 duplicate breath CO determinations improved when the result was normalized (COn) for the simultaneously measured CO2 concentration (r = 0.97 versus r = 0.87). The average COn for 18 antibody positive, ABO incompatible infants who received phototherapy was 1.2 ppm higher than the average for 32 who did not receive phototherapy (p < 0.001). There was a positive correlation between COn and the duration of phototherapy (r = 0.75). Sodium acetate infusion and breath H2 did not affect the COn results. In antibody positive infants, single breath CO to CO2 ratios provided more reproducible results than CO determinations that were not adjusted for the CO2 concentration. Therefore, a portable instrument that measures both gases on the same aliquot of exhaled air and that is not affected by H2 should have clinical utility as an indicator of heme catabolism and bilirubin production that is not distorted by hyper- or hypoventilation.

Impaired Mitochondrial Substrate Oxidation in Muscle of Insulin-Resistant Offspring of Type 2 Diabetic Patients

Insulin resistance is the best predictor for the development of diabetes in offspring of type 2 diabetic patients, but the mechanism responsible for it remains unknown. Recent studies have demonstrated increased intramyocellular lipid, decreased mitochondrial ATP synthesis, and decreased mitochondrial density in the muscle of lean, insulin-resistant offspring of type 2 diabetic patients. These data suggest an important role for mitochondrial dysfunction in the pathogenesis of type 2 diabetes. To further explore this hypothesis, we assessed rates of substrate oxidation in the muscle of these same individuals using (13)C magnetic resonance spectroscopy (MRS). Young, lean, insulin-resistant offspring of type 2 diabetic patients and insulin-sensitive control subjects underwent (13)C MRS studies to noninvasively assess rates of substrate oxidation in muscle by monitoring the incorporation of (13)C label into C(4) glutamate during a [2-(13)C]acetate infusion. Using this approach, we found that rates of muscle mitochondrial substrate oxidation were decreased by 30% in lean, insulin-resistant offspring (59.8 +/- 5.1 nmol x g(-1) x min(-1), P = 0.02) compared with insulin-sensitive control subjects (96.1 +/- 16.3 nmol x g(-1) x min(-1)). These data support the hypothesis that insulin resistance in skeletal muscle of insulin-resistant offspring is associated with dysregulation of intramyocellular fatty acid metabolism, possibly because of an inherited defect in the activity of mitochondrial oxidative phosphorylation.

Phlorizin administration does not attenuate hypophagia induced by intraruminal propionate infusion in lactating dairy cattle.

Infusion data from ruminants has shown that propionate stimulates satiety and decreases meal size, possibly because of increased propionate oxidation in the liver. In this experiment, phlorizin was used to increase glucose demand, which was expected to decrease propionate oxidation and attenuate the decrease in dry matter intake (DMI) caused by propionate infusion. Twelve multiparous, ruminally-cannulated Holstein cows (49+/-33 d in milk, 40+/-7 kg/d milk; mean+/-SD) were randomly assigned to square and treatment sequence in a replicated 4x4 Latin square experiment with a 2x2 factorial arrangement of treatments. Treatments were subcutaneous injection of phlorizin or propylene glycol in combination with intraruminal infusion of either Na acetate or Na propionate. Following a 7-d adaptation period, phlorizin (4 g/d) and control injections were administered every 6 h for 7 d. During the final 2 d of injections, Na acetate or Na propionate solutions (1 mol/L, pH 6.0) were infused continuously at the rate of 0.80 L/h. Feeding behavior data were collected during the final 2 d of treatment. Phlorizin caused urinary excretion of 400+/-40 g glucose/d across infusion treatments. Phlorizin tended to increase plasma free fatty acid and beta-hydroxybutyrate concentrations to a greater extent with Na acetate compared to Na propionate infusion (both interactions P<0.15). Phlorizin decreased and Na propionate increased plasma insulin and glucose concentrations. Infusion of Na propionate decreased DMI (18.4 vs. 21.1+/-1.4 kg/d, P<0.001) through an increase in intermeal interval (89.2 vs. 77.3+/-6.6 min, P=0.03), resulting in fewer meals per day (11.6 vs. 13.7+/-0.7, P<0.001). Phlorizin did not alter DMI (P=0.39) or measures of feeding behavior, nor were there interactions with infusion type. Increasing glucose demand does not limit the extent to which propionate decreases DMI in lactating dairy cows.

Vascular resistance and endothelial function in cyclosporine-treated lung transplant recipients

The majority of patients undergoing solid organ transplantation develop hypertension, to which vasoconstriction and impaired endothelial function have been suggested to contribute. We compared basal vascular resistance and nitric oxide-mediated endothelial-dependent and independent vasoreactivity between cyclosporine-treated lung transplant recipients and healthy subjects. Forearm blood flow was measured by venous occlusion plethysmography at rest and during acetylcholine, glyceryltrinitrate and N(G)-monomethyl-L-arginine acetate (L-NMMA) infusion in 11 lung transplant recipients 3-5 years after transplantation and in eight healthy subjects. Forearm vascular resistance (FVR) was calculated. Plasma levels of endothelin-1 (ET-1) and von Willebrand factor (vWf) were analysed. Basal vascular resistance was 40% lower in transplant recipients than in healthy subjects (P = 0.021). Endothelial-dependent and independent vasodilation did not differ. Plasma levels of ET-1 and vWf were higher in transplant recipients (P = 0.009 and P < 0.001 respectively). There was a significant correlation between ET-1 levels and FVR in healthy subjects (r = 0.83, P = 0.042), but not in transplant recipients (r = -0.14, P = 0.70). The findings oppose the theory of generalized vasoconstriction and impaired endothelial function in the pathogenesis of hypertension after transplantation. Increased plasma levels of ET-1 do not cause increased FVR in lung transplant recipients.

Lazaroid U‐83836E Improves Tolerance to Hemorrhagic Shock and Limb Ischemia and Reperfusion in Rats and Increases Cardiac Heat Shock Protein 72

Aminosteroids of the lazaroid type protect organs from ischemia-reperfusion damage. The authors hypothesized that lazaroid U-83836E may be beneficial in a shock model with hemorrhage combined with limb ischemia. Furthermore, the authors hypothesized that lazaroids induce expression of heat shock proteins (HSPs) of the 72-kDa family. Rats were divided into two groups (lazaroid and control groups, n = 8 each) and pretreated with the lazaroid U-83836E (5 mg/kg) or with vehicle intraperitoneally at 12 and 24 hours before experiments. At the time of the experiment, rats were anesthetized, and the femoral artery of each rat was cannulated. After 20 minutes of stabilization, blood was shed from each rat to bring its mean arterial pressure to 24-28 mmHg for 2 hours. Bilateral tourniquets were tightened proximally on the rat thighs during those 2 hours and then released. Shed blood plus equal amounts of Ringer acetate then were infused to restore normal blood pressure, followed by a continuous infusion of Ringer acetate, the rate of which was regulated to maintain blood pressure, until 30 minutes after start of resuscitation. Fluid resuscitation was stopped, and rats were observed for another 3.5 hours. At the end of the observation period, the rats' hearts were collected for immunoblot analysis of HSP72. Additional hearts were collected from similarly pretreated rats not undergoing the episode of hemorrhagic shock and fluid resuscitation. Pretreatment with U-83836E improved mean arterial blood pressure after hemorrhagic shock and fluid resuscitation (p = 0.02), combined with improvements in acid-base balance (improved base excess and standard bicarbonate; p = 0.02 and p = 0.01, respectively). Western blot of cardiac protein extracts demonstrated that lazaroid pretreatment increased expression of HSP72. Pretreatment with the lazaroid U-83836E improved outcome markers in this hemorrhagic shock model. The observed protection may be caused by increased expression of HSP72.

Effects of intravenous sodium bicarbonate and sodium acetate on equine acid-base status

Changes in blood gases, pH, and plasma electrolyte concentrations in response to intravenously infused sodium bicarbonate (NaHCO3) and sodium acetate (NaCH3CO2) solutions (1.34 mEq/mL) in 5 light breed mares were investigated. Jugular venous blood samples were collected before and after completion of the infusions in 20-minute intervals for 200 minutes. Infusion of sodium bicarbonate and sodium acetate caused significant (P .05) thereafter. There were significant reductions (P < .01) in plasma-ionized calcium and potassium after infusion of both sodium bicarbonate and sodium acetate. This study found that significant metabolic alkalosis in horses and corresponding shifts in electrolyte concentrations can be induced by intravenous infusion of solutions of either sodium bicarbonate or sodium acetate, and they persist for at least 3 hours. These data show that the short-term elevation in pH and bicarbonate ion concentration is momentarily higher after infusion of sodium bicarbonate. This is likely due to the direct infusion of bicarbonate ions in the sodium bicarbonate treatment, such that further metabolism is not required to be effective. However, the longer-term alkalosis did not differ between isomolar solutions of sodium bicarbonate and sodium acetate.

402 Cholesterol Synthesis in vivo in Premature Infants from Deuterated Water (D2O)

Background: Cholesterol is one of the main components of cell membranes and is required as precursor for many metabolically active compounds. To date cholesterol metabolism is uncompletely defined in premature infants. Using a 12 h i.v. constant infusion of 13C acetate, Renfurm (Pediatr Res 2004) studied de novo cholesterol synthesis in premature infants. This method required indwelling lines. Objective: To assess if cholesterol synthesis can be measured with a less invasive method based on D2O as precursor (Matthan Lipids 2000). Material and Methods: Cholesterol synthesis was studied in 15 newborns (BW<1500 gr, age<48 h) on parenteral nutrition without lipids. Subjects received orally a bolus of D2O (0.1 gr/kg), followed by 0.000625 gr/kg every 12 for 48 h. Urine and plasma samples (200 ul) were collected before study start and every 12 h thereafter till 48 h. Unesterified cholesterol was obtained from plasma lipid extraction and thin layer chromatography separation, and cholesterol deuterium enrichments measured by Gas-Chromatography-Isotope Ratio- Mass Spectrometry (GC-IRMS). Deuterium enrichment of urine samples were measured by GC-IRMS and regarded as the precursor enrichment pool. Cholesterol Fractional Synthesis Rate (FSR) and secretion time (ST) were measured as previously described (Matthan Lipids 2000). Data are given as mean±SD. Results: Study weight was 1084±546 gr, gestational age 28±2 wks, mean age 23±19 h. Energy Intake was 30±8 kcal/kg/day. Cholesterol FSR was 16±12% and ST 11±11 h. Conclusion: This method has the advantage that tracer can be given orally, steady state of precursor is easily achieved and very small amount of blood is needed. We measured cholesterol synthesis from D2O in vivo in premature infants and the calculated rate was approximately that found in adults (Jones J Lipid Res 1994). Given the high variability between patients larger study will better define factors related to endogenous cholesterol synthesis early in life.

Suppression of glial glutamine release to the extracellular fluid studiedin vivoby NMR and microdialysis in hyperammonemic rat brain

Release of glial glutamine (GLN) to the extracellular fluid (ECF), mainly mediated by the bidirectional system N transporter SN1, was studied in vivo in hyperammonemic rat brain, using (15)N-nuclear magnetic resonance (NMR) to monitor intracellular [5-(15)N]GLN and microdialysis/gradient (1)H-(15)N heteronuclear single-quantum correlation NMR to analyse extracellular [5-(15)N]GLN. GLN(ECF) was elevated to 2.4 +/- 0.2 mm after 4.5 h of intravenous ammonium acetate infusion. The [GLN(i)]/[GLN(ECF)] ratio (i = intracellular) was 9.6 +/- 0.9, compared with 17-20 in normal brain. GLN(ECF) then decreased substantially at t = 4.9 +/- 0.1 h. Comparison of the time-courses of intra- and extra-cellular [5-(15)N]GLN strongly suggested that the observed decrease reflects partial suppression of glial GLN release to ECF. Suppression also followed elevation of GLN(ECF) to 1.9 mM, resulting in a [GLN](i)/[GLN(ECF)] ratio of 8.4, upon perfusion of alpha-(methylamino)isobutyrate which inhibits neuronal uptake of GLN(ECF) mediated by sodium-coupled amino acid transporter (SAT). The results provide first evidence for bidirectional operation of SN1 in vivo, and clarify the effect of transmembrane GLN gradient on glial GLN release at physiological Na(+) gradient. Implications of the results for SN1 as an additional regulatory site in the glutamine/glutamate cycle and utility of this approach for examining the role of GLN in an experimental model of fulminant hepatic failure are discussed.

The effect of exercise on the clearance of infused acetate in the horse

This study was designed to determine if exercise affected the clearance of intravenously infused acetate in the horse. Plasma acetate concentrations were measured in 4 conditioned horses during submaximal exercise and at rest following an infusion of sodium acetate (2.5 mmol/kg body weight [BW]). The effects of acetate infusion were compared with an equivolume infusion amount of 0.9% NaCl. Plasma glucose, lactate, free fatty acid, glycerol, triglyceride, and β-hydroxybutyrate concentrations were also analyzed. After infusion, plasma acetate concentrations decreased more rapidly during exercise (t 1 /2: 4.5 minutes) than at rest (t 1 /2: 11.7 minutes; P P P P

Effect of short chain fatty acids infused intraileally on interdigestive exocrine pancreatic secretions in growing pigs

The effect of intraileally infused short chain fatty acids (SCFA) and saline as control on the exocrine pancreatic secretions during the interdigestive phase was studied using three 8-weeks-old piglets. Pigs were surgically fitted with a pancreatic duct catheter, re-entrant duodenal T-cannula for collection and subsequent return of pancreatic juice, and with an infusion T-cannula at the distal ileum. Saline as control, 5.0 and 10.0 mm butyrate, 7.5 and 15.0 mm propionate and 85.0 and 170.0 mm acetate were infused at 2 ml/kg body weight (BW) for 30 min into the ileum of overnight fasted piglets via ileal T-cannula. The calculated volume of infusates was administrated in five equal bolus at 6 min intervals over a period of 30 min. The pancreatic juice was collected 60 and 30 min before and 30, 60, 90 and 120 min after the start of infusion. The trypsin (p = 0.07, p > 0.15 respectively) and protein (p > 0.15, p = 0.05 respectively) outputs immediately decreased after the infusion of acetate at the dose of 85.0 and 170.0 mm, respectively, whereas pancreatic juice outflow (p > 0.15) was not significantly affected when compared with levels 30 min before infusion. After the infusion of butyrate at the dose of 5.0 mm, trypsin (p = 0.01) and protein (p = 0.12) outputs increased immediately whereas pancreatic juice outflow was not affected (p > 0.15) in comparison with levels 30 min before infusion. No significant differences were observed after infusion of butyrate at the dose of 10 mm for the pancreatic juice outflow, trypsin and protein outputs when compared with the level before infusion, although these values were numerically lower immediately after the infusion. The pancreatic juice outflow increased (p = 0.03) after the infusion of propionate at the dose of 7.5 mm and decreased (p = 0.005) immediately after the infusion of propionate at the dose of 15.0 mm when compared with the levels 30 min before the infusions. After the infusion of propionate at the dose of 7.5 or 15.0 mm for the output of protein and trypsin, no significant differences (p > 0.15) were observed when compared with levels 30 min before infusion. In summary, the intraileal infusion of SCFA at different doses exerts a short-term and moderate effect on the interdigestive exocrine pancreatic secretions in pigs.

Glutamine-dependent inhibition of pial arteriolar dilation to acetylcholine with and without hyperammonemia in the rat

Glutamine has been shown to influence endothelial-dependent relaxation and nitric oxide production in vitro, possibly by limiting arginine availability, but its effects in vivo have not been well studied. Hyperammonemia is a pathophysiological condition in which glutamine is elevated and contributes to depressed CO(2) reactivity of cerebral arterioles. We tested the hypothesis that acute hyperammonemia decreases pial arteriolar dilation to acetylcholine in vivo and that this decrease could be prevented by inhibiting glutamine synthetase with L-methionine-S-sulfoximine (MSO) or by intravenous infusion of L-arginine. Pial arteriolar diameter responses to topical superfusion of acetylcholine were measured in anesthetized rats before and at 6 h of infusion of either sodium or ammonium acetate. Ammonium acetate infusion increased plasma ammonia concentration from approximately 30 to approximately 600 microM and increased cerebral glutamine concentration fourfold. Arteriolar dilation to acetylcholine was intact after infusion of sodium acetate in groups pretreated with vehicle or with MSO plus methionine, which was coadministered to prevent MSO-induced seizures. In contrast, dilation in response to acetylcholine was completely blocked in hyperammonemic groups pretreated with vehicle or methionine alone. However, MSO plus methionine administration before hyperammonemia, which maintained cerebral glutamine concentration at control values, preserved acetylcholine dilation. Intravenous infusion of L-arginine during the last 2 h of the ammonium acetate infusion partially restored dilation to acetylcholine without reducing cerebral glutamine accumulation. Superfusion of 1 or 2 mM L-glutamine through the cranial window for 1 h in the absence of hyperammonemia attenuated acetylcholine dilation but had no effect on endothelial-independent dilation to nitroprusside. We conclude that 1) hyperammonemia reduces acetylcholine-evoked dilation in cerebral arterioles, 2) this reduction depends on increased glutamine rather than ammonium ions, and 3) increasing arginine partially overcomes the inhibitory effect of glutamine.

Effect of glutamine synthetase inhibition on astrocyte swelling and altered astroglial protein expression during hyperammonemia in rats

Inhibition of glutamine synthesis reduces astrocyte swelling and associated physiological abnormalities during acute ammonium acetate infusion in anesthetized rats. We tested the hypothesis that inhibition of glutamine accumulation during more prolonged ammonium acetate infusion in unanesthetized rats reduces cortical astrocyte swelling and immunohistochemical changes in astrocytic proteins. Rats received a continuous i.v. infusion of either sodium acetate or ammonium acetate for 24 h to increase plasma ammonia from about 30–400 μmol/l. Cohorts were pretreated with vehicle or l-methionine- S-sulfoximine (MSO; 0.83 mmol/kg). MSO reduced glutamine synthetase activity by 57% and glutamine synthetase immunopositive cell number by 69%, and attenuated cortical glutamine accumulation by 71%. Hyperammonemia increased the number of swollen astrocytes in cortex and MSO reduced this increase to control values. The number of glial fibrillary acidic protein immunopositive cells in cortex was greater in hyperammonemic rats and the increase in superficial cortical layers was attenuated by MSO. Immunoreactivity for the gap junction protein connexin-43 in the neuropil, assessed by optical density, was greater in the hyperammonemic group compared with controls, but this increase was not attenuated by MSO. No changes in the optical density of GLT1 glutamate transporter immunoreactivity in cortex were detected in any group. We conclude that glutamine synthetase inhibition reduces astrocyte swelling and ameliorates some of the reactive astroglial cytoskeletal alterations seen at 24 h of hyperammonemia, but that gap junction changes in astrocytes occur independently of glutamine accumulation and swelling.