Influence of age on cerebral recovery after deep hypothermic circulatory arrest in piglets.

BACKGROUND: In the first weeks of life there are important maturational changes in the central nervous system in many species in energy metabolism, synapse number, and concentration of neuronal excitatory receptors. METHODS: Four groups of 10 piglets (aged 1, 2, 4, and 10 weeks) underwent 1 hour of deep hypothermic circulatory arrest at 15 degrees C, with cooling and rewarming on cardiopulmonary bypass. Cerebral blood flow and metabolic rate measurements and electroencephalographic recordings were obtained from 5 animals per group. The remaining animals underwent cerebral magnetic resonance spectroscopy. RESULTS: Preoperative cerebral blood flow and glucose consumption were higher at 4 and 10 weeks than at 1 and 2 weeks. Cerebral adenosine triphosphate content decreased more rapidly during deep hypothermic circulatory arrest at 4 and 10 weeks. Phosphocreatine recovery was greater at 30 minutes of reperfusion at 10 weeks compared with 1 week. Recovery of cerebral phosphocreatine/ adenosine triphosphate ratio and intracellular pH was remarkably uniform at all ages. Latency to recovery of electroencephalographic activity decreased with increasing age (p = 0.04). CONCLUSIONS: Differences in acute recovery of brain energy metabolism and electroencephalogram after cardiopulmonary bypass and 1 hour of deep hypothermic circulatory arrest in piglets between 1 and 10 weeks of age are small. Further studies are required to correlate these acute findings with subsequent neurologic outcome.

Racial differences in parathyroid hormone dynamics.

Elevations in PTH levels have been reported in black subjects. Such observations have not been consistent, however, and seem paradoxical in view of the known bone-resorptive action of this hormone and the fact that black subjects have a higher bone mineral density and fewer fractures than their white counterparts. In this study, we used dynamic stimulation of the calcium-PTH axis to fully characterize potential racial differences in PTH dynamics. We, therefore, defined the inverse sigmoidal curve that describes the relationship between serum ionized calcium concentration and intact PTH levels in six normal white and six normal black volunteers and determined the four parameters that characterize this relationship. An elevation in any one of these parameters can result in hyperparathyroidism. Black subjects had higher maximal and minimal PTH responses to hypo- and hypercalcemia (mean intact PTH levels of 9.2 +/- 13 and 0.7 +/- 0.1 pmol/L respectively) than white subjects (6.9 +/- 0.6 and 0.3 +/- 0.1 pmol/L, respectively). There were no differences in the set-points or slopes of the curves. Despite the higher baseline and stimulated endogenous PTH levels in black subjects, their baseline and stimulated osteocalcin levels were lower. Our dynamic studies, therefore, document mild hyperparathyroidism in black subjects and suggest mild skeletal resistance to PTH.

Effects of cerebroplegic solutions during hypothermic circulatory arrest and short-term recovery.

UNLABELLED: Previous studies have suggested that a simple crystalloid "cerebroplegic" solution may prolong the safe duration of hypothermic circulatory arrest. We tested the hypothesis that pharmacologic modification of the cerebroplegic solution would further enhance cerebral protection. Forty-six 4-week-old miniature piglets underwent core cooling to 15 degrees C nasopharyngeal temperature and 2 hours of hypothermic circulatory arrest. Twelve animals had a 50 ml/kg dose of saline infused into the carotid artery system at the onset of hypothermic circulatory arrest and repeat doses of 10 ml/kg every 30 minutes during arrest. Eleven animals received the same initial and repeat doses of University of Wisconsin organ preservation solution and 10 received University of Wisconsin solution with 7.5 mg/L of MK-801, an excitatory neurotransmitter antagonist. In 13 control animals blood was partially drained from the piglet before 2 hours of circulatory arrest at 15 degrees C and no cerebroplegic solution was infused. All solutions were delivered at 4 degrees C. Brain temperature (n = 24) at the onset of hypothermic circulatory arrest was 15.0 degrees +/- 0.1 degrees C (mean +/- standard error). Brain temperature after cerebroplegic infusion dropped to 13.0 degrees +/- 0.3 degrees C and stayed lower than brain temperature in the control group throughout the hypothermic circulatory arrest period. Recovery of cerebral adenosine triphosphate and intracellular pH determined by phosphorus 31 magnetic resonance spectroscopy (n = 22) was significantly improved by saline infusion and was further improved with University of Wisconsin solution and University of Wisconsin solution plus MK-801 (p < 0.001). Recovery of cerebral blood flow measured by microspheres (n = 24) also was augmented by University of Wisconsin solution (p < 0.001) but not in the presence of MK-801. The vascular resistance response to acetylcholine and nitroglycerin suggested that MK-801 has a direct vasoconstrictive effect. Recovery of cerebral oxygen consumption (n = 24) was increased by University of Wisconsin solution and University of Wisconsin solution with MK-801 (p = 0.002). Brain water content (n = 46) was significantly lower in all cerebroplegia-treated groups than in controls (p < 0.001). CONCLUSION: Cerebroplegia improves short-term recovery after 2 hours of circulatory arrest in hypothermic piglets. Pharmacologic modification with University of Wisconsin solution further improves the recovery of cerebral blood flow and metabolism. MK-801 does not augment the protective effects of University of Wisconsin solution and reduces the recovery of cerebral blood flow by a direct vascular action. Modified cerebroplegia may provide a novel approach to improved cerebral protection when prolonged hypothermic circulatory arrest is necessary.

Effects of aprotinin on acute recovery of cerebral metabolism in piglets after hypothermic circulatory arrest.

Brain protection during cardiopulmonary bypass and hypothermic circulatory arrest is incomplete. Activation of blood protease cascades may contribute to cellular injury under these conditions. To test this hypothesis, effects of the protease inhibitor aprotinin on recovery of brain energy metabolism after hypothermic circulatory arrest were studied in the piglet. Twenty-four 4-week-old piglets (10 aprotinin-treated and 14 control) underwent core cooling, 1 hour of circulatory arrest at 15 degrees C, reperfusion and rewarming (45 minutes), and normothermic perfusion (3 hours) on cardiopulmonary bypass. Cerebral high-energy phosphate concentration and intracellular pH were studied by phosphorus-31 magnetic resonance spectroscopy in 12 animals. In the remaining animals cerebral and regional blood flow were measured with radioactive microspheres and carotid artery blood flow was measured with an electromagnetic flowmeter. Cerebral oxygen and glucose extraction were measured, and vascular resistance responses to endothelium-dependent (acetylcholine) and -independent (nitroglycerin) vasodilators were calculated. Recovery of cerebral adenosine triphosphate (p = 0.02) and intracellular pH (p = 0.04) in the initial 30 minutes of reperfusion was accelerated in the aprotinin-treated piglets. These piglets showed a greater in vivo cerebral and systemic endothelium-mediated vasodilation (acetylcholine response: cerebral p < 0.01, systemic p = 0.04) after reperfusion. The response to endothelium-independent vasodilation (nitroglycerin) was the same in both groups. Carotid blood flow tended to be greater at 20 minutes of reperfusion and less during 45 to 80 minutes after reperfusion in the aprotinin-treated animals. Brain water content postoperatively was 0.8077 in the aprotinin group and 0.8122 in control animals (p = 0.06).(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical shift imaging of particle filtration in sandstone cores.

Recent developments have led to increased interest in the application of borehole nuclear magnetic resonance (NMR) as a probe of petrophysical properties. Of particular importance in this connection is the measurement of the longitudinal relaxation time, T1. As T1 is controlled by the pore surface area, its value may be strongly influenced by the invasion of submicron-sized clay particles found in drilling muds. We have studied this effect by the application of phase encode magnetic resonance imaging (MRI) techniques. The extent to which T1 values are affected by particulate invasion is found to depend strongly on the mud characteristics. With thinned spud muds there is a region deep within the core where T1 values are significantly reduced due to an initial spurt of clay particles. In better formulated muds this effect is greatly reduced.

Effects of MK-801 and NBQX on acute recovery of piglet cerebral metabolism after hypothermic circulatory arrest.

Brain protection during open heart surgery in the neonate and infant remains inadequate. Effects of the excitatory neurotransmitter antagonists MK-801 and NBQX on recovery of brain cellular energy state and metabolic rates were evaluated in 34 4-week-old piglets (10 MK-801, 10 NBQX, 14 controls) undergoing cardiopulmonary bypass and hypothermic circulatory arrest at 15 degrees C nasopharyngeal temperature for 1 h, as is used clinically for repair of congenital heart defects. MK-801 (dizocilpine) (0.75 mg/kg) or NBQX [2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline] (25 mg/kg) was given intravenously before cardiopulmonary bypass. Equivalent doses were placed in the cardiopulmonary bypass prime plus continuous infusions after reperfusion (0.15 mg kg-1h-1 and 5 mg kg-1h-1). Changes in high-energy phosphate concentrations and pH were analyzed by magnetic resonance spectroscopy in 17 animals until 225 min after reperfusion. Cerebral blood flow determined by radioactive microspheres as well as cerebral oxygen and glucose consumption were studied in 17 other animals. Cerebral blood flow and oxygen consumption were depressed relative to control by both MK-801 and NBQX at baseline. Recovery of phosphocreatine (p = 0.010), ATP (p = 0.030), and intracellular pH (p = 0.004) was accelerated by MK-801 and retarded by NBQX over the 45 min of rewarming reperfusion and the first hour of normothermic reperfusion. The final recovery of ATP at 3 h and 45 min reperfusion was significantly reduced by NBQX (46 +/- 26% baseline, mean +/- SD) versus control (81 +/- 19%) and MK-801 (75 +/- 8%) (p = 0.030). Cerebral oxygen consumption recovered to 105 +/- 30% baseline in group MK-801 and 94 +/- 31% in control but only to 61 +/- 22% in group NBQX (p = 0.070). Cerebral blood flow stayed significantly lower in group NBQX relative to control. Thus, MK-801 accelerates recovery of cerebral high-energy phosphates and metabolic rate after cardiopulmonary bypass and hypothermic circulatory arrest in the immature animal. At the dosage used NBQX exerts an adverse effect.

Effects of pH on brain energetics after hypothermic circulatory arrest.

The pH management that provides optimal organ protection during hypothermic circulatory arrest is uncertain. Recent retrospective clinical data suggest that the pH-stat strategy (maintenance of pH at 7.40 corrected to core temperature) may improve brain protection during hypothermic cardiopulmonary bypass with a period of circulatory arrest in infants. The impact of alpha-stat (group A) and pH-stat (group P) strategies on recovery of cerebral high-energy phosphates and intracellular pH measured by magnetic resonance spectroscopy (A, n = 7; P, n = 5), organ blood flow measured by microspheres, cerebral metabolic rate measured by oxygen and glucose extraction (A, n = 7; P, n = 6), and cerebral edema was studied in 25 4-week-old piglets undergoing core cooling and 1 hour of circulatory arrest at 15 degrees C. Group P had greater cerebral blood flow during core cooling (54.3% +/- 4.7% versus 34.2% +/- 1.5% of normothermic baseline, respectively; p = 0.001). The intracellular pH during core cooling showed an alkaline shift in both groups but became more alkaline in group A than in group P at the end of cooling (7.08 to 7.63 versus 7.09 to 7.41, respectively; p = 0.013). Recovery of cerebral adenosine triphosphate (p = 0.046) and intracellular pH (p = 0.014) in the initial 30 minutes of reperfusion was faster in group P. The cerebral intracellular pH became more acidotic during early reperfusion in group A, whereas it showed continuous recovery in group P. Brain water content postoperatively was less in group P (0.8075) than in group A (0.8124) (p = 0.05). These results suggest that compared with alpha-stat, the pH-stat strategy provides better early brain recovery after deep hypothermic cardiopulmonary bypass with circulatory arrest in the immature animal. Possible mechanisms include improved brain cooling by increased blood flow to subcortical areas, improved oxygen delivery, and reduction of reperfusion injury, as well as an alkaline shift in intracellular pH with hypothermia in spite of a stable blood pH.

Application of missing pulse steady state free precession to the study of renal microcirculation.

Missing pulse steady state free precession (MP-SSFP), an extension of steady state free precession (SSFP), was evaluated for its ability to measure slow fluid flows. In experiments using flow phantoms, the MP-SSFP signal was sensitive to fluid velocities in the millimeters per second range. Isolated perfused rabbit kidneys were then used to determine if MP-SSFP could measure perfusion in a biological tissue. The signal intensities in the different anatomical regions of the kidney were observed to be related to the total flow to the organ. Furthermore, increasing the flow sensitivity of the pulse sequence by increasing the gradient strength resulted in decreases in the image signal intensity. The MP-SSFP signal was more sensitive to flow in the medulla than in the cortex. This can be related to slow flow sensitivity of MP-SSFP and the known differences in velocity profiles between these two regions. These results suggest that MP-SSFP may be a powerful tool for the noninvasive measurement of slow fluid flows in different regions of the kidney.

Regional 1H transverse magnetization studies in perfused rabbit kidney.

A Carr-Purcell-Meiboom-Gill imaging sequence consisting of 128 echoes is used to extract transverse magnetization decay curves (TDCs) at 1.9 T from 1.7 x 1.7 x 5-mm3 voxels within the cortex, outer medulla, and inner medulla of perfused rabbit kidneys. The spatially localized TDCs within each tissue type are found to be better approximated by biexponential, as opposed to monoexponential, functions. The biexponential parameters characterizing the TDCs demonstrate an improved degree of tissue specificity over that available from monoexponential analyses. The fraction of the quickly relaxing TDC component and the relaxation rate of this component are observed to decrease from cortex to inner medulla. A two-site exchange analysis is used to convert biexponential TDC parameters into water volume fractions and exchange rates. The exchange rates between the fast and slowly relaxing pools increased from cortex to inner medulla. All exchange rates were less than 1.5 Hz, indicating a relatively slow water exchange process. The imaging methods and subsequent analyses offer the potential to generate unconventional MR images with tissue contrast dependent upon water compartmentation and exchange.

Mitochondrial injury: an early event in cisplatin toxicity to renal proximal tubules.

Oxygen consumption (QO2) and net K+ transport were studied in rabbit proximal tubule suspensions to define the early effects of cisplatin on proximal tubule function. Cisplatin caused dose-dependent inhibition of QO2, which was delayed in onset. The concentration of cisplatin required for inhibition decreased as the duration of exposure was increased [40-min exposure, threshold concentration of 10(-4) M, inhibitor constant (Ki) of 10(-3) M; 4-h exposure, threshold concentration of 3 X 10(-5) M, Ki of 10(-4) M]. Both ouabain-sensitive and ouabain-insensitive QO2 were reduced, indicating inhibition of all adenosinetriphosphatases, including Na(+)- K(+)-ATPase activity. There was a parallel fall in ouabain-sensitive net K+ transport and cytosolic K+ content, confirming the latter observation. Na(+)-K(+)-ATPase activity was unchanged in cell membranes prepared by hypotonic lysis from cisplatin-treated tubules, indicating an indirect cytosol-dependent mechanism of enzyme inhibition. Nystatin-stimulated QO2 was reduced in cisplatin-treated tubules, excluding inhibition of Na+ entry as the mechanism of injury and suggesting mitochondrial injury. The latter was confirmed by measurement of carbonylcyanide-m-chlorophenylhydrazone (CCCP)-uncoupled QO2 in intact cells and ADP-stimulated (state 3) QO2 in digitonin-permeabilized tubules. Furthermore, by maximally stimulating mitochondrial respiration with CCCP and nystatin, it was possible to demonstrate mitochondrial injury at a time when basal QO2 and K+ transport were apparently normal. These data suggest that mitochondrial injury is a central event in cisplatin toxicity to the proximal tubule.

Characterization of the major brain osmolytes that accumulate in salt-loaded rats.

Previous studies demonstrated an accumulation of "idiogenic osmoles" in the brain with chronic salt loading. Amino acids are known to constitute a portion of these solutes, but the balance of the solutes has yet to be fully characterized. In the present study, 1H-nuclear magnetic resonance (NMR) spectroscopy and biochemical assays of rat brain were used to identify and quantify changes in organic solutes in two different animal models of hypernatremia: hypertonic salt loading and water deprivation. Five days of salt loading increased plasma sodium concentration (PNa) to 165 meq/l and 3 days of water deprivation increased PNa to 151 meq/l, compared with 141 meq/l in controls. Amino acids, methylamines, and polyols were all significantly higher in salt-loaded animals compared with controls. Specifically, higher contents of glutamine (+65%), glutamate (+27%), myo-inositol (+36%), phosphocreatine + creatine (PCr + Cr) (32%), glycerophosphorylcholine (GPC) (+75%), and choline (+114%) were observed. Sorbitol and betaine, osmolytes known to accumulate in the hypertonic inner medulla, were present in low amounts in the brain and were unchanged with salt loading. In contrast to the results with salt loading, no accumulation of brain organic solutes was detected after 3 days of water deprivation. Based on these findings, we propose that amino acids, methylamines, and polyols function as osmoregulatory solutes in the brains of salt-loaded rats in a manner similar to that observed in other biological systems, whereas 3 days of water deprivation is an insufficient stimulus for their accumulation.

Methylamine and polyol responses to salt loading in renal inner medulla.

Methylamines and polyhydric alcohols (polyols) are major organic osmolytes of the mammalian renal inner medulla and have generally been noted to change in parallel with urine osmolality. In the present study, responses of inner medullary methylamines and polyols to 5 days of salt loading were investigated. Salt loading increased plasma sodium concentration and induced a saline diuresis that resulted in a significantly lower urine osmolality (Uosmol) in salt-loaded rats (1,246 mosmol) compared with controls (2,147 mosmol). Analysis of inner medullary organic osmolytes using 1H-NMR spectroscopy and biochemical assays indicated no significant change in total methylamines, total polyols, or total osmolytes with salt loading. However, there were marked changes in individual organic osmolytes. Renal inner medullary glycerophosphorylcholine (GPC) was 41% lower in salt-loaded rats, and was the only organic osmolyte that changed in parallel with Uosmol, which was 42% lower in this group. In contrast, glycine betaine (betaine) and sorbitol contents were elevated by 286% and 33%, respectively, with salt loading, and myo-inositol (inositol) was unchanged. These findings indicate selective renal inner medullary osmolyte responses to salt loading with only GPC varying directly with changes in urine osmolality.

Methylamines and polyols in kidney, urinary bladder, urine, liver, brain, and plasma. An analysis using 1H nuclear magnetic resonance spectroscopy.

Methylamines and polyols are known to behave as organic osmolytes in the adaptation of many cells to hyperosmolar conditions. Using 1H nuclear magnetic resonance spectroscopy to analyze perchloric acid extracts we have examined several tissues in the rat for the presence of these compounds. Methylamines such as glycerophosphorylcholine, choline and betaine were observed in the renal inner medulla, urinary bladder, urine, liver, brain, and plasma. Myoinositol was relatively abundant in the renal inner medulla and brain whereas sorbitol was detected only in the inner medulla. A variety of unidentified compounds was also detected in each tissue. Although these methylamines and polyols are known to respond to osmotic changes in the renal inner medulla, their responses in other tissues remain to be investigated.

Organic osmolytes in inner medulla of Brattleboro rat: effects of ADH and dehydration.

Inner medullary methylamine [glycerophosphorylcholine (GPC) and glycine betaine (betaine)] and polyol [sorbitol and myo-inositol (inositol)] osmolytes were measured in water-restricted and antidiuretic hormone (ADH)-infused Brattleboro (DI) rats. Compared with DI rats allowed water ad libitum, rats dehydrated for 3 days had higher urinary osmolality (Uosmol) (812 vs. 239 mosmol/kgH2O) and plasma osmolality (Posmol) (333 vs. 296 mosmol/kgH2O). Dehydration reduced betaine content (36 vs. 66 nmol/mg protein) but had no significant effect on GPC, sorbitol, or inositol. In separate protocols, DI rats, allowed water ad libitum, were infused for either 3 or 12 days with either ADH in saline (+ADH) or saline alone (-ADH). Compared with -ADH controls, 3- or 12-day ADH-infused rats were antidiuretic (Uosmol, 1,000-1,300 mosmol/kgH2O) but not dehydrated (Posmol, 297-300 mosmol/kgH2O). Three days of ADH infusion caused an increase in GPC (340%), betaine (80%), and sorbitol (248%) but not in inositol. After 12 days of ADH, further increases were observed in GPC (730%) and sorbitol (870%); inositol was also elevated (170%), whereas betaine was unchanged. Consequently, the total osmolyte content was significantly higher in +ADH than in -ADH [449 vs. 256 (3 days) and 778 vs. 199 (12 day) nmol/mg protein], whereas total osmolyte levels in dehydrated and control rats were similar (222 vs. 219 nmol/mg protein).(ABSTRACT TRUNCATED AT 250 WORDS)

Protection of the kidney against ischemic injury by inhibition of 5'-nucleotidase.

We have evaluated the impact of inhibiting adenine nucleotide dephosphorylation on the metabolic and functional consequences of renal ischemia. Intramuscular injection of the ADP-analogue adenosine alpha, beta-methylene diphosphate (AMP-CP) achieved a 70% reduction in 5'-nucleotidase activity, as measured in crude extracts of rat kidney. AMPCP-treated animals had an increased residual nucleotide pool at the end of 45 min of ischemia compared with untreated rats. Assessment of renal ATP by 31P-nuclear magnetic resonance (31P-NMR) in vivo during reflow demonstrates the following: 1) higher rapid initial recovery of ATP (69.3 +/- 1.2 vs. 50.0 +/- 0.5% control value, P less than 0.005), 2) accelerated rate of ATP restoration (0.20 +/- 0.02 vs. 0.11 +/- 0.01% control/min, P less than 0.005), and 3) significantly enhanced renal ATP content after 120 min (93.6 +/- 2.0 vs. 63.1 +/- 0.7% control, P less than 0.005). Kidney function, as measured by the rate of inulin clearance 24 h after the insult, was also significantly improved in AMPCP-treated rats (725 +/- 50 vs. 313 +/- 28 microliters.min-1.100 g body wt-1). Thus inhibition of 5'-nucleotidase results in enhanced metabolic and functional recovery from a renal ischemic insult.

Metabolic and functional consequences of inhibiting adenosine deaminase during renal ischemia in rats.

The concentrations of renal ATP have been measured by 31P-nuclear magnetic resonance (NMR) before, during, and after bilateral renal artery occlusion. Using in vivo NMR, the initial postischemic recovery of ATP increased with the magnitude of the residual nucleotide pool at the end of ischemia. ATP levels after 120 min of reflow correlated with functional recovery at 24 h. In the present study the effect of blocking the degradation of ATP during ischemia upon the postischemic restoration of ATP was investigated. Inhibition of adenosine deaminase by 80% with the tight-binding inhibitor 2'-deoxycoformycin led to a 20% increase in the residual adenine nucleotide pool. This increased the ATP initial recovery after 45 min of ischemia from 52% (in controls) to 62% (in the treated animals), as compared to the basal levels. The inhibition also caused an accelerated postischemic restoration of cellular ATP so that at 120 min it was 83% in treated rats vs. 63% in untreated animals. There was a corresponding improvement in the functional recovery from the insult (increase of 33% in inulin clearance 24 h after the injury). Inhibition of adenosine deaminase during ischemia results in a injury similar to that seen after a shorter period of insult.

Chemical and functional correlates of postischemic renal ATP levels.

Renal energy metabolism was investigated before, during, and after ischemic insults of varying durations with in vivo 31P NMR spectroscopy. The postischemic recovery of renal ATP was found to be a biphasic process regardless of the length of the ischemia. This two-stage recovery consisted of a quick initial component immediately upon reflow followed by a slower, more gradual return toward preischemic levels. To characterize the source of each phase of the recovery, kidneys were extracted with perchloric acid at the end of the different periods of ischemia (before reflow). Concentrations of adenine nucleotides and breakdown products adenosine, inosine, and hypoxanthine were determined by 1H NMR spectroscopy. Excellent correlation was found between the residual nucleotide pool and the magnitude of the initial phase of ATP recovery. Additionally, the renal ATP content after 120 min of reflow was shown to have a strong correlation with subsequent functional recovery. These experiments show that in vivo 31P NMR can provide new and dynamic information concerning the biochemical recovery from ischemia. Furthermore, this data has the potential to predict the eventual functional recovery of the organ.

Postischemic ATP-MgCl2 provides precursors for resynthesis of cellular ATP in rats.

Postischemic administration of ATP-MgCl2 is known to enhance recovery of renal function and accelerate the restitution of cellular ATP levels. To differentiate between a direct and indirect effect of the exogenous nucleotide, rats were subjected to 45 min of bilateral renal ischemia and were infused with either ATP-MgCl2, AMP-MgCl2, or normal saline. The immediate recovery of the cellular ATP was similar in all three groups of animals, whereas the subsequent recovery was accelerated by the infusion of either nucleotide. Since ATP-MgCl2 and AMP-MgCl2 produced similar results, this study provides evidence that exogenous ATP may act by providing precursors for the resynthesis of the cellular adenine nucleotide pool rather than being a direct source of energy.

Hepatic gluconeogenesis from alanine: 13C nuclear magnetic resonance methodology for in vivo studies.

This paper describes an experimental protocol designed to optimize 13C NMR spectra from the liver of the living anesthetized rat at 1.9 T. The protocol involves the use of a Helmholtz NMR coil which is positioned around the liver after surgical exposure. 1H decoupling is facilitated by double tuning this coil to both the 1H and the 13C frequencies. The protocol was shown to be suitable for studying the hepatic metabolism of 13C-labeled substrates in vivo by investigating the metabolism of [3-13C]alanine. Labeled glucose, glutamate, glutamine, and aspartate were formed and detected by 13C NMR in vivo in this experiment. The labeling patterns in these metabolites provided evidence that the major flow of alanine carbon into the Krebs cycle is via the pyruvate carboxylase reaction rather than through pyruvate dehydrogenase.