Effects of intravenous insulin-like growth factor-I and insulin administration on insulin-like growth factor-binding proteins in the ovine fetus.

The insulin-like growth factors (IGF) are important anabolic hormones in the mammalian fetus; their anabolic actions are potentially modulated by alterations in the IGF-binding proteins (IGFBP). We have previously shown that the nutritional state of the fetus affects both IGF-I and the IGFBP concentrations. The present study was designed to determine the effect of alterations in insulin and IGF-I circulating concentrations on the IGFBPs. Because both insulin and IGF-I elicit decreases in glucose and amino acid concentrations, the concentrations of these substrates were clamped during the hormone infusions. Sixteen ovine fetuses were chronically catheterized at approximately 115 days of gestation, and experimental procedures performed at approximately 130 days of gestation. Insulin, IGF-I or both were infused for an 8-h period. Baseline concentrations of hormones and binding proteins were obtained, and concentrations were also obtained at the end of the infusion. Hepatic IGFBP-1 mRNA expression was also determined. Intravenous infusion of IGF-I significantly increased IGF-I concentrations in plasma in the ovine fetus. Intravenous infusion of insulin inhibited hepatic IGFBP-1 gene expression when amino acids and glucose were clamped. In contrast, intravenous infusion of recombinant human IGF-I (rhIGF-I) enhanced hepatic IGFBP-1 gene expression. Neither insulin nor rhIGF-I treatment had an effect on hepatic IGFBP-3 gene expression. Insulin did not alter plasma IGFBP-1 significantly, but it increased IGFBP-3 in plasma. rhIGF-I increased both IGFBP-1 and IGFBP-3 protein levels in plasma. The responses of IGFBP-1 and IGFBP-3 to increased plasma IGF-I and insulin may serve to protect the fetus from exaggerated anabolic effects and to blunt the hypoglycemic potential of circulating IGFs and insulin.

Glucose and amino acid kinetic response to graded infusion of rhIGF-I in the late gestation ovine fetus.

Insulin-like growth factor I (IGF-I) has anabolic effects and is thought to be important in fetal development. The present study was designed to determine the dose response of recombinant human (rh) IGF-I on ovine fetal glucose and amino acid kinetics. Chronically catheterized fetal lambs were studied at 122-127 days gestation. The kinetics of leucine, phenylalanine, and glucose were measured before and during the infusion of rhIGF-I. rhIGF-I was infused into the fetal inferior vena cava at low, medium, or high rates (9.9, 20.1, or 40.2 nmol/h, respectively). A stepwise increase in serum IGF-I was achieved (164 +/- 3, 222 +/- 7, and 275 +/- 5 ng/ml). Insulin concentrations were decreased at the medium and high rhIGF doses. The rate of appearance (Ra) of leucine and phenylalanine and leucine oxidation decreased. Phenylalanine appearance from protein breakdown was decreased, with a maximal suppression of 30% observed at the highest rate of infusion. Glucose Ra was increased at the medium and high doses; other aspects of glucose metabolism were unchanged. The change in both glucose Ra and suppression of proteolysis was significantly correlated to the rhIGF-I infusion rate. It is concluded that rhIGF-I exerts dose-related effects in the ovine fetus, increasing fetoplacental glucose turnover and causing significant suppression of both proteolysis and amino acid oxidation.

Aromatic amino acids are utilized and protein synthesis is stimulated during amino acid infusion in the ovine fetus.

The purpose of this study was to determine whether the ovine fetus is capable of increased disposal of an amino acid load; if so, would it respond by increased protein synthesis, amino acid catabolism or both? A further purpose of the study was to determine whether the pathways of aromatic amino acid catabolism are functional in the fetus. Late gestation ovine fetuses of well-nourished ewes received an infusion of Aminosyn PF alone (APF), and Aminosyn PF + glycyl-L-tyrosine (APF+GT) at rates estimated to double the intake of these amino acids. The initial study, using APF, was performed at 126 +/- 1.4 d; the APF+GT study was performed at 132 +/- 1.7 d (term = 150 d). Phenylalanine and tyrosine kinetics were determined using both stable and radioactive isotopes. Plasma concentrations of most amino acids, but not tyrosine, increased during both studies; tyrosine concentration increased only during the APF+GT study. Phenylalanine rate of appearance and phenylalanine hydroxylation increased during both studies. Tyrosine rate of appearance increased only during the APF+GT study; tyrosine oxidation did not increase during either study. Fetal protein synthesis increased significantly during both studies, producing a significant increase in fetal protein accretion. Fetal proteolysis was unchanged in response to either amino acid infusion. These results indicate that the fetus responds to an acute increase in amino acid supply primarily by increasing protein synthesis and accretion, with a smaller but significant increase in amino acid catabolism also. Both phenylalanine hydroxylation and tyrosine oxidation are active in the fetus, and the fetus is able to increase phenylalanine hydroxylation rapidly in response to increased supply.

Effect of rhIGF-I infusion on whole fetal and fetal skeletal muscle protein metabolism in sheep.

Insulin-like growth factor I (IGF-I) has been shown to have significant anabolic effects in the regulation of fetal protein metabolism. To investigate the tissue-specific effects of IGF-I on fetal skeletal muscle metabolism, we infused recombinant human (rh) IGF-I directly into the hindlimb of nine chronically catheterized, late-gestation fetal sheep. Substrate balance and amino acid kinetics were measured across the hindlimb and were compared with the effects at the whole body level before and during a 3-h infusion of rhIGF-I into the external iliac artery at 150 microgram/h. Infusion of rhIGF-I resulted in increases in IGF-I concentrations by 2- to 5. 75-fold in the ipsilateral iliac vein and by nearly 3-fold in the abdominal aorta. In the study limb, IGF-I had no effect on protein synthesis (phenylalanine rate of disposal 0.88 +/- 0.13 before vs. 0. 73 +/- 0.19 micromol/min during IGF-I) or breakdown (phenylalanine rate of appearance 0.67 +/- 0.13 before vs. 0.60 +/- 0.17 micromol/min during IGF-I) and did not alter net phenylalanine balance. IGF-I also did not affect hindlimb oxygen or glucose uptake. In contrast, at the whole body level, the rate of appearance of leucine, indicative of fetal protein breakdown, decreased during IGF-I infusion (rate of appearance of leucine 41.1 +/- 3.3 to 37.6 +/- 2.7 micromol/min) as did fetal leucine oxidation (8.4 +/- 0.8 to 6.8 +/- 0.6 micromol/min). There was no change in the umbilical uptake of leucine, and although not statistically significant, fetal leucine accretion increased 2.4-fold. These results provide further evidence that IGF-I promotes fetal protein accretion; however, its site of action is in tissues other than skeletal muscle.

Variation in long-term engraftment of a large consecutive series of lambs transplanted in utero with human hematopoietic cells.

We investigated the survival and chimeric engraftment characteristics of a large consecutive series of lambs that were transplanted with human hematopoietic cells in utero. Approximately 50% of the fetal sheep survived. Neither the transplantation of human cells into fetal sheep, nor the parity of the ewe was associated with increased mortality, as compared with the risk of surgery alone. However, a breed-associated mortality was noted. Sixty percent of surviving recipient lambs contained donor, human hematopoietic cells in blood and bone marrow (BM) cells. Chimerism ranged from 0.0001-1%. Human hematopoietic progenitors were identified in the BM in 8 of 12 chimeric sheep examined. Some lambs engrafted with human cells maintained a human chimerism for up to at least 2 years. Our data demonstrate that a large proportion of fetal sheep are capable of engrafting human cells, albeit at widely variable levels of engraftment.

Effects of circulating IGF-I on glucose and amino acid kinetics in the ovine fetus.

To investigate the role of insulin-like growth factor I (IGF-I) in the regulation of fetal metabolism, the kinetics of leucine, phenylalanine, and glucose were assessed in the chronically catheterized ovine fetus (0.85 gestation) before and during infusion of recombinant human IGF-I (rhIGF-I). Substrate kinetics were determined by tracer dilution. rhIGF-I was infused at 6.7 nmol.kg fetus-1.h-1. Fetal insulin and growth hormone concentrations were significantly decreased by 50% during rhIGF-I infusion. Net umbilical glucose uptake was unchanged, and glucose rate of appearance increased in the fed state only. There were no changes in the net umbilical uptakes of leucine or phenylalanine, but the rates of appearance of both declined during rhIGF-I infusion, indicative of decreased fetal protein breakdown (Ra,Leu 45.4 +/- 1.40 to 40 +/- 1.4 mumol/min in the fed state, 43 +/- 1.5 to 37 +/- 1.5 mumol/min in the fasted state; Ra,Phe 10.7 +/- 0.3 to 10.4 +/- 0.3 mumol/min in the fed state and from 10.7 +/- 0.3 to 9.8 +/- 0.3 mumol/min in the fasted state). Leucine oxidation was also decreased (8.90 +/- 0.76 to 6.52 +/- 0.81 mumol/min, P = 0.025), more so in the fasted than the fed state. These results indicate a significant antiproteolytic endocrine effect for IGF-I in the late-gestation mammalian fetus.

Comparison of steady-state diffusion and transit time ultrasonic measurements of umbilical blood flow in the chronic fetal sheep preparation.

OBJECTIVE: Our purpose was to measure umbilical blood flow continuously by use of a transit time ultrasonic flow transducer and to compare the blood flow measurements with the steady-state diffusion method in the chronic fetal sheep preparation. STUDY DESIGN: We compared umbilical blood flow measurements calculated by the steady-state diffusion method with ethanol as the diffusing substance and with the transit time ultrasonic flow transducer placed on the common umbilical artery in five chronically prepared fetal sheep. RESULTS: There was no statistical difference between measurements of umbilical blood flow measured by the flow transducer versus the steady-state diffusion method, 600 +/- 22 versus 664 +/- 56 ml per minute (mean +/- SEM) (p = 0.23). The mean coefficient of variation within each study was 13.6% for the steady-state diffusion method versus 4.1% for the transit time flow transducer. Umbilical blood flow variance was significantly lower as measured by the flow transducer compared with the diffusion method (p < 0.0001). There were no differences in umbilical blood flow per kilogram or fetal oxygen uptake between the two methods. CONCLUSION: We conclude that umbilical blood flow can be measured continuously under steady-state conditions by use of a transit time flow transducer. Because of the lower variability in the flow transducer-obtained measurements, we speculate that the flow transducer may differentiate alterations in umbilical blood flow with greater precision in chronic preparations. This may be advantageous for measuring absolute changes in fetal substrate uptake, especially under non-steady-state conditions.

Effect of prolonged uterine blood flow reduction on fetal growth in sheep.

The purpose of the present investigation was to study the effect of 7 days of uterine blood flow reduction on fetal growth. Reduction in uterine blood flow was accomplished by external occlusion of the terminal aorta in 20 pregnant sheep. Linear growth was monitored daily by means of a crown-rump length measuring device. The deliveries of oxygen, glucose, and lactate to the fetus, as well as their uptakes by the fetus, were determined before and after 7 days of uterine blood flow reduction and correlated with rates of fetal growth. Identical studies were conducted in nine control animals. Uterine blood flow reduction resulted in a significant decrease in fetal oxygen delivery and fetal arterial oxygen content. Linear growth rate decreased by 38% in the occluded animals during hypoxemia. In addition, there was a 20% reduction in daily weight gain in occluded animals compared with controls. There were no differences in the uptakes of oxygen, glucose, and lactate by the fetus. Positive correlations were found between linear growth rate and fetal arterial oxygen content (r2 = 0.25, P = 0.0001) and between linear growth rate and fetal oxygen delivery (r2 = 0.21, P = 0.0006). The correlations between linear growth rate and fetal oxygenation provide strong evidence of the central role of oxygen in the regulation of fetal growth.

Bilirubin index: a new standard for intervention?

The current practice for treating neonates for jaundice centers on the recommendation that bilirubin levels should be kept below 20 mg/dL. Preventing bilirubin levels from exceeding 20 mg/dL, however, does not guarantee the avoidance of kernicterus, lower IQs or neurologic abnormalities. Studies in the 1960s and 1970s reported cases of infants with clinical and pathological kernicterus whose neonatal bilirubin levels were well below 20 mg/dL. It is now well accepted that protein binding, acidosis, hypoxia, intracranial hemorrhage and hemolytic disease play a role in facilitating bilirubin toxicity. This paper reviews previously published studies that were instrumental in identifying the role of hypoxia, acidosis, hemolytic disease, intracranial hemorrhage and protein binding in bilirubin encephalopathy and identifies two key variables which contribute to bilirubin flux-free bilirubin concentration and time. The paper proposes a new approach for evaluating bilirubin levels termed 'bilirubin index'. Future research should initially focus on healthy term infants without concomitant illness and should record free bilirubin levels as a function of time. The area under the bilirubin versus time curve represents the integration of bilirubin level with respect to time, or simply termed the 'bilirubin index'. The bilirubin index could then be correlated with parameters for measuring neurological outcome. Assuming a correlation would exist, the bilirubin index may then become the number for guidance with respect to intervention therapy. Attempting to address this issue by starting with a healthy population of neonates and correlating bilirubin index with neurological outcome offers a better chance for uncovering that 'threshold of toxicity'.

Collateral arterial blood supply to the pregnant uterus in the sheep.

The common internal iliac artery in the sheep gives rise to the arteries which are the primary source of blood to the uterus. We have used a vascular occluder placed on the common internal iliac artery of the pregnant sheep to induce and study the effects of chronic fetal hypoxaemia. We have noted that uterine blood flow and fetal oxygenation improve within 5 d of occlusion of the common internal iliac artery. To investigate the basis of this observation we performed serial angiograms and postmortem examinations of the uterine arterial circulation in pregnant sheep under normal conditions and after 7 d of blood flow occlusion. We identified distal anastomoses on the surface of the uterus between the middle uterine and internal pudendal arteries by way of the urogenital arteries and their branches. Furthermore, the internal pudendal artery filled retrogradely through other branches when the vessel was ligated at its origin. The development of a collateral circulation to the uterus in response to chronic common internal iliac artery occlusion is a physiological adaptation to maintain an adequate supply of blood to the uterus and thus to maintain fetal oxygen delivery.

Amino acid uptake by the fetal ovine hindlimb under normal and euglycemic hyperinsulinemic states.

As part of an effort to establish the contribution of different fetal organs to fetal amino acid metabolism, we measured in nine sheep fetuses the uptake of 27 amino acids by the hindlimb under normal conditions and conditions by euglycemic hyperinsulinemia. The fetal hindlimb is representative of nonvisceral tissues, which in the mature fetus account for approximately 70% of fetal weight and 30% of fetal O2 consumption. In the normal condition, there was a significant uptake of 21 amino acids for a net total nitrogen uptake of 132 +/- 21 mg N.day-1 x 100 g-1. The amino acids taken up by the fetal limb included alanine and glutamine. In addition, the fetal limb had significant glutamate and serine uptakes. Because glutamate flows from fetus to placenta and there is no fetal uptake of maternal serine, this indicates production and interorgan transport of these amino acids within the fetus. Insulin infusion significantly decreased the arterial concentration of every amino acid with the exception of cystathionine and significantly increased limb blood flow and glucose uptake. It significantly increased the limb uptake of alanine, asparagine, glycine, isoleucine, methionine, and tyrosine, decreased the uptake of aspartate, and produced no significant change in the net total nitrogen uptake, which remained similar to control (137 +/- 16 mg N.day-1 x 100 g-1).

Increased fetal glucose concentration decreases ovine fetal leucine oxidation independent of insulin.

Fetal leucine oxidation rate is elevated during fasting of the ewe. Euglycemic hyperinsulinemia causes the leucine oxidation rate to decline. However, it is unclear whether this is a direct effect of insulin or is secondary to increased insulin-mediated glucose utilization. To better delineate the mechanism of decreased oxidation, we suppressed fetal insulin secretion by somatostatin infusion. Glucose was infused at a variable rate to achieve glucose concentrations 125 and 150% of basal. Leucine rate of appearance (Ra) was determined by infusion of [15N, 1-13C]leucine. Fraction of leucine appearance oxidized was determined by [1-14C]leucine infusion and determination of fetal 14CO2 excretion. Each fetus was studied during ad libitum maternal feeding and after a 5-day complete maternal fast. Changes were noted in fetal leucine oxidation, which declined from 8.4 +/- 1.2 to 5.0 +/- 0.8 mumol/min in the fed state during glucose infusion. Basal leucine oxidation was elevated during fasting (11 +/- 1.5 mumol/min, P < 0.05) and declined to 8.0 +/- 1.4 mumol/min during glucose infusion (P = 0.056). Leucine carbon Ra was unchanged by fasting and by glucose infusion; leucine nitrogen Ra declined in the fed state only. Leucine oxidation was inversely correlated with glucose concentration (oxidation = 12-0.26 x glucose concentration, r = 0.42, P = 0.004). Leucine oxidation was not correlated with insulin concentration (r = 0.2). Changes in fetal glucose concentration may alter the pattern of utilization of essential amino acids, independent of changes in insulin and insulin-mediated glucose utilization rate.

Fetal pH improvement after 24 h of severe, nonlethal hypoxia.

The accumulation of hydrogen ions in fetal blood when fetal oxygenation is acutely decreased suggests metabolic instability. However, in 6 chronically prepared fetal sheep whose arterial O2 content and pO2 were abruptly reduced and maintained at about 1.7 mM and 12.6 Torr, respectively, we observed a significant decrease in fetal arterial lactate concentrations and an increase in pH to normal values after 24 h of continuous hypoxia compared with values observed after 5 h. This demonstrates that fetal pH measurements may not detect prolonged intrauterine hypoxia.

Metabolic adaptation of fetal hindlimb to severe, nonlethal hypoxia.

To test the hypothesis that an important aspect of the fetal response to severe, nonlethal hypoxia is a relatively large reduction in oxidative metabolism and small increase in lactate production by organs whose O2 supply is selectively reduced, net fluxes of O2, glucose, pyruvate, lactate, and CO2 derived from fetal plasma lactate carbon [(CO2)PL] were measured across the hindlimb and umbilical circulations in six sheep fetuses before and at 200-260 min of hypoxia. During hypoxia, blood lactate reached a high but steady level (15.2 +/- 2.2 vs. 1.7 +/- 0.2 mM; P < 0.001). Hypoxia was induced by reducing uterine blood flow. Limb O2 uptake and (CO2)PL decreased (P < 0.01) and lactate output increased (P < 0.05) (-83.1 +/- 13.9, -28.6 +/- 5.0, and +35.7 +/- 13.7 nmol.min-1 x g-1, respectively), while pyruvate and glucose uptakes remained similar to control. The increase in limb glycolysis was approximately 10% of the value that would compensate for the decrease in oxidative energy metabolism. The ratio of limb O2 uptake to fetus O2 uptake decreased significantly (0.247 +/- 0.029 vs. 0.447 +/- 0.036; P < 0.01). In contrast to fetal limb (CO2)PL, fetal (CO2)PL did not decrease. During severe, nonlethal hypoxia, fetal survival depends on uneven and counterbalancing organ O2 uptake and lactate metabolism.

Effect of hyperinsulinemia on ovine fetal leucine kinetics during prolonged maternal fasting.

The primary effect of insulin on whole body protein metabolism in postnatal life is to reduce proteolysis. To assess the role of insulin in the regulation of protein metabolism in prenatal life, leucine kinetics were determined in the ovine fetus at baseline and in response to hyperinsulinemia. These measurements were made in each fetus in two different maternal states: ad libitum maternal feeding and after a 5-day maternal fast. Maternal fasting resulted in significant increases in baseline fetal leucine rate of appearance (Ra; 51.9 +/- 16.7 vs. 37.3 +/- 3.6 mumol/min, P < 0.05) and leucine oxidation (30.1 +/- 8.9 vs. 8.8 +/- 2.2 mumol/min, P < 0.05). Hyperinsulinemia, which was associated with significant increases in fetal glucose utilization, did not affect total fetal leucine R(a) or leucine release from fetal proteolysis in either maternal state. Under well-fed maternal conditions, hyperinsulinemia produced no changes in the fetal oxidative or nonoxidative disposal of leucine. In contrast, during maternal fasting, hyperinsulinemia reduced fetal leucine oxidation (11.0 +/- 3.7 vs. 31.1 +/- 8.9 mumol/min, P < 0.05) and increased the nonoxidative disposal of leucine (35.4 +/- 4.0 vs. 19.0 +/- 6.1 mumol/min, P < 0.05). This resulted in a change in the fetal leucine accretion rate from negative to positive (-20.9 +/- 7.5 vs. 7.5 +/- 6.7 mumol/min, P < 0.05). These results suggest that, under conditions of restricted maternal substrate intake, fetal hyperinsulinemia and the attendant increase in fetal glucose utilization are associated with increased protein synthesis rather than decreased protein breakdown, thereby improving fetal leucine carcass accretion.

Fetal hind limb oxygen consumption and blood flow during acute graded hypoxia.

Hind limb blood flow and O2 uptake, mean blood pressure, and heart rate were measured in six fetal sheep at 127-141 d gestation in the control state and at different levels of hypoxia that were induced by partial occlusion of the maternal terminal aorta. Blood flow was measured by an ultrasonic flow transducer. Control fetal arterial O2 content ([O2]a) in the descending aorta was 3.25 + 0.17 mM. In response to graded acute hypoxia, blood flow first increased (22.2 versus 19.9 mL.min-1.100 g-1, p = 0.003) and then decreased abruptly at approximately 1.5 mM [O2]a.O2 uptake decreased about 12% (14.74 versus 16.71, p = 0.03) as [O2]a decreased to 1.5 mM, and then fell markedly, tending toward zero for [O2]a = 0.9 mM. In the 2.7- to 1.5-mM [O2]a range, heart rate increased above control (194 versus 169 min-1, p = 0.0024), whereas mean blood pressure did not change significantly. For [O2]a less than 1.5 mM, heart rate decreased to 148 min-1 (p = 0.0005) and mean blood pressure increased above baseline (55 versus 47 torr, p = 0.0001). In conjunction with previous evidence, these results define a state of acute moderate hypoxia in which the whole fetus can sustain a relatively high rate of oxidative metabolism, and a state of acute severe hypoxia ([O2]a between 1.5 and 1.0 mM) in which O2 uptake by some fetal organs is selectively and markedly decreased.

Effect of neuromuscular blockade on fetal oxidative metabolism.

Because fetal neuromuscular blockade has been used during cord blood sampling procedures to prevent fetal movement, we studied the effect of pancuronium bromide on fetal arterial concentrations of glucose, lactate, and oxygen and their umbilical uptake in 6 chronically prepared fetal sheep. We observed a significant increase in the arterial concentrations and decrease in the umbilical uptakes. We suggest that cord blood sampling procedures for fetal assessment of oxidative metabolism not be performed after fetal neuromuscular blockade so that data obtained might best reflect the unperturbed physiological condition of the fetus.

Fetal neuromuscular blockade: effect on oxygen demand and placental transport.

To study mechanisms by which variations in fetal oxygen demand alter fetal oxygen saturation and PO2, we measured uterine and umbilical blood flow and transplacental oxygen diffusion rate in eight chronically prepared pregnant ewes before and during fetal neuromuscular blockade with pancuronium bromide (0.2 mg/kg). Uterine and umbilical blood flows were measured by applying the steady-state method using ethanol as the test substance. Fetal oxygen uptake decreased 7.5% (P less than 0.05). Umbilical blood flow increased 6% (P less than 0.05), whereas uterine blood flow did not change significantly. Fetal arterial oxygen saturation increased markedly (54.8-60.9%; P less than 0.001). There were also significant increases in umbilical vein oxygen saturation (83.6-86.9%; P less than 0.01), uterine vein oxygen saturation (70.7-72.2%; P less than 0.01), umbilical vein PO2 (29.4-32.1 Torr; P less than 0.001), and uterine vein PO2 (49.4-50.7 Torr; P less than 0.01). The uterine-umbilical venous PO2 difference decreased significantly (20.0-18.6 Torr; P less than 0.001), whereas there was no significant change in the uterine-umbilical venous PCO2 difference or in the umbilical ethanol shunt. The data indicate that follows a small decrease in fetal oxygen demand is caused by two aspects of placental oxygen transport: 1) umbilical and uterine blood flow do not react homeostatically to prevent the rise of PO2 in the placental circulation, and 2) the decrease in oxygen flux from placenta to fetus is associated with a decrease in the transplacental PO2 gradient.

Measurement of blood flow and oxygen consumption in the pelvic limb of fetal sheep.

In order to determine blood flow and oxygen consumption in the pelvic limb of fetal sheep, we applied the Fick principle of measurement of oxygen consumption in seven paired experiments in seven fetal sheep under normal conditions and after treatment with pancuronium bromide. Catheterization procedures, which minimized interference with the study limb circulation, avoided changes of catheter tip position during fetal movements,n and prevented collateral circulation to and from tissues not located in the pelvic limb, were utilized. Blood flow through the external iliac artery was measured by means of a transit time ultrasonic method. Six sample sets for oxygen content were drawn from the external iliac artery and vein during 45-min control period and repeated after neuromuscular blockade. Normal oxygen consumption under these experimental conditions was determined to be 20.7 +/- 1.9 (mean +/- SEM) mumole.min-1.100 g-1. Neuromuscular blockade caused oxygen consumption to decrease significantly (P less than 0.01) by 12% to 18.1 +/- 2.1 mumole.min-1.100 g-1 and decreased the average coefficient of variation from 15 to 8%. The data demonstrate that spontaneous skeletal muscle activity accounts for a significant amount of oxygen consumption, the level of which can vary widely over brief periods of time. These results suggest that such tissues with significant spontaneous changes in metabolic activity require repeated blood flow measurements with simultaneous determination of substrate arteriovenous differences to best describe metabolism under normal conditions.