Helen Moorehead

Fetal and Maternal Hormonal Response to Starvation in the Ewe

Summary: Five ewes 90–130 days of gestation with chronically indwelling fetal catheters were studied. Blood samples were drawn each morning simultaneously from the maternal femoral artery, fetal femoral artery, and fetal umbilical vein. Total starvation was begun after blood samples had been drawn on the 3rd morning, but water was given ad libitum. The mean maternal arterial plasma glucose concentration before starvation (73.4 ± 31.0 (SEM) mg/100 ml plasma H2O) fell to 54.8 ± 5.2 mg/100 ml (P < 0.01) after 24 hr of fasting and to 37.5 ± 1.6 mg/100 ml (P < 0.001) by 72 hr of fasting. The maternal arterial fructose concentration was low in both the fed (2.9 ± 0.4 mg/100 ml) and fasted states (1.8 ± 0.5 mg/100 ml, day 3). The maternal arterial insulin concentration fell early in fasting from a mean of 27.9 ± 3.9 uU/ml on 2 days before starvation to 12.4 ± 1.8 uU/ml after 24 hr of fasting (P < 0.005) and remained low for the duration of the 7-day fasting period. The mean maternal arterial glucagon concentration in the fed state was 112 ± 20 pg/ml and did not change significantly throughout the 7 days of maternal fasting.Fetal arterial plasma glucose was much lower than maternal levels (mean 18.4 ± 1.1 mg/100 ml plasma H2O in the fed state), decreased to 12.1 ± 1.4 mg/100 ml after 24 hr of fasting (P < 0.05) and did not change significantly after the 1st day of fasting. The whole blood fructose concentration in the fetal artery was high in the fed state (71.7 ± 11.5 mg/100 ml, mean 2 days before fasting) and fell quickly during starvation (34.2 ± 3.4 mg/100 ml after 48 hr). The fructose concentration did not change further for the duration of the fasting period. The fetal arterial insulin concentration was lower than the maternal (19.4 ± 1.0 uU/ml in the fed state and did not fall dramatically after fasting as the mother did. After 24 hr of fasting, the fetal insulin was 15.0 ± 1.5 uU/ml (P < 0.005) and remained low throughout the fast. The umbilical venous-arterial differences of insulin were statistically different from 0 in both fed (mean – 1.4 ± 0.3 uU/ml; P < 0.005) and fasted state (mean – 1.4 ± 0.5 uU/ml; P < 0.02), with the fetal artery greater than the umbilical vein in 25 of 28 measurements. The fetal arterial concentration of glucagon was lower than in the ewe (32 ± 7 pg/ml) and also did not change significantly during starvation.There was a good correlation of the plasma glucose and insulin concentrations for both the fetus and the ewe. Analysis of pooled fetal and maternal regression lines for insulin-glucose relationships reveals no difference in the slope (maternal 0.48, fetal 0.48), but a statistically significant difference in the y intercept (maternal – 7.70, fetal 8.65, P < 0.005), with the fetal curve shifted to the left.The excellent correlation between plasma glucose and insulin concentration throughout the fed and fasted periods suggests that the decrease in fetal plasma glucose and the decreased utilization of exogenous glucose by the fetus is mediated by fetal insulin level. Although the response of insulin to the change in glucose in the fetus is the same as in the mother, the threshold is set at a lower glucose concentration in the fetus. The data in this study would suggest that glucagon is not an important regulator of fetal metabolism during maternal starvation because there was no significant change in the glucagon concentration in the fetus throughout the study.Speculation: Fetal growth and metabolism appear to be influenced by both substrate availability from the mother and fetal hormonal mechanisms. The role of alanine and the possibility of altering fetal growth by manipulating substrate availability need to be investigated to further understand the fetal metabolic response to maternal nutritional deprivation.

Effects of Fasting on Gluconeogenic Enzymes in the Ovine Fetus

ABSTRACT. Fetal and maternal sheep were studied to determine whether changes in gluconeogenic enzyme activities could be detected in the liver and/or kidney associated with maternal nutritional deprivation. Thirteen ewes and 16 fetuses were sacrificed in the fed state, while 13 ewes with 17 fetuses were sacrificed after 5 days of fasting, all at 125 days gestation (term = 147 days). Fetal weight was decreased in the fasted versus fed group (2.86 ± 0.56 versus 3.61 ± 0.58 kg, p < 0.001). Tissues were analyzed for glucose-6-phosphatase, fructose-1,6-diphosphatase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, glutamate oxaloacetate aminotransferase, and glutamate pyruvate aminotransferase. In maternal liver, four of the six enzymes increased significantly during fasting, whereas none of the enzymes increased in maternal kidney. In fetal hepatic tissue, five of the six enzymes (with the exception of pyruvate carboxylase) increased during maternal fasting and three of the enzymes increased in renal tissue. These data are consistent with the potential for increased rates of gluconeogenesis in the ovine fetus during periods of compromised maternal nutrition.

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 μg/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 μmol/min during IGF-I) or breakdown (phenylalanine rate of appearance 0.67 ± 0.13 before vs. 0.60 ± 0.17 μmol/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 μmol/min) as did fetal leucine oxidation (8.4 ± 0.8 to 6.8 ± 0.6 μmol/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.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.

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.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.

Determination of the specific activity of sheep plasma amino acids using high-performance liquid chromatography: Comparison study between liquid scintillation counter and on-line flow-through detector

A method was developed for the determination of the specific activities of leucine and phenylalanine in plasma using a flow-through scintillation counter coupled with high-performance liquid chromatography components. Results were compared with those obtained from liquid scintillation counting. Differences in the specific activities of leucine and phenylalanine between the two methods were not statistically significant. We concluded that flow-through radioactivity detection can be used for quantitative amino acid assays. However, the minimum activity that can be detected may be prohibitively low in certain applications.

Effects of Exogenous Glucagon on Concentrations of Glucose, Fructose and Insulin in Plasma of Sheep Fetus

Exogenous glucagon infused into the fetal sheep resulted in an increase in the concentration of glucose and insulin in fetal arterial plasma without a significant change in the concentration of fructose. Lack of any significant changes in glucagon, insulin, glucose and fructose concentrations in maternal plasma suggests that the alterations in the fetus are secondary to fetal metabolic and hormonal mechanisms rather than reflecting effects of maternal metabolism.

1226 THE EFFECT OF FASTING ON OVINE FETAL AND MATERNAL GLUCONEOGENIC ENZYMES

Fetal and maternal sheep were studied to determine whether changes in gluconeogenic enzyme activities could be detected in the liver and/or kidney associated with nutritional deprivation. Thirteen ewes and 16 fetuses were sacrificed in the fed state while 13 ewes with 17 fetuses were sacrificed after five days of fasting, all at 125 days gestation (term = 147 days). Fetal weight was decreased in the fasted versus fed group (2.86 ± .56 versus 3.61 ± .58 kg, p <.001). Weights of fetal liver (66 ± 16 versus 105 ± 27 gm), fetal kidney (9.3 ± 1.9 versus 12.4 ± 1.9 gm) and maternal kidney (74.9 ± 8.3 versus 88.0 ± 9.3 gm) were all significantly decreased with fasting (p < .001). Tissues were analyzed for glucose-6-phosphatase (G-6-P), fructose-1, 6-diphosphatase (FDP), glutamate-oxaloacetate aminotransferase (GOT) and glutamate-pyruvate aminotransferase (GPT). In both fetus and mother, G-6-P and FDP were increased during fasting when measured per gm liver tissue and per mg protein (p <.001). GOT and GPT were also increased in fetal liver but not in kidney with fasting. However, activities of the enzymes per whole fetal liver or kidney were not different between fed and fasted states. Therefore, although gluconeogenic enzyme activities increased in relative activity per unit weight in fetal tissues during fasting, total enzyme activity is unchanged due to a decrease in liver and kidney mass.

263 INCREASED AMINO ACID CATABOLISM IN THE FETAL LAMB DURING MATERNAL FASTING

The effect of maternal fasting upon fetal nitrogen uptake in the form of free amino acids (QN), fetal oxygen consumption (QO2) glucose utilization (QG), urea excretion (QU) and umbilical blood flow (UBF) was assessed in the chronic fetal lamb preparation. Five animals (mean gestational age 118 days) were studied in the fed state 7 days after surgery and again 12 days post-operatively after 5 days of maternal fasting. QG decreased from 6.88 ± 1.04 to 2.69 ± .30 mg/kg/min during the fasted state while QO2 remained unchanged (7.38 ± .53 vs. 7.41 ± .70 ml/kg/min). The glucose:oxygen quotient thus decreased from .69 to .28 during fasting. QU increased from 448 to 691 mg/kg/day, although nitrogen uptake from the placenta did not change during fasting (802 vs. 768 mg/kg/day). These findings indicate that the fetal metabolic rate remains unchanged during the fasted state while exogenous glucose utilization decreases to <50% of fed state values. An augmented utilization of amino acids is reflected by the increased urea excretion rate, but without a detectable increase in nitrogen uptake from the placenta. Therefore, the ovine fetus becomes increasingly reliant on amino acid substrate during fasting and may be required to catabolize its own protein stores, or to direct exogenous amino acids from growth to catabolism to maintain adequate energy substrate.

290 THE EFFECTS OF EXOGENOUS GLUCAGON ON FETAL METABO-LISM IN THE SHEEP

The effect of exogenous glucagon on fetal and maternal glucose, fructose, insulin and glucagon concentrations was studied in the chronic fetal sheep model. Eight near-term fetuses were infused with glucagon (40 ng/min) for 60 min via a fetal femoral vein catheter. Fetal plasma glucagon increased from 37.66±7.86 (SEM) to 420.50±38.59 pg/ml (p<.001) within 15 min of beginning the infusion, reaching 531.80±117.54 pg/ml (p<.001) by 60 min and returning to the baseline level 60 min after stopping the infusion. Fetal arterial glucose increased from 12.06±0.84 to 15.50±0.68 mg % (p<.0001) after 15 min of infusion (p<.001), and reached a maximum level of 17.18±0.94 mg % by 60 min (p<.001). Fetal arterial plasma insulin was significantly increased within 60 min of infusion (16.45±3.73 to 29.38±4.41 uU/ml, p<.001). Fetal fructose did not change. During the 60 min infusion and postinfusion period, there were no changes in the maternal concentrations.These results, in addition to our previous studies demonstrating an increase in fetal glucagon concentration with prolonged maternal fasting, provide further evidence that glucagon is an important regulator of fetal metabolism.

1021 DETERMINATION OF SERUM BILIRUBIN BY SKIN REFLECTANCE

A non-invasive method to determine neonatal serum bilirubin concentration from skin reflectance was developed. Spectral reflectance measurements using a novel fiberoptic reflectometer (400-750 nm, 8 sec scan time) were performed on the blanched skin (3 cm2 area) on the back of 108 infants. The predicted bilirubin value from the skin reflectance was calculated as a function of the percent reflectance at 420, 460 and 510 nm. This 3-wavelength function was derived from the Kubelka-Monk theory relating reflectance to the absorption and scattering properties of the skin.The correlation of the bilirubin value predicted from the triple wavelength function and the actual serum bilirubin value for 217 determinations in 108 black and white infants showed a correlation coefficient (R) of 0.78 (P<.00001). The 95% confidence limit for the predicted bilirubin value compared to the actual bilirubin value was ±1.6 mg%. The 103 samples in the 48 black patients showed an R of 0.86 (P<.00001) with a 95% confidence limit of ±1.5 mg%. The 114 determinations in the 50 white infants showed a 95% confidence limit of ±1.4 mg%.Skin reflectance can serve as a screening tool for predicting serum bilirubin concentration in black and white neonates.