Prevention of the normal expansion of maternal plasma volume: a model for chronic fetal hypoxaemia

The effects of inadequate expansion of maternal blood volume on uterine blood flow, fetal oxygen levels and vasoactive mediators during the third trimester were studied in 8 pregnant sheep. Results were compared to those obtained during 15 normal pregnancies. Prevention of the normal (20 ml/day) increase in maternal plasma volume was achieved by repeated haemorrhage and injections of furosemide. These treatments also reduced the rise in blood flow to the pregnant uterine horn that normally occurs during this period of gestation: at term flow was only 508 +/- 61 (SEM) compared to 838 +/- 83 ml/min in the control group (P greater than 0.01). This reduction in uterine blood flow caused a gradual fall in fetal PaO2, and rise in fetal levels of plasma renin activity, vasopressin, catecholamines and angiotensin II without change in pHa or base excess. Four to 5 days prior to delivery, the difference from control in PaO2 was -3.9 +/- 0.5 mmHg, plasma renin activity +2.9 +/- 1.7 ng/ml.h, vasopressin +4.2 +/- 1.1 pg/ml, catecholamines +957 +/- 145.3 pg/ml and angiotensin II +243 +/- 108.2 pg/ml. Furthermore, the fall in PaO2 and rise in vasoactive mediators that normally occur 3-5 days prior to the onset of labour was either absent (PaO2 and plasma renin activity) or blunted. Thus when expansion of blood volume during pregnancy is inadequate, blood flow to the uterus is adversely affected. This leads to various degrees of chronic fetal hypoxaemia and stimulation of vasoactive mediator systems. However, the normal stimulation of vasoactive mediator systems that occurs 3-5 days before delivery appears to be blunted. Experimental prevention of blood volume expansion during pregnancy produces an excellent model for the study of chronic mild fetal hypoxaemia.     

Determination of insulin-like growth factor-I in normal subjects and in patients with growth hormone disorders by radioimmunoassay using biosynthetic homologous peptide

A highly sensitive and specific RIA for IGF-I has been developed using recombinant DNA-derived IGF-I of very high purity and specific antiserum to it. This assay system could detect IGF-I at as low concentrations as 20-30 ng/ml. The intra-assay and interassay coefficients of variation at various concentrations of IGF-I were 4.9 to 6.5% and 5.4 to 8.0%, respectively. The recovery rate of pure IGF-I added to plasma was 77.0 +/- 3.7%. The antiserum did not cross-react with porcine insulin, biosynthetic human insulin, hGH, hEGF, the synthetic C-domain of IGF-I or that of IGF-II, but reacted equally with an analog, Thr59-IGF-I. Plasma IGF-I was extracted by the acid-ethanol method before assay to separate IGF-I from its binding protein. When plasma IGF-I was assayed without extraction, the inhibition curves of serial dilution of plasma samples from several individuals were not parallel to the standard curve of IGF-I. The plasma concentration of IGF-I was 147 +/- 49 ng/ml (mean +/- SD) in 156 normal adults aged from 20-59 years. As reported by others, the IGF-I levels were low in cord plasma (41.8 +/- 23.5 ng/ml) and plasma of patients with GH deficiency (64.6 +/- 42.0 ng/ml), while its levels were high in normal children of pubertal ages (12-13 yr, 365 +/- 126 ng/ml) and in patients with active acromegaly (562 +/- 115 ng/ml). This RIA system is a simple and useful method for determining plasma IGF-I in normal and diseased states.     

Identification and content of insulin-like growth factors in porcine oviductal fluid.

Oviductal fluid (OvF) was collected from gilts by indwelling catheters during the estrous cycle and analyzed for content of insulin-like growth factors (IGF-I, IGF-II). Group 1, composed of 9 gilts in a summer environment near a boar, yielded mean daily fluid volumes of 1.18 +/- 0.16 ml during estrus and 0.69 +/- 0.03 ml post-estrus. Group 2, composed of 7 gilts in a moderate-temperature, light-regulated room, yielded 1.20 +/- 0.18 ml during estrus and then OvF flow essentially stopped. Serum samples were also collected 2 times daily during the cycle and analyzed along with the OvF for IGF-I and IGF-II. Serum was also analyzed for estradiol-17 beta (E2). For group 1, OvF content (concentration x fluid volume) of IGF-I and IGF-II was greater (p less than 0.05) at estrus than pre- and post-estrus. Daily mean content values (ng/day) for IGF-I and IGF-II during peri-estrus were 30.9 +/- 6.3 and 62.2 +/- 12.3, respectively. During nonestrus, values were 6.8 +/- 6.3 and 11.7 +/- 12.3, respectively. For group 2, OvF content of IGF-I and IGF-II during estrus was similar to that of group 1. Whereas IGF content differed between estrus and nonestrus periods, IGF concentrations were similar (p greater than 0.05), a finding that results from the difference in OvF produced. Compared with OvF concentrations of IGF for a given pig, blood plasma concentrations of IGF-I and IGF-II were 2- to 5-fold higher in the plasma sample collected the same day.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-like growth factors and insulin-like growth factor binding proteins in adult patients with severe liver disease before and after orthotopic liver transplantation

INTRODUCTION: The liver is the main source of serum insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) and the concentration of these proteins might reflect liver function. METHODS: In a retrospective longitudinal study we examined serum levels of total and free IGF-I, IGF-II, IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-6 in 21 adult patients with end-stage liver disease before and after orthotopic liver transplantation (LTX) by sensitive and specific RIAs. In each patient, the mean value of at least three measurements before and after LTX was calculated. RESULTS: Before LTX, serum levels of total and free IGF-I, IGF-II, IGFBP-3 were low and showed a rapid and significant increase in almost all patients after successful LTX (total IGF-I: 30 +/- 7 vs. 256 +/- 30 ng/ml, p < 0.001; free IGF-I: 1.3 +/- 0.3 vs. 3.5 +/- 0.6 ng/ml, p < 0.01; IGF-II: 177 +/- 28 vs. 618 +/- 30 ng/ml, p < 0.001; IGFBP-3: 1,230 +/- 136 vs. 3,665 +/- 264 ng/ml, p < 0.001). In contrast, IGFBP-1 was found to be high immediately before LTX, and declined to normal levels after LTX (210 +/- 40 vs. 90 +/- 15 ng/ml, p < 0.01), while IGFBP-2 did not show any significant changes (1,154 +/- 296 vs. 1,303 +/- 192 ng/ ml). Positive correlations were found between IGF-I, IGF-II or IGFBP-3, and serum pseudocholinesterase (R = 0.50, 0.72 and 0.61 respectively, p < 0.001). Negative correlations were found between IGF-I, IGF-II or IGFBP-3, and prothrombin time (R = 0.50, 0.59 and 0.51 respectively, p < 0.001). CONCLUSION: Patients with severe liver disease show decreased levels of total and free IGF-I, IGF-II and IGFBP-3, and increased levels of IGFBP-1. These abnormalities are promptly normalized after successful LTX. Thus, serum levels of IGF-I, IGF-II and IGFBP-3 might be useful parameters for the assessment of liver function.     

Longitudinal data for intrauterine levels of fetal IGF-I and IGF-II

OBJECTIVE: An increasing body of evidence supports a major role for the insulin-like growth factors (IGFs) in the control of human fetal growth. Individual data at various times of pregnancy suggest that IGF-I and IGF-II levels remain stable up to the 33rd week of pregnancy. Thereafter, both increase to reach values 2-3 times higher at term. In order to provide an accurate reflection of fetal IGFs in utero, we sampled fetal blood from the umbilical cord by cordocentesis. METHODS: We measured IGF-I and IGF-II in 12 fetuses longitudinally for up to 5 times between the 21st week of gestation and delivery. RESULTS: All patients showed a progressive increase in IGF-I and IGF-II levels. Data determined during different time intervals (before 29th, 29th to 32nd, after 32nd week) were compared and the main increase was found after the 32nd week. The median for IGF-I before the 29th week was 33.5 ng/ml (range 19-40.5) and increased to 41 ng/ml (32-59) between the 29th to 32nd and further to 54.1 ng/ml (range 17-70) thereafter. During the same time interval, the median for IGF-II increased from 217 ng/ml (86-326) to 349 ng/ml (227-467). In 7 patients, cord blood after delivery was available. For IGF-II a further increase was consistently found after birth (from 282 ng/ml (175-511) to 393 ng/ml (297-513)), whereas only 2 fetuses showed an increase in IGF-I. CONCLUSION: We conclude that in human fetuses, IGF-I and IGF-II levels increase longitudinally throughout pregnancy. Therefore, they may become important markers of healthy fetal development.     

Studies on the growth of the fetal guinea pig. Effects of reduction in uterine blood flow on the plasma sulphation-promoting activity and on the concentration of insulin-like growth factors-I and -II

The effects of prenatal growth restriction caused by uterine artery ligation at midgestation has been studied in pregnant guinea pigs. Ligation of a uterine artery at day 30 of pregnancy commonly caused a reduction in fetal growth of greater than 45% by days 40-65 of gestation. This was associated with substantial delays in the development of a number of fetal tissues and in particular that of the skeleton which remained cartilagenous for longer than normal. Hence normally by day 50 of pregnancy clear evidence of epiphyseal ossification in the long bones of the fore- and hindlimbs was present, but in growth retarded fetuses of less than 50% of normal size such evidence was sparce. Delayed skeletal development and the slowing of fetal growth rate correlated well with marked depression of plasma sulphation-promoting activity. Indeed plasma from fetuses that were less than 40% of normal size inhibited sulphate incorporation into pig costal cartilage. This indicated the presence of inhibitory factors in the plasma of such fetuses, an interpretation that was re-inforced by the observation that plasma IGF-II concentrations were 2-4 times above normal. In contrast plasma IGF-I concentration was depressed upto 50% by growth retardation in line with the fall in fetal plasma insulin concentration. The changes in plasma sulphation-promoting activity and of IGF-I are consistent with slowing of DNA, RNA and protein synthesis and of gene expression in tissues of the growth-retarded fetus. The elevated fetal plasma IGF-II concentration provided further evidence that in the fetal guinea pig this hormone has a potentially glyconeogenic action and maintains essential glycogen stores.     

Effect of reduced uterine blood flow on fetal and maternal cortisol

We have measured the changes in fetal and maternal plasma concentrations of cortisol in relation to blood gases and percent oxygen saturation during 2- and 4-h episodes of reversibly reduced uterine blood flow in sheep between 120 days gestation and term. During that period of reduced uterine blood flow there was a significant decrease in fetal arterial percent oxygen saturation (SaO2), PO2 and pH. Fetal SaO2 decreased from 59.5 +/- 3.2% to 31.8% +/- 2.8% by 15 min, 32.9 +/- 2.9% by 60 min, and 33.5 +/- 2.9% by 120 min. Fetal PO2 decreased from 3.2 +/- 0.1 KPa to 2.0 +/- 0.2 KPa by 15 min, 2.2 +/- 0.2 KPa by 60 min and 2.3 +/- 0.1 KPa by 120 min. Fetal pH decreased from 7.36 +/- 0.01 to 7.30 +/- 0.03 by 15 min, 7.27 +/- 0.02 by 60 min and 7.25 +/- 0.03 by 120 min. During the period of reduced uterine blood flow, fetal plasma concentrations of cortisol increased from 37.1 +/- 10.8 nmol/l to 53.3 +/- 9.2 nmol/l by 15 min, 49.2 +/- 11.4 nmol/l by 60 min and 43.3 +/- 9.0 nmol/l by 120 min. The greatest percentage increase in fetal plasma concentrations of cortisol occurred in fetuses of 126-139 days gestation. There was no significant change in maternal blood gases, SaO2 or plasma concentrations of cortisol. These experiments demonstrate that there is a significant increase in fetal plasma concentrations of cortisol in response to reductions in uterine blood flow from as early as 120 days gestation.     

Radioimmunoassay for insulin-like growth factor II (IGF-II)

Insulin-like growth factor II (IGF-II) levels in human plasma were measured in physiological and pathological conditions by radioimmunoassay (RIA) with biosynthetic IGF-II. This RIA was specific for IGF-II and cross-reactivity with IGF-I was 1%. The sensitivity was 15 pg/tube with 50% displacement at 50 pg/tube. The intra- and inter-assay coefficients of variation for IGF-II were 6.3 and 9.3%, respectively. The plasma IGF-II levels in normal adults, patients with hypopituitarism and patients with active acromegaly were 589.6 +/- 15.8, 800.9 +/- 45.6 and 330.3 +/- 24.3 ng/ml, respectively. After human growth hormone (hGH) treatment in hypopituitarism, IGF-II slightly increased, but not significantly. After adenomectomy in patients with acromegaly, IGF-II significantly decreased. These data indicate that IGF-II concentrations in plasma were partially GH dependent. This GH dependency was less than that of IGF-I. IGF-II was low in patients with anorexia nervosa and with liver cirrhosis and high in patients with renal failure. In two cases with extrapancreatic tumor-associated hypoglycemia, plasma IGF-II was increased to 1123.8 and 843.5 ng/ml, and returned to normal after tumor resection. These data showed that IGF-II was partly dependent on GH and nutritional conditions and that IGF-II was the most likely cause of some cases of hypoglycemia with extrapancreatic tumor. This specific and sensitive RIA of IGF-II would be useful in evaluating its physiological and pathological role in plasma and tissue.     

Fetal asphyxia stimulates an increase in fetal plasma catecholamines and [Met]-enkephalin-arg6-phe7 in the late-gestation sheep fetus

We have investigated whether enkephalin-containing peptides and catecholamines are increased in fetal plasma during periods of reduced uterine blood flow which produce moderate fetal asphyxia (i.e. hypoxemia, hypercapnia and acidemia). Experiments (n = 16) were performed in 11 ewes between 121-139 days gestation. In 8 experiments a clamp placed around the common iliac artery of the ewe was adjusted to produce a 50% reduction in the partial pressure of arterial oxygen (PO2) in fetal plasma for 30 min between 121-125 days gestation (n = 4) and between 131-139 days gestation (n = 4). Control (n = 8) experiments were performed when the arterial clamp was not adjusted. There was no significant effect of asphyxia on fetal plasma noradrenaline concentrations before 126 days gestation. After 130 days gestation during asphyxia, fetal plasma noradrenaline concentrations increased significantly from 2.20 +/- 0.72 pmol/ml (-15 min) to 14.06 +/- 0.75 pmol/ml (+5 min). The fetal adrenaline response to asphyxia did not change with increasing gestational age and after 130 days gestation fetal plasma adrenaline increased significantly from 1.48 +/- 0.46 pmol/ml (-15 min) to 4.05 +/- 1.22 pmol/ml (+10 min). Met-enkephalin-arg6-phe7 immunoreactivity was measurable (25-117 pg/ml) in all pre-experimental fetal sheep plasma samples collected between 121-139 days gestation. There was no specific effect of asphyxia on fetal plasma [Met]-enkephalin-arg6-phe7-IR before 130 days gestation. However after 130 days gestation, there was a significant increase in fetal plasma (Met-enkephalin Arg-6-phe7-IR above baseline values, when compared to control experiments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development and characterization of a competitive polyclonal antibody enzyme-immunoassay for salmon insulin-like growth factor-II

This paper describes the development and validation of a competitive, polyclonal antibody enzyme-immunoassay (EIA) for the measurement of salmon and trout insulin-like growth factor-II (IGF-II). A polyclonal antiserum was raised against a synthetic peptide epitope, corresponding to amino acid residues 1-9 of the N-terminus of mature Atlantic salmon (Salmo salar) IGF-II. The antiserum was purified by hydrophobic charge induction chromatography (HCIC). The partially purified immunoglobulins were used in an enzyme-immunoassay system (EIA) resulting in a highly specific assay for salmon IGF-II with cross-reactivity of less than 0.01% for recombinant salmon IGF-I and recombinant salmon growth hormone (GH), and 5.57% for salmon insulin (sIns). The recombinant salmon IGF-II (rsIGF-II) standard curve limit of detection was 1.37 ng/ml with an EC(50) of 44.97+/-0.82 ng/ml. Intra- and interassay coefficients of variation were determined at 7.47% (n=15) and 7.42% (n=15), respectively. Added rsIGF-II was adequately recovered from acid-treated Atlantic salmon and rainbow trout (Oncorhynchus mykiss) plasma samples. Parallel dose-response inhibition curves were demonstrated for the plasma of both fish species tested. Circulating IGF-II levels of 22.26+/-2.66 and 18.24+/-1.43 ng/ml were determined for acid-treated plasma of normal adult Atlantic salmon and rainbow trout, respectively. This EIA should prove to be useful in the study of factors which influence circulating plasma levels of IGF-II in these fish species.     

Retinal photocoagulation does not influence intraocular levels of IGF-I, IGF-II and IGF-BP3 in proliferative diabetic retinopathy-evidence for combined treatment of PDR with somatostatin analogues and retinal photocoagulation?

Retinal photocoagulation reduces the incidence of severe visual loss in proliferative diabetic retinopathy (PDR). Reduced levels of VEGF/VPF might result in an improved function of the blood-retina barrier and cause a decrease of blood derived intraocular growth factors such as IGF-I. This study investigates whether retinal photocoagulation is able to normalize the concentrations of IGF-I, IGF-II and IGF-BP3 in the vitreous humor of patients undergoing vitrectomy. Levels of IGFs and the permeability marker, albumin, were measured in serum and vitreous of 52 patients. Three groups were compared: controls without proliferating eye disease (n = 19) and patients with PDR with (PDR+; n = 25) and without (PDR-; n = 8) previous retinal photocoagulation. IGF-I, IGF-II, IGF-BP3 and albumin were determined by immunological methods and were confirmed to be increased in patients with PDR compared to controls. Retinal photocoagulation influenced neither the intraocular concentration of the permeability marker albumin (PDR+: 253.2 +/- 46 mg/dl; PDR-: 256.4 +/- 66.5 mg/dl) nor the levels of IGFs (PDR+: IGF-I: 1.2 +/- 0.1 ng/ml; p = 0.38; IGF-II: 34.8 +/- 2.2 ng/ml; p = 0.1; IGF-BP3: 75.7 +/- 9.7 ng/ml; p = 0.27; PDR-: IGF-I: 1.1 +/- 0.2ng/ml; IGF-II: 29.3 +/- 5.2 ng/ml; IGF-BP3: 61.5 +/- 18.3 ng/ml). Systemic levels of albumin and IGFs were not changed significantly by retinal photocoagulation. These results demonstrate that previous retinal photocoagulation in patients undergoing vitrectomy does not functionally reestablish the blood-retina barrier despite decreases in VEGF/VPF. The lack of influence on intraocular concentrations of the serum-derived growth factors, IGF-I, IGF-II and IGF-BP3, might in part explain the failure of previous photocoagulation in the investigated patients. These results suggest that a combined treatment with retinal photocoagulation and growth hormone-lowering drugs, such as somatostatin analogues, could be a useful treatment, which may prevent further loss of visual acuity in patients with PDR.     

Effects of sc administration of recombinant human insulin-like growth factor I (IGF-I) on normal human subjects

Recombinant human insulin-like growth factor I (IGF-I) was administered subcutaneously to each of 5 normal human subjects at doses of 0 mg/kg (control), 0.06 mg/kg, or 0.12 mg/kg successively at one week intervals. After 0.06 mg/kg or 0.12 mg/kg IGF-I injections, plasma IGF-I levels increased from 185 +/- 17 ng/ml (mean +/- SEM) to maximal levels of 396 +/- 21 ng/ml at 3 hours and from 169 +/- 14 ng/ml to 480 +/- 27 ng/ml at 4 hours, respectively. These two peak values were statistically different (p less than 0.05). After 0.06 mg/kg and 0.12 mg/kg IGF-I administration, blood glucose levels decreased from 85 +/- 2 mg/dl to minimal levels of 73 +/- 3 mg/dl at 3 hours and from 83 +/- 1 mg/dl to 50 +/- 4 mg/dl at 2 hours, respectively. These two minimal values were statistically different (p less than 0.001). Serum insulin and C-peptide levels were decreased in a dose dependent manner after IGF-I administration. There were no changes between blood urea nitrogen levels before and 4 hours after IGF-I administration. The urinary GH concentration decreased after 0.06 mg/kg IGF-I administration, but increased and maintained normal values after 0.12 mg/kg IGF-I administration.     

Up-regulation of IGF-I receptors on IM-9 cells by IGF-II peptides

To examine a possible role for IGF-II in the regulation of IGF-I receptors we measured 125I-IGF-I binding on IM-9 cells following pre-incubation with IGF-II/IGF-I mixtures, purified MSA (a rat IGF-II-like peptide), pure IGF-I, or insulin. Whereas all preparations tested induced down-regulation of IGF-I binding after 20 hours, distinct differences were noted after six hour pre-incubation: IGF-I (100 ng/ml) and insulin (1 microgram/ml) both induced down-regulation of IGF-I binding (15 +/- 2% and 19 +/- 2% respectively). However, a mixture of IGF-II and IGF-I (100 ng/ml each) induced consistent up-regulation of IGF-I binding (16 +/- 2%) (mean +/- SE, n = 14), and a preparation enriched in IGF-II (250 ng/ml IGF-II and 75 ng/ml IGF-I) induced 20 +/- 5% (n = 3) up-regulation at six hours. Purified MSA (200 ng/ml) induced 15% up-regulation of IGF-I binding at six hours. Scatchard analysis of displacement curves showed that increased binding was due to loss of low affinity binding, with enhancement of high affinity sites. The up-regulation of IGF-I binding was unaffected by treatment with 0.1 mM cycloheximide, but was blunted by 5 microM colchicine. It is concluded that 1. IGF-II induces up-regulation of IGF-I receptors on IM-9 cells following 6 hour pre-incubation; 2. This phenomenon is not mimicked by the structurally-related peptides IGF-I or insulin; The up-regulation is due to enhanced high affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Systemic levels contribute significantly to increased intraocular IGF-I, IGF-II and IGF-BP3 [correction of IFG-BP3] in proliferative diabetic retinopathy.

Increased intraocular levels of angiogenic growth factors such as insulin-like growth factor I (IGF-I) have been demonstrated in proliferative diabetic retinopathy (PDR). It is unclear whether increased leakage of the blood retina barrier or local synthesis primarily determine intraocular levels of IGFs in man, which is of special interest regarding possible therapeutic options with somatostatin analogues in PDR. This is the first study investigating parallelly serum and vitreous levels of IGF-I/II, IGF-BP3 and the liver-derived permeability marker albumin to determine in vivo the amount of circulation-derived intraocular IGFs. A control group without retinal proliferation and patients with PDR were compared. Levels of IGF-I/II, IGF-BP3 and albumin were determined by immunological methods. Vitreous levels of albumin were 2.2-fold elevated in patients with PDR (254.1 +/- 37.2mg/dl; n = 27; p = 0.0027) compared to controls (115.7 +/- 36.2mg/dl; n =10), whereas serum levels were slightly decreased in diabetes patients (5049 +/- 196 mg/dl vs. 4330 +/- 186 mg/dl; p = 0.0283). This was comparable to an increase of IGF-I/11 and IGF-BP3 in vitreous from PDR patients (IGF-I: 2.3 +/- 1.1 ng/ml p = 0.005. IGF-II: 37.9 +/- 4.9 ng/ml; p = 0.0003. IGF-BP3: 97.9 +/- 26.9 ng/ml; p = 0.0001; n = 34) compared to controls (IGF-I: 0.7 +/- 0.1 ng/ml. IGF-II: 21.3 +/- 4.2 ng/ml. IGF-BP3: 31.3 +/- 4.9 ng/ml: n = 19). Serum levels did not differ significantly among the groups regarding IGF-I, II and IGF-BP3. Intraocular albumin and IGF-I levels calculated as percentage of the respective serum levels correlated significantly (r = 0.42; p = 0.012). This study demonstrates that influx of IGF-I, II and IGF-BP3 in PDR quantitatively parallels influx of the liver derived serum protein albumin suggesting that leakage of the blood retina barrier and serum levels of IGF primarily determine intravitreal IGF levels rather than local synthesis. Suppression of systemic IGF levels by new, highly effective somatostatin-analogues therefore provides a promising approach to prevent PDR.     

The effects of pancreatectomy on the plasma concentrations of insulin-like growth factors 1 and 2 in the sheep fetus

The effects of hypoinsulinaemia and altered metabolite concentrations on the fetal plasma concentrations of insulin-like growth factors (IGF) have been investigated in chronically catheterized fetal sheep made insulin deficient by pancreatic ablation. Fetal pancreatectomy reduced significantly the plasma IGF-1 concentration and increased plasma IGF-2 activity in comparison with the values observed in sham operated fetuses. Mean plasma IGF-1 concentrations in the sham operated and pancreatectomized fetuses were 18.6 +/- 3.1 ng/ml (n = 7) and 13.4 +/- 1.4 ng/ml (n = 13) respectively. When all the data were combined, there was a significant positive correlation between the plasma concentrations of IGF-1 and insulin in utero. The mean IGF-2 activity was 2349 +/- 83 ng/ml (n = 7) in the sham operated fetuses and 3800 +/- 532 ng/ml in the pancreatectomized animals (n = 13). Plasma IGF-2 activity was correlated positively with plasma glucose, fructose and alpha-amino nitrogen levels and inversely related to the plasma insulin concentration in utero. These observations demonstrate that the fetal pancreas is essential for normal IGF production in the fetus and suggest that insulin, substrate availability and the IGFs may interact in the regulation of fetal growth.     

Effects of endogenous angiotensin II on the fetal circulation

The role of endogenous angiotensin II in the regulation of the circulation was investigated by infusion of [sar1],[ala8]-angiotensin II, a competitive antagonist of angiotensin II, into fetal sheep with chronically-maintained intravascular catheters. The thesis considered was that angiotensin II may have a greater role in the fetus than in the adult since the autonomic nervous system does not develop fully until late in gestation. Fetal cardiac output and its distribution to various organs and actual blood flows to fetal tissues were determined by the radionuclide-labelled microsphere technique. Intravenous infusion of [sar1], [ala8]-angiotensin II at a rate of 13.95-42.15 microgram/min per kg fetal body weight increased plasma renin activity from a control value of 8.9 +/- 1.6 to 18.9 +/- 3.9 ng/ml per h (SEM). Mean arterial blood pressure fell significantly from a control level of 47 +/- 1.6 to 41 +/- 1.1 mmHg. Blood flow to the unbilical-placental circulation decreased from 239 +/- 27.0 to 198 +/- 20.2 ml/min per kg, but the calculated vascular resistance in the umbilical-placental circulation did not change. Although cardiac output did not change, blood flow to the peripheral circulation, which includes the fetal skin, muscle and and bone and constitutes 75 +/- 0.9% of the total fetal body weight, increased as did flow to the thyroid and adrenal circulations. Endogenous angiotensin II appears to be important in maintaining blood flow to the umbilical-placental circulation by maintaining fetal arterial blood pressure. Angiotensin II exerts this effect by mediating a tonic vasoconstriction primarily in the peripheral circulation.     

Insulin-like growth factors and binding proteins in early milk from mothers of preterm and term infants

Breast-fed preterm infants often show a better outcome, partly ascribed to the benefit of insulin-like growth factors (IGFs) and their binding proteins (IGFBP). We compared IGF-I, IGF-II, IGFBP-2 and IGFBP-3 levels, measured by radioimmunoassays in milk samples from 30 mothers of preterm (<31 weeks) and from 19 mothers of term (>37 weeks) infants at days 7 and 21 postpartum. Proteolysis of IGFBP-2 within mother's milk and digestion of (125)I-IGF-II and (125)I-IGFBP-2 by gastric juice from neonates were assessed by electrophoretic techniques. Mean concentrations did not differ between preterm and term milk: IGF-I (2.8 +/- 0.2 vs. 2.3 +/- 0.1 ng/ml), IGF-II (12.0 +/- 0.4 vs. 12.2 +/- 0.5 ng/ml), IGFBP-3 (100.0 +/- 5.1 vs. 80.0 +/- 5.8 ng/ml), but did so for IGFBP-2 (3,144 +/- 172 vs. 2,428 +/- 188 ng/ml, p < 0.02). Immunoblots revealed 42% (p < 0.05) more IGFBP-2 fragments of 14 and 25 kDa in preterm milk. Incubation with gastric juice caused cleavage of (125)I-IGFBP-2 and partial cleavage of (125)I-IGF-II. Mutual complexation protected IGF-II and IGFBP-2 from cleavage, suggesting that both are likely to arrive in the bowel in an intact form to exert promotive effects. The results provide further evidence that IGFBP-2 and IGF-II in breast milk are relevant factors for the early development of preterm infants.     

Effects of chronic reduction in uterine blood flow on fetal and placental growth in the sheep

Pregnancy is associated with a significant increase in uteroplacental blood flow (UBF), which is responsible for delivering adequate nutrients and oxygen for fetal and placental growth. The present study was designed to determine the effects of vascular insufficiency on fetal and placental growth. Thirty-nine late-term pregnant ewes were instrumented to investigate the effects of chronic UBF reduction. Animals were split into three groups based on uterine blood flow, and all animals were killed on gestational day 138. UBF, which began at 851 +/- 74 ml/min (n = 39), increased in controls (C) to 1,409 +/- 98 ml/min (day 138 of gestation) and in the moderately restricted (R(M)) group to 986 +/- 69 ml/min. In the severely restricted (R(S)) group, UBF was only 779 +/- 79 ml/min on gestational day 138. This reduction in UBF significantly affected fetal body weight with R(M) fetuses weighing 3,685 +/- 178 g and R(S) fetuses weighing 2,920 +/- 164 g compared with C fetal weights of 4,318 +/- 208 g. Fetal brain weight was not affected, whereas ponderal index was significantly reduced in R(M) (2.94 +/- 0.09) and R(S) fetuses (2.49 +/- 0.08) compared with the value of the C fetuses (3.31 +/- 0.08). Placental weight was also significantly reduced in the R(M) group, being 302 +/- 24 g, whereas the R(S) group placenta weighed 274 +/- 61 g compared with the C values of 414 +/- 57 g. Fetal heart, liver, lung, and thymus were all significantly smaller in the R(S) group. Thus the present study shows a clear relationship between the level of UBF and both fetal and placental size. Furthermore, the observation that fetal brain weight was not affected, whereas fetal body weight was significantly reduced suggests that this experimental preparation may provide a useful model in which to study asymmetric fetal growth restriction.     

Enkephalin peptides in fetal sheep, changes with gestation, origin and production by the placenta.

Enkephalin-containing peptides have been followed in the circulation of fetal sheep between 118-143 days gestation. Using a combination of radioimmunoassay and hplc met5-enkephalin was found in the concentration range 60-500 pg/ml and proenkephalins containing met5-enkephalin had a concentration of 150-4000 pg/ml. The concentration of both increased towards term. The sources of the enkephalin peptides was investigated by measurement of differences across the umbilical circulation and by studying the effects of fetal adrenal demedullation and chemical sympathectomy. The placenta showed a continuous net output of enkephalin peptides which increased close to term. This placental output was increased sharply by reduction of uterine blood flow either using compression of the uterine artery or through infusion of adrenaline at 35 micrograms/min into the maternal circulation. Maternal hypoxia caused by breathing 9% O2 plus 3% CO2 also increased fetal plasma enkephalin levels, although not output from the placenta. Adrenal demedullation, particularly if accompanied by chemical sympathectomy depressed fetal plasma enkephalin concentrations and sharply suppressed the fetal peptide responses to maternal hypoxia. It is concluded that the placenta and the fetal adrenal are important sources of met5-enkephalin-containing peptides in the fetal circulation. The placental production appears to be closely tied to changes in uterine perfusion and adrenal output changes in response to fetal oxygenation.