Urinary desmosine as a biomarker in acute lung injury.

Acute lung injury (ALI) is a complex disorder associated with an acute inflammatory response thought to contribute to tissue injury. Desmosine, a cross-linking amino acid present in elastin, is released during matrix degradation and cleared by the kidney. Results from animal models and human disease studies have suggested that ALI is associated with the release of desmosine, resulting in increased urinary desmosine. A radioimmunoassay was used to monitor urinary desmosine levels over 10 days in ten patients with ALI. The concentration of desmosine was measured with and without acid hydrolysis. Baseline urinary desmosine was increased in two of ten patients. The concentration of desmosine at baseline did not appear to be related to age, gender, neutrophil elastase (NE)/alpha(1)-antiprotease complex concentration or P(a)O(2)/F(i)O(2) ratio. No meaningful changes in desmosine levels were noted after removal from mechanical ventilation. Baseline desmosine concentrations did not appear to correlate with the risk of death. The limited sensitivity, predictive correlations and dynamic modulation would suggest that urine desmosine has a limited role as a biomarker for ALI. Hydrolysis of urine samples appears necessary for optimal measurement of urine desmosine.

A sensitive chemiluminescent enzyme immunoassay for the bioanalysis of carboxyl-terminal B-chain analogues of human insulin.

Quantification of analogues of human insulin in biological matrices is complicated by differences in their immunoreactivity and the presence of both the analogue and endogenous concentrations of insulin in test samples. To facilitate pharmacokinetic comparisons of carboxyl-terminal B-chain analogues of human insulin, we undertook development of a sensitive ELISA. The ELISA detection method was optimized systematically to permit routine analysis of 10-microl serum samples. Accordingly, a noncompetitive 'sandwich' chemiluminescent ELISA was validated for the quantification of carboxyl-terminal B-chain insulin analogues in human serum over a concentration range from 5 to 3125 pM. The mean bias (RE%) within the validated range varied from -10.3 to 4.3%, with an intermediate precision (inter-assay CV%) from 4.2 to 11.5%. The two-sided 90% expectation tolerance interval for total measurement error was within +/-25% of the nominal concentration for all levels of validation samples. Insulin lispro, human insulin, proinsulin, despentapeptide insulin (DPI) and porcine insulin displayed comparable crossreactivity in the ELISA. Potential utility of the new assay for insulin bioanalysis in nonhuman species was investigated by assessing the pharmacokinetic profile of DPI in rats following administration of a single subcutaneous dose. The sensitive chemiluminescent detection method is simple to perform and should be readily adaptable for ELISAs of other therapeutic proteins.

Phase I and pharmacokinetic study of LY309887: a specific inhibitor of purine biosynthesis.

PURPOSE: In this phase I trial in humans the safety and pharmacology of LY309887 on a weekly schedule combined with daily oral 5-mg doses of folic acid were evaluated. BACKGROUND: LY309887 is an inhibitor of folate-dependent enzymes involved in de novo purine biosynthesis and has a broad preclinical antitumor activity. In murine systems, combining this agent with exogenous folic acid results in an enhanced therapeutic index. METHODS: This study was a single-institution, open-label, clinical trial of dose escalation with toxicity and pharmacokinetic parameters determined. The dose range studied was 0.5-4 mg/m2 per week x6 and then a modified schedule weekly x3 every 6 weeks. RESULTS: Dose-limiting toxicities were of delayed onset and associated with hematologic, neurologic, and mucosal effects. Pharmacokinetic parameters revealed dose linearity for AUC and Cmax. Low circulating levels of drug persisted for over 200 h. Urinary excretion accounted for approximately 50% of the parent drug but was highly variable. The urinary excretion was near maximal within 24 h of dosing. CONCLUSIONS: The modified dosing schedule allowed repetitive dosing in patients. Further evaluation of the 2 mg/m2 per week x3 every 6 weeks with daily oral folate supplement as a potential phase II dose may be warranted.

An immunoadsorption strategy to produce specific antisera against analogs of human proteins: development of sensitive and specific radioimmunoassays for two analogs of human leptin.

Immunoassay technology is routinely used to measure concentrations of proteins and polypeptides in biological matrices. Increasingly, research efforts have sought to create analogs of human proteins with the aim of improving efficacy or pharmaceutical properties relative to the native protein. Pharmacokinetic assessment of these polypeptide analogs, however, can be greatly confounded by the presence of endogenous native protein. This report describes an immunization and immunoabsorption strategy that was used to create monospecific polyclonal antibodies against analogs of human leptin (LY355101 and LY396623, one and two amino acid changes relative to native human leptin, respectively). Rabbits were immunized with either LY355101 or LY396623. Antisera were screened to determine if any showed increased specificity for the analog relative to native human leptin. Antisera showing increased specificity for the leptin analog were then treated by immunoabsorption against native human leptin, thus depleting human leptin cross-reactivity. The antibodies developed in this process were used in radioimmunoassays. which were validated for use in clinical studies. Both assays proved to be highly specific for LY355101 or LY396623 in the presence of native human leptin. Use of this procedure permitted the measurement of LY355101 and LY396623 pharmacokinetics that were not confounded by the high levels of endogenous human leptin found in obese subjects. This technique has the potential for broad application in the development of assays capable of specifically measuring protein analogs without cross-reactivity to an endogenous substance.

Validation of immunoassays for bioanalysis: a pharmaceutical industry perspective.

Immunoassays are bioanalytical methods in which quantitation of the analyte depends on the reaction of an antigen (analyte) and an antibody. Although applicable to the analysis of both low molecular weight xenobiotic and macromolecular drugs, these procedures currently find most consistent application in the pharmaceutical industry to the quantitation of protein molecules. Immunoassays are also frequently applied in such important areas as the quantitation of biomarker molecules which indicate disease progression or regression, and antibodies elicited in response to treatment with macromolecular therapeutic drug candidates. Currently available guidance documents dealing with the validation of bioanalytical methods address immunoassays in only a limited way. This review highlights some of the differences between immunoassays and chromatographic assays, and presents some recommendations for specific aspects of immunoassay validation. Immunoassay calibration curves are inherently nonlinear, and require nonlinear curve fitting algorithms for best description of experimental data. Demonstration of specificity of the immunoassay for the analyte of interest is critical because most immunoassays are not preceded by extraction of the analyte from the matrix of interest. Since the core of the assay is an antigen-antibody reaction, immunoassays may be less precise than chromatographic assays; thus, criteria for accuracy (mean bias) and precision, both in pre-study validation experiments and in the analysis of in-study quality control samples, should be more lenient than for chromatographic assays. Application of the SFSTP (Societe Francaise Sciences et Techniques Pharmaceutiques) confidence interval approach for evaluating the total error (including both accuracy and precision) of results from validation samples is recommended in considering the acceptance/rejection of an immunoassay procedure resulting from validation experiments. These recommendations for immunoassay validation are presented in the hope that their consideration may result in the production of consistently higher quality data from the application of these methods.

Pharmacokinetic and glucodynamic comparisons of recombinant and animal-source glucagon after IV, IM, and SC injection in healthy volunteers.

The structure of the hormone glucagon is identical among humans and several species of other mammals. Equivalence of recombinant glucagon (rG) to animal-source glucagon (aG) was assessed in this two-part, open-label, randomized study. Part I was a four-way crossover intravenous dose-ranging study of rG (pH 2.8) involving 12 subjects. Part II was a six-way crossover study of 29 subjects comparing rG (diluent pH 2.0 and 2.8) with aG administered subcutaneously (sc) and intramuscularly (im). Maximum glucagon plasma concentrations (C(max)) and area under the glucagon concentration curve (AUC) were calculated. Additionally, maximum blood glucose concentrations (BG(max)), maximum absolute BG excursion (MAE), and area under the glucose concentration curve from time of dosing to return to baseline (AUC(rtb)) were calculated. The primary focus was equivalence of the formulation intended for marketing (rG pH 2.0) to aG. Administration of rG pH 2.0 through the im route demonstrated equivalence to aG for all pharmacokinetic and glucodynamic comparisons. Subcutaneous administration of rG pH 2.0 demonstrated standard bioequivalence for AUC (5.87 versus 6.63 ng x h/mL; NS) and near equivalence for C(max) (7.94 versus 9.12 ng/mL; p < 0.05). rG pH 2.0 showed glucodynamic equivalence to aG (BG(max), 136 versus 133 mg/dL; MAE, 50.0 versus 47.4 mg/dL, respectively) and statistically greater AUC(rtb) values (151 versus 126 mg x h/dL, p < 0. 05). rG and aG were equally safe and well tolerated. In conclusion, rG provides equivalent safety and efficacy to aG.

Metabolic and immunologic effects of insulin lispro in gestational diabetes.

OBJECTIVE: To compare the immunologic response to insulin lispro with that to regular human insulin, thereby assuring its safety for use in women with gestational diabetes, and to verify that it is effective. RESEARCH DESIGN AND METHODS: We compared the metabolic and immunologic effects of insulin lispro and regular human insulin in 42 women >18 years of age diagnosed with gestational diabetes by oral glucose tolerance testing at 14-32 weeks of gestation. Patients were randomized to receive regular human insulin or insulin lispro before consuming a test meal. Serum insulin, blood glucose, and C-peptide concentrations were measured. Throughout the remainder of gestation, patients received premeal insulin lispro or regular human insulin combined with basal insulin and performed blood glucose self-monitoring before and after each meal. Insulin antibodies and HbA1c were determined at enrollment and 6 weeks later. In addition, 10 patients received continuous intravenous insulin (4 lispro, 6 regular human insulin) and dextrose infusions intrapartum to assess placental insulin transfer. RESULTS: Anti-insulin antibody levels were similar in the two groups. Insulin lispro was not detectable in the cord blood. During a meal test, areas under the curve for glucose, insulin, and C-peptide were significantly lower in the lispro group. Mean fasting and postprandial glucose concentrations and end point HbA1c were similar in the two groups. The lispro group demonstrated fewer hypoglycemic episodes (symptoms and blood glucose concentrations <55 mg/dl). No fetal or neonatal abnormalities were noted in either treatment group. CONCLUSIONS: Insulin lispro may be considered a treatment option for women with gestational diabetes.

Sensitive RIA for the specific determination of insulin lispro.

Insulin lispro is an insulin analog in which the primary sequence has been altered by the inversion of amino acids B28 and B29. To date, it has not been possible to specifically measure insulin lispro in the presence of endogenous insulin because of the high degree of homology between these peptides. However, the specific determination of insulin lispro offers advantages over quantifying total concentrations of immunoreactive insulin. We therefore immunized guinea pigs and screened for antibodies with increased affinity and selectivity for insulin lispro. We prepared a monospecific antiserum by a novel immunoadsorption strategy using despentapeptide insulin. The antiserum was used to develop a competitive RIA for insulin lispro. The RIA has a low limit of quantification (17.2 pmol/L); has no interference from insulin, proinsulin, or C-peptide; and has interassay CVs of 2.6-13.4%. The new RIA is useful for measuring serum concentrations of insulin lispro.

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.

Antiviral activity and toxicity of fialuridine in the woodchuck model of hepatitis B virus infection.

Woodchucks were used to study the antiviral activity and toxicity of fialuridine (FIAU; 1,-2'deoxy-2'fluoro-1-beta-D-arabinofuranosyl-5-iodo-uracil). In an initial experiment, groups of six chronic woodchuck hepatitis virus (WHV) carrier woodchucks received daily doses of FIAU by intraperitoneal injection for 4 weeks. At 0.3 mg/kg/d, the antiviral effect was equivocal, but at 1.5 mg/kg/d, FIAU had significant antiviral activity. No evidence of drug toxicity was observed during the 4-week period of treatment or during posttreatment follow-up. In a second experiment, groups of nine WHV carriers or uninfected woodchucks were given 1.5 mg/kg/d of FIAU orally for 12 weeks, and the results compared with placebo-treated controls. After 4 weeks, the serum WHV-DNA concentration in the FIAU-treated carrier group was two to three logs lower than that in the placebo-treated group. After 12 weeks of FIAU treatment, serum WHV DNA was not detectable by conventional dot-blot analysis, hepatic WHV-DNA replicative intermediates (RI) had decreased 100-fold, and hepatic expression of WHV core antigen was remarkably decreased. No evidence of toxicity was observed after 4 weeks, but, after 6 to 7 weeks, food intake decreased and, after 8 weeks, the mean body weights of woodchucks treated with FIAU were significantly lower than controls. Anorexia, weight loss, muscle wasting, and lethargy became progressively severe, and all FIAU-treated woodchucks died or were euthanized 78 to 111 days after treatment began. Hepatic insufficiency (hyperbilirubinemia, decreased serum fibrinogen, elevated prothrombin time), lactic acidosis, and hepatic steatosis were characteristic findings in the final stages of FIAU toxicity in woodchucks. The syndrome of delayed toxicity in woodchucks was similar to that observed previously in humans treated with FIAU, suggesting that the woodchuck should be valuable in future investigations of the molecular mechanisms of FIAU toxicity in vivo and for preclinical toxicological evaluation of other nucleoside analogs before use in patients.

A sensitive and specific radioimmunoassay for LY309887, a potent inhibitor of glycinamide ribonucleotide formyltransferase.

LY309887, a reduced analogue of folic acid, is a potent inhibitor of glycinamide ribonucleotide formyltransferase and possesses a broad spectrum of antitumor activity. During preclinical studies using supplementation with oral folic acid, this second-generation inhibitor displayed both the desired safety profile and the pharmacology to warrant clinical investigation. A sensitive analytical method was needed to assess the pharmacokinetics of LY309887 due to the low doses planned for Phase I studies and the potential for low concentrations in plasma long after i.v. administration. We therefore undertook the development of a competitive RIA. A highly specific antiserum was raised in rabbits following immunization with LY309887 coupled to BSA. A RIA tracer was prepared by radioiodination of compound 389753, the adduct of LY309887 with p-tyramine. We developed a competitive-binding RIA procedure and used superparamagnetic particles coated with goat antirabbit IgG as a method for separating the bound and free forms of LY309887. The RIA is sensitive (0.5 ng/ml in serum and 25 ng/ml in urine), specific (negligible interference from endogenous folates), and reproducible (interassay coefficients of variation ranging from 8.1 to 15.4% and 7.6 to 8.3% for serum and urine controls, respectively). We used the RIA to assess the i.v. pharmacokinetics of LY309887 in both patients with metastatic cancer and dogs. The sensitivity of the RIA permitted the demonstration that serum concentrations of LY309887 decline in a multiexponential manner with a prolonged terminal elimination phase. We conclude that the RIA is a valid method for quantifying LY309887 in biological fluids.

Relation of race, age, and sex hormone differences to serum leptin concentrations in children and adolescents.

We explored the effects of race, age, and sex hormones on the serum leptin concentrations in 203 white and 88 black children and adolescents (ages 9.3-20.5 years). A significant sex by race interaction on serum leptin levels (p = 0.0301) was observed with lower serum leptin concentrations, adjusted for subscapular thickness and age, in black boys than in white boys. Girls had serum leptin levels that were on average 2.15 times those of boys (p < 0.0001). There was an age by sex interaction (p < 0.0001) with serum leptin concentrations decreasing in boys but not in girls with age. A strongly inverse relationship of serum testosterone levels with serum leptin levels in boys (p = 0.0067) appeared to explain this effect of age. In conclusion, the serum leptin concentration is slightly lower in black boys. A higher testosterone level in boys appears to account for an age-related decline in serum leptin in boys and the overall lower levels in boys than in girls.

Leptin and body composition of Nigerians, Jamaicans, and US blacks.

The role of leptin in humans remains controversial. Leptin concentrations are highly correlated with body fat stores. We tested whether or not this relation was consistent across the range of body composition encompassing the lean as well as the obese. Individuals participating in community-based comparative research in Nigeria (n = 363), Jamaica (n = 372), and the United States (Maywood, IL; n = 699) had their plasma leptin concentrations and body compositions (with bioelectrical impedance analysis) measured. All participants identified themselves as being black. Body mass index (in kg/m2) ranged from 14 to 62. Large differences in mean plasma leptin were noted across populations for both men and women in Nigeria, Jamaica, and the United States, respectively (men: 2.8, 3.9, and 6.8 microg/L; women: 10.3, 18.6, and 27.7 microg/L). An exponential function fit the relation between percentage body fat or total fat mass and leptin for men and women at each site. For women and men the exponential function with either percentage body fat or total fat mass was of the same shape, but increased by a constant in women, yielding higher leptin concentrations than in men at every level of body fat. On the basis of this broad distribution of body composition, the data suggest an exponential response of leptin to increases in body fat stores, consistent with the development of leptin resistance in individuals developing obesity. These findings likewise confirm that men and women exhibit different set points in terms of leptin production.

Leptin and body composition of Nigerians, Jamaicans, and US blacks

The role of leptin in humans remains controversial. Leptin concentrations are highly correlated with body fat stores. We tested whether or not this relation was consistent across the range of body composition encompassing the lean as well as the obese. Individuals participating in community-based comparative research in Nigeria (n = 363), Jamaica (n = 372), and the United States (Maywood, IL; n = 699) had their plasma leptin concentrations and body compositions (with bioelectrical impedance analysis) measured. All participants identified themselves as being black. Body mass index (in KG/m2) ranged from across populations for both men and women in Nigeria, Jamaica, and the United States, respectively (men: 2.8, 3.9, and 6.8 microg/L; women: 10.3, 18.6, and 27.7 microg/L). An exponential function fit the relation between percentage body fat or total fat mass and leptin for men and women at each site. For women and men the exponential function with either percentage body fat or total fat mass was of the same shape, but increased by a constant in women, yielding higher leptin concentrations than in men at every level of body fat. On the basis of this broad distribution of body composition, the data suggest an exponential response of leptin to increase in body fat stores, consistent with the development of leptin resistance in individuals developing obesity. These findings likewise confirm that men and women exhibit different set points in terms of leptin production(AU)

A radioimmunoassay for LY315902, an analog of glucagon-like insulinotropic peptide, and its application in the study of canine pharmacokinetics.

Glucagon-like insulinotropic peptide (GLP-1) and its analogs are of interest because of their therapeutic potential in type II diabetes. LY315902 is a GLP-1-(7-37)-OH analog with a modified N-terminus (IP7), an octanoic acid (C8) acylated on the lysine residue at position 34, and a substitution with arginine at position 26. We developed a sensitive and specific radioimmunoassay (RIA) for the determination of immunoreactive LY315902 in the plasma of animals. A homobifunctional cross-linker was used to couple the nonacylated form of LY315902 [IP7-R26-GLP-1-(7-37)-OH] to carrier proteins to enhance its immunogenicity. Following immunization, animal antisera were screened by RIA for the presence of LY315902 antibodies. One rabbit produced a high-affinity antiserum that display insignificant cross-reactivity against two forms of native GLP-1 and possible major metabolites of LY315902. In this RIA method, plasma samples were combined with radioiodinated LY315902 and rabbit anti-IP7-R26-GLP-1-(7-37)-OH serum, and then incubated overnight at room temperature. The bound forms of LY315902 were separated by polyethylene glycol assisted second antibody precipitation. The sensitivity of the assay was estimated to be 19 pM. Inter-assay precision (%CV) and accuracy (recovery) for quality control samples in dog plasma ranged from 8.0% to 14.7% and 92.8% to 107.3%, respectively. By applying this assay to measure plasma concentrations of immunoreactive LY315902 in dogs following twice daily subcutaneous injections of LY315902, we determined that the plasma half-life of LY315902 is significantly longer than that of native GLP-1-(7-37)-OH. We concluded that the structural modifications which were made to produce LY315902 prolonged its plasma half-life. The extended plasma half-life of LY315902 correlated well with its prolonged pharmacology in dogs.

Nutritional regulation of circulating insulin-like growth factors (IGFs) and their binding proteins in the ovine fetus.

Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) are important for fetal and postnatal development, but the regulation of circulating IGFs and IGFBPs has not been as thoroughly investigated in the maternal/fetal unit as in the adult animal where nutrition status plays a regulatory role. We used the chronically-catheterized, late-gestation ovine model and compared circulating IGFs and IGFBPs levels, and hepatic IGF-I mRNA levels. Following a five-day maternal fast, both IGF-I and IGF-II levels were decreased in the maternal and fetal circulation (P < 0.05), accompanied by a decrease in fetal hepatic IGF-I mRNA levels, but the IGFBP2 level was increased and the IGFBP3 level was decreased in maternal circulation, whereas the IGFBP1 level was increased in fetal circulation. In both fed and fasting states, the infusion of glucose (150% of baseline) did not alter IGFs or IGFBPs in either maternal or fetal circulation. To understand the regulation of the endogenous IGF system, rhIGF-I was infused (6.7 nmol/kg fetus/h) into the fetal circulation. While maternal IGFs or IGFBPs remained unchanged, IGF-I infusion into fetal circulation resulted in an increase in IGF-I, a decrease in IGF-II, and an overall increase in the IGFBPs (P < 0.05). Taken together, circulating IGFs and IGFBPs in the ovine fetus are more sensitive to prolonged nutrient deficit than to a brief glucose increase. The nutrition status therefore regulates the IGF system in maternal and fetal circulation which, in turn, may regulate the nutrient utilization for fetal growth.

Evidence of free and bound leptin in human circulation. Studies in lean and obese subjects and during short-term fasting.

Little is known about leptin's interaction with other circulating proteins which could be important for its biological effects. Sephadex G-100 gel filtration elution profiles of 125I-leptin-serum complex demonstrated 125I-leptin eluting in significant proportion associated with macromolecules. The 125I-leptin binding to circulating macromolecules was specific, reversible, and displaceable with unlabeled leptin (ED50: 0.73 +/- 0.09 nM, mean +/- SEM, n = 3). Several putative leptin binding proteins were detected by leptin-affinity chromatography of which either 80- or 100-kD proteins could be the soluble leptin receptor as approximately 10% of the bound 125I-leptin was immunoprecipitable with leptin receptor antibodies. Significantly higher (P < 0.001) proportions of total leptin circulate in the bound form in lean (46.5 +/- 6.6%) compared with obese (21.4 +/- 3.4%) subjects. In lean subjects with 21% or less body fat, 60-98% of the total leptin was in the bound form. Short-term fasting significantly decreased basal leptin levels in three lean (P < 0.0005) and three obese (P < 0.005) subjects while refeeding restored it to basal levels. The effects of fasting on free leptin levels were more pronounced in lean subjects (basal vs. 24-h fasting: 19.6 +/- 1.9 vs. 1.3 +/- 0.4 ng/ml) compared with those in obese subjects (28.3 +/- 9.8 vs. 14.7 +/- 5.3). No significant (P > 0.05) decrease was observed in bound leptin in either group. These studies suggest that in obese individuals the majority of leptin circulates in free form, presumably bioactive protein, and thus obese subjects are resistant to free leptin. In lean subjects with relatively low adipose tissue, the majority of circulating leptin is in the bound form and thus may not be available to brain receptors for its inhibitory effects on food intake both under normal and food deprivation states.

Difference in the influence of maternal and paternal NIDDM on pancreatic beta-cell activity and blood lipids in normoglycaemic non-diabetic adult offspring.

The 75-g oral glucose tolerance test was performed in 38 normoglycaemic (World Health Organization criteria) non-diabetic volunteers, aged 31-40 years, of whom 20 had a non-insulin-dependent diabetic (NIDDM) mother and 18 had an NIDDM father. At the time of the study the offspring of NIDDM mothers had a somewhat higher body mass index (BMI) (males: 26.5 +/- 1.0 (mean +/- SEM), females: 27.5 +/- 1.5 kg/m2) than the offspring of NIDDM fathers (males: 23.4 +/- 0.9, females: 24.2 +/- 1.2 kg/m2). There was no difference in the time-course of glycaemia; however the serum concentrations of immunoreactive insulin (IRI), C-peptide and proinsulin were significantly higher in offspring of NIDDM mothers than in offspring of NIDDM fathers: area under the curve (AUC) serum IRI: 0.928 +/- 0.091 vs 0.757 +/- 0.056 nmol.l-1.h-1, p = 0.019; serum C-peptide: 6.379 +/- 0.450 vs 4.753 +/- 0.242 nmol.l-1.h-1, p = 0.004; serum proinsulin: 172 +/- 40 vs 51 +/- 7 pmol.l-1.h-1, p = 0.008). Serum IRI correlated with BMI, but C-peptide and proinsulin did not, and after accounting for BMI by covariance analysis they remained significantly higher in offspring of NIDDM mothers. In this group serum proinsulin was significantly higher in male than in female offspring (AUC serum proinsulin: 289 +/- 68 vs 77 +/- 27 pmol.l-1.h-1, P = 0.015). Male offspring of NIDDM mothers also had significantly higher serum triglyceride levels than females of the same group and than offspring of NIDDM fathers. The offspring (male and female) of NIDDM mothers had slightly lower serum apolipoprotein A-I levels than the offspring of NIDDM fathers. Significant correlations were found between serum triglycerides, HDL-cholesterol and apolipoprotein B, and serum concentrations of pancreatic beta-cell peptides, mostly in the offspring of NIDDM mothers; however, they did not display unequivocal association with gender within this group. The data are consistent with clinical observations of a greater risk of NIDDM transmission from the mother than from the father, and may suggest that male offspring are more exposed to this risk than female offspring.

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.