An enzyme-linked immunosorbent assay for erythropoietin using monoclonal antibodies, tetrameric immune complexes, and substrate amplification

We recently reported the development of several monoclonal antibodies (MoAbs) to native human erythropoietin (Ep). In the present study we have used the two antibodies with highest affinity to develop a two- sided or sandwich enzyme-linked immunosorbent assay (ELISA) to measure Ep in human serum. In this assay Ep is incubated in microtiter wells precoated with the first (IgE) anti-Ep antibody. Assay wells are then incubated with the second (IgG1) anti-Ep antibody, which is labeled noncovalently with the enzyme alkaline phosphatase (AP) by means of bispecific tetrameric antibody complexes consisting of IgG1 anti-Ep cross-linked to IgG1 anti-AP using rat MoAbs specific for mouse IgG1. Application of this noncovalent labeling procedure, in combination with substrate amplification, results in a detection sensitivity of 0.5 to 1.0 mU/sample (5 to 10 mU/mL), which makes this assay suitable for measuring normal serum Ep levels. The validity of this ELISA for quantitating Ep in biological fluids was demonstrated by the parallelism obtained between pure recombinant Ep dose-response curves and those obtained with plasma and serum from healthy donors and patients with various hematologic disorders. Normal plasma Ep levels detected with this ELISA ranged from 9 to 101 mU/mL with a mean of 32 +/- 23 (SD) mU/mL. Ep levels in sera from patients with polycythemia vera were in the low to normal range, whereas Ep levels in sera from patients with secondary polycythemia and patients with aplastic anemia were moderately to strongly elevated. These results demonstrate that the Ep-ELISA is a sensitive, reliable, and nonradioactive immunologic method for quantitating Ep levels and should prove useful in a variety of clinical and laboratory settings.     

Immunoreactive erythropoietin concentrations in fetal and neonatal rats and the effects of hypoxia

Immunoreactive erythropoietin (Ep) was measured in normoxic and hypoxic (0.5 atm; 18 hours) fetal rats from day 14 to day 21 of gestation and in neonatal rats from birth to weaning, and was compared to the adult rat. Amniotic fluid (AF) Ep was approximately 100 mU/mL on day 14 and 15, and decreased to 20 mU/mL on day 20, with no difference between the hypoxic and normoxic mothers. Only on day 21 did the Ep in the AF increase slightly in the hypoxic group, while the Ep in the control group continued to fall to 15 mU/mL on day 21, the last day of pregnancy. Before day 17 of gestation the rat fetus appears to have hypoxia-independent, extrahepatic Ep available which is followed by hepatic and renal Ep production, both of which become sensitive to maternal hypoxia during the last days of pregnancy. In the neonatal rat plasma and tissue, Ep levels varied greatly during the first three weeks of life regardless of whether the animals were hypoxic or not. With the exception of the first and ninth days of life, circulating Ep levels were higher than adult levels in normal newborn rats. Neonatal rats responded to hypoxia with increasing Ep levels, and the response increased with age such that during the third week of life the plasma Ep levels were significantly higher than in adult hypoxic rats. No sex difference in male and female response to hypoxia could be documented until sexual maturity (day 42). In the normoxic neonatal rat more Ep originated from the liver than the kidneys until day 10, while under hypoxic conditions the switch occurred as early as two days after birth.     

O2-supply to the kidneys and the production of erythropoietin

The concentration of erythropoietin (Ep) in blood increases little following a reduction of the renal blood flow (rbf). In the present study we examined whether a reduction of rbf in rats induces a lowering of the renal venous PO2. In addition, the combined effects of reduced rbf and hypobaric hypoxia on the production of Ep were studied. To lower rbf, silver clips with stepwisely reduced inner diameters were applied on both renal arteries. The PO2 of renal venous blood decreased gradually to values below 10 mm Hg, when rbf was reduced from 80 to 10% of normal. Under these conditions plasma Ep increased only moderately from 20 +/- 6 to 69 +/- 24 mU/ml within 18-20 h. However, plasma Ep of rats exposed to hypobaric hypoxia (stimulated altitude of 6000 m for 18-20 h) was 431 +/- 68 mU/ml, when rbf was 100-80% of normal, compared to 931 +/- 91 mU/ml, when rbf was reduced to 40-10%. Thus, a reduction of the blood flow to the kidney appears to be no major stimulus for the production of Ep, even when the PO2 in the kidney becomes very low. However, plasma levels of Ep increase markedly, when the whole body O2 offer is lowered. These results support the view that Ep production is not only dependent on the PO2 in the kidney but also under the control of extrarenal O2 sensitive mechanisms. For example, the hypothalamic-hypophyseal system is thought to influence the production of Ep.     

Hyperinsulinemia in the physiologic range is not superior to short-term fasting in suppressing insulin secretion in obese men

The negative-feedback control exerted by plasma insulin on beta-cell insulin release in normal-weight and obese subjects is still a matter of debate. Subjects submitted to a euglycemic insulin clamp undergo a suppression of insulin secretion that is due to both the infused insulin and the 2- to 3-hour fast during the procedure. We elected to elucidate the role of physiologic hyperinsulinemia per se in the insulin negative autofeedback in obese men. Ten men with massive uncomplicated obesity (age, 18 to 37 years; body mass index [BMI], 41 +/- 1.15 kg/m2) and 6 normal-weight healthy men (age, 22 to 30 years; BMI, 22 +/- 0.28 kg/m2) underwent 2 studies in random order: (1) a euglycemic-hyperinsulinemic glucose clamp with an insulin infusion rate of 1 mU/kg/min and (2) a control study with saline infusion. Serum C-peptide concentrations were significantly higher in obese versus control subjects at baseline (2.54 +/- 0.178 v 1.63 +/- 0.256 ng/mL, P < .05). Exogenous insulin infusion significantly suppressed serum C-peptide at steady state ([SS] last 30 minutes of insulin or saline infusion) in controls (mean of the last 4 measurements from 120 minutes to 150 minutes, 0.86 +/- 0.306 ng/mL, P < .05 vbaseline) but not in obese patients (2.03 +/- 0.26 ng/mL, nonsignificant [NS] v baseline). During the saline infusion studies, C-peptide levels slightly and similarly declined over time in both groups (2.71 +/- 0.350 at baseline v 2.31 +/- 0.300 ng/mL at SS in obese patients, NS, and 1.96 +/- 0.189 v 1.62 +/- 0.150 ng/mL in controls, NS). This study shows that in obese men hyperinsulinemia within the postprandial range is not superior to a 2.5-hour fast for the suppression of beta-cell activity, suggesting an impairment of the insulin negative autofeedback in this clinical condition.     

Plasma levels of plasminogen activator inhibitor type 1, beta- thromboglobulin, and fibrinopeptide A before, during, and after treatment of acute myocardial infarction with alteplase

Plasma levels of plasminogen activator inhibitor type-1 (PAI-1), beta- thromboglobulin (beta TG), and fibrinopeptide A (FPA) were followed over 24 hours in 30 patients treated with alteplase for acute myocardial infarction. Samples were taken at baseline (T Oh), after 90 minutes (under alteplase, no heparin, T 1.5h), after 120 minutes (under alteplase and heparin, T 2h), 30 minutes after thrombolytic therapy (T 3.5h), as well as 12 hours (T 12h) and 24 hours (T 24h) after baseline. PAI-1 antigen levels (55 +/- 9 ng/mL at T Oh, mean +/- SEM) decreased to 35 +/- 5 (T 1.5h) and 40 +/- 6 (T 2h) ng/mL under alteplase, before increasing to 84 +/- 22 (T 3.5h), 130 +/- 30 (T 12h), and 64 +/- 7 (T 24h) ng/mL after therapy, P less than .001. A high baseline PAI-1 activity (18 +/- 3 ng/mL) decreased to 2.0 +/- 0.4 (T 1.5h) and 1.7 +/- 0.2 (T 2h) under alteplase and increased to 32 +/- 5 (T 12h) and 19 +/- 3 (T 24h) ng/mL after therapy (P less than .0001). beta TG levels (339 +/- 105 ng/mL at T Oh) decreased to 203 +/- 48 (T 2h), 154 +/- 51 (T 3.5h), 187 +/- 40 (T 12h), and 142 +/- 32 (T 24h) ng/mL under heparin (P less than .01). FPA levels (34 +/- 9 ng/mL at T Oh) increased to 85 +/- 15 ng/mL under alteplase alone (T 1.5h) and normalized under heparin (11 +/- 4, 6 +/- 2, 4 +/- 2, and 3 +/- 1 ng/mL at T 2h, T 3.5h, T 12h, and T 24h, respectively). A high level of FPA at T 3.5h correlated with reocclusion (33 +/- 12 ng/mL, n = 4 v 2.9 +/- 0.5 ng/mL, n = 21, P less than .005). We conclude that plasma levels of PAI- 1 antigen as well as activity markedly increase after alteplase therapy of acute myocardial infarction. The high activity of PAI-1 and decreasing beta TG levels suggest that platelets do not contribute significantly to this phenomenon. The marked increase of FPA levels under recombinant tissue-type plasminogen activator alone and its normalization under heparin emphasize the important role of concomitant anticoagulation in controlling further intravasal fibrin generation under alteplase.     

Relationship between changes in glucose production and gluconeogenesis during mild hypoglycemia in humans

We measured 14C-alanine conversion to 14C-glucose (an index of gluconeogenesis) and glucose production in six healthy volunteers during low-dose insulin infusion (0.3 mU/kg.min for four hours). Insulin rose from 7 +/- 2 to 20 +/- 2 microU/mL, and plasma glucose fell to a plateau of 65 to 70 mg/dL after 60 minutes. Glucagon and catecholamines increased after 60 minutes, whereas C-peptide decreased immediately. Glucose production decreased transiently by 43% and then returned to baseline after 45 minutes. In contrast, 14C-alanine conversion to 14C-glucose was unchanged for 120 minutes, but then rose twofold above baseline by 240 minutes. Our data suggest that early recovery of glucose production during mild hyperinsulinemia occurs independent of changes in gluconeogenesis. However, gluconeogenesis plays an increasingly more important role in maintaining glucose production when mild hypoglycemia is prolonged.     

Angiotensinogen concentrations and renin clearance : implications for blood pressure regulation

Renin (REN) requires seconds to convert angiotensinogen (AGT) to angiotensin I. We tested the hypothesis that this long catalytic cycle might indicate an influence of AGT concentrations on REN clearance. We studied 2 transgenic rat (TGR) strains for human (h) AGT; one strain has hAGT values approximately 7-fold higher than the other (68+/-18 versus 10+/-4 microg angiotensin I/mL). hREN (30 000 pg) was bolus-infused into both lines and into nontransgenic controls. The terminal half-life (T1/2beta) was increased (130 versus 82 minutes) and the metabolic clearance rate (MCR) was decreased (0.83+/-0.29 versus 2.2+/-0.66 microL. min(-1). g(-1)) in the high hAGT strain compared with the low hAGT strain. The difference was not related to volume of distribution at steady state. Infused hREN blocked with remikiren resulted in T1/2beta and MCR values that were not different from control values. Infused unblocked and blocked radiolabeled hREN was distributed similarly in the hAGT TGR strains. Infused mouse REN, which cannot convert hAGT, had similar T1/2beta and MCR values in hAGT TGR. Measuring REN with direct radioimmunoassay or by enzyme kinetic assay gave similar results. We next crossed homozygous hAGT TGR from both strains with homozygous hREN TGR. Heterozygous offspring from the low hAGT TGR strain had plasma REN activity, hREN concentration, and rat AGT values that were no different from those of their parents. However, TGR offspring with high hAGT values had massively elevated plasma REN activity and hREN concentration as well as elevated blood pressure, even though both the hREN and rREN genes are downregulated. We conclude that increased AGT concentrations decrease REN MCR and increase REN T1/2beta. The REN-AGT complex may stabilize plasma REN concentration and regulate plasma REN activity independent of renal REN secretion and angiotensin II-mediated feedback. These effects could augment angiotensin I generation and influence blood pressure. The notion that AGT is merely a passive substrate reservoir for REN should be revised.     

Plasma norepinephrine kinetics, dopamine-beta-hydroxylase, and chromogranin-A, in hypothyroid patients before and following replacement therapy

Whether the increased plasma norepinephrine level reported in hypothyroidism is the result of impaired norepinephrine (NE) clearance or increased NE release by nerve terminals is unknown. We, therefore, measured plasma NE levels and clearance in 11 hypothyroid patients before [T4 index, 41.2 +/- 7.7 nmol/L (mean +/- SEM); TSH, 71.4 +/- 23.0 mU/L] and 4 +/- 0.5 months after thyroid replacement (T4 index, 136.4 +/- 24.4 nmol/L; TSH, 3.2 +/- 1.2 mU/L) and in 8 healthy volunteers. Plasma dopamine-beta-hydroxylase and chromogranin-A, which are coreleased with NE by sympathetic nerve endings, were also measured. Plasma NE was higher in the hypothyroid (2.37 +/- 0.24 nmol/L) than in the euthyroid state (1.86 +/- 0.24 nmol/L; P less than 0.02) or in the controls (1.87 +/- 0.27 nmol/L). Plasma clearance of NE, however, was not affected after thyroid replacement (hypothyroid, 2.08 +/- 0.31 L/min; euthyroid, 1.94 +/- 0.21 L/min; controls, 1.86 +/- 0.15 L/min). There was no significant change in plasma dopamine-beta-hydroxylase (hypothyroid, 720 +/- 139 nmol/mL.h; euthyroid, 553 +/- 97 nmol/mL.h) or plasma chromogranin-A (hypothyroid, 48.9 +/- 7.1 ng/mL; euthyroid, 42.9 +/- 5.3 ng/mL) after thyroid replacement. We conclude that the increased plasma NE in hypothyroid patients is not due to a change in plasma clearance, but is more likely secondary to increased NE release.     

Administration of human recombinant insulin-like growth factor-I to patients following major gastrointestinal surgery

OBJECTIVE: The aim was to study the pharmacokinetic parameters and biological activity of a single dose of human recombinant IGF-I (rhIGF-I) administered to patients following major gastrointestinal surgery. DESIGN: A double blind placebo controlled externally randomized study of 30 patients; the study commencing 24 hours after major colonic or gastric surgery. MEASUREMENTS: After a baseline blood sampling day, IGF-I (40 micrograms/kg by single subcutaneous dose, n = 20) or placebo (n = 10) was administered and serum and urine samples collected over the ensuing 72 hours. Serum IGF-I, IGF-II, IGF binding proteins (IGFBP-1, IGFBP-3), GH and insulin were measured by radioimmunoassay. Serum IGF bioactivity was assessed using a validated porcine cartilage bioassay. Serum and urinary electrolytes were measured by standard methodology. RESULTS: Serum immunoreactive IGF-I levels peaked at 4 hours following injection of IGF-I (1.09 +/- 0.12 U/ml mean +/- SEM), remained elevated for 15 hours and returned to basal levels by 24 hours after injection. IGF bioactivity was increased by 57% 6 hours after IGF-I injection. Mean levels of IGFBP-1 and IGFBP-3, IGF-II and GH were unaffected by IGF-I administration. Insulin levels were suppressed at 30 minutes following injection of IGF-I compared with the placebo group (16.9 +/- 3.0 mU/I vs 32.3 +/- 7.1, P = 0.02); thereafter, there were no differences in insulin levels. The mean change in serum creatinine following IGF-I (-6.3 +/- 3.0 mmol/l) was significantly different from that in the control group (+7.2 +/- 6.2, P = 0.03). Creatinine clearance rose from a mean of 71.6 +/- 7.5 ml/min to 83.2 +/- 7.6 ml/min after IGF-I treatment (P = 0.02). In the IGF treated patients, cholesterol levels consistently fell (-0.20 +/- 0.05 mmol/l); this was not observed in the placebo group (+0.20 +/- 0.14, P = 0.006). Basal serum potassium levels in the IGF treatment group (4.1 +/- 0.1 mmol/l) fell to 3.8 +/- 0.1 at 4 hours (P = 0.002) and 3.6 +/- 0.1 at 10 hours (P = 0.001) returning to a level of 4.0 +/- 0.1 (P = 0.293) at 24 hours after injection. There were no other observed differences in serum or urinary electrolytes or serum free fatty acids and triglycerides. Pharmacokinetic parameters derived from baseline adjusted IGF-I measurements revealed a slow absorption of the administered dose with a Tmax of 5.0 +/- 0.43 hours and an elimination half-life of 10.8 +/- 1.2 hours. The computed volume of distribution was 0.33 +/- 0.05 I/kg and the clearance on average 25 ml/min. CONCLUSION: A single subcutaneous dose of IGF-I normalized circulating IGF-I levels in post-operative patients, was well tolerated and without side-effects. IGF bioactivity was increased and associated with a fall in serum cholesterol, potassium and creatinine levels and a rise in creatinine clearance. Further long-term studies are now required to assess the anabolic effects of rhIGF-I in this type of patient group.     

The in vivo metabolism of recombinant human erythropoietin in the rat

We compared the in vivo plasma clearance and organ accumulation in anesthetized rats of 125I-labeled, recombinant human erythropoietin and 125I-labeled, desialylated recombinant erythropoietin. The immediate volume of distribution of 125I-labeled, recombinant erythropoietin approximated that of the plasma volume. Its plasma clearance was multiexponential, with an initial rapid distribution phase (t1/2 = 53 minutes) and a slower elimination phase (t1/2 = 180 minutes). Organ accumulation of labeled recombinant erythropoietin, as compared with 125I-labeled human albumin, was negligible until 30 minutes after injection when small amounts appeared in the kidneys and bone marrow. Only 24% of the 125I-labeled, desialylated recombinant erythropoietin was recovered immediately after injection, and 96% of the hormone was cleared from the plasma with a t1/2 of 2.0 minutes. The bulk of the desialylated hormone accumulated in the liver where it was rapidly catabolized and its breakdown products released back into the plasma. Significantly, in contrast to unmodified erythropoietin, there was also early accumulation of desialylated hormone in the kidneys, marrow, and spleen. Desialylated orosomucoid but not orosomucoid, yeast mannan, or dextran sulfate 500 inhibited the rapid plasma clearance and hepatic accumulation of desialylated erythropoietin. Oxidation of the desialylated hormone restored its plasma recovery and clearance to normal but rendered it biologically inactive, and accumulation in organs other than the kidney was negligible.     

Improvement of glucose tolerance by nateglinide occurs through enhancement of early phase insulin secretion.

Nateglinide is a new, fast-onset, short-acting hypoglycemic agent, which increases early phase insulin secretion and the total amount of insulin secreted. However, it is not clear which of these effects contribute more to the decrease in postprandial plasma glucose (PG). To further clarify the pharmacologic actions of nateglinide, we investigated the changes in PG and insulin levels during meal tolerance tests with and without nateglinide. Subjects were 10 newly diagnosed and untreated inpatients with type 2 diabetes. After diet and exercise therapy for 1 week, nateglinide at 270 mg divided 3 times a day, was started. Meal tolerance tests were performed before (baseline) and after a single nateglinide administration (day 1), after 7 days of repeated administration (day 7), and after cessation of nateglinide on day 8. Mean fasting PG was 146 +/- 6 mg/dl (mean +/- SEM) at baseline and 130 +/- 6 mg/dL on day 7 (P =.0004). The 2-hour postprandial PG level was 226 +/- 10 mg/dL at baseline, 145 +/- 11 mg/dL on day 1 (P =.0008), and 190 +/- 15 mg/dL on day 8 (P =.08, baseline; P =.01, day 7). The mean fasting insulin level was 5.4 +/- 1.0 microU/mL at baseline and did not change significantly during the study. The 30-minute postprandial insulin level was 14.4 +/- 1.9 microU/mL at baseline, 39.5 +/- 4.5 microU/mL on day 1 (P =.0004), and 23.6 +/- 3.6 microU/mL on day 8 (P =.045, baseline; P =.010, day 7). The total insulin amount, in terms of area under the curve (AUC. IRI), was 3.99 +/- 0.7 x 10(3) microU/mL. min at baseline, 5.47 +/- 0.8 microU/mL. min on day 1 (P =.029), and 6.01 +/- 1.9 microU/mL. min on day 8 (P =.047 v baseline). The early phase of insulin secretion, based on the ratio of delta IRI to delta PG from fasting to 30 minutes after a meal was 0.15 +/- 0.13 at baseline, 1.44 +/- 0.26 on day 1 (P =.0009) and 0.26 +/- 0.06 on day 8 (P =.05 v day 1). After cessation of nateglinide, the postprandial PG level increased immediately. Although early phase insulin secretion returned nearly to the baseline level, total insulin secretion remained at a high level. These results suggested that early phase insulin secretion contributes more than total insulin secretion to the improvement of postprandial hyperglycemia in type 2 diabetes.     

Glucose and amino acid metabolism in aging man: differential effects of insulin

Insulin is a major regulator of glucose and body protein homeostasis, both of which demonstrate age-related changes. To clarify insulin's role in these age-related changes and to compare age-related glucose and protein homeostatic responses, insulin-mediated aspects of glucose and amino acid metabolism were simultaneously examined in healthy postabsorptive young (n = 5, mean age, 25 years) and elderly (n = 5, mean age, 76 years) men. Primed constant infusions of L-[1-13C]leucine and L-[15N]alanine were administered during a basal period (0 to 180 minutes) and during four separate single rate euglycemic insulin infusions (180 to 360 minutes). Steady state insulin concentrations were 16 +/- 1, 29 +/- 3, 75 +/- 5, and 2407 +/- 56 microU/mL in the young and 23 +/- 4, 37 +/- 8, 96 +/- 11 and 3,357 +/- 249 microU/mL in the elderly at the different insulin infusion rates of 6, 10, 30, and 400 mU mU.m-2.min-1, respectively. For the 6 and 10 mU insulin infusion rates, a primed, constant infusion of [6,6 - 2H2]glucose permitted quantitation of hepatic glucose production. Glucose disposal rates adjusted for lean body mass (LBM) were lower in the elderly than in the young at the 6, 10, and 30 mU insulin infusion rates and similar in the two age groups in the 400 mU studies. Insulin dose-dependent reductions occurred in eight of ten plasma amino acids and were not influenced by age. There was an insulin dose-dependent reduction in plasma leucine flux which was similar in both age groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deconvolution analysis of cardiac natriuretic peptides during acute volume overload

Cardiac natriuretic peptides, especially amino terminal pro-Brain Natriuretic Peptide (NT-proBNP), are emerging as powerful circulating markers of cardiac function. However, the in vivo secretion and elimination (t1/2) of these peptides during acute volume overload have not been studied. We present the first report of the secretion and elimination of cardiac natriuretic peptides, based on deconvolution analysis of endogenous ovine plasma levels measured by specific radioimmunoassay. Four normal, conscious sheep underwent rapid right ventricular pacing (225 bpm) for 1 hour to stimulate acute cardiac natriuretic peptide release. Plasma samples and right atrial pressure measurements were taken at regular intervals 30 minutes before, during, and 4 hours after pacing. Baseline right atrial pressure significantly increased (P:=0.02) during the 1 hour of pacing in association with a prompt increase in plasma BNP (P:=0.03), atrial natriuretic peptide (P:=0.01), and NT-proBNP (P:=0.02). Deconvolution analysis showed that the t1/2 of NT-proBNP (69.6+/-10.8 minutes) was 15-fold longer than BNP (4.8+/-1. 0 minutes). Despite sustained increases in atrial pressure, cardiac secretion of natriuretic peptides (particularly atrial natriuretic peptide) fell during the pacing period, suggesting a finite source of peptide for secretion. Size-exclusion high-performance liquid chromatography revealed NT-proBNP to be a single immunoreactive peak, whereas BNP comprised at least 2 immunoreactive forms. These findings, especially the prompt secretion of BNP and the prolonged t1/2 of NT-proBNP, clarify the metabolism of BNP forms and help to explain the diagnostic value of NT-proBNP measurement as a sensitive marker of ventricular function.     

Acute cortisol excess results in unimpaired insulin action on lipolysis and branched chain amino acids, but not on glucose kinetics and C-peptide concentrations in man

To assess whether acute cortisol excess impairs insulin action on lipolysis, plasma amino acids, endogenous insulin secretion, and glucose kinetics, nine normal subjects were studied after acute cortisol excess (80 mg hydrocortisone by mouth) and after placebo. Insulin sensitivity was assessed 6 hours after hydrocortisone using the glucose clamp technique (insulin infusion of 20 mU/m2 X minute for 120 minutes, plasma insulin levels of approximately equal to 50 mU/L). Hyperinsulinemia suppressed plasma free fatty acids (FFA) similarly by 75 and 76%, respectively. Most plasma amino acid concentrations were increased after hydrocortisone; however, the insulin-induced decrease of branched chain amino acids, serine, threonine, and tyrosine was unimpaired after hydrocortisone. Plasma C-peptide concentrations were less suppressed during hyperinsulinemia after hydrocortisone than after placebo (by 0.15 +/- 0.03 v 0.25 +/- 0.02 nmol/L, P less than 0.01), suggesting diminished insulin-induced suppression of insulin secretion. The glucose infusion rates required to maintain euglycemia were 35% lower (P less than 0.01) after hydrocortisone due to decreased insulin effects on metabolic clearance rate of glucose and diminished suppression of hepatic glucose production (0.4 +/- 0.1 v -0.1 +/- 0.1 mg/kg X minute, p less than 0.05, 3-3H-glucose infusion method). The data demonstrate that acute elevation of plasma cortisol to levels near those observed in severe stress results in insulin resistance of peripheral and hepatic glucose metabolism but in unimpaired insulin effects on plasma FFA and branched chain amino acids, suggesting that cortisol's lipolytic and proteolytic effects are antagonized by elevated plasma insulin levels.     

In vivo and in vitro complexes of activated protein C with two inhibitors in baboons.

In vivo complex formation of activated protein C with protein C inhibitor (APC-PCI) and with alpha 1-antitrypsin (APC-alpha 1AT) following infusion of 0.25 or 1.0 mg APC/kg in 1 hour into baboons was studied using immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA)s. Before APC infusion, detectable plasma levels (about 30 ng/mL) of APC-alpha 1AT complex were found in the baboon plasma. At the lower APC dose, APC-PCI and APC-alpha 1AT complex levels were 1.4 +/- 0.3 (mean +/- SD) and 0.8 +/- 0.1 microgram/mL after 1 hour of infusion. At the higher APC dose, the APC-PCI level was similar to the APC-alpha 1AT level during the first 30 minutes, but after 1 hour of infusion the APC-alpha 1AT level was higher than the APC-PCI level, reaching 4.1 +/- 1.2 and 2.9 +/- 1.2 microgram/mL, respectively. After 24 hours, complex levels had returned to basal conditions. During infusion of protein C (1.0 mg/kg in 1 hour), both complexes were detected in low concentrations. Following bolus injection of APC, half-lives (t1/2) for APC and APC-PCI and APC-alpha 1AT complexes of 10, 40, and 140 minutes, respectively, were observed. After 1-hour incubation with 2.5 micrograms/mL APC, baboon plasma contained 1.0 +/- 0.2 and 0.8 +/- 0.1 microgram/mL of APC-PCI and APC-alpha 1AT, respectively. Addition of 10 micrograms/mL APC to baboon plasma yielded 2.5 and 2.4 micrograms/mL APC-PCI and APC-alpha 1AT after 1 hour, respectively. Immunoblotting analysis also showed in vivo formation of complexes of APC with an auxilliary inhibitor but not in vitro in citrated plasma. These data show that both PCI and alpha 1AT are physiologic inhibitors of APC and suggest that when PCI is depleted by a high dose of APC, alpha 1AT becomes the major inhibitor of APC.     

Alcohol and glucose counterregulation during acute insulin-induced hypoglycemia in type 2 diabetic subjects

To investigate the influence of alcohol on glucose counterregulation and recovery during acute insulin-induced hypoglycemia in type 2 diabetic subjects, 8 diet-treated type 2 diabetic subjects were examined twice after an overnight fast. A graded hyperinsulinemic (1 mU/kg/min, 60 to 195 minutes) euglycemic/hypoglycemic clamp was performed with concomitant infusion of 3-(3)H-glucose to assess glucose turnover. After a euglycemic baseline period (150 to 180 minutes), 200 mL of water was taken either alone or with alcohol (0.4 g/kg body weight). Hypoglycemia (plasma glucose nadir, 2.8 mmol/L) was subsequently induced, and the recovery period followed after discontinuation of insulin and the variable glucose infusion. On both study days, circulating concentrations of insulin and glucose were comparable. Alcohol intake markedly increased plasma lactate (area under the curve [AUC], recovery period) (244 +/- 30 v 12 +/- 4 mmol/L x 240 minutes; P = .00009) and suppressed plasma nonesterified fatty acids (NEFA) (AUC, recovery period) (95 +/- 13 v 161 +/- 18 mmol/L x 240 minutes; P = .0008). No differences were found in the counterregulatory response of catecholamines, cortisol, and growth hormone (GH). However, alcohol intake decreased peak glucagon significantly (155 +/- 12 v 200 +/- 17 pg/mL; P = .038). In diet-treated, mild type 2 diabetic subjects, alcohol does not modify recovery from insulin-induced hypoglycemia.     

Interactions of galanin and arginine on growth hormone, prolactin, and insulin secretion in man.

Galanin (GAL), a 29 amino acid neuropeptide, is known to increase both basal and growth hormone-releasing hormone (GHRH)-induced growth hormone (GH) secretion while not significantly increasing prolactin (PRL) secretion in man. GAL is also endowed with an inhibiting effect on glucose-stimulated insulin release in animals, but not in man. We studied the effect of GAL (80 pmol/kg/min infused over 60 minutes) on the arginine- (ARG, 30 g infused over 30 minutes) stimulated GH, PRL, insulin, and C-peptide secretion in eight healthy volunteers (age, 20 to 30 years). GAL induced an increase of GH (GAL v saline, area under curve [AUC], mean +/- SEM: 316.5 +/- 73.9 v 93.2 +/- 20.9 micrograms/L/h, P less than .05), but failed to modify both PRL and insulin secretion. GAL enhanced the ARG-induced stimulation of both GH (1,634.1 +/- 293.1 v 566.9 +/- 144.0 micrograms/L/h, P less than .02) and PRL secretion (1,541.9 +/- 248.8 v 1,023.8 +/- 158.7 micrograms/L/h, P less than .02). On the contrary, GAL blunted the ARG-stimulated insulin (816.3 +/- 87.7 v 1,322.7 +/- 240.9 mU/L/h, P less than .05), as well as C-peptide secretion (105.1 +/- 9.8 v 132.8 +/- 17.3 micrograms/L/h, P less than .02). ARG administration induced a transient increase of glucose levels (P less than .01 v baseline) followed by a significant decrease (P less than .05 v baseline). This latter effect was prevented by the coadministration of GAL. In conclusion, these results show that in man GAL potentiates the GH response to ARG, suggesting that these drugs act at the hypothalamic level, at least in part, via different mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blood-borne collagenous debris complexes with plasma fibronectin after thermal injury

Plasma fibronectin augments the clearance of blood-borne foreign and effete complexes by mononuclear phagocytes. The release of a "gelatin-like" ligand into plasma after thermal injury has been reported. We quantified the release of this collagenous debris from thermally injured skin, and its potential interaction with soluble fibronectin in plasma using anesthetized rats. Collagen-like material debris in the plasma was detected by assay of hydroxyproline. Fibronectin was measured by a double antibody enzyme-linked immunosorbent assay (ELISA) technique. Over a 24-hour postburn interval, plasma hydroxyproline increased from 6.7 +/- 0.6 micrograms/mL to a maximum of 19.0 +/- 3.3 micrograms/mL at 60 minutes postburn, and normalized by 6 hours. A direct correlation existed between the magnitude of burn injury and the increase in plasma hydroxyproline. In parallel, plasma fibronectin declined over a 15-minute to 2-hour period postburn, and normalized by 3 to 4 hours with rebound hyperfibronectinemia observed at 24 hours. The elevation in total plasma hydroxyproline was not due to an increase in plasma Clq (zero time, 26.2 +/- 1.4 micrograms/mL; 60 minutes, 23.9 +/- 1.1 micrograms/mL). Tracer studies with 125I-fibronectin showed that the acute decline of plasma fibronectin was due to its uptake by the liver and binding to sites of tissue injury. Total hydroxyproline in extracts of burn skin, used as an index of soluble collagenous material, rose from 15 +/- 3.3 micrograms/g skin at zero time to 129.3 +/- 43.7 micrograms/g skin by 5 minutes postburn, with a decline to 38 +/- 22 micrograms/g skin by 24 hours. The formation of circulating fibronectin-gelatin complexes in vivo was documented by cross-immunoelectrophoresis coupled with autoradiography using 125I-gelatin as a model ligand. Thus, collagenous tissue debris from burned skin may enter the plasma after thermal injury and directly complexes with soluble fibronectin before hepatic phagocytic clearance.     

Metabolism of 125 I-luteinizing hormone-releasing hormone

With the use of prepared iodine-125-monoiodo-LH-RH, the disappearance rates of LH-RH (luteinizing hormone-releasing hormone) in man and in the rat were investigated, as well as the distribution of LH-RH in the rat. After 10 microcuries of iodine-125-LH-RH were injected as an iv bolus into 3 male volunteers, blood samples from the opposite arm were obtained through an indwelling venous catheter at 2, 5, 8, 11, 15, 20, 30, 45, and 60 minutes postinjection and at 6 and 24 hours postinjection. To describe the disappearance curve a 3-term exponential equation was necessary and sufficient. The ranges of the t1/2s for the first, second, and third components were 2-4 minutes, 30-55 minutes, and starting at more than 10 hours. The initial distribution space was 3-4.7 1 or 37-47 ml/kg body weight. Similar procedures in the rat (45) resulted in an equation with 2 exponentials, with the ranges of the t1/2s for the first and second components being 5-10 minutes and 150-600 minutes. In both man and rat the distribution volume approximated estimated plasma volume. Following the injection of iodine-125-LH-RH in the rat, pituitary radioactivity increased, as expected, reaching a maximum tissue/serum ratio of 1.5 at 90 minutes. 2 iodinated oligomers of LH-RH were found to have different disappearance rates and distributions in the rat than did iodine-125-LHrh. the disappearance rate of iodine-125-LH-RH in this study is consistent with values for other small peptide hormones.     

Effect of insulin antibodies and their kinetic characteristics on plasma free insulin dynamics in patients with diabetes mellitus

To determine the influence of insulin antibodies (and their equilibrium kinetic properties) on the pharmacokinetics of insulin, we examined the relationship between insulin antibody binding and the initial rate of increase, time to peak, and return to baseline of therapeutic doses of insulin injected subcutaneously (0.15 U/kg) and the half-life, distribution space, and metabolic clearance rate of intravenously infused insulin (2 mU/kg/min) in insulin-treated patients with diabetes mellitus. Compared to age-weight-matched nondiabetic subjects, the diabetic subjects had reduced initial rates of increase (0.33 +/- 0.2 v 0.44 +/- 0.03 microU/mL/min, P less than 0.05), delayed time to peak (130 +/- 12 v 86 +/- 8 min, p less than 0.02), and prolonged return to baseline (485 +/- 37 v 313 +/- 13 min, P less than 0.01) of plasma free insulin levels after subcutaneous injection of insulin, and a prolonged half-life (19.8 +/- 5.8 v 4.3 +/- 0.3 min, P less than 0.02), increased distribution space (904 +/- 284 v 109 +/- 10 mL/kg, P less than 0.001), and augmented metabolic clearance rate (28.5 +/- 1.8 v 17.3 +/- 0.7 mL/kg/min, P less than 0.001) after intravenously infused insulin. All of these abnormal parameters were significantly correlated with binding of insulin to insulin antibodies at tracer insulin concentrations (Bo) and with the high affinity of insulin antibody binding sites as determined by Scatchard analysis. However, patients with 125I insulin antibody binding (Bo) less than 10 percent had normal or near normal plasma free insulin pharmacokinetics.(ABSTRACT TRUNCATED AT 250 WORDS)