Estimation of the secretion rate of insulin from the urinary excretion rate of C-peptide. Study in obese and diabetic subjects.

Direct methods for measuring the secretion rate of insulin are too cumbersome for clinical application. Since C-peptide is secreted in an equimolar ratio with insulin and is excreted into the urine, measuring the urinary excretion rate of C-peptide (U-C) could serve as an indicator of its secretion rate (SR-C) if its urinary clearance (UCI-C) is constant and unaffected by plasma C-peptide concentration, body mass, or diabetes. We measured clearance ratios of C-peptide/creatinine (CR) in the fasting state and integrated 0-1, 1-3, and 3-5 h after 100 g of glucose p.o. as well as over a full 24-h in eight obese, eight lean, and six maturity-onset diabetic subjects. CR did not differ significantly when values in the fasting state were compared with those in the postprandial periods and was therefore unaffected by plasma C-peptide concentration. Furthermore, CR was similar in the lean, obese, and diabetic subjects. SR-C, determined as the product of the metabolic clearance rate of C-peptide and its fasting or integrated plasma concentrations, correlated significantly with U-C in all the subjects (r = 0.87, P less than 0.0001). The correlation of U-C with SR-C in the diabetic subjects alone was also significant (r = 0.88, P less than 0.0001). In conclusion, our data support the use of U-C as an indirect measure of SR-C and therefore of SR-I.

Increased integrated concentration of norepinephrine, epinephrine, aldosterone, and growth hormone in patients with uncontrolled juvenile diabetes mellitus.

The 24-h integrated plasma concentration of glucose (IC-glucose), norepinephrine (IC-NE), epinephrine (IC-E), cortisol (IC-F), growth hormone (IC-GH), aldosterone (IC-ALDO), and plasma renin activity (IC-PRA) were measured in 11 nonobese juvenile-onset nonketotic diabetic patients exhibiting hyperglycemia and glycosuria and 34 matched control subjects using a portable pump, drawing blood at a constant rate through a nonthrombogenic i.v. catheter. The diabetic patients had a noticeable rise of their IC-NE, IC-E, IC-GH, and IC-ALDO. There was no significant difference between the IC-F and IC-PRA of the patients and the control subjects.

Correlation of urinary excretion of C-peptide with the integrated concentration and secretion rate of insulin.

The secretion rate of insulin (SR-I) of 50 normal subjects was calculated from the 24-h integrated concentration of insulin (IC-I), the peripheral metabolic clearance of insulin (pMCR-I), and the mean fractional hepatic insulin extraction (fhMCR-I) that was derived from our data. fhMCR-I was determined as the difference in the molar secretory rate of C-peptide (SR-C) and the molar peripheral clearance of insulin (pMCR-I x IC-I) divided by SR-C. The IC-I in our 50 subjects was 1.19 +/- 0.38 ng/ml and the IC-C was 2.93 +/- 0.58 ng/ml. Based on these data, the fhMCR-I was 0.40 and the Sr-I was estimated to be 54.8 +/- 18.0 U/24 h. The 24-h urinary C-peptide excretion (U-C), 44.9 +/- 20.4 micrograms/24 h, had a statistically significant correlation with SR-I (r = 0.838, P less than 0.0001), while the IC-I correlated significantly with the 24-h urinary C-peptide/g of creatinine (r = 0.838, P less than 0.0001). The U-C may thus serve as a practical method for estimating the SR-I.

Evidence for elevated glucose threshold in patients with impaired glucose tolerance and symptoms of hypoglycemia during OGTT.

We evaluated the relationship between hypoglycemic symptoms, glucose nadir levels, and hormone changes in patients with impaired glucose tolerance (IGT) after an oral glucose tolerance test (OGTT). The peak counterregulatory hormone response was determined at the glucose nadir identified by continuous glucose monitoring. Eight patients with IGT who had symptoms and signs typical of hypoglycemia at the glucose nadir were compared with completely asymptomatic subjects (5 IGT patients and 13 patients who had normal glucose tolerance [NGT]). The mean glucose nadir of symptomatic IGT patients was 3.50 +/- 0.46 mM, which was not statistically different from the mean of asymptomatic NGT patients (4.10 +/- 0.56 mM) but was significantly lower than that for asymptomatic IGT patients (5.10 +/- 0.81 mM, P less than 0.001). Seven of 8 symptomatic IGT patients had glucose levels that never fell below the range of glucose nadirs for asymptomatic NGT patients. However, the symptomatic IGT group had significantly higher levels of growth hormone, cortisol, epinephrine, and norepinephrine than the asymptomatic groups in response to the nadir. We conclude that patients with IGT are capable of experiencing signs and symptoms of hypoglycemia at physiological glucose levels during OGTT with reflex stimulation of counterregulatory hormone release. This may indicate that symptomatic IGT patients have a higher glucose threshold for eliciting characteristic hypoglycemic symptom episodes than individuals with NGT.

Effect of testosterone on insulin-like growth factor-I (IGF-I) and IGF-I receptor gene expression in the hypophysectomized rat.

Circulating levels of insulin-like growth factor-I (IGF-I) increase during puberty, concurrent with an increase in the levels of GH and the gonadal steroids. The relationship between the changes observed in IGF-I and testosterone (T) levels are not understood. This study was designed to determine whether T has a direct effect on IGF-I serum levels, liver IGF-I gene expression, and epiphyseal growth plate IGF-I and IGF-I receptor gene expression. Hypophysectomized castrated rats were divided into four groups of six animals. The T group was treated with sc T for 5 days. The GH group was treated with a single dose of GH. The GH plus T group was treated with T for 5 days and with GH on the last day of treatment. The control group was injected for 5 days with vehicle alone. Serum IGF-I levels in the T group were not significantly different from those in the control group, and the levels in the GH plus T group were not significantly different from those in the GH group. There was an 11-fold increase in liver IGF-I mRNA abundance in the GH group compared to the control group (P less than 0.01). Liver IGF-I mRNA levels in the T group were not significantly different from those in the control group. When liver IGF-I mRNA levels in the GH plus T group were compared to those in the GH-treated group, no significant differences were found. In the epiphyseal growth plate region, there was a 12-fold increase in IGF-I mRNA levels in the GH group compared to those in the control group, but there was no statistical difference between the control and T groups. IGF-I mRNA levels in the GH plus T group were not significantly different from those in the GH-treated group. IGF-I receptor mRNA abundance was not significantly different in the T group compared to that in the control group. GH decreased IGF-I receptor mRNA by 2.3-fold, but T treatment before GH injection did not change this effect. We conclude that in castrated hypophysectomized rats, T does not stimulate IGF-I gene expression in the liver, nor does it increase IGF-I serum levels. T alone also does not have a stimulatory effect on IGF-I or IGF-I receptor gene expression in the epiphyseal growth plate region.(ABSTRACT TRUNCATED AT 400 WORDS)

Normal integrated concentration of aldosterone and plasma renin activity: effect of age.

The integrated concentration of aldosterone (IC-ALDO) and PRA (IC-PRA) was studied in 78 normal subjects ranging in age from 9--50 yr. Whereas the IC-ALDO to IC-PRA were found to decrease with age, the ratio of IC-ALDO to IC-PRA was not affected by age. A significant lowering of the normal range and variance was achieved by measuring the 24-h integrated concentration instead of the concentration in discrete samples.

Integrated concentration of epinephrine and norepinephrine in normal subjects and in patients with mild essential hypertension.

Since the level of plasma catecholamines fluctuates rapidly during the day, measurement in a single blood sample could be misleading. A portable nonthrombogenic blood withdrawal system, permitting normal activity and sleep, was used for obtaining the 24-h integrated concentration (IC) of epinephrine (E) and norepinephrine (NE) in 46 normal control subjects, 30 patients with mild essential hypertension, and 1 patient with pheochromocytoma. The mean IC of E (ICE) and the mean IC of NE (ICNE) of the control subjects were 31 +/- 15 and 194 +/- 106 pg/ml, respectively (mean +/- 1 SD). The mean ICE and ICNE of the essential hypertensive patients were 30 +/- 21 and 224 +/- 90 pg/ml, respectively. No significant difference could be found between the levels found in essential hypertensive and normal control subjects. The levels of ICE and ICNE in the patient with pheochromocytoma were 1350 and 882 pg/ml, respectively, which are 88 and 6.5 SD above the mean of the normal control subjects.

Effect of food intake on the metabolic clearance rate of aldosterone.

The effect of eating on the metabolic clearance rate (MCR) of aldosterone was investigated in 10 adult individuals. 3H-aldosterone was infused continuously over a period of 5 h while the subjects remained supine. Three h after the start of the infusion, each subject ate a bowl of soup. The MCR of aldosterone, before and after the intake of food, was calculated by dividing the rate of infusion of 3H-aldosterone by the mean concentration of under the same conditions but without eating. The MCR of aldosterone (mean +/- 1 SD) was 1284 +/- 513 L/24 h before food intake and 2182 +/- 180 L/24 h after food intake in the 10 individuals who ate. The MCR in the 11 subjects who did not eat was 1363 +/- 446 and 1357 +/- 434 during the same periods (p greater than 0.05). The 29% increase in the MCR induced by eating was highly significant (p less than 0.001); it was similar in magnitude and duration to a previously reported effect of food intake on the hepatic blood flow (13).

Evaluation of the 6-hour integrated concentration of cortisol as a diagnostic procedure for Cushing's syndrome.

The diagnostic value of the 24-h integrated concentration (IC) test of cortisol (IC-24-F) was found to be superior to the value of both urinary 17OHCS and urinary free-cortisol tests. The IC-24-F test is too cumbersome for widespread clinical use. The purpose of the present study was to evaluate the diagnostic value of an abbreviated and practical 6-h IC of cortisol (IC-6-F) test. The IC of cortisol (IC-F) was measured in 68 normal subjects and 13 patients with surgically proven Cushing's syndrome. A portable nonthrombogenic constant blood withdrawal system was used over a 24-h period. The IC-F was measured in plasma withdrawn during each 1/2-h period (IC-1/2-F). The mean of 12 consecutive measurements of IC-1/2-F yielded the IC-6-F. The mean of all the IC-1/2-F collected over a 24-h period constituted the IC-24-F. The IC-1/2-F of the patients and their IC-6-F from 0800-1400 h, 1400-2000 h, and 2000-0200 h overlapped the corresponding levels in the control subjects. There was no overlap between the IC-24-F and the IC-6-F (from 2000-0200 h) of the patients and the control subjects. It was concluded that the diagnostic value of a 6-h IC-F test conducted during the afternoon and early part of the might is equal to the diagnostic value of the 24-h IC-F test.

Hypocortisolemia in children undergoing evaluation for growth hormone deficiency.

The group of children who have clinical manifestations of GH deficiency may potentially contain a large number of patients with secretory defects of cortisol. We assessed physiological cortisol secretion by measuring the 24-h integrated concentration of cortisol (IC-F) in a series of 105 patients, aged 7-19 yr, undergoing endocrinological evaluation for growth impairment possibly due to GH deficiency. The reference value for IC-F, established from 30 normal stature, normal weight children (controls), aged 7-18 yr, was 157 +/- 41 nmol/L (mean +/- 1 SD). There was no effect of age, gender, or pubertal status on IC-F in controls. The IC-F of patients was 150 +/- 72 nmol/L. Twelve patients (11%) had IC-F values more than 2 SD below the mean (i.e. less than 75 nmol/L) of the controls (P less than 0.001). An IC-F below 75 nmol/L was associated with a blunted peak cortisol response to insulin-induced hypoglycemia (367 +/- 160 nmol/L compared to 464 +/- 155 nmol/L in the other patients; (P less than 0.05). None of the patients had obvious clinical symptoms of hypocortisolemia at the time of testing. In general, IC-F levels were not correlated with IC-GH. However, 10 patients who had subnormal IC-F values also had laboratory evidence of GH secretory defects; 7 had subnormal IC-GH levels but normal stimulated GH responses, and 3 had both subnormal responses to stimulation as well as subnormal IC-GH. The long term prognosis and management implications of hypocortisolemia diagnosed in this patient group require further evaluation.

Reproducibility of growth hormone testing procedures: a comparison between 24-hour integrated concentration and pharmacological stimulation.

The purpose of this study was to compare the reproducibility of two approaches to the evaluation of GH secretion: the integrated concentration of GH (IC-GH), a physiological test of GH secretion, and pharmacological stimulation tests. IC-GH was determined in 40 poorly growing children twice within 4 weeks. The first and second IC-GH were highly correlated r = 0.859, P less than 0.001. One hundred and thirteen poorly growing children underwent pharmacological GH stimulation tests twice within 6 weeks. A moderate correlation was found between the first and second pharmacological test r = 0.524, P less than 0.01. Among the three pharmacological stimuli studied, clonidine (n = 81) had the highest reproducibility followed by arginine (n = 20), and insulin (n = 12). We conclude that IC-GH is more consistently reproducible than the GH response to repeated pharmacological stimulation.

Metabolic clearance rates of synthetic human growth hormone in lean and obese male rhesus monkeys.

The MCR of synthetic human GH was studied in eight adult male rhesus monkeys (Macaca mulatta). Four monkeys were lean (less than 20% body fat), and four were obese (greater than 35% body fat). The monkeys were given a single bolus injection of GH (2.5 micrograms/kg BW), followed by a constant infusion of GH (250 micrograms/h) for 2.5 h. Venous blood samples were collected before the infusion and every 10 min during the infusion. In both groups a plateau of the plasma GH concentrations, indicating a steady state, was reached 70 min after the start of the infusion. The MCR of GH was calculated from the ratio of the constant GH infusion rate and the plateau plasma GH concentration in each monkey. The MCR of synthetic GH was 12.7 +/- 1.7 (+/- SD) L/24 h in the lean group and 19.5 +/- 2.9 L/24 h in the obese group (P less than 0.007). However, the MCR/kg ratio in the lean monkeys was the same as that in the obese animals. We conclude that 1) MCR of GH is directly proportional to body weight; and 2) the lower plasma GH levels in obesity may be due to an increase in its MCR not compensated for by an appropriate increase in the rate of GH secretion.

Detection of primary aldosteronism by the 6-hour integrated aldosterone/renin ratio.

An outpatient diagnostic procedure measuring the 6-hour integrated plasma concentration of aldosterone and plasma renin activity was used to detect primary aldosteronism in 12 patients with low renin hypertension, including six with mild hypertension and normal urinary excretion and spot plasma levels of aldosterone. The ratio of integrated plasma concentration of aldosterone to plasma renin activity in the 12 patients (mean, 339; range, 116-700; p less than 0.0001) did not overlap with that measured in 105 normotensive controls (mean, 27.8; range, 5-97) or in 87 subjects with essential hypertension (mean, 29.2; range, 4-67). Eight patients had surgically proven adenomas (3 of which measured less than 5 mm) with normalization of blood pressure following adrenalectomy. The four remaining patients had bilateral hyperplasia. The 6-hour integrated plasma concentration of aldosterone to plasma renin activity ratio was found to be a useful new outpatient diagnostic tool for evaluation of primary hyperaldosteronism.

The influence of age on the 24-hour integrated concentration of growth hormone in normal individuals.

We examined changes in spontaneously secreted growth hormone with aging by studying the 24-h integrated concentration of GH (IC-GH) of 173 nonobese subjects (height, greater than or equal to 5%; 7-65 yr of age). There was no significant difference in IC-GH on repeat testing of 13 men or in 23 women studied in the follicular and again in the luteal phase of the menstrual cycle. The level of IC-GH was strongly effected by age; children had the highest mean IC-GH, and there was a decline in IC-GH with increasing age after the second decade of life. The correlation of IC-GH with age was highly significant (r = 0.73; P less than 0.0001). There was no difference in IC-GH between males and females when matched for age. The mean IC-GH at Tanner stage 5 of puberty (7.4 +/- 2.0 ng/ml) was higher than that at stages 2-4 (5.7 +/- 1.4; P less than 0.0005) or that in prepubertal children (5.8 +/- 1.4; P less than 0.001). Thus, age and pubertal status must be carefully considered when interpreting the IC-GH for patients suspected of having deficient or excessive secretion of GH.

Growth hormone (GH) response to GH-releasing hormone in children with subnormal integrated concentrations of GH.

We determined the GH responses to human GH-releasing hormone-40 (GHRH) in poorly growing children who had either normal or deficient GH secretion, as measured by pharmacological stimulation and integrated concentration of GH (IC-GH). Ten patients had both normal pharmacologically stimulated GH and IC-GH (GH-normal), 15 patients had normal pharmacologically stimulated GH but deficient IC-GH [GH neurosecretory dysfunction (GHND)], and the remaining 7 patients had both subnormal stimulated GH and IC-GH [GH deficiency (GHD)]. The mean peak plasma GH response to GHRH was 11.7 +/- 8.5 (+/- SD) ng/ml in GHD patients, significantly lower than the responses of both the GHND (49.2 +/- 39.2 ng/ml; P less than 0.0001) and GH-normal (51.8 +/- 44 ng/ml; P less than 0.0001) groups. The range of peak GH responses to GHRH in GHD patients overlapped the lower end of the range of responses in the GHND and GH-normal patients. Three GH-normal and eight GHND patients had greatly enhanced GH responses to GHRH (greater than 50 ng/ml); no GHD patients had a response over 24.2 ng/ml. There was no difference between the GH responses of male and female patients within groups to GHRH. There was a significant correlation between the log of the peak GH response to GHRH and the log of the maximal GH response to standard pharmacological stimuli (r = 0.51; P less than 0.005). Because of the variability of GH responses to GHRH encountered among the patients, the response to GHRH cannot be used as a test for identifying patients with inadequate spontaneous GH secretion. The IC-GH is the only method that can identify children with GHND.

Twenty-four hour integrated concentrations of progesterone, 17-hydroxyprogesterone and cortisol in normal male subjects.

The 24 h integrated concentrations of progesterone, 17-hydroxyprogesterone (17-OHP) and cortisol were determined in 5 male subjects ranging in age from 25 to 36 years. Using a nonthrombogenic catheter and a constant withdrawal pump, blood was collected for a period of 24 h in 30 min aliquots. All five subjects had similar 24 h integrated concentrations of cortisol (9.2 +/- 5.4). One subject had elevated 24 h integrated concentrations of progesterone (55.2 +/- 18.7) and 17-OHP (352 +/- 122) when compared to the mean integrated concentrations (progesterone: 24.9 +/- 4.7; 17-OHP: 109 +/- 33) of the 4 other subjects. The 30 min integrated concentrations showed a diurnal variation for both 17-OHP and cortisol but not for progesterone. The best correlation between cortisol and 17-OHP occurred when the concentration of 17-OHP 90 or 120 min earlier. No significant correlation occurred between cortisol and progesterone.

Growth failure with normal serum RIA-GH and low somatomedin activity: somatomedin restoration and growth acceleration after exogenous GH.

Two three-year-old boys with dwarfism (height ages 1-4/2 and 1-11/12 years) and delayed bone ages (1-4/12 and 1-9/12 years) had normal growth hormone (GH) responses after stimulation and low levels of somatomedin. Unlike patients with Laron syndrome, the two patients generated normal levels of somatomedin after administration of exogenous hGH. Treatment with hGH (2 IU every other day) brought about a significant increase in the growth rate of both patients. The growth rate of the first patient increased from 2 cm/year before treatment to 12 cm/year on therapy. The growth rate of the second patient was 4.5 cm/year before treatment, and 8.3 cm/year while on treatment. The two cases represent a new syndrome of dwarfism which may be caused by secretion of a biologically inactive but immunoreactive GH.

Improved bioassay for insulin by continuous glucose monitoring.

The USP bioassay for insulin currently used by industry and government requires a sc injection of insulin to rabbits and multiple assays of blood glucose. One hundred and forty-four rabbits are usually assayed, each assay taking at least 2 weeks. A bioassay for insulin is described. The new assay is based on a continuous monitoring of blood glucose and repeated iv injection of insulin to dogs. The test is completed in 1 day and is carried out on one dog. The interassay variability of seven repeated tests of an off the shelf soluble insulin was +/- 8%.

Comparison of estimates of gonadotropin levels by isolated blood samples, integrated blood concentrations, and timed urinary fractions.

Gonadotropin levels in isolated blood samples, integrated plasma concentrations (IC), and timed urinary collections have been compared in 5 males with delayed puberty and 7 normal adult males. There was a significant correlation between urinary levels in 24-h collection and those in each of four shorter timed collections for both LH and FSH. Similarly, 24-h integrated plasma concentration and 4-h (0800--1200 h) integrated plasma concentration obtained on 10 additional subjects showed significant correlation. The 4-h integrated plasma concentrations correlated with single blood samples or the mean of three samples obtained at 0800, 1200, and 1600 h. These 4-h plasma samples also correlated significantly with all urine collections for FSH but only with the 2200--0800 h urine collection for LH. The study suggests that LH and FSH levels in urine samples collected over several hours correlate with 24-h urinary excretion and that levels in single blood samples estimate the 24-h plasma integrated concentration.

Comparison of physiological and pharmacological tests of growth hormone function in children with short stature.

We studied 30 short-statured children to investigate their 24-h integrated serum concentration of GH (ICGH) and its diurnal pattern and to compare this data with their responses to standard stimulation tests for GH release. Eight patients with normal stimulation tests had a mean (+/- SD) ICGH value of 8.0 +/- 3.8 ng/ml (range, 4.5--16.4), and all had several secretory peaks greater than 12 ng/ml. Nineteen patients with abnormal responses (peak, less than 10 ng/ml) had a mean ICGH value of 2.7 +/- 1.9 ng/ml (range, less than 0.8 to 7.0); this was significantly lower than that of control subjects and patients with normal stimulation tests. However, 5 of these patients had normal ICGH values. Three patients with borderline stimulation test responses (10--12 ng/ml) had normal ICGH values (3.2, 3.4, and 11.9 ng/ml). Of the 22 patients with either abnormal or borderline stimulation tests, 14 had an ICGH result below the range of normal. Of these 14, 11 had no secretory peaks greater than 10 ng/ml, whereas 3 had peaks between 10--12 ng/ml. The 8 other patients had ICGH results in the normal range. Despite significant correlation (r = 0.668), integrated GH values did not correlate with stimulation tests in a minority of the patients, the status of whom remains to be determined by long term response to exogenous GH therapy.