Cholecystokinin in the brains of obese and nonobese mice.

Extracts of the cerebral cortes of genetically obese (ob/ob) mice with hyperphagia contain 0.05 +/- 0.02 microgram (mean +/- standard error) of cholecystokinin octapeptide equivalent per gram of wet weight compared to 0.15 +/- 0.01 microgram per gram for their nonobese littermates and 0.20 +/- 0.01 microgram per gram for normal LAF1 mice. These findings are suggestive of a causal relation between the diminished brain immunoreactive cholecystokinin content and the unrestrained appetite of the obese mice.

Iodoinsulin used to determine specific activity of iodine-131.

Anodal mobility of iodoinsulin on starch-gel electrophoresis increases progressively as the number of iodine atoms substituted in the molecule increases. The iodine content of iodine-131 solutions is determined by comparison of autoradiographic patterns of insulin heavily labeled with iodine-131 and of insulins lightly labeled with iodine-131 and known quantities of iodine-127.

Parathyroid hormone in plasma in adenomatous hyperparathyroidism, uremia, and bronchogenic carcinoma.

The concentration of parathyroid hormone (measured by radioimmunoassay) in plasma of patients with severe chronic uremia is frequently much higher than it is in the majority of cases having adenomatous hyperparathyroidism. igher-than-normal concentrations of parathyroid hormone in plasma are found in a significant percentage of unselected patients with bronchogenic carcinoma.

Molluscan gastrin: concentration and molecular forms.

Blood and gastrointestinal tissues of the sea hare Aplysia californica and the land snail Otala lactea contain immunoreactive gastrin in heterogeneous forms similar to those of mammals. The observation that blood concentrations in terms of porcine gastrin standard are comparable to those of pig, man, and dog suggests significant homology between the structures of molluscan and mammalian gastrins.

Radioimmunoassay of ACTH in plasma.

Techniques are described in detail for a radioimmunoassay of plasma adrenocorticotropin (ACTH) that is capable of detecting hormone in unextracted normal human plasma at 1:5 dilution under the conditions described. The sensitivity of the assay is at the level of 1 mumug/ml (equivalent to 0.014 mU/100 ml). In normal subjects ACTH concentrations averaged 22 mumug/ml (equivalent to 0.308 mU/100 ml) plasma at 8-10 a.m. In a smaller group the concentrations averaged 9.6 mumug/ml (equivalent to 0.134 mU/100 ml) at 10-11 p.m. Although a circadian rhythm in normal subjects was not always well marked throughout the daytime hours, plasma ACTH usually fell to its lowest value in the late evening. In hospital patients who were not acutely ill, concentrations were infrequently above 100 mumug/ml in the morning and usually fell to significantly lower levels in the late evening. Severely ill hospital patients occasionally exhibited a.m. concentrations above 200 mumug/ml. In a group of subjects showing frequent spiking of plasma 17-OHCS concentrations throughout the day parallel spiking of plasma ACTH as well was generally observed.Metyrapone produced marked increases in plasma ACTH within 24 hr in all cases and generally within 3-6 hr except when started late in the day. Dexamethasone brought about a persistent reduction in plasma ACTH in a patient under continued treatment with metyrapone.Hypoglycemia, electroshock, surgery under general anesthesia, histalog and vasopressin administration were usually followed by significant increases in plasma ACTH concentration. Prior administration of dexamethasone blocked the response to hypoglycemia. Marked elevations in plasma ACTH were observed in patients with adrenal insufficiency off steroid therapy, in Cushing's disease after adrenalectomy even in the presence of persistent hypercortisolemia, and in some untreated patients with Cushing's disease. Umbilical cord blood contained higher plasma ACTH concentrations than maternal blood at delivery in seven of eight cases. After suppression of ACTH secretion by dexamethasone or cortisol. ACTH disappeared from plasma with half-times ranging from 22 min to 30 min in three cases studied.

Radioimmunoassay of somatomedin B. Application to clinical and physiologic studies.

A radioimmunoassay has been developed for Somatomedin B, a growth hormone-dependent factor that stimulates DNA synthesis in human glia-like cells. The sensitivity permits detection of this factor in human plasma diluted 1: 20,000 and in monkey plasma diluted 1: 5,000. It is not measurable in nonprimate plasma diluted 1: 20. The concentration in growth hormone-deficient adult patients is equivalent to 6.6plus or minus0.5 ug/ml of a highly purified somatomedin preparation. In acromegaly the concentration is 19.3plus or minus2.3 ug/ml and falls after definitive therapy that results in a decrease in plasma growth hormone. In unextracted human plasma the immunoreactive Somatomedin B is associated with a plasma protein at least as large as gamma-globulin and with an electrophoretic mobility on paper resembling the alpha-globulins. The level of Somatomedin B in the bound form in human plasma under steady-state conditions may depend on the rate of production of the peptide and/or the concentration of the plasma-binding protein. At present there is no information concerning which of these is modulated by growth hormone. Immunoreactive Somatomedin B is found predominantly in Cohn plasma fractions III and IV, largely dissociated from the plasma-binding protein. The disappearance curves of labeled purified Somatomedin B and of immunoreactive Somatomedin B from acromegalic plasma administered intravenously to a dog were superposable; the terminal portion of the disappearance curve having a half time of almost an hour.

Ectopic ACTH production in carcinoma of the lung.

Immunoreactive ACTH was found in almost all tissue extracts of lung carcinoma from patients without clinical evidence of Cushing's syndrome; i.e. 14 of 15 primary tumors, nine of nine metastatic lymph nodes, and four of four metastatic liver nodules contained immunoreactive ACTH. The incidence of ACTH in extracts of other tumor types was much lower. Comparable normal tissues contained no detectable ACTH. Immunoreactive growth hormone, parathyroid hormone, or gastrin was not found in the same carcinoma tissue. The predominant form of ACTH in the tumor extracts was big ACTH. In pituitary extracts little ACTH predominated.53% of 83 patients with lung carcinoma had afternoon plasma ACTH levels greater than 150 pg/ml; more than 90% of plasmas containing less than 150 pg/ml were obtained from patients who had received radiation therapy or chemotherapy. 31% of 45 patients with chronic obstructive pulmonary disease (COPD), 28% of 25 patients with other severe lung disease, and 6% of 33 controls had elevated values. Big ACTH predominated in the plasma of patients with lung carcinoma or COPD having elevated ACTH levels. Tissue from the lung of a smoking dog with atypical histologic changes contained immunoreactive ACTH, almost exclusively in the big form, while tissue from another smoking dog that was histologically normal contained no ACTH. Thus ACTH may be present even in precancerous lung lesions. These studies suggest that serial plasma ACTH levels may be of value in screening for, and/or management of, patients with carcinoma of the lung.

Heterogeneity of parathyroid hormone. Clinical and physiologic implications.

When immunoreactive human parathyroid hormone (hPTH), extracted by three different solvents (20% acetone in 1% acetic acid, 8 M urea, or normal saline) from parathyroid glandular tissue was subjected to Sephadex G-100 gel filtration and immunoassay using two different antisera (273 and C-329), four distinct fractions were observed. The first (I), a void volume peak, was detected by both antisera with similar immunoreactivity, as was a second (II), which had the elution and sedimentation properties of highly purified bovine parathyroid hormone (bPTH); a third (III) eluted between [(125)I]growth hormone and [(125)I]insulin, sedimented with the velocity of a molecule of approximately 6,000 mol wt, and was detected primarily by antiserum 273; a final fraction (IV), detected primarily by C-329, eluted just prior to [(125)I]insulin. The elution profiles of the acetone-acetic acid and 8 M urea extracts were similar and contained fraction II as their major component. In saline extracts, however, fraction III predominated. Three fractions, having gel filtration and immunologic characteristics similar to fractions II, III, and IV, respectively, of saline glandular extracts, were detected in the plasma of patients with both primary (adenomatous or carcinomatous) and secondary hyperparathyroidism. The predominant component in every plasma was the intermediate fraction that, like III, was detected primarily by antiserum 273, while the least abundant form was consistently the final fraction, detected primarily by antiserum C-329. The first fraction, like II, was detected with about equal potency by both antisera and had an elution volume on Sephadex corresponding to that of intact bPTH. It bore a reciprocal relationship to serum calcium and disappeared from the plasma of a uremic patient during calcium infusion or following parathyroidectomy with a half-time of no more than 20 min. This component therefore probably represents biologically active hormone. The intermediate and final fractions had turnover times in the plasma of a uremic patient more than 100 times greater than the active form, remained elevated even in the presence of post-parathyroidectomy hypoparathyroidism in this patient and were presumed to be biologically inactive. The ratio of biologically inactive fragments to the active form was greater in secondary hyperparathyroidism. The evidence presented favors a glandular origin for the fragments. Comparison of hormonal assays with the two antisera reveals a striking advantage in the preoperative diagnosis of primary hyperparathyroidism with antiserum 273 that is due to the enhanced sensitivity occasioned by its detection of a biologically inactive as well as the biologically active hormonal form.

Immunologic potency of recombined A- and B-chains of synthetic human and pancreatic pork insulins.

Pork insulin recombined from cleaved A- and B-chains of pancreatic insulin was shown to be identical with the pancreatic hormone both in erythrocyte receptor assays and in immunoassays employing four different antisera, including two species-specific human anti-insulin sera. Pancreatic human insulin and that prepared from recombined bacterially synthesized A- and B-chains were also indistinguishable in the same systems. This study demonstrates that the prohormone stage of either human or porcine insulin is not required to preserve potency of membrane binding or immunochemical reactivity, even employing an antiserum with marked species specificity.

Influence of the age of erythrocytes on their insulin receptors.

Specific binding of 125I-insulin to erythrocyte receptors increased linearly with reticulocyte count in routine blood specimens from hospitalized patients. Specific binding as high as 30-35% is observed in patients with reticulocyte counts in the range of 20-25% compared with 9.0 +/- 0.5% specific binding in healthy control subjects with normal reticulocyte counts. It is concluded that care must be exercised in interpreting elevations of specific 125I-insulin binding to erythrocyte receptors as due to specific disease processes without considering how the disease itself may alter mean erythrocyte age at the time of sampling.

Some considerations in the preparation of raioiodoinsulin for radioimmunoassay and receptor assay.

125I-insulins, prepared by iodination with chloramine T in marked excess or by stepwise, stoichiometric addition of the oxidizing agent, were compared with respect both to their molecular distribution of iodine and to their suitability for use in a cultured lymphocyte receptor assay. Iodination of insulin in aqueous solution results in the same distribution of iodine atoms, independent of experimental method and dependent only on the average iodine number. This distribution can be calculated on the basis of a Monte Carlo simulation, For insulin iodinated at an average of 0.8 I atoms per molecule, approximately 50 per cent of the radioactivity is in other than monoiodoninsulin. Purification methods that separate on the basis of charge, such as starch-gel electrophoresis, are then required, to obtain monoiodoinsulin. More highly iodinated insulin do bind to the lymphocyte receptor, although, as in radioimmunoassay, the overiodinated species are less satisfactory for use as tracers. The shelf life of iodinated insulin appears to be related better to the average iodine content than to any other factor, presumably because of decay catastrophe. There is no evidence to suggest that exposure to chloramine T in marked excess for a few seconds is deleterious to insulin.

Evidence against insulin synthesis BY IM-9 lymphocytes.

Insulin concentrated from acid/ethanol extracts of IMg lymphocytes by the use of octadecylsilyl (ODS) silica cartridges averaged 0.13 ng/ml cell volume compared with the 0.12 ng insulin/ml cell culture medium. The insulin content of these cells appears to be less than 1% of previously reported values and probably could be accounted for by concentration of insulin from the incubation medium on cell receptors.

Guinea pig glucagon differs from other mammalian glucagons.

Mammalian glucagon is thought to be highly conserved. Glucagons from pig, cow, human, rat, and hamster have identical amino acid sequences, whereas the amino acid contents of rabbit and camel glucagons are consistent with this 29-amino acid sequence. It had earlier been reported that guinea pig (GP) glucagon contains 40 amino acids. In the current study, glucagon was purified from two GP pancreata by a series of three HPLC steps after acid-alcohol extraction and acetone precipitation. GP glucagon is a 29-amino acid peptide that differs from other mammalian glucagons by substitution of Gln for Asp in position 21, Leu for Val in position 23, Lys for Gln in position 24, Leu for Met in position 27, and Val for Thr in position 29. In view of the marked changes in the COOH-terminal of GP glucagon, receptor binding studies were performed using both rat and GP liver membranes. Labeled synthetic porcine glucagon has similar binding in the two systems and its binding is inhibited to a similar degree by synthetic porcine glucagon, whereas GP glucagon is 10-fold less potent at inhibiting binding in both systems. This suggests that glucagon receptor binding sites in the GP are evolutionarily more conserved than is GP glucagon.

Preparation, purification, and stability of high specific activity 125I-labeled thyronines.

A modified chloramine-T method is described for the preparation of several radioiodothyronines by an exchange reaction, which results in low specific activity preparations, or by an addition reaction, which yields radioiodothyronines with specific activities up to 7 mCi/microgram. Purification by paper chromatography is shown to be more convenient than by LH-20 chromatography and provides better resolution among the various thyronines. Radioiodothyronines with only a single iodine atom in the outer (3,5,3'-triiodothyronine and 3,3'-diiodothyronine) are stable for several months when stored in organic solvents. The least stable radioiodothyronines are those with two 125I atoms in the outer ring (3,3',5'-triiodothyronine (rT3) produced from 3-iodothyronine and thyroxine (T4) from 3,5-diiodothyronine). The stability of rT3 and T4 stored in human plasma at 4 C is much greater than when stored in buffer at the same pH. The use of high specific activity [125I]rT3 has permitted the development of a radioimmunoassay with a sensitivity of 1 pg rT3/ml incubation volume.

Brain/gut peptides in fed and fasted rats.

The concentrations and contents of vasoactive intestinal peptide (VIP) and cholecystokinin (CCK) in the brain and of these peptides along with secretin and glucagon-like immunoreactivity (GLI) in the gut were compared in a group of 16 5-day fasted adult Sprague-Dawley rats with the corresponding peptides in a group of 16 nonfasted littermates. The mean weight of the fasted rats at the beginning of the study was 263 +/- 10 g (+/- SEM) and was 177 +/- 7 g before killing, for a net loss of 33% of initial body weight; the 16 fed rats increased their mean weight from 225 +/- 11 to 284 +/- 12 g, for a net gain of 12%. During the 5-day fast there was no change in the weight of the cortex, hypothalamus, or brain stem. However, the weight of tissues from the gut decreased to about half the weight of the corresponding tissues in the fed animals. There was no significant change in brain VIP or CCK. VIP content in the gut was unchanged. However, because of the decrease in organ weight, its concentration almost doubled. Secretin concentrations in the gut of fasted rats did not change significantly, but organ contents fell to about half. The gut content of GLI also fell by half or more. The concentrations of CCK in methanol extracts of the duodenum and jejunum remained relatively constant, but those in acid extracts fell by 40% in the fasted animals. This represents an approximately 70% decrease in organ content of CCK. These findings are interpretable as demonstrating that during a prolonged fast neuronal CCK and VIP are well conserved, but endocrine CCK, secretin, and GLI are markedly decreased because of loss of intestinal mucosa.

Ontogeny of immunoreactive cholecystokinin, vasoactive intestinal peptide, and secretin in guinea pig brain and gut.

The ontogeny of cholecystokinin (CCK), vasoactive intestinal peptide (VIP), and secretin (SEC) as well as other brain and gut peptides has been extensively studied in the rat. However, since the guinea pig (GP) appears to be functionally more mature at birth than the rat, we hypothesized that the concentrations of brain peptides, but not necessarily of gut peptides, in GP neonates would be closer to adult concentrations than those in the rat. The concentration and organ content of these three peptides were determined at birth, at 7, 14, 21, and 28 days of age, and in the adult in various portions of the gastrointestinal tract and brain. VIP and CCK in the brain at birth are 75% of adult levels compared to less than 10% in the rat. VIP concentrations in the stomach, duodenum, and jejunum are relatively constant from birth through adult life, whereas in the rat, the intestinal VIP concentration increased 3- to 4-fold from birth to adulthood. SEC, which in the gut is found only in mucosal tissues, peaked at birth, remained low from 7-28 days of age, and rose again in the adult. CCK, which in the gut is found in both the mucosa and muscle wall, like SEC peaks at birth, then remains low from 7-28 days of age, and rises in the adult. It is concluded that the GP neuronal tissues are much more mature at birth than are those in the rat.

Characterization of mouse ACTH in plasma and in extracts of pituitary and of adrenotropic pituitary tumor.

The predominant component of immunoreactive ACTH in the plasma of adrenalectomized normal mice and of mice bearing the adrenotropic mouse pituitary tumor, AtT-20, and in extracts of the normal mouse pituitary and pituitary tumor, has an elution volume on Sephadex G-50 gel filtration approximately midway between the void volume and the elution volume of human ACTH (1-39 peptide). The tumor extracts are shown to contain, in addition to this intermediate ACTH, 2 other components of immunoreactive ACTH, one which coelutes with 131I-labeled albumin (big ACTH) and the other with [125I]hACTH (little ACTH). Big and intermediate ACTH are urea-stable. Controlled tryptic digestion of mouse-tumor big ACTH results within 10 seconds in conversion to an intermediate component followed by continued loss of immunoreactivity. Under the same conditions of tryptic digestion of intermediate ACTH, there is only continuous loss of immuno-reactivity with no change of hormonal form. These findings strengthen the hypothesis that mouse intermediate ACTH is not a precursor for little ACTH.

Immunoreactive somatomedin B in urine.

Urinary excretion by 8 normal adult subjects of immunoreactive somatomedin B was 27.6 +/- 4.4 mug between 1000 h and 1400 h compared to a mean plasma concentration at 1200 h of 5.9 +/- 0.9 mug/ml. Free somatomedin B in urine averaged 85.9%, although in the plasma of the same subjects all but less than 5% was bound to serum proteins.

Insulin as a potential factor influencing blood pressure in amputees.

War-injured, bilateral above-knee amputees are known to be at increased risk for cardiovascular mortality. To evaluate possible risk factors, we compared blood pressures and plasma glucose and insulin responses to orally administered glucose in 19 above-knee amputees from the Vietnam War (mean age, 36 +/- 1 years) with those of 12 age-matched unilateral below-elbow amputees. Body composition by densitometry and maximal oxygen consumption during arm or leg exercise were also determined. Nine of 19 leg amputees were hypertensive compared with one of 12 arm amputees. Their 3-hour average insulin responses were markedly increased (260 +/- 60 microU/ml) compared with those of normotensive leg (125 +/- 24 microU/ml) and arm amputees (101 +/- 20 microU/ml), and their mean body fat content (37.2%) also was elevated compared with that in both of these groups (23.2 and 22.6%, respectively). A unique finding was that both insulin response and body fat content were strongly and independently correlated with diastolic blood pressure (r = 0.55, p less than 0.01, and r = 0.62, p less than 0.01, respectively). We conclude that insulin may be a major factor in blood pressure regulation in the maturity-onset obesity that develops following traumatic leg amputation in young, healthy men.

Hyperinsulinemia.

Patients with mild or early non-insulin-dependent diabetes mellitus often display a delay in insulin response followed by late hyperinsulinemia during oral glucose tolerance testing. Those patients with long-standing disease or elevations of fasting plasma glucose in excess of 140 mg/dl are generally hypoinsulinemic in response to an oral glucose tolerance test. Diabetic patients who do not have an acute response to intravenous glucose may have normal responses to intravenous tolbutamide or intravenous arginine, suggesting that delayed responsiveness to glucose is not due to decreased pancreatic insulin content. An association between hyperinsulinemia and hypertension has been suggested by recent studies from several laboratories. In a homogeneous population of men who suffered traumatic bilateral above-the-knee amputation in the Vietnam War with subsequent development of obesity, it was shown that there was strong correlation between hypertension and hyperinsulinemia during oral glucose tolerance testing despite only mild glucose intolerance. In addition, a subset of hypertensive women who were in their third trimester of pregnancy were markedly hyperinsulinemic during oral glucose tolerance testing in the absence of any abnormalities of glucose tolerance. Thus, the relationship between hyperinsulinemia and hypertension, and the possible reasons for this relationship, are fields of active investigation at present.