Rosalyn S. Yalow

RADIOIMMUNOASSAY OF GASTRIN.

Summary A highly specific radioimmunoassay for endogenous human plasma gastrin was developed using guinea pig antisera to crude porcine gastrin. Highly purified 125 I-porcine gastrin I, further purified on starch gel electrophoresis after labeling, was employed as tracer, and an anionic exchange resin was used to separate bound and free labeled gastrin. Sensitivity is better than 5 pg per ml. Cholecystokinin-pancreozymin and amino-terminal and carboxyl-terminal gastrin peptides cross-react only poorly in the gastrin-antigastrin system. Plasma gastrin and standard gastrin show parallel inhibition curves against the labeled porcine gastrin. Reproducibility of determinations in different assays is demonstrated. Plasma gastrin is unaffected by boiling for 5 to 10 min. High values of plasma gastrin were observed in 5 patients with Zollinger-Ellison syndrome and in 17 patients with pernicious anemia. Administration of HCl per os or into the stomach in 5 patients with pernicious anemia produced an acute fall in plasma gastrin concentration; the half-time for disappearance of gastrin from plasma was as short as 7 min.

Hypoglycemia: a potent stimulus to secretion of growth hormone.

In normal subjects, hypoglycemia produces an abrupt and sustained rise in levels of human growth hormone in plasma. This effect is independent of insulin, glucagon, or epinephrine. Prolonged fasting is accompanied by a rise in the hormone level in plasma. Measurement of this hormone after induced hypoglycemia is a specific test for pituitary somatotropic function.

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 Cushings disease after adrenalectomy even in the presence of persistent hypercortisolemia, and in some untreated patients with Cushings 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.

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.

Plasma Insulin Concentrations in Nondiabetic and Early Diabetic Subjects: Determinations by a New Sensitive Immuno-assay Technic

For many years insulin was regarded as only weakly antigenic, or even, according to some, as essentially nonantigenic. However, a few years ago, during the course of investigations on the metabolism of I-labeled insulin, it was observed that the tagged insulin showed a prolonged retention in the blood stream of all subjects who had received insulin therapy, and it was demonstrated that this behavior was attributable to the presence of insulin-binding antibodies. Since the concentration of antibody is extremely low in most such patients and since the insulin-antibody complexes do not precipitate, certain special technics, including the use of an isotopically labeled insulin, are required for the detection of the antibody. Further extension of these technics has made possible an immunologic method for the assay of very small amounts of insulin.

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 Cushings 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.

Size heterogeneity of immunoreactive human ACTH in plasma and in extracts of pituitary glands and ACTH-producing thymoma

Abstract Immunoreactive ACTH in plasma and in extracts of pituitary glands and ACTH-producing thymoma was demonstrated using Sephadex gel filtration and separatory ultracentrifugation to be composed of at least two major components, one ACTH-like and the other of considerably greater molecular size. The relative distribution between the two major components varied greatly among plasmas. It is concluded that the larger molecular weight fraction originates in the pituitary and represents ACTH bound in covalent linkage to a larger peptide.

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.

Release of cholecystokinin peptides from a synaptosome-enriched fraction of rat cerebral cortex☆

Abstract Cholecystokinin (CCK) and its C-terminal fragments have been shown previously to be concentrated in synaptosome-rich subcellular fractions from rat brain. In this report we demonstrate that release of immunoreactive CCK is increased by 200% in solutions containing K + and Ca ++ according to the paradigms usually employed to evaluate neuronal chemicals purported to have a role in synaptic physiology. This strengthens our hypothesis that CCK has a role in synaptic function.

Size and Charge Distinctions Between Endogenous Human Plasma Gastrin in Peripheral Blood and Heptadecapeptide Gastrins

The major fraction of immunoreactive plasma gastrin in peripheral blood, cross-reacting in a radioimmunoassay system with 125I-porcine gastrin I, exhibits less acidic behavior on starch gel and paper electrophoresis and a larger molecular size on Sephadex gel filtration than that shown by heptadecapeptide gastrins. The plasma hormone is not altered by boiling and could not be shown to be converted by antral aqueous extracts to heptadecapeptide-like gastrin. From the electrophoretic and filtration behavior of plasma gastrin, it is suggested that the major fraction of the plasma hormone is a molecule of approximately 7000 mol wt containing heptadecapeptide gastrin linked to a more basic peptide. A small fraction of plasma hormone exhibits characteristics similar to heptadecapeptide gastrin.