Arnold W. Lindall

Bioassay of parathyroid hormone.

Summary There is an increase in excretion of administered P32 by thyroparathyroidectomized male rats under the influence of parathyroid hormone. Amounts as small as 0.5 U.S.P. unit may be detected, an increase in sensitivity of 25 times over the method of Tepperman et al.(4), and of 200 times over the standard U.S.P. assay method. In addition, there are no tedious chemical determinations of blood phosphate levels to be done.A method is disclosed for extraction, concentration, and bioassay measurement of the concentration of bioactive parathyroid peptides in biological or other fluids. Parathyroid peptides are extracted from the fluid by a support-antibody matrix specific to the N-terminal region. The bioassay is directed to the extracted parathyroid peptides. The volume of the medium containing the extracted parathyroid peptides and tissue, cells or tissue extracts with adenylate cyclase-coupled parathyroid hormone receptors may be chosen to be significantly less than the volume of biological or other fluid being assayed.

Studies on the Isolated Goosefish Insulin Secretion Granule

Goosefish islet secretion granules were isolated from sucrose homogenates by centrifugation. The morphology of the fractions was determined by electron microscopy. Studies on the stability of the secretion granules revealed them to be sensitive to pH and deoxycholate. The secretion granule proteins were subjected to electrophoresis in polyacrylamide gels, and the resultant bands were characterized by a number of stains and by immunoassay of insulin. These procedures showed that the granule fraction had a heterogeneous configuration consisting of lipid, carbohydrate and protein. One band containing much of the insulin activity did not show the ability to stain with aldehyde fuchsin, possibly indicating a different character of insulin.

The Effect of Dietary Fats on the Serum Lipoproteins of Normal Dogs

Summary The serum lipoproteins of normal dogs were studied while on different dietary regimens including low fat and various high fat diets containing coconut, olive, safflower, or menhaden oil. The serum low density (beta lipoprotein) fraction was increased nearly threefold by feeding coconut oil in contrast to the other diets which, except for a small increase with olive oil, showed little effect on the lipoproteins. High density lipoproteins, while constituting the major fraction in dogs, did not respond to the diets. Reasonably good separation into alpha and beta lipoprotein by ultracentrifugation was demonstrated by electrophoresis of the fractions on polyacrylamide gels.

Column chromatography of human serum parathyroid immunoreactive peptides.

Recently various reports have appeared attempting to define the size of serum parathyroid hormone (PTH). Canterbury and Reiss have shown that their assay measured, predominantly, a peptide of about 7000 mol wt and less of those peptides with 9500 and 5000 mol wt (1). They show, however, that the 9500 mol wt peptide is sensitive to calcium perturbation and decays rapidly after parathyroidectomy. Habener et al. (2) showed evidence that 9500 mol wt PTH is secreted and concluded that peripheral cleavage results in a 7000 mol wt peptide being predominant in peripheral serum. Martin et al. (3) have shown in monolayer cultures of human parathyroid glands that a peptide similar to beef PTH is secreted. Arnaud et al. (4) showed evidence that both 7000 and 9500 mol wt peptides are present in uremic serum and parathyroid adenomas, and concluded that the former peptide is a better steady state indicator or parathyroid dysfunction while the large form is related to acute changes of parathyroid function. In this communication we will show evidence that uremic serum has principally a peptide of 10,000 mol wt and that, in contrast, the pattern of serum PTH peptides from patients with parathyroid adenomas can be quite variable. These data support the hypothesis presented previously, relating to the synthesis and secretion of human parathyroid hormone (5). Methods. Measurement of parathyroid hormone. An antibody to beef parathyroid hormone was prepared in chickens by injecting them for 9 mo with 75% pure beef PTH of 9500 mol wt. The antibody recognizes the intact beef PTH molecule but does not recognize the N-terminal 1-34 segment of beef PTH. This antibody is unique in that it is multivalent in its ability to recognize a number of human parathyroid peptides ranging from > 25,000 to 3000 mol wt (5).

Effect of pH on Conversion of Proinsulin to Insulin by a Subcellular Fraction of Rat Islets

Previous studies on insulin biosynthesis in rats indicate that insulin is formed by way of a precursor (proinsulin) and one or more intermediates (1). A delay of 10–20 min. between proinsulin synthesis and onset of conversion has been demonstrated. Within this same period of time proinsulin is rapidly accumulated into the secretion granule containing fraction of the cell. The T1/2 for conversion of proinsulin to insulin is approximately 1 hr and the newly synthesized insulin appears to be secreted after 1 hr (2). Cell fractionation studies (3, 4) and electron microscopic radioautography indicate that proinsulin is synthesized on the rough endoplasmic reticulum and sequestered into the secretion granules presumably by way of the Golgi apparatus. These findings have suggested that the conversion of proinsulin to insulin occurs within the Golgi apparatus and/or the developing secretion granules. The distribution of labeled proinsulin and insulin in subcellular fractions of isolated rat islets is consistent with this hypothesis (4). Also several inhibitors of energy metabolism have demonstrated an inhibition of the conversion of proinsulin to insulin, when added prior to the presumed transport of the proinsulin from the endoplasmic reticulum to the Golgi region (5). It has been suggested that the enzyme necessary for conversion of proinsulin to insulin is trypsin-like, possibly acting in conjunction with a protease having a specificity similar to carboxypeptidase B (6). Studies with thiol inhibitors indicate that the transforming enzyme may belong to a group of proteases having a thiol in the active center, such as cathepsin B. We report here studies on the proteolysis of endogenous and exogenous proinsulin as a function of pH and on the concentration of proteolytic activity in the secretion granule-containing fraction. Methods and Materials. Islets were isolated from (350–400 g) male Holtzman rats by a modification of the method of Moskalewski (7) developed by Lacy and Kostianovsky (8). This method has previously been reported (9).

The Biosynthesis of C14- and H3-Labeled Insulin

In recent years, there has been much interest in the biosynthesis of insulin because of its role in diabetes. In addition, the exact amino acid sequence of the two peptide chains is known [1] and, therefore, the insulin molecule has become a model protein for research in synthetic mechanisms. Many techniques for the purification and characterization of insulin have been developed [2,3,4]. In the study of insulin biosynthesis, several investigators have incubated mammalian pancreas slices in the presence of radioactive amino acids in vitro, and then isolated the labeled insulin [3,5,6] This approach is limited because the beta cells of the islets of Langerhans (which manufacture and store insulin) comprise only a small fraction of the total pancreas. During the development of the pancreas in certain teleost fish, however, the islet tissue becomes separated from the exocrine pancreas and it appears in the adult as a macroscopic aggregate of endocrine tissue called the principal islet or Brockmann body. The principal islet contains cells which are functionally and anatomically analogous to the B-cells of mammalian islets; fish insulin is similar to mammalian insulin in structure and in biological activity [7,8]. Our studies on insulin biosynthesis using fish islets were begun in 1959; this report is a summary of these investigations.

A CRITICAL STUDY OF PYRIDINE NUCLEOTIDE CONCENTRATIONS IN NORMAL FED, NORMAL FASTED, AND DIABETIC RAT LIVER.

Abstract Rat liver pyridine nucleotide concentrations were determined in diabetes and starvation by the use of a further modified fluorimetric technic, which corrects for auto-oxidation of reduced pyridine nucleotides. Studies of rat liver pyridine nucleotides in starvation and diabetes showed that NAD NADH 2 ratios decreased in both conditions. In contrast, NADP NADPH 2 ratios were found to be increased. The conflicting observations by earlier workers on pyridine nucleotides in diabetes were discussed in view of the difficulties in measuring pyridine nucleotides by whole tissue analysis. The significance of whole cell pyridine nucleotide analysis was discussed in the light of values obtained for pyridine nucleotides in subcellular fractions.

Plasma immunoreactive calcitonin in lung cancer.

We have measured plasma calcitonin in 135 untreated eucalemic men with lung cancer and a control/smoker population. Calcitonin levels were determined by radioimmunoassay and validated by immunoextraction. Plasma immunoreactive calcitonin moieties were purified by immunoadsorbent chromatography, treated with mercaptoethanol and urea, and characterized by gel filtration. Artifacts in human calcitonin radioimmunoassays of cancer-patient plasmas were detected by parallel plasma incubations in a salmon calcitonin radioimmunoassay system which does not detect human calcitonin and by immunoprecipitation of tracer at the end of radioimmunoassay incubations. Heating fresh plasmas to 65 degrees C for 1.5 hours reduced radioimmunoassay artifacts without loss of calcitonin moieties. Such characterization of hypercalcitoninemia in each of the histopathological types of lung cancer has raised some important questions about the interpretation of plasma calcitonin radioimmunoassay measurements in lung cancer. Based on inhibition of tracer-antibody binding, plasma calcitonin seemed to be elevated in 18% (14/80) of basal plasma samples obtained from patients with epidermoid or with anaplastic lung cancer. Unequivocal hypercalcitoninemia (heat stable, causing no inhibition of antibody-tracer binding in the salmon calcitonin radioimmunoassays, and immunoextractable with human calcitonin antibodies) was not found in any of the apparently hypercalcitoninemic plasmas from persons with epidermoid or anaplastic lung cancer. By contrast, unequivocal hypercalcitoninemia was found in 27% (15/55) of plasmas from patients with small cell carcinoma or adenocarcinoma. Most of the immunoreactive calcitonin recovered from small cell and adenocarcinoma lung cancer plasmas with unequivocally elevated calcitonin is much larger than calcitonin monomer.

Comparison of insulin and proinsulin storage in an islet adenoma and adjacent pancreas.

Abstract The storage of insulin and proinsulin in various subcellular compartments of an islet adenoma and islets from the adjacent pancreas was compared. The tumor had a greater concentration of proinsulin relative to insulin in the secretion granule fraction than the adjacent islets. The microsome fraction from both the adenoma and islets had a larger percentage of proinsulin compared to insulin in contrast to other fractions. In a tolbutamide tolerance test before surgery, the percentage of serum proinsulin relative to insulin was 53% before and 17% after tolbutamide. Proinsulin secretion was temporally separated from insulin secretion in the early phase.

A comparative study of the insulin content of islet tissue of various species using anti-insulin serum and the epididymal adipose tissue methods.

Summary The insulin content of microdissected rat islets when measured by the two-antibody immunoassay method was approximately one-fourth that obtained when the epididymal fat pad method was used. Insulin content of the subcellular goosefish islet fractions after acid-alcohol extraction was approximately 103 U/g when measured by the fat pad method, as compared to 0.063 U/g when measured by the immunoassay method. Thus there is a 1600-fold difference in the values obtained using these two methods. About 84% of the total insulin contained in goosefish islets (estimated by the immunoassay method, after acid-alcohol extraction) was recovered in the mitochondria plus secretion granule fraction. Using the fat pad, 74% of the total insulin was found in this subcellular fraction. Acid-alcohol extraction of the secretion granule plus mitochondria, produced an eightfold increase in apparent insulin content when measured by the immunoassay method; thus most of the insulin contained in the secretion granule was not available to react with the insulin-antibody until it was extracted or modified by the acid-alcohol extracted and unextracted subcellular fraction. One microliter of antiserum (to beef-pork insulin) was able to completely neutralize the effect of 500 μU of either bovine or rat (islet) insulin as measured by the fat pad method. However, five times this amount of antiserum was needed to neutralize the same quantity of goosefish insulin. The observed 1600-fold difference in insulin content of goosefish islet tissue when measured by the fat pad and immunoassay methods indicates that goosefish insulin is hardly able to displace beef 131I insulin from its combination with the (beef-pork) antibody, whereas in the absence of a competing insulin only five times the quantity of antiserum is required to neutralize the effects of goosefish insulin.