Arnold Lazarow

Immunoassay of insulin using a two-antibody system.

Summary Tracer amounts of I131-insulin are precipitated by specific antibodies. The change in percent precipitated, as increasing amounts of unlabeled insulin are added, forms the basis of this assay. The method is sensitive to less than one microunit.

Further Studies of an Inhibitor of the Two Antibody Immunoassay System

Previous studies with the two antibody immunoassay method have demonstrated the presence of an “inhibitor” in fresh plasma which interferes with the precipitating step of the two antibody system. Immunoelectrophoretic studies described here demonstrated no cross-reactivity of rat or human gamma globulin with anti-guinea pig plasma (rabbit source), which had been suggested by other workers. Concomitant inactivation of complement and “inhibitor” by heat, antigen-antibody complex, EDTA, or ammonia indicates that complement is the inhibitor of the two antibody immunoassay system. EDTA (0.01 M) inactivates the plasma “inhibitor,” permitting assay of undiluted plasma by the two antibody method.

COMPONENT QUANTITATION OF TISSUE SECTIONS. I. CHARACTERIZATION OF THE INSTRUMENTS

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STAINING OF INSULIN WITH ALDEHYDE FUCHSIN

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Isotransplantation of organ-cultured neonatal pancreas: reversal of alloxan diabetes in the rat.

Pancreases from neonatal rats four to 16 days postpartum were grown in organ culture for from two to nine days. Approximately 10-20 explants, each measuring 1 mm.3 (1 mg.), were grown on a single Millipore filter placed at the gas-liquid interface of a medium consisting of 50 per cent horse serum and 50 per cent chick embryo extract. Following organ culture, an estimated 9-20 mg. of cultured islet tissue were dissociated with collagenase and isotransplanted into the peritoneal cavity of alloxan-diabetic recipients. In seven of eight recipients the diabetes was reversed between 11 and 53 days of posttransplantation. Animals receiving 12-16 mg. of cultured islet attained normoglycemia in 11-20 days; animals receiving 9-10 mg. of cultured islet tissue recovered between 45 and 53 days. These animals have remained symptom-free for over six months. Biopsies of grafts taken from the peritoneal cavity following reversal of diabetes contained well-vascularized islets compared primarily of heavily granulated beta cells. Quantitative analysis of host pancreases by the linear scanning method (biopsied at one to two weeks and four to five months following reversal of the diabetes) demonstrated that the total beta-cell mass was 3 per cent and the total insulin content was 6 per cent of the normal values. Little or no evidence of regeneration of host beta cells was observed. These studies show that a period of organ culture prior to isotransplantation does not impair the ability of islet tissue to reverse alloxan diabetes in the rat.

Citrate metabolism in diabetes. I. Plasma citrate in alloxan-diabetic rats and in clinical diabetes.

Abstract A “bimodal” increase in plasma citrate levels was observed in alloxan-diabetic rats. Three days following alloxan injection, the plasma citrate levels in cardiac puncture blood was 9.40 mg. 100 ml. as compared to 4.95 for the control. At 14 days the value approximated that in the normal rats, whereas at 28 days (and subsequent time periods), the citrate levels were markedly elevated. In a second series of experiments in which the animals were killed by decapitation, a similar “bimodal” change in plasma citrate values was noted; however, the plasma citrate levels in both diabetic and control rats were generally higher than was the case when blood was drawn by cardiac puncture. No relationship could be established between the blood sugar level and plasma citrate values. In treated human diabetics, plasma citrate values were not significantly different from the normal, i.e., 2.70 as compared to 2.41 mg. 100 ml. However, in 2 cases of untreated human diabetes exhibiting severe ketoacidosis, the plasma citrate values were markedly elevated. In one, the initial value was 44.4 mg. 100 ml. serum; the level rapidly returned toward normal; at 4 weeks it was 1.56 mg. 100 ml. The increased serum citrate levels were not the result of citrate mobilization from bone, since there was no concomitant increase in the serum calcium levels. These results suggest the possibility that there may be an alteration in the tricarboxylic acid cycle in diabetes (both experimental and clinical), and that this alteration results in the accumulation of plasma citrate.

Cell Types of the Islets of Langerhans and the Hormones They Produce

The pancreatic islet tissue was first described by Paul Langerhans in 1869 while he was still a medical student. Langerhans identified a new cell type in the pancreas which had not heretofore been observed. He noted that these cells were grouped together in rounded cell masses measuring between 120 and 240 microns in diameter and that these were distributed throughout the parenchyma of the pancreas. Laguesse, some twentyfive years later, named these structures the islets of Langerhans. Laguesse studied the cytology and development of the islets.3 He observed that the islet cells contained granules which could be seen in the living cell examined in serum and that these granules could be stained by safranine or gentian violet.

Citrate metabolism in diabetes: II. Tissue citrate content, citrate synthase and oxidation in alloxan-diabetic rat☆

Abstract The alloxan-diabetic rat is characterized by (1) decreased citrate synthase (citrogenase) activity of the liver; (2) a two- to threefold increase in the citrate content of the tissues and (3) a decreased rate of citrate oxidation by the muscle (but not liver). The possible relationship of these changes is discussed.

STUDIES ON THE ISOLATED ISLET TISSUE OF FISH. IV. IN VITRO INCORPORATION OF C14- AND H8-LABELED AMINO ACIDS INTO GOOSEFISH ISLET TISSUE PROTEINS

1. Goosefish islet tissue, incubated in vitro with C14- or H3-labeled amino acids showed significant labeling of the protein fractions.2. The purified alcohol-soluble fraction is presumed to contain the insulin which is synthesized in vitro.3. The rate of amino acid incorporation into the alcohol-soluble fraction increases progressively with increasing time of incubation; it is decreased in the absence of oxygen.4. The addition of glucose decreases the number of counts incorporated into the alcohol-soluble fraction.5. In the C14-amino acid incorporation experiments, the specific activity of the purified alcohol-soluble fraction (128,000 cpm/mg. protein) is three times greater than that of the trichlor-precipitable protein residue.6. These studies Support the thesis that amino acids, added to the islet tissue in vitro, are incorporated into insulin.

Insulin Content of Fetal Rat Pancreases Grown in Organ Culture and Subsequently Transplanted into Maternal Hosts

Portions of pancreases from 19.5-day fetal rats were grown in organ culture on two types of liquid media: low glucose, containing 162 to 170 mg. glucose/100 ml.; high glucose, containing 1,031 to 1,073 mg. glucose/100 ml. Following four days of organ culture, the tissues were extracted and assayed for insulin content (immunoassay). Other cultures were transplanted to maternal hosts at two sites: beneath the capsule of the kidney and into the anterior chamber of the eye. Ten days later, the transplants were removed and assayed for insulin content. Large quantities of immunologically active insulin were present in the culture media following forty-eight and ninety-six hours of incubation. Expiants exhibited a four- to sixfold increase in insulin content following four days in vitro. Pancreatic expiants cultured on the high glucose medium contained less extractable insulin than similar ex-plants cultured on the low glucose medium. When cultured expiants were transplanted to hosts with normal blood glucose levels, the total extractable insulin content of the grafts was 55 per cent to 130 per cent greater than that at the time of transplantation. When cultures were transplanted to alloxan diabetic hosts, the total extractable insulin content of the grafts was less than that found in grafts to normal hosts. A small but statistically significant drop in blood glucose level was observed in hyperglycemie diabetic maternal hosts receiving transplants of organ cultured pancreas. These results indicate the continued growth and functional responsiveness of the islet beta cells during organ culture and following subsequent transplantation.