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Higher serum iron and ferritin levels noted in hepatitis B antigen (HBAg) carriers than in noncarriers suggests that virus might actively replicate in hepatocytes, stimulate ferritin synthesis, and result in increased liver iron stores. A comparative semiquantitative study of immunohistochemical ferritin (0-12) and hemosiderin (0-9) was performed on 54 normal, 13 cirrhotic, and 70 nonneoplastic livers from patients with hepatocellular carcinoma, in each group, comparing amounts in HBAg-positive and HBAg-negative patients. Mean scores for ferritin and hemosiderin were high in all three groups, normal livers averaging 8.3 and 6, respectively, cirrhotic livers, 8.5 and 7.4, respectively, and carcinoma livers, 5.6 and 6.1, respectively. In each group, there was no significant difference in ferritin and hemosiderin mean scores in HBAg-positive and HBAg-negative patients. The large liver iron stores do not appear to be a consequence of hepatitis B virus infection alone. Their role in the development of hepatocellular carcinoma is still to be elucidated.

Serum ferritin concentrations are elevated in 35% to 100% of patients with hepatocellular carcinoma (HCC). With an immunoperoxidase technique, ferritin was demonstrated in tumor tissue from 32 of 74 (43%) black southern African patients, and from 12 of 19 (63%) American patients with HCC (P greater than 0.1). Ferritin was present in nonneoplastic liver in 82% of African and 100% of American patients (P greater than 0.1). Moderate to large amounts of stainable hepatic storage iron (hemosiderin) were present in 76% of African and 67% of American patients (P greater than 0.1). Fifty-two (70%) African patients had macronodular cirrhosis. In the literature, 80% to 90% of American patients with HCC have cirrhosis. High serum ferritin levels in patients with HCC may be due to ferritin production by the tumor, or related to the associated iron overload and/or cirrhosis.

Electrophoretic and filtration analysis of immunoreactive plasma gastrin extends our previous finding of two immunoreactive components of plasma gastrin. One component has the characteristics of Gregory-Tracy heptadecapeptide gastrin; the other component has a less acidic charge than heptadecapeptide gastrin but emerges from a Sephadex gel column in a zone consistent with a molecular weight of about 7000, which is 3 1/2 times the molecular weight of heptadecapeptide gastrin. The larger, more basic component (BG) usually represents the major fraction of plasma gastrin; the heptadecapeptide-like component (H-LG) is virtually undetectable in some samples. However, only (H-LG) was found in the urine of a patient whose plasma contained nearly equal amounts of both components. There is no spontaneous interconversion of the two components in vitro, and on refractionation each component retains its integrity. However, trypsin converts BG into an immunoreactive component with characteristics similar to those of heptadecapeptide gastrin. The separated plasma components individually are indistinguishable from each other and from heptadecapeptide porcine gastrin I in their immunochemical reaction with guinea pig antiporcine gastrin I antibodies. The secretion of both components is stimulated by feeding.

Plasma gastrin concentrations were determined by radioimmunoassay in 8 subjects given a test meal with and without prior atropinization. Gastrin levels increased in all subjects following feeding. Integrated increments in plasma gastrin responses over the 2-hr period following feeding were always greater under atropine than in the control studies. Particularly marked differences between atropine and control studies were observed in 3 patients with duodenal ulcer. One patient with pernicious anemia and high fasting plasma gastrin concentrations showed no significant sustained changes in concentration in the fasting state over a 2-hr period following atropine administration. It is concluded that the tonic release of gastrin in patients with anacidity is atropine-resistant and that the reduction in gastric acid secretion produced by atropine in acid-secreting subjects serves to blunt the normal inhibitory effect of antral acidification on gastrin secretion, thereby overcoming any possible atropine blockade of gastrin release in response to feeding.

Gastric acid secretion and plasma gastrin concentrations were determined during fasting and following sham feeding or feeding in dogs with Pavlov pouches or gastric fistulae. Sham feeding caused immediate elevations of plasma gastrin concentrations that rapidly returned to basal levels following the onset of acid secretion although acid was continuously secreted for several hours. Sham feeding with the gastric fistula open produced higher gastrin concentrations and acid output than with the fistula closed. Feeding raised the plasma gastrin concentrations considerably higher than sham feeding in the same dogs although the peak acid output equaled that following sham feeding with gastric fistula open. The delay in onset of acid secretion was more pronounced during feeding than during sham feeding. Atropinization did not significantly influence basal plasma gastrin concentration but virtually abolished the gastrin response to sham feeding. Plasma gastrin responses were observed following feeding despite atropinization.

The preparation of I131-labeled human insulin from a lot of human insulin containing approximately 25% insulin by weight and its purification from labeled contaminants are described. The reaction of human insulin-I133 with insulinbinding antibodies in the serums of human subjects treated with commercial mixtures of animal insulins is demonstrated directly. Comparison of the binding of human insulin and beef insulin in antiserums from eight insulinresistant and nonresistant diabetic subjects revealed a lesser affinity' of antibody for human than for beef insulin in most cases, but considerable variability in this respect was encountered among different antiserums.

Extracts of mucosa from human antrum, duodenum, and proximal jejunum obtained at surgery and at autopsy contain two immunoreactive components of gastrin, separable on the basis of size and charge. These components exhibit the same characteristics as the two plasma gastrin components identified earlier. The major plasma component (BG) has a less acidic charge and larger molecular size than heptadecapeptide gastrin; the minor plasma component (H-LG) resembles heptadecapeptide gastrin in both respects. H-LG predominates in most extracts of antral mucosa, but the relative abundance of BG increases distally, being the only detectable component (although at very low concentration) in proximal jejunal mucosal extracts. Both components are immunochemically indistinguishable from the two plasma components and from heptadecapeptide porcine gastrin.

Separation of Antibody-Bound and Unbound Peptide Hormones Labelled with Iodine-131 by Talcum Powder and Precipitated Silica

Five patients with active Hodgkin’s disease were treated for a period varying from four to eighteen days with an antiguanine preparation, guanazolo (5-amino-7-hydroxy-1H-v-triazolo[d]pyrimidine) with a total dosage of 400 mg. to 800 mg. intramuscularly. No therapeutic effect was observed. The only toxic manifestations were pain and tenderness at the site of injection in 4 of the 5 cases. The effect of guanazolo on tissue cultures of an involved node was tested. No inhibition of growth was observed, thus paralleling the lack of in vivo effect.

The role of serum albumin in the causation of cardiac edema has been disputed. Starling (1) postulated that circulatory stasis resulting from cardiac insufficiency is responsible for anoxia and increased permeability of the capillary membrane, the consequence of which is the passage of albumin and water into the extravascular spaces. Although Smirk (2) observed evidence of increased capillary permeability to water and crystalloids in congestive heart failure, Stead and Warren (3) demonstrated, by direct analysis, that the protein concentration of cardiac edema fluid was not elevated above that of the edema fluid produced by tourniquet stasis in normal subjects. Other studies have directed attention to the low serum albumin concentrations occasionally encountered in heart failure (4-7), and have suggested or implied (4, 8) a causal relationship to the pathogenesis of cardiac edema. Abnormalities in liver function tests and morphologic evidence of liver injury in severe congestive failure (9-14) have appeared to strengthen the possibility that hepatic synthesis of albumin may be impaired in this condition. Alternatively, low serum albumin concentrations have been attributed to inadequate dietary protein intake or poor absorption from a congested gastrointestinal tract (15, 16). Since the plasma concentration of albumin is not an adequate measure of the total quantity of circulating and extravascular albumin stores, it was of interest to measure the amount of total exchangeable albumin in subjects with heart failure employing I'8l labeled albumin as a tracer. The rate of metabolism of I18l labeled albumin was also studied with the aim of evaluating the ability of the subject in heart failure to synthesize albumin. Although there may yet remain some reservations regarding the validity of this tracer in the

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.

Australia antigen (Au) has been identified in the blood of patients with "long-incubation hepatitis" by use of agar-gel diffusion or complement fixation (CF) [1-6]. Characterization of the antigen as a viruslike particle and its localization in the nuclei of liver cells of patients with hepatitis have provided evidence that Au may be the agent of long-incubation hepatitis [7-11]. A precipitating and complement-fixing antibody to Au (anti-Au) has been identified in the serum of patients who have received multiple transfusions but rarely, if ever, in serum from patients convalescing from hepatitis [1-3, 5-12]. It has been shown that the presence of anti-Au does not always prevent the development of hepatitis [13]. Transfusion of blood containing Au may result in hepatitis in the recipient [3, 12]. With agar-gel diffusion, it has been possible to identify Au in some but not all units of blood that have induced

Porcine insulin, which is distinguished from human insulin only in the amino acid at the C terminal of the B chain, is antigenic in man. Even if the last amino acid or the last eight amino acids are removed from the C terminus of the B chain of insulin, the altered insulin still reacts with human antibodies to porcine insulin; thus, the antigenic determinant of porcine insulin is located in a part of the molecule where the amino acid sequence is the same as it is in the corresponding part of the human insulin molecule.

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.

The technical methods employed and the schematic model on which the analyses are based are common to all the determinations and are presented first. The specific analyses and results obtained for the various phases of iodine metabolism are then considered in separate sections. An attempt has been made to follow a similar order of presentation in each of these sections. The theoretical basis for the methods employed in the measurement of each aspect of iodine metabolism is followed by a sample calculation from the observed data in one of the subjects, R. D. Reference is then usually made to the results of the measurements, and the section is concluded with an analysis of the validity of the determinations and the sources of error.

VIRTUALLY all human subjects treated with commercial mixtures of beef-pork insulin develop circulating insulin-binding antibodies that are readily detectable with insulin labelled with iodine-131 by means of paper or starch eleetrophoresis, paper chromatography or ultracentrifugation1. These antibodies react not only with the specific immunizing insulins but also with horse, sheep and human insulins2, and with dog, monkey, rabbit and whale insulins (unpublished work) in greater or lesser degree. Harris, Sanger and Naughton3 have shown that the amino-acid sequences of insulins from four different ungulate species (cattle, pig, horse and sheep) differ only in the 8–10 positions of the A chains. Although some human antisera react quite similarly with these four insulins, certain antisera can distinguish clearly among insulins from the different species, generally reacting much more strongly with beef and sheep insulins than with pork and horse insulins2. These differences in reaction of the ungulate insulins have been related2 to the known differences in the 8–10 sequence of the A-chain or to possible differences in secondary or tertiary structure.

For years investigators have sought an assay for insulin which would combine virtually absolute specificity with a high degree of sensitivity, sufficiently exquisite for measurement of the minute insulin concentrations usually present in the circulation. Methods in use recently depend on the ability of insulin to exert an effect on the metabolism of glucose in vivo or in excised muscle or adipose tissue. Thus, the insulin concentration in plasma has been estimated: a) from the degree of hypoglycemia produced in hypophysectomized, adrenalectomized, alloxan-diabetic rats (1); b) from the augmentation of glucose uptake by isolated rat hemidiaphragm (2); or c) from the increased oxidation of glucose-1-C14 by the rat epididymal fat pad (3). Since there have been reports indicating the presence, in plasma, of inhibitors of insulin action (4) and of noninsulin substances capable of inducing an insulin-like effect (5,6), these procedures, while yielding interesting information regarding the effects of various plasmas on glucose metabolism in tissues, are of doubtful specificity for the measurement of insulin per se (5).

WE have previously reported on the immuno-assay of beef insulin and certain other animal insulins, employing antiserums from human subjects treated with commercial mixtures of beef and pork insulin1. The insulin-binding antibodies present in these anti-serums do not form precipitable complexes with insulin, but with the use of insulin labelled with iodine-131 the complexes are readily demonstrable by paper chromatography and electrophoresis2. Beef, pork, sheep and horse insulins can be assayed quantitatively by measurement of the degree of competitive inhibition of binding of any insulin labelled with iodine-1311–3. As might have been anticipated, however, human insulin competes too weakly in systems employing human antiserum to be measurable at concentrations which obtain in vivo. Furthermore, the lack of availability of significant quantities of pure human insulin precludes its use as an antigen for animal immunization. However, in the present work, it has been found that human insulin cross-reacts strongly with insulin-binding antibodies in guinea pigs immunized with crystalline beef insulin, and that guinea pig anti-beef insulin serum has characteristics suitable for the detection and measurement of human insulin at concentrations which exist in the plasma of normal fasting subjects.

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.

In the method presented here for the correction of sample absorption of C(14) activity, the only requirement is the availability of any C(14)-labeled compound of sufficiently high specific activity to permit addition, in negligible mass, of a number of counts equal to or greater than that in unknown samples and with solubility characteristics that exclude preferential layering during drying of samples. The principle may be applied to liquid scintillation counting. Absorption curves are dispensed with, and the weights of the assayed samples need not be determined.