Human Prolactin: Measurement in Plasma by In Vitro Bioassay

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Abstract

Prolactin has been measured in unextracted human plasma by a sensitive and specific in vitro bioassay. Secretory activity of breast tissue fragments from mid-pregnant mice, incubated in organ culture with human plasma, serves as the histologic end point. Sensitivity is 5 ng/ml (0.14 mU/ml) or somewhat better for ovine prolactin, and approximately 0.42 mU/ml for prolactin activity of human plasma at the dilutions used in the assay. Human growth hormone as it circulates in blood, like the material extracted from pituitary glands, is strongly lactogenic. Antisera to human growth hormone are capable of completely neutralizing the prolactin effect of large amounts (600 ng/ml) of human growth hormone added to the system. Plasma prolactin activity is less than 0.42 mU/ml in normal men and women. Of 26 patients with nonpuerperal galactorrhea, 14 had elevated prolactin activities ranging from 0.42 to 3.5 mU/ml. Growth hormone levels by radioimmunoassay were far too low, in general, to account for the observed prolactin activity. All of 14 nursing mothers, 1-30 days post partum, had elevated prolactin activity with a mean of 2.29 and a total range of 0.56-4.5 mU/ml. Growth hormone was in the low normal range in all of these subjects. Seven patients on psychoactive drugs of the phenothiazine series similarly had elevated prolactin activity with low growth hormone. Antiserum to human growth hormone, when preincubated with plasma samples from each of these three groups of subjects, produced no significant inhibition of prolactin activity. In two acromegalic patients with markedly elevated growth hormone levels, antiserum to growth hormone produced complete inhibition of prolactin activity in one and partial inhibition in the other. These studies indicate that human growth hormone and human prolactin are separate molecules, with little if any immunologic cross-reactivity, at least as demonstrated by the antisera used in this study, and that their release is governed by different physiologic mechanisms.

Authors

David L. Kleinberg, Andrew G. Frantz

Colonic Secretion of Water and Electrolytes Induced by Bile Acids: Perfusion Studies in Man

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Abstract

Each of the three major bile acids of man was tested for its influence on electrolyte and water absorption in the human colon. Transport from isotonic solutions, with or without added bile acids, was compared in 35 studies on 20 healthy volunteers by colonic perfusions under steady-state conditions. Electrolytes and water were always absorbed from control solutions, but dihydroxy bile acid solutions induced continuous secretion or inhibition of sodium, potassium, and water absorption, which was reversible. Deoxycholic acid caused consistent secretion at 3 mm concentrations, whereas chenodeoxycholic acid did not induce secretion until the concentration was 5 mm. The trihydroxy bile acid (cholic acid) produced no significant change in absorption at 10 mm. Inhibition of absorption was also induced by mixtures of the glycine or taurine conjugated bile acids. Secretion of sodium and chloride, induced by bile acid perfusion, was linearly correlated with secretion of water; potassium secretion was relatively constant regardless of the volume of secretion. These results establish a striking influence of bile acids on colonic absorptive activity, provide an explanation in part for the diarrhea that frequently accompanies ileal disease or resection, and imply that diarrhea should occur in other disease states that produce elevated concentrations of dihydroxy bile acids in the colonic lumen.

Authors

Hagop S. Mekhjian, Sidney F. Phillips, Alan F. Hofmann

Effects of Nitroglycerin on Regional Myocardial Blood Flow in Coronary Artery Disease

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Abstract

Regional myocardial blood flow before and after sublingual nitroglycerin was measured in 10 patients with coronary artery disease. During thoracotomy, 133Xe was injected directly into the subepicardium in diseased regions of the anterior left ventricular wall, and washout rates were recorded with a scintillation counter. All disappearance curves were closely approximated by two exponential decays analyzed as two parallel flow systems by the compartmental method. The appearance of a double exponential decay pattern in diseased regions suggests that the slow phase was associated with collateral blood flow, although nonhomogeneous myocardium-to-blood partition coefficients for xenon cannot be excluded. Nitroglycerin increased the rapid phase flow in 9 of 10 patients and the slow flow in 7 of 10 patients. Average flow increased in 9 of the 10 patients (P < 0.01). Mean rapid phase flow in the control state was 110 ml/100 g per min and after nitroglycerin increased to 132 ml/100 g per min (P < 0.01); slow phase flow increased from 12 ml/100 g per min to 15 ml/100 g per min (P < 0.05). It is concluded that, under these conditions, nitroglycerin improves perfusion in regions of diseased myocardium in patients with coronary artery disease.

Authors

Lawrence D. Horwitz, Richard Gorlin, Warren J. Taylor, Harvey G. Kemp

The Regulation of Aldosterone Secretion in Anephric Man

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Abstract

The regulation of aldosterone secretion in anephric man was investigated in studies on nephrectomized patients who were being intermittently hemodialyzed while awaiting renal transplantation. The effects of supine and upright posture on the concentration of plasma aldosterone on the 1st day postdialysis and on a 3rd or 4th day postdialysis were compared to the effects of postural variation in normal subjects who were on a low sodium intake and on a high sodium intake. In contrast with the normal subjects who exhibited higher concentrations of plasma aldosterone after 2 hr of upright posture than in the supine position and low concentrations of plasma aldosterone on a high sodium intake, the anephric patients showed less consistent variations in plasma aldosterone due to changes in posture and exhibited higher concentrations of plasma aldosterone on the 3rd or 4th day postdialysis, despite an increase in body weight, than on the 1st day postdialysis.

Authors

Francis Bayard, C. Robert Cooke, David J. Tiller, Inese Z. Beitins, Avinoam Kowarski, W. Gordon Walker, Claude J. Migeon

On the Mechanism of Inhibition in Fluid Reabsorption by the Renal Proximal Tubule of the Volume-Expanded Rat

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Abstract

We undertook to determine the extent to which the inhibition in absolute proximal fluid reabsorption in response to expansion of extracellular volume with noncolloid-containing solutions is the result of concomitant reductions in postglomerular (efferent arteriolar) protein concentration. Selective elevation of efferent arteriolar oncotic pressure in volume-expanded rats (Ringer's 10% body weight) to levels slightly in excess of normal by microperfusion with 9-10% albumin-Ringer's solution nearly completely reversed the inhibition in absolute and fractional reabsorption in adjacent proximal tubules. In contrast, during similar microperfusion with a 6-7% albumin solution, no increase in proximal reabsorption was measured. We interpret these findings to indicate that the bulk of the inhibition in absolute proximal reabsorption in response to volume expansion with colloid-free solutions is causally mediated by the accompanying parallel decline in postglomerular vascular protein concentration.

Authors

Barry M. Brenner, Julia L. Troy, Terrance M. Daugharty, I. F. Ueki, D. P. Nicholas, C. F. Wong

Evidence for a Humoral Mechanism in Volume Expansion Natriuresis

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Abstract

The role of a humoral mechanism in the natriuresis induced by volume expansion was evaluated using an isolated dog kidney perfused by a second dog which had been pretreated with desoxycorticosterone acetate (DOCA). Expansion of the perfusion dog with an equilibrated volume of blood from a reservoir, resulted in an increase in UnaV (sodium excretion) from 153.6±27.9 (sem) to 345.5±57.8 μEq/min, P<0.001. FEna (fractional sodium excretion) increased from 3.4±0.6 to 8.1±1.2%, P<0.01. The natriuresis occurred in the face of a significant decrease in Cin, RBF, and renal arterial pressure, and in the absence of any change in plasma protein concentration or packed cell volume. In a control group of experiments, sodium excretion did not change when the perfusion dog was not volume expanded, although Cin (inulin clearance) and RBF (renal blood flow) decreased to the same degree as in the expanded group. These data support the conclusion that volume expansion of the perfusion dog either stimulated the release of a natriuretic factor or suppressed the release of an antinatriuretic factor which was manifested by an increase in sodium excretion in the isolated kidney.

Authors

George J. Kaloyanides, Maher Azer

Effects of Ca⁺⁺ and Prostaglandin E₁ on Vasopressin Activation of Renal Adenyl Cyclase

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Abstract

Adenyl cyclase activity was assayed in crude homogenates of the renal cortex, medulla, and papilla of the golden hamster. The specific activity (moles C-AMP/unit of time per mg protein of tissue) of the enzyme under basal conditions, was greatest in papilla, somewhat lower in medulla, and least in cortex. On an absolute scale, the sensitivity to vasopressin was greater in the medullary and papillary than in the cortical homogenates. In addition, at concentrations of 0.1-1.0 mm, CaCl2 inhibited the enzyme in the order papilla > medulla > cortex. These results imply the existence of distinct differences in the composition of the adenyl cyclase-receptor complex in various parts of the kidney. We proposed that Ca++ inhibits the core enzyme directly since at the minimally inhibitory concentration (0.1 mm), CaCl2 reduced to an equivalent extent (a) basal activity, (b) the response to graded doses of vasopressin (0.5 to 50.0 mU/ml) and (c) the response to maximal stimulatory concentrations of NaF (10 mm). Prostaglandin E1 (PGE1 = 10−7m) had no effect on either basal adenyl-cyclase activity or the response to 10 mm NaF in medullary and papillary homogenates. 7-Oxa-13-prostynoic acid (10−4m) similarly had no effect under basal conditions or on stimulation with NaF in medullary homogenates. Both fatty acids, however, inhibited the enzymic response to vasopressin, particularly at low concentrations of the peptide. The straight-chain fatty acid, 11-eicosanoic acid (10−7m), was inactive on basal activity or on the response to vasopressin. The possibility that PGE1 modifies the coupling mechanism between the core enzyme and the hormone-specific receptor is discussed.

Authors

Fumiaki Marumo, Isidore S. Edelman

The Source of Plasma Dihydrotestosterone in Man

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Abstract

The source of plasma dihydrotestosterone (DHT) (17β-hydroxy-5α-androstan-3-one) in humans has been investigated by infusing two potential peripheral precursors, testosterone (T) and androstenedione (A). Metabolic clearance rates (MCR), conversion ratios (CR), transfer constants (ρ), and blood production rates (PB) were calculated. Plasma testosterone and dihydrotestosterone were measured by competitive binding techniques. The MCRDHT was 652 ±35 (SD) liters/day in five males and 314 ±63 (SD) liters/day in four adult females. In each individual, the MCRDHT was significantly lower than MCRT as predicted by testosterone-binding protein affinity studies. The PBDHT was 302 ±65 (SD) μg/day in males and 56 ±26 μg/day in females. Testosterone and androstenedione are precursors (prehormones) for plasma dihydrotestosterone. The conversion ratio CRBBT-DHT, calculated as the ratio of counts per minute per liter of plasma of product to precursor after infusion of labeled precursor, was 5.6 ±0.6 (SD)% (six subjects) in the male and 3.5 ±0.4 (SD)% (four subjects) in the female. CRBBA-DHT after androstenedione infusion to three female subjects averaged 9.2%. No dihydrotestosterone back conversion was detected (< 0.2%). The transfer constants were [ρ]BBT-DHT, 3.9 ±1.0% (male) and 1.7 ±0.6% (female), and [ρ]BBA-DHT average was 13.3% in three female subjects. Using either plasma testosterone and dihydrotestosterone values from our subjects and mean androstenedione values as reported in the literature, approximate contributions can be calculated. Testosterone conversion accounts for at least 70% of plasma DHT in the male, but less than 20% in the normal female. Androstenedione appears to be a major prehormone of plasma dihydrotestosterone accounting for at least two-thirds plasma dihydrotestosterone by peripheral conversion in adult females. In three normal women undergoing tubal ligation, there was an unimpressive gradient between ovarian vein and peripheral plasma dihydrotestosterone. It is suggested that dihydrotestosterone in the blood does not arise from direct secretion but may reflect events occurring in peripheral androgen target tissues.

Authors

T. Ito, R. Horton

An Unusual Hemoglobin Anomaly and Its Relation to α-Thalassemia and Hemoglobin-H Disease

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Abstract

A Chinese family with hemoglobin H in the propositus has been reinvestigated. Although the original propositus is now deceased, a sister has the same hematological manifestations. Her hemoglobin, like that of the deceased sister, contains hemoglobins A, H, and Bart's. In addition, however, two minor components have been detected. These minor components appear to have abnormal α-chains and are also present in the maternal grandmother, the mother, a maternal aunt, and three other siblings but only in about one-tenth the amount. One of the minor components may be the same as Hb-Thai (25). The father has the characteristics of classical α-thalassemia. These results are discussed in relation to current concepts of α-thalassemia as they relate to “silent” and “classical” α-thalassemia and to possible multiple α-chain loci.

Authors

G. D. Efremov, Ruth N. Wrightstone, T. H. J. Huisman, W. A. Schroeder, Carol Hyman, Jorge Ortega, Kenneth Williams

Glutathione Synthesis in Human Erythrocytes: II. PURIFICATION AND PROPERTIES OF THE ENZYMES OF GLUTATHIONE BIOSYNTHESIS

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Abstract

The two enzymes required to synthesize glutathione de novo have been purified from human erythrocytes. Glutamylcysteine synthetase was purified 4300-fold and was approximately 80% pure based on polyacrylamide gel electrophoresis. The purified enzyme catalyzes the formation of 30.5 μmoles of γ-glutamyl-cysteine per mg of protein per hr and is inhibited by sulfhydryl inhibitors.

Authors

Philip W. Majerus, M. J. Brauner, M. B. Smith, Virginia Minnich

Effects of Norethandrolone on the Transport and Peripheral Metabolism of Thyroxine in Patients Lacking Thyroxine-Binding Globulin: OBSERVATIONS ON THE PHYSIOLOGICAL ROLE OF THYROXINE-BINDING PREALBUMIN

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Abstract

Studies of the effect of norethandrolone on the transport and peripheral metabolism of thyroxine were carried out in four patients lacking thyroxine-binding globulin. Before norethandrolone administration, values for serum protein-bound iodine (PBI) were decreased (1.8 ±0.5 μg/100 ml) and the proportion of free thyroxine increased (0.036 ±0.008%). As a result, values for the absolute concentration of free thyroxine iodine were at the lower end of the normal range (0.63 ±0.12 mμg/100 ml). During the control thyroxine-turnover study, the thyroxine distribution space was strikingly increased (18.2 ±7.9 liters) and the fractional rate of thyroxine turnover moderately increased (17.1 ±11.3%/day), as compared to the expected mean values for normal subjects. Therefore, calculated values for the daily rate of thyroxine clearance were increased even more, ranging between 255 and 500% of normal values. However, owing to the low PBI in these patients, the daily disposal of thyroxine iodine was similar to that expected in normals on the basis of age and weight. During the administration of norethandrolone, the thyroxine-binding capacity of the thyroxine-binding prealbumin increased strikingly in all patients, values averaging 162% of those found during the control period. This increase was associated with a highly significant increase in PBI (133% of control values) and a small but significant decrease in the proportion of free thyroxine, resulting in no significant change in the absolute concentration of free thyroxine iodine. In all four patients, administration of norethandrolone was associated with a pronounced decrease in the thyroxine distribution space to values which averaged 69% of those found during the control period. Values for the fractional rate of thyroxine turnover increased slightly. As a result, thyroxine-clearance rate decreased in all patients. Owing to the reciprocal changes in clearance rate and PBI, no significant change in total daily thyroxine disposal was observed. The present studies reveal that when the thyroxine-binding prealbumin is increased in patients lacking thyroxine-binding globulin, several indices of peripheral thyroxine transport and metabolism are altered. However, these changes were small, even in the absence of thyroxine-binding globulin. It is suggested, therefore, that the effect of changes in thyroxine-binding prealbumin would be even smaller in individuals in whom thyroxine-binding globulin is present.

Authors

Lewis E. Braverman, Theodore AvRuskin, Michael J. Cullen, Apostolos G. Vagenakis, Sidney H. Ingbar

The Glycogenolytic Activity of Immunoreactive Pancreatic Glucagon in Plasma

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Abstract

Conclusions concerning the physiologic role of pancreatic glucagon in health and its contribution to disorders of carbohydrate metabolism, such as diabetes mellitus, are based entirely on measurements of plasma glucagon by radioimmunoassay. The changes in plasma immunoreactive glucagon can have the metabolic and clinical significance which has been implied, only if the glucagon detected by immunoassay has biological activity. The present study was designed to determine if a relationship between the immunoassayable glucagon and glycogenolytic activity of plasma could be demonstrated.

Authors

Jose Marco, Gerald R. Faloona, Roger H. Unger

Left Ventricular Performance and Coronary Flow after Coronary Embolization with Plastic Microspheres

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Abstract

Coronary flow, left ventricular circumference, and left ventricular pressure were observed in the isovolumically contracting, isolated canine heart supported with arterial blood from a donor. Systolic pressure, heart rate, and coronary perfusion pressure were held constant while the coronary bed was progressively embolized with either large (average 865 μ) or small (average 10 μ) polystyrene microspheres. During embolization with large microspheres, coronary flow diminished progressively. After sufficient embolization, decreased ventricular performance was indicated by a rise in end-diastolic pressure. During embolization with small microspheres, coronary flow initially increased, which suggests the effective release of a vasodilator substance. Return of coronary flow to control levels occurred only after the end-diastolic pressure rose, on the average, to above 30 mm Hg. After embolization with both sizes of microspheres, ventricular diastolic pressure-volume relationships showed decreased ventricular compliance. This was attributed, in part, to edema of the ventricular wall and, in part, to focal shortening of the sarcomeres where the circulation was compromised. Embolization with both sizes of microspheres ultimately caused a decrease in ventricular performance, although when the systolic pressure was increased the usual relationship between peak developed wall stress, and end-diastolic pressure showed less of a descending limb than that found in the nonembolized, isolated heart.

Authors

R. G. Monroe, C. G. LaFarge, W. J. Gamble, A. E. Kumar, F. J. Manasek

The Protein and Lipid Composition of Arterial Elastin and Its Relationship to Lipid Accumulation in the Atherosclerotic Plaque

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Abstract

Elastin preparations from intimal layers and the media of normal and atherosclerotic human aortae were analyzed for protein and lipid content. In atherosclerotic aortae, elastin from plaques was compared with elastin from adjacent normal appearing areas of the same aorta.

Authors

Dieter M. Kramsch, Carl Franzblau, William Hollander

Leukokinetic Studies: XIV. BLOOD NEUTROPHIL KINETICS IN CHRONIC, STEADY-STATE NEUTROPENIA

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Abstract

The kinetics of blood neutrophils was investigated by means of the in vitro radioactive diisopropyl fluorophosphate method in 35 patients with a chronic, steady-state neutropenia. There were 17 patients in whom the half disappearance time of neutrophils was normal. In 10 of these patients, the production of neutrophils was low and in 7, production was normal. In 18 patients the half disappearance time of neutrophilic granulocytes was shorter than normal. The production of neutrophilic granulocytes was low in five of these patients, normal in eight patients, and increased in five. An attempt was made to correlate other laboratory measurements with the kinetic picture, but no relationship was found; the marrow neutrophil reserve as measured by endotoxin or cortisol injection; marrow cellularity on aspiration or biopsy; in vitro-labeling index with 3HTdR; or serum lysozyme concentration proved of no value in identifying the various kinetic groups. The only finding that seemed to correlate with the kinetic picture was the presence or absence of splenomegaly. In 12 of the 18 patients with a short half disappearance time, splenomegaly was present whereas in 15 of 17 patients with a normal half disappearance time, there was no splenomegaly. Of 20 patients with greater than 1000 neutrophils per mm3, 17 were found to have a normal total-blood neutrophil pool. Thus these patients, with many of their cells marginated, agree to have a “shift neutropenia.”

Authors

C. R. Bishop, G. Rothstein, H. E. Ashenbrucker, J. W. Athens

Metabolism of Fibrinogen in Cirrhosis of the Liver

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Abstract

The metabolism of human fibrinogen labeled with radioactive iodine was studied in 50 patients with documented cirrhosis of the liver and in 35 healthy control subjects. Results in cirrhotic subjects were the following: plasma volume 47 ± 10 ml/kg; plasma fibrinogen concentration 250 ± 102 mg/100 ml; total plasma fibrinogen pool 118 ± 59 mg/kg, representing 0.73 ± 0.10 of the total body pool; fibrinogen half-life 2.99 ± 0.59 days; fractional catabolic rate 0.34 ± 0.09 of the plasma pool per day; absolute catabolic rate 39 ± 20 mg/kg per day; fractional transcapillary efflux rate 0.82 ± 0.30 of the plasma pool per day. Results in the control subjects were the following: plasma volume 42 ± 7 ml/kg; plasma fibrinogen concentration 284 ± 71 mg/100 ml; total plasma fibrinogen pool 119 ± 40 mg/kg, representing 0.72 ± 0.07 of the total body pool; fibrinogen half-life 4.14 ± 0.56 days; fractional catabolic rate 0.24 ± 0.04 of the plasma pool per day; absolute catabolic rate 28 ± 9 mg/kg per day; fractional transcapillary efflux rate 0.60 ± 0.26 of the plasma pool per day.

Authors

G. N. Tytgat, D. Collen, M. Verstraete

Influence of Endogenous Insulin Secretion on Splanchnic Glucose and Amino Acid Metabolism in Man

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Abstract

Splanchnic exchange of glucose, 20 individual amino acids, lactate, and pyruvate was studied in normal subjects in the postabsorptive state and after stimulation of endogenous insulin secretion by infusion of glucose at two dose levels. In the basal state, mean splanchnic glucose production was 3.4 mg/kg per min. A net uptake of lactate, pyruvate, and nine amino acids was observed, with alanine accounting for half of the total splanchnic-amino acid extraction.

Authors

Philip Felig, John Wahren

The Glycosaminoglycans of Normal and Arthritic Cartilage

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Abstract

The cartilages from the hip joints of 13 normal and 15 osteoarthritic humans were analyzed for glycosaminoglycan content and distribution. The GAGs were separated by elution with CPC on a short cellulose column by the technique of Svejcar and Robertson after digestion of the tissue with pronase and papain. The eluates were identified by a variety of methods including determination of molar ratios, N-acetyl-hexosamine determinations after hyaluronidase treatment and thin-layer chromatography of unhydrolyzed and hydrolyzed GAGs.

Authors

Henry J. Mankin, Louis Lippiello

Bile Acid Synthesis in the Isolated, Perfused Rabbit Liver

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Abstract

These experiments were carried out to demonstrate the usefulness of the perfused rabbit liver for studies of bile acid metabolism, and to determine the rate-limiting enzyme of bile acid synthesis. Rabbits were fed a semisynthetic diet, with or without the addition of 1% cholestyramine, under controlled conditions. At the end of 2-5 wk, the livers were removed and perfused for 2.5 hr employing various 14C-labeled precursors to measure de novo cholic acid synthesis. The livers were then analyzed for cholesterol, and the bile collected during the perfusion was analyzed for cholesterol and bile acids. Control bile contained, on the average, 0.34 mg of glycocholate, 7.4 mg of glycodeoxycholate, and 0.06 mg of cholesterol. After cholestyramine treatment of the donor rabbits, the bile contained 3.3 mg of glycocholate, 3.7 mg of glycodeoxycholate, and 0.05 mg of cholesterol. It was assumed that in cholestyramine-treated animals the enterohepatic circulation of the bile acids had been interrupted sufficiently to release the feedback inhibition of the rate-controlling enzyme of bile acid synthesis. Therefore, a given precursor should be incorporated into bile acids at a more rapid rate in livers of cholestyramine-treated animals, provided that the precursor was acted upon by the rate-controlling enzyme. It was found that the incorporation of acetate-14C, mevalonolactone-14C, and cholesterol-14C into cholate was 5-20 times greater in the livers of cholestyramine-treated animals than in the controls. In contrast, there was no difference in the incorporation of 7α-hydroxycholesterol-14C into cholate regardless of dietary pretreatment. It was concluded that given an adequate precursor pool, the 7α-hydroxylation of cholesterol is the rate-limiting step in bile acid formation.

Authors

E. H. Mosbach, M. A. Rothschild, I. Bekersky, M. Oratz, J. Mongelli

Energy Metabolism in Human Erythrocytes: I. EFFECTS OF SODIUM FLUORIDE

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Abstract

Exposure of red cells to fluoride produces a variety of metabolic alterations, most of which are based upon the secondary effects of enolase inhibition, which reduces pyruvate synthesis and interferes with the regeneration of diphosphopyridine nucleotide (NAD). Adenosine triphosphate (ATP) is consumed in the hexokinase and phosphofructokinase reactions but is not regenerated since the deficiency of NAD limits glyceraldehyde phosphate dehydrogenase. ATP depletion in the presence of fluoride and calcium induces a massive loss of cations and water.

Authors

Stephen A. Feig, Stephen B. Shohet, David G. Nathan

Reversal of Digoxin Toxicity with Specific Antibodies

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Abstract

To determine whether digoxin-specific antibodies can reverse established digoxin toxicity in the dog, digoxin intoxication was produced by the intramuscular administration of digoxin, 0.09 mg/kg, on each of 3 consecutive days. All animals developed toxic arrhythmias (atrioventricular block, ventricular premature contractions and/or ventricular tachycardia). In control animals not receiving antidigoxin antibodies, the arrhythmias persisted throughout a 6 hr study period. Seven of the nine control dogs were dead within 24 hr and one moribund animal was sacrificed at that time; the last animal died within 48 hr.

Authors

Donald H. Schmidt, Vincent P. Butler Jr.

Isolation and Properties of Phagocytic Vesicles from Polymorphonuclear Leukocytes

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Abstract

A method for the isolation of intact phagocytic vesicles from guinea pig peritoneal-exudate granulocytes and human peripheral-blood leukocytes is presented. After leukocytes ingested the particles of a stable emulsion of paraffin oil, the uningested emulsion was washed away and the cells were homogenized. The homogenate was placed in the middle of a three-step discontinuous sucrose gradient and centrifuged for 1 hr at 100,000 g. The phagocytic vesicles, containing the low density paraffin-oil particles, were simultaneously washed and collected by floatation, while the other organelles, chiefly granules, sedimented through the lower wash layer, and the particle-free supernatant remained in the middle of the gradient.

Authors

Thomas P. Stossel, Thomas D. Pollard, Robert J. Mason, Martha Vaughan

Preparation and Properties of Thyroxine-Binding Alpha Globulin (TBG)

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Abstract

Thyroxine-binding alpha globulin (TBG) in human serum was isolated from Cohn fractions IV-5,6 and IV-4 by (1) chromatography on carboxymethyl (CM) cellulose, (2) gel filtration on Sephadex G-200, (3) chromatography on diethylaminoethyl-Sephadex, (4) a novel procedure of “double-gel” electrophoresis, and (5) preparative polyacrylamide gel electrophoresis. The protein was homogeneous by analytical disc gel electrophoresis, immunoelectrophoresis, and ultracentrifugal analyses (sedimentation velocity and sedimentation equilibrium), and after addition of thyroxine-125I showed a constant specific radioactivity on polyacrylamide electrophoresis. The sedimentation and diffusion coefficients were s20, w, 3.0 × 10−13 sec, and D20, w, 8.05 × 10−7 cm2·sec−1, and the molecular weight obtained by sedimentation equilibrium was 36,500. Gel filtration studies on Sephadex G-200 demonstrated that the protein had the same elution volume as that of native TBG in serum, apparently excluding the possibility of a subunit of the native protein. Chemical composition was ascertained by amino acid and carbohydrate analyses. The maximal thyroxine (T4)-binding capacity measured by reverse flow paper electrophoresis was 15,000 μg per g of protein, representing more than 2100 times that of the starting material, or about 5000 times that of whole serum. Based on the molecular weight obtained, the TBG preparation could bind 0.7 mole T4 per mole of protein, suggesting a single binding site. The association constant for T4 was estimated to be of the order of 1010 by competitive binding studies employing TBG and T4-binding prealbumin (TBPA).

Authors

Kenneth Sterling, Satoshi Hamada, Yoshihiro Takemura, Milton A. Brenner, Edward S. Newman, Mitsuo Inada

Ligand Kinetics in Hemoglobin Hiroshima

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Hemoglobin Hiroshima is an electrophoretically fast-moving variant with a fourfold increase in oxygen affinity and a decreased Bohr effect. Based on a decreased rate of dissociation of O2 in the presence of dithionite and an increased rate of binding of CO by the deoxy form, we have concluded that the kinetic basis of the high affinity exhibited by Hb Hiroshima is the concurrence of a faster combination rate and a slower dissociation rate for ligands.

Authors

Ronald L. Nagel, Quentin H. Gibson, Howard B. Hamilton

The Dynamics of Glomerular Ultrafiltration in the Rat

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Abstract

Using a unique strain of Wistar rats endowed with glomeruli situated directly on the renal cortical surface, we measured glomerular capillary pressures using servo-nulling micropipette transducer techniques. Pressures in 12 glomerular capillaries from 7 rats averaged 60 cm H2O, or approximately 50% of mean systemic arterial values. Wave form characteristics for these glomerular capillaries were found to be remarkably similar to those of the central aorta. From similarly direct estimates of hydrostatic pressures in proximal tubules, and colloid osmotic pressures in systemic and efferent arteriolar plasmas, the net driving force for ultrafiltration was calculated. The average value of 14 cm H2O is lower by some two-thirds than the majority of estimates reported previously based on indirect techniques. Single nephron GFR (glomerular filtration rate) was also measured in these rats, thereby permitting calculation of the glomerular capillary ultrafiltration coefficient. The average value of 0.044 nl sec−1 cm H2O−1 glomerulus−1 is at least fourfold greater than previous estimates derived from indirect observations.

Authors

Barry M. Brenner, Julia L. Troy, Terrance M. Daugharty