Isolation and Characterization of Heparin from Human Lung

• Text
• PDF

Abstract

Heparin as measured by azure A metachromasia and anticoagulant activity has been extracted with 1 M NaCl from 35S-labeled human lung fragments or dispersed human lung cells enriched for mast cells. The 35S-labeled metachromatic material in the 3 M NaCl eluate from Dowex-1 chromatography of the extract from lung fragments exhibited an average mol wt of 20,000 by Sepharose 4B gel filtration. The 35S-labeled metachromatic material with the charge characteristics of commercial porcine heparin on DEAE cellulose chromatography was entirely heparin by the criteria of resistance to degradation by chondroitin ABC lyase and complete degradation by purified heparinase. Antithrombin affinity chromatography of purified heparin with an anticoagulant activity of 137 U/mg, revealed that the one-third that was bound and eluted had a 273 U/mg sp act, whereas the unbound activity was 31 U/mg. Thus, the previously observed heterogeneity of commercial porcine heparin for binding to human antithrombin was also observed with human heparin. The mast cell-enriched human lung cell preparations yielded [35S]mucopolysaccharides with an average mol wt of 60,000 by Sepharose 4B gel filtration. Approximately 30% of this fraction was degraded by chondroitin ABC lyase, and the residual 70% was degraded by purified heparinase. When the chondroitin ABC lyase-resistant fraction was subjected to alkali degradation the average mol wt was reduced to 20,000. The calculated human lung mast cell heparin content of 2.4-7.8 μg/106 cells gave a ratio to histamine on a weight basis similar to that of intact lung fragments, thereby implying that heparin in the lung fragments was largely restricted to the mast cells.

Authors

Dean D. Metcalfe, Robert A. Lewis, Jeremiah E. Silbert, Robert D. Rosenberg, Stephen I. Wasserman, K. Frank Austen

Inhibition by propionyl-coenzyme A of N-acetylglutamate synthetase in rat liver mitochondria. A possible explanation for hyperammonemia in propionic and methylmalonic acidemia.

• Text
• PDF

Abstract

In the search for the mechanism by which hyperammonemia complicates propionic and methylmalonic acidemia the effects of a series of acyl-coenzyme A (CoA) derivatives were studied on the activity of N-acetylglutamate synthetase in rat liver mitochondria using acetyl-CoA as substrate. Propionyl-CoA was found to be a competitive inhibitor. The inhibition constant of 0.71 mM is in the range of concentrations of propionate found in the serum of patients with propionic and methylmalonic acidemia. Propionyl-CoA was also found to be a substrate for N-acetylglutamate synthetase, forming N-propionylglutamate. This compound was a weak activator of rat liver carbamoylphosphate synthetase; the activation constant was 1.1 mM as compared with 0.12 mM for N-acetylglutamate. A decreased level of N-acetylglutamate in liver mitochondria that would follow inhibition of N-acetylglutamate synthetase by propionyl-CoA would be expected to lead to hyperammonemia. Methylmalonyl-CoA, tiglyl-CoA, and isovaleryl-CoA at a concentration of 3 mM caused 30-70% inhibition of N-acetylglutamate synthetase. 3the latter two compounds are readily detoxified by the formation of N-acylglycine conjugates in liver, which may prevent large accumulations and could explain why hyperammonemia is not characteristic of patients with beta-ketothiolase deficiency or isovaleric acidemia in whom these compounds would be expected to be elevated.

Authors

F X Coude, L Sweetman, W L Nyhan

Attenuation of Angiotensin II- and III-induced Aldosterone Release by Prostaglandin Synthesis Inhibitors

• Text
• PDF

Abstract

The effect of two prostaglandin synthesis inhibitors, indomethacin and meclofenamate, on angiotensin II (AII)- and III (AIII)-induced aldosterone release was studied in normal and sodium-depleted conscious rats and in adrenal capsular cell suspensions obtained from normal rats. In normal rats, in vivo AII and AIII were equipotent in causing dose-related increases in serum aldosterone concentrations. Indomethacin decreased the basal serum aldosterone levels by 50% and serum renin levels by 43%. In addition, the steroidogenic effects of AII and AIII were reduced by 45 and 63% with 3 mg/kg of indomethacin and 63 and 73% with 10 mg/kg, respectively. In contrast, meclofenamate failed to alter basal serum levels of aldosterone or AII-stimulated aldosterone release but inhibited serum renin levels by 27% and the aldosterone-stimulating effect of AIII by 99%. Indomethacin (3 mg/kg) and meclofenamate (2 mg/kg) inhibited urinary prostaglandin (PG)E2 and PGF2α excretion by 63 and 52% and 37 and 31%, respectively. Both inhibitors significantly decreased the adrenal capsular PGE2 and PGF2α content and the conversion of [14C]arachidonate to [14C]PGE2 and [14C]PGF2α. In sodium-depleted rats, indomethacin produced similar effects reducing the control serum aldosterone levels by 29%, AII-stimulated aldosterone by 47%, and completely suppressing the aldosterone response to AIII without altering serum renin activity. In adrenal cell suspensions, similar results were observed with indomethacin inhibiting basal and AII- and AIII-stimulated aldosterone release by 29, 81, and 93%, respectively. Meclofenamate failed to alter basal and AII-stimulated aldosterone release but inhibited that stimulated by AIII by 86%. The present findings suggest that prostaglandins modulate the effects of the renin-angiotensin system by stimulating the release of renin from the kidney and augmenting the steroidogenic effects of AII and AIII in the adrenal cortex.

Authors

William B. Campbell, Celso E. Gomez-Sanchez, Beverley V. Adams, James M. Schmitz, Harold D. Itskovitz

Mechanisms of Killing of Newborn Larvae of Trichinella spiralis by Neutrophils and Eosinophils: KILLING BY GENERATORS OF HYDROGEN PEROXIDE IN VITRO

• Text
• PDF

Abstract

Eosinophil and/or neutrophil leukocytes appear to have important roles in host defense against invasive, migratory helminth infestations, but the mechanisms of larval killing by leukocytes are uncertain. This study examines killing of newborn (migratory phase) larvae of Trichinella spiralis during incubation with granule preparations of human eosinophils or neutrophils and generators of hydrogen peroxide (glucose-glucose oxidase) (G-GO) or superoxide and hydrogen peroxide (xanthine-xanthine oxidase). Larvae were killed by either hydrogen peroxide-generating system in a concentration-dependent manner. Direct enumeration of surviving larvae after incubation in microtiter wells containing the appropriate reagents was used in assess larval killing. Verification of the microplate assay was demonstrated by complete loss of larval ability to incorporate [3H]deoxyglucose and loss of infectivity after incubation in comparable concentrations of G-GO. Larvae were highly sensitive to oxidative products; significant killing occurred after incubation with 0.12 mU glucose oxidase and complete killing occurred with 0.5 mU. Comparable killing of bacteria required over 60 mU glucose oxidase. At 5 mU glucose oxidase, killing was complete after 6 h of incubation. Killing by G-GO was inhibited by catalase but not by boiled catalase or superoxide dismutase and was enhanced by azide. Addition of peroxidase in granule pellet preparations of eosinophils or neutrophils did not enhance killing by G-GO. These data indicate a remarkable susceptibility of newborn larvae of T. spiralis to the hydrogen peroxide generated by neutrophil and eosinophil leukocytes.

Authors

David A. Bass, Pamela Szejda

Role of the Liver in Regulation of Ketone Body Production during Sepsis

• Text
• PDF

Abstract

During caloric deprivation, the septic host may fail to develop ketonemia as an adaptation to starvation. Because the plasma ketone body concentration is a function of the ratio of hepatic production and peripheral usage, a pneumococcal sepsis model was used in rats to measure the complex metabolic events that could account for this failure, including the effects of infection on lipolysis and esterification in adipose tissue, fatty acid transport in plasma and the rates of hepatic ketogenesis and whole body oxidation of ketones. Some of the studies were repeated with tularemia as the model infection. From these studies, it was concluded that during pneumococcal sepsis, the failure of rats to become ketonemic during caloric deprivation was the result of reduced ketogenic capacity of the liver and a possibly decreased hepatic supply of fatty acids. The latter appeared to be a secondary consequence of a severe reduction in circulating plasma albumin, the major transport protein for fatty acids, with no effect on the degree of saturation of the albumin with free fatty acids. Also, the infection had no significant effect on the rate of lipolysis or release of fatty acids from adipose tissue. Ketone body usage (oxidation) was either unaffected or reduced during pneumococcal sepsis in rats. Thus, a reduced rate of ketone production in the infected host was primarily responsible for the failure to develop starvation ketonemia under these conditions. The liver of the infected rat host appears to shuttle the fatty acids away from β-oxidation and ketogenesis and toward triglyceride production, with resulting hepatocellular fatty metamorphosis.

Authors

Robert W. Wannemacher Jr., Judith G. Pace, Francis A. Beall, Richard E. Dinterman, Vance J. Petrella, Harold A. Neufeld

Erythropoiesis in vitro. Role of calcium.

• Text
• PDF

Abstract

The in vitro plasma clot technique was employed to examine the role of calcium during the interaction of erythropoietin and mouse erythroid progenitor cells. Erythropoietin-induced erythroid colony formation was increased 24% by the carboxylic ionophore A23187 (10 nM), whereas a 35% increase was produced by the carboxylic ionophore Ro 2-2985/1 (1 nM). EGTA (3 mM) inhibited erythropoietin-induced erythroid colony formation. Inhibition of erythroid colony formation by EGTA could be reversed by Ca2+, but not by Mn2+, Mg2+, Zn2+, or Fe2+. At least 30 min exposure of marrow cells to erythropoietin in vitro was required for production of erythroid colonies. EGTA substantially inhibited erythropoietin-induced erythroid colony formation even when the marrow cells were exposed to the hormone for up to 2 h before addition of the chelator. Marrow cells incubated first in calcium-free medium with erythropoietin and then cultured in the presence of calcium but not erythropoietin, failed to form erythroid colonies although colony formation occurred when erythropoietin was provided. Taken together, the data indicate that calcium is required for both extracellular and intracellular events during the interaction of erythropoietin with its target cells.

Authors

J Misiti, J L Spivak

Ethylmalonic-Adipic Aciduria: IN VIVO AND IN VITRO STUDIES INDICATING DEFICIENCY OF ACTIVITIES OF MULTIPLE ACYL-CoA DEHYDROGENASES

• Text
• PDF

Abstract

The mechanisms underlying ethylmalonic-adipic aciduria were studied in a 5-yr-old girl. Oxidation of radioactive substrates by cultured skin fibroblasts from the proband and asymptomatic family members was also determined and compared to that by normal fibroblasts and that by cells from a patient with glutaric aciduria type II.

Authors

Stephanos Mantagos, Myron Genel, Kay Tanaka

Studies of Relationships among Bile Flow, Liver Plasma Membrane NaK-ATPase, and Membrane Microviscosity in the Rat

• Text
• PDF

Abstract

Liver plasma membrane (LPM) NaK-ATPase activity, LPM fluidity, and bile acid-independent flow (BAIF) were studied in rats pretreated with one of five experimental agents. Compared with controls, BAIF was increased 24.6% by thyroid hormone and 34.4% by phenobarbital, decreased by ethinyl estradiol, but unchanged by propylene glycol and cortisone acetate. Parallel to the observed changes in BAIF, NaK-ATPase activity also was increased by thyroid hormone (40.8%) and decreased by ethinyl estradiol (26.2%). In contrast, NaK-ATPase activity failed to increase after phenobarbital but did increase 36% after propylene glycol and 34.8% after cortisone acetate. Thus BAIF and NaK-ATPase activity did not always change in parallel. The NaK-ATPase Km for ATP was not affected by any of these agents.

Authors

Emmet B. Keeffe, Bruce F. Scharschmidt, Nancy M. Blankenship, Robert K. Ockner

Effect of Central Catecholamine Depletion on the Osmotic and Nonosmotic Stimulation of Vasopressin (Antidiuretic Hormone) in the Rat

• Text
• PDF

Abstract

The central nervous system (CNS) mechanism(s) for the release of antidiuretic hormone (ADH) by various stimuli is unknown. In this study, the role of CNS catecholamines in effecting ADH release was examined in conscious rats 10-14 d after the cerebroventricular injection of 6-hydroxydopamine (6-OHDA). This dose of 6-OHDA caused a 67% depletion of brain tissue norepinephrine and only 3% depletion of heart norepinephrine, as compared with controls, which were injected with the vehicle buffer alone. Either intravenous 3% saline (osmotic stimulus) or intraperitoneal hyperoncotic dextran (nonosmotic stimulus) was administered to water-diuresing rats through indwelling catheters. Neither of these maneuvers changed arterial pressure, pulse, or inulin clearance in control or 6-OHDA rats. The 3% saline caused similar increases in plasma osmolality (15 mosmol/kg H2O) in control and 6-OHDA rats. The control rats, however, increased urinary osmolality (Uosm) to 586 mosmol/kg H2O, whereas 6-OHDA rats increased Uosm only to 335 mosmol/kg H2O (P < 0.005). These changes in Uosm were accompanied by an increase in plasma ADH to 7.6 μIU/ml in control animals vs. 2.9 μIU/ml in 6-OHDA rats (P < 0.005). All waterdiuresing animals had undetectable plasma ADH levels. Dextran-induced hypovolemia caused similar decrements (− 10%) in blood volume in both control and 6-OHDA animals, neither of which had significant changes in plasma osmolality. This nonosmotic hypovolemic stimulus caused an increase in Uosm to 753 mosmol/kg H2O in control rats, whereas Uosm in 6-OHDA rats increased to only 358 mosmol/kg H2O (P < 0.001). At the same time, ADH levels also were significantly greater in Cont rats (2.4 μIU/ml) than in the 6-OHDA animals (0.69 μIU/ml; P < 0.05). These results therefore suggest that CNS catecholamines may play an important role in mediating ADH release in response to both osmotic and nonosmotic (hypovolemic) stimuli.

Authors

Thomas R. Miller, William A. Handelman, Patricia E. Arnold, Keith M. McDonald, Perry B. Molinoff, Robert W. Schrier

Induction of suppressor activity in the autologous mixed lymphocyte reaction and in cultures with concanavalin A.

• Text
• PDF

Abstract

T lymphocytes that are activated in the autologous mixed lymphocyte reaction (MLR) have suppressor activity. Concanavalin A (Con A) augments the suppressor activity generated in cultures containing both T and non-T lymphocytes and can induce suppressor activity in T-lymphocyte preparations that contain too few (10%) non-T cells to generate a significant autologous MLR. However, when such T-lymphocyte preparations are further depleted of adherent cells and contain less than 2% non-T cells, Con A fails to induce suppressor activity. These findings support the concept that an autologous MLR may play an important role in generation of suppressor cells by Con A.

Authors

J B Innes, M M Kuntz, Y T Kim, M E Weksler

Regulation of the Conversion of Thyroxine to Triiodothyronine in the Perfused Rat Liver

• Text
• PDF

Abstract

This study was undertaken to determine what factors control the conversion of thyroxine (T4) to triiodothyronine (T3) in rat liver under conditions approximating those found in vivo. Conversion of T4 to T3 was studied in the isolated perfused rat liver, a preparation in which the cellular and structural integrity is maintained and that can perform most of the physiologic functions of the liver. The perfused liver readily extracted T4 from perfusion medium and converted it to T3. Production of T3 by the perfused liver was a function of the size of the liver, the uptake of T4 by the liver, and the presence of T4-5′-deiodinase activity. Production of T3 was increased by increasing the uptake of T4 by liver, which could be accomplished by increasing the liver size, by increasing the perfusate T4 concentration, or by decreasing the perfusate albumin concentration. These changes occurred without altering the conversion of T4 to T3. The liver had a large capacity for extracting T4 and for T4-5′-deiodination to T3, which was not saturated at a T4 concentration of 60 μg/dl. Production of T3 was decreased by inhibiting hepatic T4-5′-deiodinase with propylthiouracil, which decreased T3 production by decreasing the conversion of T4 to T3. Propylthiouracil did not alter hepatic T4 uptake.

Authors

Anthony S. Jennings, Duncan C. Ferguson, Robert D. Utiger

Testicular Feminization Associated with a Thermolabile Androgen Receptor in Cultured Human Fibroblasts

• Text
• PDF

Abstract

Evidence for a qualitative abnormality in the androgen receptor was obtained by studies of temperature sensitivity. The binding of [3H]dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) was studied in monolayers of cultured genital skin fibroblasts from genetic males with abnormal sexual differentiation resulting from androgen resistance. Binding in cells from eight patients with a female phenotype (complete and incomplete testicular feminization) fell from half-normal levels at the usual assay temperature of 37°C to levels <20% of normal when cells were incubated at 42°C. This thermal inactivation was rapidly reversed when the assay temperature was lowered to 37°C, was not associated with altered dihydrotestosterone metabolism, and was also demonstrable with [3H]methyltrienolone as the binding ligand. Binding increased to overlap the normal range when the assay temperature was lowered to 26°C. The patients with receptor-deficient testicular feminization include three pairs of siblings; the pedigrees in two of these families are compatible with X-linkage.

Authors

James E. Griffin

T-lymphocyte differentiation in vitro in severe combined immunodeficiency. Defects of stem cells.

• Text
• PDF

Abstract

A study of T-lymphocyte differentiation was made on fractionated bone marrow cells from normal volunteers and from 11 patients with severe combined immunodeficiency (SCID) using normal thymic epithelial monolayers and their culture supernates as inducing agents. Normal marrow cells could regularly be induced to bear the human T-lymphocyte antigen (HTLA), to form rosettes with sheep erythrocytes (E rosettes), and to respond to the mitogen concanavalin A (Con A) after coculture with the thymic epithelial monolayers or their culture supernates. In contrast, studies of T-cell differentiation on the marrow cells of patients with SCID revealed varying defects, ranging from a complete "absence" of definable T-cell precursors to partial differentiation resulting in acquisition of one (HTLA) or two (HTLA and E rosettes) markers for T lymphocytes. Only in one patient was there induction of all three T-cell markers, namely, HTLA, E rosettes, and responsiveness to Con A. These observations indicate that SCID is a heterogeneous disorder in which defects of differentiation can occur at one or more multiple sites of differentiation leading the the clinical expression of T- and B-cell dysfunction. Further, our studies indicate that in T-cell differentiation, HTLA probably appears before the capacity to form E-rosettes, and development of the latter capacity is followed by a state of responsiveness to mitogens. A scheme of normal differentiation along with the defects of precursor T cells seen in SCID is presented.

Authors

R N Pahwa, S G Pahwa, R A Good

Generation of Hydroxyl Radical by Enzymes, Chemicals, and Human Phagocytes In Vitro: DETECTION WITH THE ANTI-INFLAMMATORY AGENT, DIMETHYL SULFOXIDE

• Text
• PDF

Abstract

Methane (CH4) production from the anti-inflammatory agent, dimethyl sulfoxide (DMSO), was used to measure ·OH from chemical reactions or human phagocytes. Reactions producing ·OH (xanthine/xanthine oxidase or Fe++/EDTA/H2O2) generated CH4 from DMSO, whereas reactions yielding primarily O-2̇ or H2O2 failed to produce CH4. Neutrophils (PMN), monocytes, and alveolar macrophages also produced CH4 from DMSO. Mass spectroscopy using d6-DMSO showed formation of d3-CH4 indicating that CH4 was derived from DMSO. Methane generation by normal but not chronic granulomatous disease or heat-killed phagocytes increased after stimulation with opsonized zymosan particles or the chemical, phorbol myristate acetate. Methane production from DMSO increased as the number of stimulated PMN was increased and the kinetics of CH4 production approximated other metabolic activities of stimulated PMN. Methane production from stimulated phagocytes and DMSO was markedly decreased by purportedly potent ·OH scavengers (thiourea or tryptophane) and diminished to lesser degrees by weaker ·OH scavengers (mannitol, ethanol, or sodium benzoate). Superoxide dismutase or catalase also decreased CH4 production but urea, albumin, inactivated superoxide dismutase, or boiled catalase had no appreciable effect. The results suggest that the production of CH4 from DMSO may reflect release of ·OH from both chemical systems and phagocytic cells. Interaction of the nontoxic, highly permeable DMSO with ·OH may explain the anti-inflammatory actions of DMSO and provide a useful measurement of ·OH in vitro and in vivo.

Authors

John E. Repine, John W. Eaton, M. W. Anders, John R. Hoidal, Richard B. Fox

Circulating Immune Complexes in Cutaneous Vasculitis: DETECTION WITH Clq AND MONOCLONAL RHEUMATOID FACTOR

• Text
• PDF

Abstract

To investigate the pathogeneic significance of immune complexes in cutaneous vasculitis, 107 patients with various forms of cutaneous vasculitis, including 59 patients with necrotizing (leukocytoclastic) vasculitis (group 1), and 48 patients with lymphocytic vasculitis, or a predominately lymphocytic perivascular infiltrate (group 2), were studied. Immunoglobulins or complement components in cutaneous blood vessels were detected by direct immunofluorescence in high frequency in both groups (91 and 88%, respectively). Using two radioassays for circulating immune complexes, Clq or monoclonal rheumatoid factor (mRF) reactive material was detected in 68% of the patients with necrotizing vasculitis but only 44% of the patients in the lymphocytic-perivascular group. The mRF radioassay was elevated in 58% of the first group of patients and 41% of the patients in group 2, although Clq binding activity was increased in 54% of the patients with necrotizing vasculitis but only in 9% of the patients with a lymphocytic vasculitis or lymphocytic perivascular infiltrate. By using both sucrose density gradient ultracentrifugation and Sepharose 6B gel filtration, the Clq and mRF reactive material detected in some patients with necrotizing vasculitis eluted in high molecular weight fractions that were also anticomplementary. In one patient with necrotizing vasculitis and hepatitis B antigenemia, these heavy molecular weight Clq and mRF reactive fractions contained a two- to three-fold increase in hepatitis B surface antigen when compared with lighter molecular weight fractions. Heavy and light molecular weight mRF reactive material could be detected in selected patients in the lymphocytic-perivascular group as well as in the necrotizing vasculitis group. These studies suggest that cutaneous vasculitis, including acute necrotizing (leukocytoclastic) vasculitis and some forms of lymphocytic vasculitis, and perhaps some diseases characterized by a lymphocytic perivascular infiltrate, may represent cutaneous expressions of immune complex disease.

Authors

Susan E. Mackel, Gerhard Tappeiner, Hilton Brumfield, Robert E. Jordon

Serum and Urine Polyamines in Normal and in Short Children

• Text
• PDF

Abstract

The serum and urine polyamines putrescine, spermidine, and spermine were measured in 112 normal subjects from 0 to 70 yr of age, and in three groups of short children from 7 to 20 yr: 21 growth hormone (GH) deficient patients, 20 normal variant short stature children, and 9 girls with 45, X Turner's syndrome. Urine polyamines were expressed as micromoles per gram of creatinine or per kilogram body weight, and serum polyamines were expressed as nanomoles per milliliter.

Authors

Daniel Rudman, Michael H. Kutner, Rajender K. Chawla, Martin A. Goldsmith, R. Dwain Blackston, Raymond Bain

Metabolic clearance and production rates of prolactin in man.

• Text
• PDF

Abstract

Metabolic clearance rates (MCR) and production rates (PR) of prolactin (PRL) have been determined by the constant infusion to equilibrium technique in 11 normal subjects, 6 patients with hyperthyroidism, 4 patients with hypothyroidism, and 9 patients with hyperprolactinemia. PRL MCR was also determined tin four patients during dopamine infusion. Mean PRL MCR was 46 +/- 1 ml/min per m2 in women and 44 +/- 3 ml/min per m2 in men, and was significantly correlated with body mass (r = 0.84, P less than 0.001). In contrast with controls, PRL MCR was higher in hyperthyroidism (MCR = 52 +/- 8 ml/min per m2, P less than 0.05), was slightly lower in hypothyroidism (MCR = 38 +/- 10 ml/min per m2, P = NS), and was significantly correlated with serum thyroxine (r = 0.46, P less than 0.02). PRL MCR was lower than controls in hyperprolactinemia (MCR = 40 +/- 5 ml/min per m2, P less than 0.01) and was inversely correlated with serum PRL (r = -0.72, P less than 0.001). PRL MCR was not significantly changed by dopamine infusion. Mean PRL PR for women and men was 211 +/- 74 and 187 +/- 44 micrograms/d per m2, respectively (P = NS). In hyperthyroidism the PRL PR was elevated (PR = 335 +/- 68 micrograms/d per m2, P less than 0.02), but in hypothyroidism the increase (PR = 233 +/- 159 micrograms/d per m2) was not significant. In hyperprolactinemia the PRL PR was extremely high (PR = 31,000 +/- 29,000 micrograms/d per m2). Dopamine infusion decreased RPL PR from 270 to 66 micrograms/d per m2 indicating that its effect was on pituitary PRL secretion and not PRL metabolism. To evaluate possible circulating PRL heterogeneity that might arise during infusion, gel filtration of infusate and serum obtained during the MCR procedure was performed. Labeled monomeric PRL (peak III, Kav (partition coefficient) = 0.4) was partially converted to two larger forms (peaks I and II) in vivo. Peak I (Kav = 0) was 30--40% immunoprecipitable, although peak II (Kav = 0.2) was not immunoprecipitable. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of peak I resulted in greater than or equal to 90% conversion to peak III and restoration of full immunoactivity. Thus, peak I is a noncovalently linked aggregate that is partially immunoactive, and therefore able to alter MCR determinations. These studies demonstrate the impact of hormone heterogeneity on MCR estimations and suggest that gel filtration of immunoprecipitable material be an integral part of future MCR measurements.

Authors

D S Cooper, E C Ridgway, B Kliman, R N Kjellberg, F Maloof

Renal Tubular Sites of Altered Calcium Transport in Phosphate-depleted Rats

• Text
• PDF

Abstract

Increased calcium (Ca) excretion is characteristic of chronic phosphate (PO4) depletion (PD). To study the changes in tubular transport and the site of the hypocalciuric effect of PO4 administration, clearance and micropuncture experiments were performed in intact rats pair fed either a control diet (0.5% PO4) or a PO4-depleted (PD) diet (0.01% PO4) plus Al(OH3) and in parathyroidectomized (PTX) PD rats, infused either with saline or with neutral sodium PO4.

Authors

Kai Lau, Zalman S. Agus, Martin Goldberg, Stanley Goldfarb

Importance of Hepatic Portal Circulation for Insulin Action in Streptozotocin-Diabetic Rats Transplanted with Fetal Pancreases

• Text
• PDF

Abstract

The importance of the hepatic portal circulation in the response to insulin was assessed in streptozotocin-diabetic rats transplanted with syngeneic fetal pancreases. Partial reversal of diabetes was accomplished by transplantation of two or three fetal pancreases beneath the capsule of the kidney; complete reversal followed shunting of the venous drainage from the transplants to the liver. Plasma glucose after streptozotocin of 509±31 mg/dl (mean±SEM) fell after transplantation to 395±23 and after the shunt to 143±5 mg/dl. Urine volume fell from 84±4 to 50±5 ml/d and then to normal (17±1 ml/d) after the shunt. Glucose excretion which was 8.1±0.3 g/d after streptozotocin fell after transplantation to 4.8±0.3 g/d and after the shunt completely disappeared from the urine. The disappearance rate of glucose injected into the circulation, which was 0.50±0.07%/min in untreated diabetes, increased to 1.39±0.38%/min after transplantation and to 2.52±0.31%/min after the shunt, not different from normal controls (2.79±0.25). Plasma immunoreactive insulin (IRI) was below normal (25-35 μU/ml) and unresponsive to glucose in untreated diabetic rats. After transplantation IRI levels ranged from 73-223 μU/ml and there was no rise after glucose injection. After the shunt both the basal IRI (36±5 μU/ml) and the peak response to glucose at 10 min (58±7 μU/ml) were the same as in normal controls (42±4 and 62±7 μU/ml, respectively). The fall in IRI after the shunt is explained by increased extraction of insulin passing into the liver and also diminished secretion. After removal of the transplants plasma glucose and urine values returned almost to pretransplant levels.

Authors

Josiah Brown, Yoko Mullen, William R. Clark, I. Gabriella Molnar, Diane Heininger

Biotin-response Organicaciduria: MULTIPLE CARBOXYLASE DEFECTS AND COMPLEMENTATION STUDIES WITH PROPIONICACIDEMIA IN CULTURED FIBROBLASTS

• Text
• PDF

Abstract

Fibroblast cultures from two individuals with biotin-responsive organicacidemia were found to have a pleiotropic deficiency of propionyl-CoA carboxylase, β-methylcrotonyl-CoA carboxylase, and pyruvate carboxylase activities after growth in biotin limited culture medium, conditions which do not affect the carboxylase activities of normal cells. All three enzyme activities were restored to normal levels after transferring the mutant strains to biotin-rich medium. Both patients excreted abnormal levels of an array of metabolic intermediates, including β-methylcrotonate, β-hydroxyisovalerate, β-hydroxypropionate, and lactate, which reflect metabolic blocks at all three carboxylase sites.

Authors

M. Saunders, L. Sweetman, B. Robinson, K. Roth, R. Cohn, R. A. Gravel

Appearance and Characterization of Lipoprotein X during Continuous Intralipid Infusions in the Neonate

• Text
• PDF

Abstract

The development of hyperphospholipidemia and hypercholesterolemia was studied in infants that required total parenteral nutrition and given a continuous infusion of Intralipid, (1-4 g/kg body wt per 24 h. Detailed studies were carried out on infusion periods lasting 1-10 d. After 24 h there was a marked increase in plasma free cholesterol (68%) and phospholipid (77%) concentrations. Based on the amount of cholesterol in Intralipid, and the rate of infusion, it was estimated that at least 50% of the plasma cholesterol increment during 64-h infusions was derived from endogenous sources. By contrast, the hyperphospholipidemia could be attributed to the Intralipid as the rise in plasma was calculated to be equivalent to only 16% of the exogenous phospholipid infused. Approximately 10% of the phospholipid in Intralipid was in a triglyceride-free mesophase form with a free cholesterol:phospholipid molar ratio of 0.063. There were no systematic changes in plasma concentrations of cholesterol ester or triglyceride during Intralipid infusions. The increase in free cholesterol and phospholipid was localized in the low density lipoproteins (d = 1.006-1.063 g/ml). The presence of lipoprotein X (Lp-X) in the low density lipoprotein fraction was demonstrated by electrophoresis in agar and by isolation and chemical characterization with hydroxylapatite chromatography. Isoelectric focusing of urea-soluble protein of Lp-X revealed that albumin and apolipoproteins CII and CIII were major components, whereas apolipoprotein E and AI were minor constituents. The abnormal lipoprotein was apparent by 16 h during 64 h of infusion. After 6 d of continuous infusions the free cholesterol in Lp-X was 30±10 mg/dl (mean±SD), which represents a total Lp-X mass of 90 mg/dl. After cessation of the infusion, Lp-X, as monitored by electrophoresis in agar, disappeared within 72-96 h. Thus, during infusion of Intralipid in infants at rates commonly employed, the capacity of the clearance mechanisms for phospholipid are exceeded, which causes the accumulation of phospholipid and free cholesterol in the form of Lp-X particles. It is suggested that mesophase phospholipids in Intralipid may play a significant role in this process.

Authors

E. Griffin, W. C. Breckenridge, A. Kuksis, M. H. Bryan, A. Angel

Humoral factor that specifically regulates factor X levels in rabbits (coagulopoietin-X).

• Text
• PDF

Abstract

A heat-stable humoral substance (coagulopoietin-X) is present in rabbits partially depleted of Factor X, which is capable of raising Factor X levels when injected into recipient rabbits. Rabbits were partially depleted of Factor X by slow infusion of a globulin fraction of goat anti-rabbit Factor X antibody. This resulted in the reduction of Factor X to 40--50% of normal at 1 h and 60--70% of normal at 6 h. No effect was noted on levels of Factors II, V, or VII. Plasma from these animals, when injected into 10 recipients, specifically raised Factor X levels when measured by four different assay: one-stage assay with bovine VII- and X-deficient plasma and Russell's viper venom; one-stage assay with human X-deficient plasma and thromboplastin; chromogenic substrate assay with Russell's viper venom; and an immunologic assay (Laurell technique). No rise was noted in two control experiments in which normal plasma was injected into recipient rabbits from 2 rabbits injected with a globulin fraction of normal goat serum, nor in 12 rabbits injected with plasma from normal rabbits, nor in 5 rabbits injected with boiled plasma from normal rabbits. The rise in biologic activity of 120--150% of base line was significantly greater than the rise in immunologic activity of 114--117% of base line (P less than 0.05) on 3 different days, suggesting the production of a molecule with greater specific activity rather than increased protein synthesis.

Authors

D Trauber, K Hawkins, M Karpatkin, S Karpatkin

Lactoferrin acts on Ia-like antigen-positive subpopulations of human monocytes to inhibit production of colony stimulatory activity in vitro.

• Text
• PDF

Abstract

The relationship between Ia-like antigens (Ia-antigens) on human monocytes and the ability of lactoferrin (LF) to inhibit the production of colony stimulatory activity (CSA) for granulocyte and macrophage colony formation was investigated. Complement-dependent cytotoxicity of human monocytes by antiserum to Ia-antigen-reduced CSA production by 50%. LF decreased CSA production by monocytes but had no influence on monocytes insensitive to anti-Ia and complement. Anti-Ia in the absence of complement had no effect on production of CSA but blocked the inhibitory action of LF. This suggsts that LF inhibits production of CSA from an Ia-antigen-positive subpopulation of human blood monocytes. This may be of relevance to the regulation of myelopoiesis.

Authors

H E Broxmeyer

Regulation of Calcitonin Secretion in Normal Man by Changes of Serum Calcium within the Physiologic Range

• Text
• PDF

Abstract

To examine the relative importance of calcium and gastrin in regulation of calcitonin secretion, we administered graded oral doses of calcium to 10 normal men, ages 23-29 yr. Each subject had previously shown an appropriate increase in calcitonin secretion in response to a pharmacologic (0.5 μg/kg) pentagastrin injection. On separate days and in random order, each man drank 250 ml of distilled water containing 0.0, 0.5, 1.5, and 3.0 g of elemental calcium as the gluconate salt. Blood samples were drawn before and at 30, 60, 90, 120, 180, and 240 min after the oral calcium dose. The samples were analyzed for calcium by atomic absorption spectroscopy, and for gastrin and calcitonin by radioimmunoassays of established sensitivity and specificity. Ingestion of water (control) caused no change in any of the three variables. Calcium ingestion resulted in dose-related increases, within the normal range, of all three variables. Immunoreactive gastrin rose promptly, peaking at 30 min, and returning to basal levels or below by 120 min. In contrast, calcium and immunoreactive calcitonin levels rose slowly and in parallel, peaking at 120-240 min. Changes in calcitonin and changes in calcium were strongly and positively correlated, r = 0.73, when all data were pooled. Furthermore, individual linear regressions for changes in calcitonin and calcium levels (calculated separately for the three oral calcium doses in each subject) had positive slopes in 28 out of 30 sets (P < 0.01). The changes in calcitonin concentrations were much more poorly correlated with the corresponding changes in serum gastrin levels; in fact, the regression coefficient was weakly negative, r = −0.20. These results show that, at least in young adult men, changes of ambient calcium concentration within the normal range may be of major importance in physiologic regulation of calcitonin secretion. The findings are consistent with the hypothesis that calcitonin functions to prevent excessive postprandial hypercalcemia.

Authors

Lynn A. Austin, Hunter Heath III, Vay Liang W. Go

Hydroxyl radical generation by polymorphonuclear leukocytes measured by electron spin resonance spectroscopy.

• Text
• PDF

Abstract

Electron spin resonance spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was employed to detect the formation of hydroxyl radicals (OH.) by phagocytosing polymorphonuclear leukocytes (PMN). An electron spin resonance signal with the identical g value and splitting characteristics of the DMPO/OH). adduct was detected on incubation of normal PMN with opsonized zymosan. Adduct formation was strongly inhibited by superoxide dismutase and by the OH. scavenger mannitol, but catalase had little or no effect. (DMPO/OH). was not formed by PMN from a patient with chronic granulomatous disease; in contrast, adduct formation by PMN which lack myeloperoxidase was greater than normal. These findings are discussed in relation to the formation of OH. by PMN.

Authors

H Rosen, S J Klebanoff