Hyperlipidemia in Coronary Heart Disease I. LIPID LEVELS IN 500 SURVIVORS OF MYOCARDIAL INFARCTION

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Plasma cholesterol and triglyceride levels were measured after an overnight fast in 500 consecutively studied 3-mo survivors of myocardial infarction. Virtually all patients under 60 yr of age (95% ascertainment) and a randomly chosen group of older survivors admitted to 13 Seattle hospitals during an 11 mo period were included. A comparison of their lipid values with those of 950 controls demonstrated that 31% had hyperlipidemia. These lipid abnormalities were most commonly found in males under 40 yr of age (60% frequency) and in females under 50 yr of age (60% frequency). Elevation in triglyceride levels with (7.8%) or without (15.6%) an associated elevation in cholesterol levels was three times more common in survivors than a high cholesterol level alone (7.6%). These results raise the possibility that hypertriglyceridemia may be as an important a risk factor for coronary atherosclerosis as hypercholesterolemia. The identification of hyperlipidemic survivors of myocardial infarction provided a unique source of probands for family studies designed to disclose the genetic origin of hyperlipidemia in coronary heart disease.

Authors

Joseph L. Goldstein, William R. Hazzard, Helmut G. Schrott, Edwin L. Bierman, Arno G. Motulsky

Hyperlipidemia in Coronary Heart Disease II. GENETIC ANALYSIS OF LIPID LEVELS IN 176 FAMILIES AND DELINEATION OF A NEW INHERITED DISORDER, COMBINED HYPERLIPIDEMIA

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To assess the genetics of hyperlipidemia in coronary heart disease, family studies were carried out in 2520 relatives and spouses of 176 survivors of myocardial infarction, including 149 hyperlipidemic and 27 normolipidemic individuals. The distribution of fasting plasma cholesterol and triglyceride values in relatives, together with segregation analyses, suggested the presence of five distinct lipid disorders. Three of these—familial hypercholesterolemia, familial hypertriglyceridemia, and familial combined hyperlipidemia—appeared to represent dominant expression of three different autosomal genes, occurring in about 20% of survivors below 60 yr of age and 7% of all older survivors. Two other disorders—polygenic hypercholesterolemia and sporadic hypertriglyceridemia—each affected about 6% of survivors in both age groups.

Authors

Joseph L. Goldstein, Helmut G. Schrott, William R. Hazzard, Edwin L. Bierman, Arno G. Motulsky

Hyperlipidemia in Coronary Heart Disease III. EVALUATION OF LIPOPROTEIN PHENOTYPES OF 156 GENETICALLY DEFINED SURVIVORS OF MYOCARDIAL INFARCTION

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Although analysis of lipoprotein phenotypes is widely used to diagnose and classify the familial hyperlipidemias, an evaluation of this system as a method for genetic classification has hitherto not been published. The present study of 156 genetically defined survivors of myocardial infarction was therefore designed to examine the relationship between lipoprotein phenotypes and genetic lipid disorders. The lipoprotein phenotypes of each survivor was determined primarily by measurement of his plasma triglyceride and low density lipoprotein (LDL)-cholesterol concentrations; his genetic disorder was identified by analysis of whole plasma cholesterol and triglyceride levels in relatives.

Authors

William R. Hazzard, Joseph L. Goldstein, Helmut G. Schrott, Arno G. Motulsky, Edwin L. Bierman

Evidence for a Common, Saturable, Triglyceride Removal Mechanism for Chylomicrons and Very Low Density Lipoproteins in Man

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Hypertriglyceridemic subjects were fed diets in which dietary fat calories were held constant, but carbohydrate calories were varied. Three subjects with fasting chylomicronemia (Type V) were given less carbohydrate and four subjects without fasting chylomicronemia (Type IV) were fed diets with more calories as carbohydrate. The restricted carbohydrate intake led to disappearance of chylomicronemia in those subjects who had chylomicronemia on a normal diet (Type V to IV). In those subjects without chylomicronemia, chylomicronemia appeared in response to increased carbohydrate intake (Type IV to V). Thus chylomicron concentrations in plasma were altered even though fat intake and presumably chylomicron input into plasma was kept constant. These findings provide evidence for saturation of chylomicron removal mechanisms by alteration of endogenous triglyceride-rich lipoprotein concentrations. They suggest that chylomicrons compete with very low density lipoproteins for similar removal mechanisms. The relationship between endogenous triglyceride concentration and the lipolytic activity in plasma following heparin was then evaluated with the use of long-term heparin infusions to release and maintain lipolytic activity in the circulation. 10 subjects were placed on fatfree diets to remove circulating dietary fat. The plasma lipolytic rate during the heparin infusion was measured consecutively on different days in individuals whose triglyceride concentrations were varied by either increasing or decreasing calories. The lipolytic rate was curvilinearly related to the plasma triglyceride concentrations. This curvilinear relationship followed Michaelis-Menton saturation kinetics over a wide range of triglyceride concentrations on fat-free, high-carbohydrate diets, in multiple studies in a group of individuals. These studies suggest that endogenous and exogenous triglyceride compete for a common, saturable, plasma triglyceride removal system related to lipoprotein lipase.

Authors

John D. Brunzell, william R. Hazzard, Daniel Porte Jr., Edwin L. Bierman

Protein Digestion in Human Intestine as Reflected in Luminal, Mucosal, and Plasma Amino Acid Concentrations after Meals

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Normal human volunteers were intubated with either aspiration tubes or a biopsy capsule placed in the small intestine. The subjects were then fed a test meal containing 50 g of purified bovine serum albumin which served as the model dietary protein. Electrophoretic analysis of intestinal fluids showed that for at least 4 h the fed albumin was detectable in jejunal and ileal fluids. On separate occasions, subjects were fed the same meal without the protein. No protein was detected in intestinal fluids when the protein-free meal was fed. After the protein-rich meal, total concentrations of measured free and peptide amino acids rose from 3.21 to 29.29, and 15.94 to 117.97 μmol/ml, respectively, (P values < 0.02) in the jejunum. Similarly, total concentrations of measured free and peptide amino acids rose from 5.45 to 19.74, and 13.59 to 65.39, respectively, (P values < 0.05) in the ileum. In contrast, concentrations of free and peptide amino acids in intestinal fluids did not increase after the protein-free meal. While intracellular concentrations of amino acids in the jejunal mucosa did not show significant changes, plasma concentrations of each individual free amino acid were increased after the protein-rich meal and were either decreased or unaltered after the protein-free meal. The amino acid composition of the fed protein was reflected in the increases in intraluminal and plasma concentrations of individual amino acids after the protein-rich meal. It is concluded that after the ingestion of a test meal containing a substantial amount of protein which is within the usual range of dietary intake; (a) the exogenous protein is the principal source of the increased free and peptide amino acids in the intraluminal contents and in the plasma; (b) there are greater amounts of amino acids present as small peptides than in the free form in the gut lumen; (c) the ingested protein can be recovered as late as 4 h both in the jejunum and in the ileum.

Authors

Siamak A. Adibi, Donald W. Mercer

Identification of Inorganic Pyrophosphate in Human Platelets and Its Release on Stimulation with Thrombin

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Serum inorganic pyrophosphate (PPi) levels were consistently two- to threefold higher than plasma PPi prepared from the same blood. PPi was found in platelets in amounts ranging from 1.4 to 3 nmol/108 cells, using three different techniques for quantification. These levels are approximately 800 times higher than the mean PPi concentration in normal plasma and approximate the levels of ADP found in platelets by other workers.

Authors

Donald C. Silcox, Sergio Jacobelli, Daniel J. McCarty

C2 Deficiency DEVELOPMENT OF LUPUS ERYTHEMATOSUS

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The study of serum from a patient with C2 deficiency is described. The patient had an episode of pneumococcal meningitis at 5 mo of age with seizures and transient hemiparesis and apparent purpuric skin lesions. He was first admitted to the University of Minnesota Hospitals at 10 yr of age following the discovery of proteinuria accidentally by his mother. Since then he has been admitted repeatedly to this hospital with numerous clinical findings including arthralgia, recurrent abdominal pain, proteinuria, membranous nephropathy, malar butterfly rash, seizures, personality aberrations, and recurrent fever. In June 1971, the patient developed positive DNA and DNP antibodies and positive LE cells. When the C profile was studied before and after recognition of lupus, C1q, C1s, and C4 dropped. C3 levels were elevated as were C5, C6, and C7, C3 proactivator had been reduced in the patient even before he developed lupus. Also because of a traumatic renal biopsy leading to a perirenal hematoma, he required surgery and a blood transfusion. 1 h after blood transfusion, a C2 titer of 23 hemolytic units was detected. Almost immediately levels of C3, C5, C6, and C7 dropped, C8 and C9 remained elevated. The addition of C2 from normal blood permitted dramatic activation of C3.

Authors

Noorbibi K. Day, H. Geiger, R. McLean, A. Michael, R. A. Good

Insulin Sensitivity of Forearm Tissues in Prediabetic Man

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In genetic prediabetic subjects (the glucose tolerant offspring of two diabetic parents or the identical twin of a known diabetic) serum insulin concentrations after glucose administration are subnormal. Maintenance of glucose tolerance in this setting is apparently paradoxical, suggesting increased tissue insulin sensitivity. Accordingly, forearm tissue insulin sensitivity in nine genetic prediabetic males was compared with that of seven males without familial diabetes. Diabetes was excluded in all subjects by preliminary oral glucose tolerance testing.

Authors

Thomas Pozefsky, Martin R. Santis, J. Stuart Soeldner, Robert G. Tancredi

Metabolism and Excretion of Exogenous Thyrotropin-Releasing Hormone in Humans

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To study the metabolism of thyrotropin-releasing hormone (TRH) in vivo, 400 μg TRH was administered intravenously to eight normal male subjects. Multiple plasma and urine samples were obtained before and after TRH administration. Serum TSH concentrations increased after TRH administration in all subjects. Plasma TRH levels, measured by radioimmunoassay, were undetectable (< 0.4 ng/ml) before TRH administration. Plasma TRH concentrations averaged 33±7 ng/ml (mean ±SEM) 2 min after TRH injection. Thereafter, they decreased rapidly so that the mean plasma TRH level was 2.9 ng/ml 20 min after TRH administration. The fall in plasma TRH levels was linear during this interval. Thereafter TRH levels declined more slowly. The mean half-life (t½) of TRH was 5.3±0.5 min. The mean distribution volume was 15.7±3.8 liters, an average of 16.5% of body weight in these subjects. In the urine, 5.5±0.9% of the administered TRH was recovered in the 3 h after TRH administration. Of the total urinary TRH recovered, 84.9% was excreted in the first 30 min. These results indicate that TRH is distributed in a large volume, that it is rapidly metabolized and that a significant quantity of administered TRH is excreted in the urine.

Authors

Rahim M. Bassiri, Robert D. Utiger

Accelerated Triglyceride Secretion A METABOLIC CONSEQUENCE OF OBESITY

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A new animal model was developed to determine the effect of obesity upon endogenous triglyceride secretion. Desert sand rats (Psammomys obesus), rodents which become spontaneously obese and hyperinsulinemic when given ad lib. chow, were given intravenous Triton to allow in vivo measurement of triglyceride secretion rates (TGSR). In a group of 18 fasted animals of varying body weight and degrees of obesity, TGSR correlated significantly with body weight (r=0.68, P < 0.01) indicating that obesity was associated with accelerated endogenous release of triglyceride. In these same animals, basal plasma insulin levels correlated significantly with body weight (r=0.78, P < 0.001) and TGSR correlated significantly with mean plasma insulin levels (r=0.73, P < 0.001), suggesting that hyperinsulinemia may have been the mechanism through which obesity enhanced TGSR. No correlation was found between basal triglyceride level and either body weight, basal insulin, or TGSR which suggested that individual triglyceride removal rates among the animals may have been variable. To test this hypothesis, seven animals were studied prospectively before and after induction of obesity. There were significant increases (P < 0.02) in all parameters, i.e., weight, plasma insulin level, TGSR, and basal triglyceride level. Thus, when each animal was used as its own control, thereby minimizing the postulated factor of variable individual triglyceride removal, increments in basal triglyceride were shown to accompany the development of obesity, hyperinsulinemia, and accelerated triglyceride secretion. These data from studies in the sand rat offer in vivo evidence that obesity leads to accelerated triglyceride secretion, an effect which may be mediated by hyperinsulinemai, and which can be invoked as one possible mechanism to explain hypertriglyceridemia associated with obesity in man.

Authors

R. Paul Robertson, David J. Gavareski, James D. Henderson, Daniel Porte Jr., Edwin L. Bierman

Comparative Study of the In Vitro Proliferative Responses of Blood and Synovial Fluid Leukocytes of Rheumatoid Arthritis Patients

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Lymphocyte-rich suspensions from blood and synovial fluid (SF) of 20 patients with rheumatoid arthritis (RA) and from blood of 12 normal subjects, were cultured with heat-aggregated, aggregate-free, and native human gamma globulin (HGG), with autologous IgG separated from RA-SF by anion-exchange chromatography and with phytohemagglutinin (PHA). No significant differences were noted between the in vitro proliferative responses of blood lymphocytes of RA and normal controls to any of these preparations. Significant differences were noted between blood and SF lymphocytes of RA patients with respect to their responses to the aggregate-free HGG and to PHA. Incubation of RA-SF cells but not RA-blood cells with aggregate-free HGG before their culture with the aggregated HGG markedly suppressed the in vitro proliferative response to the latter. The observed differences between blood and SF lymphocytes and the suppression of blastogenic response of SF cells by exposure to the aggregate-free preparation raise the possibility of modulating the immune and/or the inflammatory responses in RA.

Authors

Michael D. Reynolds, Nabih I. Abdou

Phagocytic Function of Polymorphonuclear Leukocytes in Rheumatic Diseases

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Phagocytosis of yeast particles by peripheral blood and synovial fluid neutrophils was compared in the sera and synovial fluids from 16 osteoarthritis, 23 rheumatoid arthritis, and 12 miscellaneous arthritis patients. Phagocytosis by normal peripheral blood neutrophils was decreased equally and significantly in all synovial fluids. All synovial fluid neutrophils demonstrated decreased phagocytic capacity in all media. Rheumatoid arthritis synovial fluid neutrophils showed significantly less phagocytosis than miscellaneous arthritis synovial fluid neutrophils. Normal peripheral blood neutrophils which in vitro had previously ingested monosodium urate crystals or oil red O, subsequently exhibited a normal yeast phagocytic capacity. Normal peripheral blood neutrophils, which had ingested preformed immunoglobulin G-rheumatoid factor complexes exhibited significantly less yeast phagocytic capacity than control cells or cells preincubated with the individual complex components. There was a significant correlation between the log of the reciprocal of the rheumatoid factor titer in sera used to produce complexes and the phagocytic capacity exhibited by test neutrophils. Ingestion of immunoglobulin G-rheumatoid factor complexes may be important in the production of the cellular phagocytic defect which this study has demonstrated in rheumatoid arthritis synovial fluid neutrophils.

Authors

Robert A. Turner, H. Ralph Schumacher, Allen R. Myers

Endocrine Studies in Anencephaly

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Endocrine function has been investigated in four anencephalic neonates to determine the influence of absence of cortical and hypothalamic tissue and of hypoplasia of the pituitary. Intravenous glucose administration resulted in higher peak values for blood sugar and more rapid glucose disposal rates than reported in normals. Intravenous insulin tolerance tests on two of the infants failed to evoke elevations in plasma growth hormone, and the infants showed a remarkable resistance to the hypoglycemic effect of insulin. Administration of lysine-vasopressin caused an active growth hormone release. Similarly, there was a large increase in serum thyrotropin after administration of synthetic thyrotropin-releasing hormone. Basal levels of both thyrotropin and growth hormone were low as compared with values reported for normal newborns. Prolactin values obtained on three of the infants were in the normal range. The results strongly suggest that anterior pituitary function mediated by the hypothalamus and its releasing factors is deficient in anencephaly. However, the anterior pituitary can release growth hormone and thyrotropin when stimulated directly and, in the case of thyrotropin release, may function autonomously. The normal prolactin values presumably reflect the absence of the hypothalamic prolactin inhibitory factor.

Authors

Alberto Hayek, Shirley G. Driscoll, Joseph B. Warshaw

Effect of Volume Expansion on Sodium Excretion in the Presence and Absence of Increased Delivery from Superficial Proximal Tubules

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The role of the proximal tubule in the natriuresis after volume expansion was investigated by evaluating sodium excretion both in the presence and absence of increased delivery from the proximal tubule. Proximal delivery was calculated from fractional reabsorption in superficial proximal tubules determined by micropuncture and glomerular filtration rate of the micropunctured kidney. Infusion of Ringer's solution in six dogs increased delivery from the proximal tubule 4.7±1 ml/min (P < 0.01) and increased fractional sodium excretion 3.6±1.1% (P < 0.025). Infusion of hyperoncotic albumin into the renal artery during sustained volume expansion decreased delivery from the proximal tubule 6.5±0.9 ml/min (P < 0.01). Although proximal delivery was restored to below control levels, fractional sodium excretion was significantly increased 2.5±0.5% (P < 0.01) as compared with the hydropenic control period. Fractional phosphate excretion was increased 15.5±3.7% (P < 0.01) after Ringer's infusion and was decreased 10.5±1.6% (P < 0.005) after intrarenal albumin infusion, suggesting that changes in superficial nephron reabsorption were paralleled by changes in reabsorption in deeper nephrons. Similar results were found in six additional dogs in which other factors known to affect phosphate reabsorption were controlled; however, these studies cannot completely eliminate a role for deep nephrons in the natriuresis after intrarenal albumin infusion. Since 70% of the natriuresis after volume expansion was present without increased delivery from superficial proximal tubules, it is likely that increased delivery from the proximal tubule contributes a relatively minor fraction to the natriuresis of volume expansion.

Authors

Franklyn G. Knox, Edward G. Schneider, L. R. Willis, Jack W. Strandhoy, Cobern E. Ott

Plasmin-Induced Platelet Aggregation and Platelet Release Reaction. EFFECTS ON HEMOSTASIS

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Trypsin-activated pig plasmin and human plasmin activated by streptokinase (SK) caused aggregation of a suspension of washed platelets from human, rabbit, or pig blood. The platelet aggregation was reversible, but it was accompanied by a significant release of adenine nucleotides, serotonin, and platelet fibrinogen. Platelet fibrinogen was eventually digested. The effect of plasmin on platelets was inhibited by soybean trypsin inhibitor, epsilon aminocaproic acid, Persantin, prostaglandin E1, and phenylbutazone. Short treatment of platelets with plasmin enhanced their sensitivity to ADP; however, this sensitivity was lost during longer incubation with plasmin. This enzyme also made platelets less sensitive to collagen and thrombin.

Authors

S. Niewiarowski, A. F. Senyi, P. Gillies

Trypsin-Like Nature of the Pancreatic Factor That Corrects Vitamin B₁₂ Malabsorption Associated with Pancreatic Dysfunction

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Hog pancreas was subfractionated and assessed for its ability to correct vitamin B12 malabsorption in patients with pancreatic dysfunction and in rats with partial pancreatic extirpation. The constituent obtained from the pancreas that increased vitamin B12 absorption in both humans and rats was soluble at 50,000 g, heat labile, acid stable, and approximately 20,000-25,0000 in molecular weight. The active subfractions contained tryptic and chymotryptic but no amylase or lipase activity. Thrice-crystallized trypsin corrected the vitamin B12 malabsorption in both patients with pancreatic insufficiency and in rats with subtotal pancreatectomy. These data indicate that pancreatic proteolytic enzymes—in particular, trypsin—are necessary for optimal vitamin B12 absorption.

Authors

Phillip P. Toskes, Julius J. Deren, Marcel E. Conrad

Granulocyte Alkaline Phosphatase. STUDIES OF PURIFIED ENZYMES FROM NORMAL SUBJECTS AND PATIENTS WITH POLYCYTHEMIA VERA

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To characterize the biological changes which result in increased granulocyte alkaline p-nitrophenyl phosphatase activity in patients with polycythemia vera, the enzyme was purified from granule fractions of sucrose homogenates made from dextran-sedimented leukocytes of normal subjects and patients with polycythemia vera. Polycythemic blood yielded 3-10 times as much granulocyte alkaline phosphatase per 109 leukocytes as did normal blood. Sodium dodecyl sulfate extracts of granules were purified by DEAE-cellulose chromatography and sucrose gradient centrifugation to apparent homogeneity as judged by polycarylamide disk gel electrophoresis. Granulocyte alkaline phosphatase from normal subjects was purified 6910-fold with a 60% yield and a specific activity of 47 U/mg. Granulocyte alkaline phosphatase from polycythemic patients was purified 1.166-fold with a 50% yield and a specific activity of 70 U/mg. The two enzymes did not differ in molecular weight; both appeared to be about 160,000 daltons by sucrose gradient centrifugation. Both appeared to be zinc metalloenzymes, in that they were specifically inhibited by o-phenanthroline. Their elution requirements when adsorbed to DEAE-cellulose suggested they were lipoproteins although the content of phosphorus was below the threshold of detection. The identity of the two enzymes was suggested by immunological studies in which antibody prepared against purified polycythemia vera enzyme gave a precipitation reaction of identity with another polycythemia vera enzyme and two pools of normal enzyme. It is possible to account for the difference in alkaline phosphatase activity between the granulocytes of patients with polycythemia vera and normal subjects by differences in the quantity of enzyme synthesized.

Authors

Daniel Rosenblum, Shirley J. Petzold

Interaction of Intraleukocytic Bacteria and Antibiotics

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Bacteria that survive inside polymorphonuclear neutrophils (PMN) following phagocytosis are protected from the bactericidal action of most antibiotics. Two possible explanations are altered metabolism by intraleukocytic bacteria or failure of antibiotics to enter the phagosome. The oxygen consumption of intraleukocytic and extraleukocytic bacteria was measured as an index of bacterial metabolism. PMN respiration and bactericidal activity were suppressed with large doses of hydrocortisone and extraleukocytic bacterial oxygen consumption was abolished by the addition of lysostaphin. Intraleukocytic bacterial continued to consume oxygen suggesting that surviving ingested micro-organisms are metabolically active. Neither penicillin (which cannot kill intraleukocytic bacteria) nor rifampin (which can kill intraleukocytic bacteria) was bactericidal for staphylococci at 5°C. Thus, rifampin is not uniquely able to kill “resting” bacteria.

Authors

Gerald L. Mandell

Effects of 1,25-Dihydroxycholecalciferol on Intestinal Calcium Transport in Cortisone-Treated Rats

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The administration of glucocorticoids may decrease intestinal calcium absorption in vivo and the active transport of calcium in rat duodenum in vitro. It has been suggested that this apparent “anti-vitamin D-like” effect of steroid hormones may be related to alterations in vitamin D metabolism. In order to test this hypothesis, vitamin D-deficient control and cortisone-treated rats were given an intraperitoneal injection of 5.5 IU of 1,25-dihydroxycholecalciferol (1,25-DHCC), the probable end-organ active vitamin D metabolite in the intestine, and 16 h later studies of duodenal calcium transport were performed in modified Ussing chambers. In the vitamin D-deficient state, cortisone administration was associated with a diminution in JMS, JNet, and the flux ratio (JMS/JSM). While the magnitude of the increases in JMS and JNet that resulted from 1,25-DHCC treatment were approximately the same in control and cortisone-treated animals, 1,25-DHCC failed to restore these parameters to “normal levels” in the steroid-treated rats. Furthermore, contrary to the results obtained in the saline-treated controls, 1,25-DHCC failed to reduce JSM in the duodenum from cortisone-treated rats. The cortisone-related defect in calcium transport was due to alterations in both unidirectional calcium fluxes (decrease in JMS and increase in JSM), such that the JNet and the flux ratio (JMS/JSM) were only approximately 50% of the levels achieved in vitamin D-deficient control animals repleted with the same dose of 1,25-DHCC.

Authors

Murray J. Favus, Marlin W. Walling, Daniel V. Kimberg

Transport of Monosaccharides I. ASYMMETRY IN THE HUMAN ERYTHROCYTE MECHANISM

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Transport of D-glucose across human erythrocyte membranes occurs via a facilitated diffusion process which demonstrates influx-efflux asymmetry. The mechanism of the asymmetry has been studied by estimating unidirectional fluxes in the presence or absence of trans equilibrium hexose. In the absence of transhexose, the half-saturation constant for efflux at 15°C was approximately 10 mM as compared with 27 mM for influx; the corresponding values for maximal transfer rates (μmol/min per ml cell H2O) were approximately 51 vs. 18. The estimation of kinetic parameters, including the constant Fs, which is the ratio of maximal transfer rate/half-saturation constant, indicates a unique effect of intracellular hexose on the transfer system. Further evidence to support this conclusion was obtained by studying the effects of noncompetitive inhibitors on efflux vs. influx. N-ethylmaleimide, p-chloromercuribenzenesulfonate, and dichloroallyldiethylstilbestrol all inhibited efflux much more than influx. Glucose rendered the transport system more reactive to N-ethylmaleimide as assayed by efflux, whereas influx was much less affected. The results support the hypothesis that the transport system exists in two states. Transition from one state to the other is dependent on the presence of intracellular hexose.

Authors

Ellen R. Batt, David Schachter

The Association of Respiratory Infection, Recurrent Hematuria, and Focal Glomerulonephritis with Activation of the Complement System in the Cold

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The study of the activation of C3, C5, and C7 associated with the conversion of C3 in the serum of a 9-yr old girl after incubation at 0°C for 6-8 h without utilization of C1, C4, and C2 is described. The patient has upper respiratory infections associated with recurrent gross hematuria, focal glomerulonephritis, and transient renal insufficiency. Histological lesions demonstrated the presence of B1c globulin IgA and properdin in the glomeruli. The activation of complement (C) in the cold requires the patient's IgA. Removal of IgA from the serum by immunoadsorption prevents activation and conversion of C3. Bactericidal and phagocytic activity is also impaired after incubation. C3 proactivator (C3PA) level is reduced before and after incubation. Properdin level drops after incubation. These findings suggest that the activation of C3 which is demonstrable in vitro may be a continuous process in vivo.

Authors

Noorbibi K. Day, H. Geiger, R. McLean, J. Resnick, A. Michael, R. A. Good

Effect of Escherichia coli on Fluid Transport across Canine Small Bowel MECHANISM AND TIME-COURSE WITH ENTEROTOXIN AND WHOLE BACTERIAL CELLS

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An Escherichia coli strain isolated from a patient with severe cholera-like diarrhea elaborates a partly heat-labile enterotoxin shown to cause prompt adenyl cyclase stimulation and isotonic fluid secretion by canine jejunum. Both responses disappear upon removal of the enterotoxin. The duration of action of a submaximal dose of this E. coli enterotoxin was brief, despite sustained exposure to the jejunum, suggesting inactivation of the enterotoxin by its interaction with the mucosa.

Authors

R. L. Guerrant, U. Ganguly, A. G. T. Casper, E. J. Moore, N. F. Pierce, C. C. J. Carpenter

Glucocorticoid-Binding Proteins in Human Acute Lymphoblastic Leukemic Blast Cells

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The first known step in steroid hormone action is the association of the steroid with specific cytoplasmic steroid-binding proteins (SBP). Using a competitive binding assay, we detected, quantified, and partially characterized such a SBP in cytosol from glucocorticoid-sensitive human lymphoblastic leukemic blasts. The affinity of steroids for the SBP was directly related to their known killing potency. For example, steroids without glucocorticoid effect such as androstenedione, etiocholanolone, and tetrahydrocortisol were unable to displace radiolabeled dexamethasone from the SBP in the binding reaction. The dose-response curve for in vitro inhibition of [3H]thymidine uptake in leukemic blasts correlated closely with the binding affinity of glucocorticoids to the SBP, providing additional support for an essential physiologic role for SBP in steroid action. SBP activity was either greatly diminished or absent in glucocorticoid-resistant cells. Six patients who intially had SBP in their blasts and were responsive to combinations of drugs including glucocorticoids no longer had SBP activity detectable at a time when they no longer responded to combinations of drugs including glucocorticoids. In vitro [3H]thymidine uptake was not inhibited by steroids in leukemic blast cells lacking SBP activity. Other patients who had received some antileukemic therapy including glucocorticoids and who still had SBP in their leukemic blasts, were still responsive to drug combinations that included glucocorticoids. This appears to be the first study demonstrating glucocorticoid receptors in a human tissue.

Authors

Marc E. Lippman, Roger H. Halterman, Bridgid G. Leventhal, Seymour Perry, E. Brad Thompson

Identification and Characterization of Subpopulations of Lymphocytes in Human Peripheral Blood after Fractionation on Discontinuous Gradients of Albumin THE CELLULAR DEFECT IN X-LINKED AGAMMAGLOBULINEMIA

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Normal human peripheral blood lymphocytes were separated on discontinuous gradients of 17-35% bovine serum albumin (BSA) into nine fractions. Three subpopulations of lymphocytes were obtained. One occupies the top third of the gradient (fractions 1-3, 17-23% BSA) and is rich in cells characterized by a high spontaneous rate of DNA synthesis and by the ability to give rise to colony-forming units. The middle portion of the gradient (fractions 4 and 5, 23-27% BSA) is rich in thymus-derived (T) lymphocytes identified by their vigorous response to mitogens and by their ability to form rosettes with sheep erythrocytes (E). The third subpopulation at the bottom of the gradient (fractions 6-9, 27-35% BSA) is rich in bone marrow-derived (B) lymphocytes capable of staining with fluorescent antiimmunoglobulin antisera and of forming rosettes with EAC1423.

Authors

R. S. Geha, F. S. Rosen, E. Merler

Hemoglobin Messenger RNA from Human Bone Marrow ISOLATION AND TRANSLATION IN HOMOZYGOUS AND HETEROZYGOUS β-THALASSEMIA

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A method for isolating human hemoglobin messenger RNA (mRNA) from bone marrow cells was developed to investigate the molecular basis for the defect in globin synthesis in beta thalassemia. Active mRNA was isolated from the bone marrow cells and peripheral reticulocytes of patients with homozygous beta thalassemia, heterozygous beta thalassemia, sickle cell trait, double heterozygosity for beta thalassemia and sickle cell trait, as well as from a patient with normal hemoglobin synthesis but with an elevated reticulocyte count secondary to hereditary spherocytosis. The mRNA was prepared for assay in an mRNA-dependent rabbit reticulocyte cell-free system and the amount of alpha and beta globin chains synthesized was determined by carboxymethylcellulose column chromatography. The relative synthesis of alpha to beta chains in response to normal hemoglobin mRNA was found to be a function of the amount of mRNA added to the assay system: increasing the amount of mRNA led to a decrease in the alpha-to-beta-chain synthetic ratio. Therefore, assays were carried out at limiting concentrations of mRNA.

Authors

A. W. Nienhuis, P. H. Canfield, W. F. Anderson

Hemoglobin Abraham Lincoln, β32 (B14) Leucine → Proline AN UNSTABLE VARIANT PRODUCING SEVERE HEMOLYTIC DISEASE

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An unstable hemoglobin variant was identified in a Negro woman with hemolytic anemia since infancy. A splenectomy had been performed when the patient was a child. The anemia was accompanied by erythrocyte inclusion bodies and excretion of darkly pigmented urine. Neither parent of the proposita demonstrated any hematologic abnormality, and it appeared that this hemoglobin variant arose as a new mutation. Erythrocyte survival in the patient was greatly reduced: the erythrocyte t½ using radiochromium as a tag was 2.4 days, and a reticulocyte survival study performed after labeling the cells with L-[14C]leucine indicated a t½ of 7.2 days. When stroma-free hemolysates were heated at 50°C, 16-20% of the hemoglobin precipitated. The thermolability was prevented by the addition of hemin, carbon monoxide, or dithionite, suggesting an abnormality of heme binding. An increased rate of methemoglobin formation was also observed after incubation of erythrocytes at 37°C. The abnormal hemoglobin could not be separated from hemoglobin A by electrophoresis or chromatography, but it was possible to isolate the variant β-chain by precipitation with p-hydroxymercuribenzoate. Purification of the β-chain by column chromatography followed by peptide mapping and amino acid analysis demonstrated a substitution of proline for β32 leucine. It appears likely that a major effect of this substitution is a disruption of the normal orientation of the adjacent leucine residue at β31 to impair heme stabilization.

Authors

George R. Honig, David Green, Mir Shamsuddin, Loyda N. Vida, R. George Mason, David J. Gnarra, Helen S. Maurer

Biologic and Immunologic Characterization and Physical Separation of ACTH and ACTH Fragments in the Ectopic ACTH Syndrome

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Abstract

Extracts of tumors from 32 patients with the ectopic ACTH syndrome were subjected to simultaneous bioassay and radioimmunoassays for ACTH. Radioimmunoassays were performed using three antisera, one of which reacts with the extreme N-terminal 1-13 amino acid sequence of ACTH, the second with the N-terminal 1-23 sequence of the ACTH molecule, and the third with the C-terminal 25-39 amino acid sequence of ACTH. There was, in general, good correlation between bioactivity and N-terminal ACTH immunoreactivity. However, there were large excesses of both extreme N-terminal and C-terminal immunoreactive materials in most tumor extracts, which were not found in extracts of three human pituitaries. Three tumor extracts were subjected to molecular sieve chromatography on Sephadex G-50 fine resin. The bioactive ACTH eluted in the same fractions as pituitary ACTH (mol wt≃4,500 daltons) and reacted equally in all three ACTH radioimmunoassay systems. The bioactive tumor ACTH was neutralized by incubation with the C-terminal antiserum, indicating it has an intact C-terminal sequence of amino acids. The next several fractions from the Sephadex column contained a material, mol wt≃3,100, which was biologically inactive and had C-terminal immunoreactivity but no N-terminal or extreme N-terminal immunoreactivity. Incubation with the N-terminal 1-23 ACTH antiserum did not adsorb these C-terminal fragments, indicating they lacked an intact sequence of amino acids in this region. A smaller ACTH fragment (mol wt≃1,800 daltons) eluted in still later fractions and reacted with the extreme N-terminal antiserum but not with the N-terminal or C-terminal antisera. It had no steroidogenic activity, but appeared to have significant melanocyte-stimulating activity. It is concluded that, in addition to an ACTH similar, if not identical, to pituitary ACTH, tumors of patients with the ectopic ACTH syndrome contain both N-terminal and C-terminal ACTH fragments.

Authors

David N. Orth, Wendell E. Nicholson, William M. Mitchell, Donald P. Island, Grant W. Liddle

Effect of Inhibition of Lipolysis on Infarct Size After Experimental Coronary Artery Occlusion

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Abstract

Recent studies have demonstrated a depressant effect of increased delivery of FFA to the hypoxic heart. Because catecholamines are released in acute myocardial infarction, it is likely that lipolytic activity is increased. The purpose of this study was to determine whether inhibition of hormone-sensitive lipases influence the extent and severity of myocardial ischemic injury produced by coronary occlusion. Myocardial infarction was produced by occlusion of the left anterior descending coronary artery in open-chest dogs. 15 min later a surface map of S-T segments was obtained with the use of 10-14 epicardial leads in the distribution area of the occluded artery. Average S-T segment elevation of all sites was used as an index of myocardial ischemic injury. Before coronary occlusion, the average S-T segment elevation was 0.3±0.2, which increased to 4.1±0.7 mV (SEM, 12 dogs) after occlusion. Inhibition of lipolytic activity by β-pyridyl-carbinol before repeated coronary occlusion reduced the occlusion-induced S-T segment elevation to 2.1±0.6 mV (P < 0.001). When arterial concentrations of FFA were raised by i.v. infusion of a triglyceride emulsion and heparin, average S-T segment elevation after coronary occlusion increased from 1.2±0.7 to 2.2±0.8 mV (P < 0.05) in animals treated with β-pyridyl-carbinol, which suggests an unfavorable effect of circulating FFA in this setting. Isoproterenol given before a repeated occlusion increased the severity and extent of the ischemic injury. The effect of isoproterenol on the occlusion-induced S-T segment elevation was reduced, however, when the lipolytic effect of the drug was inhibited by β-pyridyl-carbinol.

Authors

John K. Kjekshus, Ole D. Mjøs

Evidence for Complement Activation via the Alternate Pathway in Skin Diseases I HERPES GESTATIONIS, SYSTEMIC LUPUS ERYTHEMATOSUS, AND BULLOUS PEMPHIGOID

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Abstract

A patient with herpes gestationis, 6 of 6 patients with bullous pemphigoid, and 5 of 25 patients with systemic lupus erythematosus were found to have properdin deposited along the skin basement membrane.

Authors

Thomas T. Provost, Thomas B. Tomasi Jr.

Studies of T- and B-Lymphocytes in Patients With Connective Tissue Diseases

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Abstract

Authors