Mannan antigenemia in the diagnosis of invasive Candida infections.

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Abstract

Because it is often difficult to diagnose invasive Candida infections, a sensitive hemagglutination inhibition assay to detect the surface antigen, mannan, was developed. Mannan antigenemia was detected early in the course of infection in 4 of 14 patients with systemic candidiasis and 2 of 5 patients with invasive gastrointestinal candidiasis. Mannan was not detected in 48 patients with noninvasive Candida or other systemic mycotic infections or in 99% of 234 patients in other control groups. Mannan antibodies were almost universally present in both candidiasis and control groups. In four patients with systemic candidiasis, an early period of mannan antigenemia was followed by a rapid rise in mannan antibody titer. These findings suggest that antemortem diagnosis would be improved in one-third of cases of invasive Candida infection detected by the hemagglutination inhibition assay. A positive test for serum mannan would be an early and specific signal of invasive disease.

Authors

M H Weiner, W J Yount

Mechanism of sodium and chloride transport in the thin ascending limb of Henle.

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Abstract

Our previous in vitro studies have disclosed that the thin ascending limb of Henle (tALH) possesses some unique membrane characteristics. In those studies we failed to demonstrated active transport of sodium chloride by the tALH, although it was shown that the isotopic permeability to sodium and chloride was unusually high. However, we did not examine the mechanisms by which the apparent high permeation of sodium chloride occurs. Thus the purpose of the present studies was to elucidate the mechanism of sodium chloride transport across the isolated tALH of the rabbit by conducting four different types of studies: (1) comparison of the observed chloride and sodium flux ratios to those predicted by Ussing's equation under imposed salt concentration gradients; (2) kinetic evaluation of chloride and sodium fluxes; (3) examination of the effect of bromide on the kinetics of chloride transport; and (4) experiments to test for the existence of exchange diffusion of chloride. In the first set of studies the predicted and the theoretical flux ratios of sodium were identical in those experiments in which sodium chloride was added either to the perfusate or to the bath. However, the observed chloride flux ratio, lumen-to-bath/bath-to-lumen, was significantly lower than that predicted from Ussing's equation when 100 mM sodium chloride was added to the bath. In the second set of experiments the apparent isotopic permeability for sodium and for chloride was measured under varying perfusate and bath NaCl concentrations. There was no statistical change in the apparent sodium permeability coefficient when the NaCl concentration was raised by varying increments from 85.5 to 309.5 mM. However, permeation of 36Cl decrease significantly with an increase in Cl from 73.6 to 598.6 mM. These events could be explained by a two component chloride transport process consisting of simple diffusion and a saturable facilitated diffusion process with a Vmax = 3.71 neq mm-1 min-1. In the third set of studies it was shown that bromide inhibits transport of chloride and that the magnitude of inhibition is dependent on chloride concentrations. The fourth set of studies ruled out the existence of exchange diffusion. In conclusion, these studies indicate that sodium transport across tALH is by simple passive diffusion, while chloride transport across tALH involves at least two mechanisms: (1) simple passive diffusion; and (2) a specific membrane interaction process (carrier-mediated) which is competitively inhibited by bromide.

Authors

M Imai, J P Kokko

Androgen binding proteins of testis, epididymis, and plasma in man and monkey.

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Abstract

Androgen-binding protein (ABP) has been found in the cytosol of testicular and epididymal homogenates of several sub-primate species. In those species which had the plasma androgen binding protein, testosterone-estradiol-binding globulin (TeBG), ABP and TeBG were found to be physically similar. We investigated the possibility that ABP might exist in monkey and man using the cytosol of testicular and epididymal homogenates and aspirates obtained by direct micropuncture of the rete testis. In polyacrylamide gel electrophoresis, pH 7.8, testicular and epididymal cytosols of monkey and man were found to contain several binding proteins of different size and net charge that bind dihydrotestosterone. These binding proteins were either indistinguishable from TeBG or could be related to TeBG as size and/or charge isomers. No ABP was detectable in up to 200 mul of monkey rete testis fluid obtained by direct micropuncture, though ABP is detectable in as little as 5 mul of rat rete testis fluid. The data suggest that the ABP's detected in the testicular and epididymal cytosols in monkey and man represent isomeric forms of plasma TeBG, and their presence in testicular cytosol most likely derives from blood contamination.

Authors

R A Vigersky, D L Loriaux, S S Howards, G B Hodgen, M B Lipsett, A Chrambach

Pulmonary abnormalities in intermediate alpha-1-antitrypsin deficiency.

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Abstract

Pulmonary function studies were carried out in a group of asymptomatic nonsmoking adults with intermediate alpha-1-antitrypsin deficiency who were attending an early disease detection unit in Rochester, N. Y. All subjects were identified by specific protease inhibitor (Pi) typing. Fifteen MZ and 14 MS subjects who had never smoked cigarettes were matched by sex and age to MM controls. Spirometry, static lung volumes, and single breath-diffusing capacity were identical in all Pi type groups with no statistically significant differences noted. Maximal expiratory flow volume curves were obtained in all subjects. MZ subjects demonstrated statistically impaired maximal flow rates at 75%, 50%, and 25% of vital capacity compared to their MM controls. Total pulmonary resistance by the oscillometric method was measured at 3, 5, 7, and 9 cycle/s in the same subjects. Increased frequency dependence of resistance (defined as the difference between total pulmonary resistance at 3 cycle/s and 9 cycle/s) was observed in MZ subjects compared to MM controls. No differences were noted by this method in MS-MM pairs. The data suggest that detectable mechanical abnormalities are present in subjects with the MZ phenotype, even in the absence of established risk factors such as cigarette smoking and high air pollution.

Authors

W J Hall, R W Hyde, R H Schwartz, G S Mudholkar, D R Webb, Y P Chaubey, P L Townes

Decreased basal, noninsulin-stimulated glucose uptake and metabolism by skeletal soleus muscle isolated from obese-hyperglycemic (ob/ob) mice.

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Abstract

Insulin resistance of diaphragms of ob/ob mice has been repeatedly demonstrated previously both in vitro and in vivo. In the present study, transport and metabolism of glucose with and without insulin stimulation were compared in a skeletal muscle more likely than diaphragm or heart to be representative of the overall striated muscle mass, i.e. isolated soleus muscle. Compared with soleus muscle from lean controls, unstimulated lactate release in the presence of exogenous glucose was depressed from 16.2 to 12.3 nmol/60 min per mg wet wt in soleus from ob/ob mutants; glycolysis was decreased from 6.6 to 3.7 and [14C]glucose oxidation to 14CO2 from 0.90 to 0.33 nmol glucose/60 min per mg wet wt. Uptake of 2-deoxyglucose (2-DOG), both with and without insulin, was very much less for soleus from ob/ob than from lean mice, at 2-DOG concentrations ranging from 0.1 to 10 mM, and in mice of 6-15 wk. When 2-DOG concentration was 1 mM, its basal uptake was 0.53 nmol/30 min per mg wet wt for soleus of ob/ob as against 0.96 for soleus of lean mice. The absolute increment due to 1 mU/ml insulin was 0.49 in muscle of ob/ob as against 1.21 in that of lean mice. When the resistance to insulin action was decreased by pretreatment in vivo by either streptozotocin injection or fasting, the decreased basal 2-DOG uptake of subsequently isolated soleus muscle was not improved. Inhibition of endogenous oxidation of fatty acids by 2-bromostearate, while greatly increasing 14CO2 production from [14C]glucose, did not affect basal [5-3H]glucose metabolism or 2-DOG uptake. It is suggested that transport and/or phosphorylation of glucose under basal, unstimulated conditions are depressed in soleus muscle of ob/ob mice, whether or not resistance to insulin and hyperinsulinemia are also present. Although the origin of the decreased basal glucose uptake remains unknown it might be related to a similar decrease in basal glucose uptake by ventromedial hypothalamic cells, an event presumably resulting in a tendency to hyperphagia. Decreased basal glucose uptake by soleus muscle of ob/ob mice might explain the hyperglycemia, and hence partly the hyperinsulinemia and excessive fat deposition of those animals.

Authors

G S Cuendet, E G Loten, B Jeanrenaud, A E Renold

An inherited enzymatic defect in porphyria cutanea tarda: decreased uroporphyrinogen decarboxylase activity.

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Abstract

Uroporphyrinogen decarboxylase activity was measured in liver and erythrocytes of normal subjects and in patients with porphyria cutanea tarda and their relatives. In patients with porphyria cutanea tarda, hepatic uroporphyrinogen decarboxylase activity was significantly reduced (mean 0.43 U/mg protein; range 0.25-0.99) as compared to normal subjects (mean 1.61 U/mg protein; range 1.27-2.42). Erythrocyte uroporphyrinogen decarboxylase was also decreased in patients with porphyria cutanea tarda. The mean erythrocyte enzymatic activity in male patients was 0.23 U/mg Hb (range 0.16-0.30) and in female patients was 0.17 U/mg Hb (range 0.15-0.18) as compared with mean values in normal subjects of 0.38 U/mg Hb (range 0.33-0.45) in men and 0.26 U/mg Hb (range 0.18-0.36) in women. With the erythrocyte assay, multiple examples of decreased uroporphyrinogen decarboxylase activity were detected in members of three families of patients with porphyria cutanea tarda. In two of these families subclinical porphyria was also recognized. The inheritance pattern was consistant with an autosomal dominant trait. The difference in erythrocyte enzymatic activity between men and women was not explained but could have been due to estrogens. This possibility was supported by the observation that men under therapy with estrogens for carcinoma of the prostate had values in the normal female range. It is proposed that porphyria cutanea tarda results from the combination of an inherited defect in uroporphyrinogen decarboxylase and an acquired factor, usually siderosis associated with alcoholic liver disease.

Authors

J P Kushner, A J Barbuto, G R Lee

Functional and physicochemical studies of hemoglobin St. Louis beta 28 (B10) Leu replaced by Gln: a variant with ferric beta heme iron.

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Abstract

Studies have been performed on a 20-yr-old man exhibiting methemoglobinemia and a severe hemolytic anemia involving formation of Heinz bodies. This condition was due to an abnormal Hb present in the red cells of the proband: Hb St. Louis, beta 28 (B10) replaced by Gln, whose structural characteristics have been previously reported. This unstable Hb represented 30% of the total and was isolated by starch block electrophoresis at pH 8.6. Electrophoretic and spectral studies showed Hb St. Louis to be a valency hybird, alpha 2 beta 2+. The presence of hemichrome in this Hb was detected by electron paramagnetic resonance studies. During this study, an electrophoretic technique was developed that allows study of the mobility of hemichrome. Oxygen equilibria performed on purified Hb St. Louis revealed a high oxygen affinity and a markedly reduced cooperativity. The Bohr effect was normal, but the interaction of this hemoglobin with 2,3-diphosphoglycerate was decreased. The oxidation rate of Hb St. Louis was normal. Hb St. Louis was completely reduced by dithionite and ferrous citrate, and the functional properties of this reduced form were normal. In contrast, Hb St. Louis was only partially reduced by diaphorase. The mechanism of the oxidation of Hb St. Louis therefore appears to differ markedly from that postulated for other Hbs M.

Authors

J Thillet, M Cohen-Solal, M Seligmann, J Rosa

Hemoglobin St. Louis [beta 28 (B10) Leu replaced by Gln]: crystal structure of the fully reduced (deoxy) form.

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Abstract

The three-dimensional structure of fully reduced Hb St. Louis has been determined to 3.5 A resolution. The difference electron density map clearly shows the site of the mutation and the effects it produces. Glutamine B10 and histidine E7 (the distal histidine) swing towards each other and, between them, stabilize a water molecule in the normally hydrophobic heme pocket. This creation of an aqueous microenvironment near the heme accounts for the thermal instability, high rate of methemoglobin formation, and increased oxygen affinity observed in solution studies of the mutant as described in the preceeding paper. Other than a small increase in tilt of the heme, virtually no further stereochemical disturbances result.

Authors

N L Anderson

Mechanism of NaCl and water reabsorption in the proximal convoluted tubule of rat kidney.

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Abstract

The role of chloride concentration gradients in proximal NaCl and water reabsorption was examined in superficial proximal tubules of the rat by using perfusion and collection techniques. Reabsorptive rates (Jv), chloride concentrations, and transtubular potential difference were measured during perfusion with solutions (A) simulating an ultrafiltrate of plasma; (B) similar to (A) except that 20 meq/liter bicarbonate was replaced with acetate; (C) resembling late proximal fluid (glucose, amino acid, acetate-free, low bicarbonate, and high chloride); and (D) in which glucose and amino acids were replaced with raffinose and bicarbonate was partially replaced by poorly reabsorbable anions (cyclamate,sulfate, and methyl sulfate). In tubules perfused with solutions A and B, Jv were 2.17 and 2.7 nl mm-1 min-1 and chloride concentrations were 131.5 +/- 3.1 and 135 +/- 395 meq/liter, respectively, indicating that reabsorption is qualitatively similar to free-flow conditions and that acetate adequately replaces bicarbonate. With solution C, Jv was 2.10 nl mm-1 min-1 and potential difference was +1.5 +/- 0.2 mV, indicating that the combined presence of glucose, alanine, acetate, and bicarbonate per se is not an absolute requirement. Fluid reabsorption was virtually abolished when tubules were perfused with D solutions; Jv was not significantly different from zero despite sodium and chloride concentrations similar to plasma; chloride concentration was 110.8 +/- 0.2 meq/liter and potential difference was -0.98 mV indicating that chloride was close to electrochemical equilibrium. These results suggest the importance of the chloride gradient to proximal tubule reabsorption in regions where actively reabsorbable solutes (glucose, alanine, acetate, and bicarbonate) are lacking and provide further evidence for a passive model of NaCl and water transport.

Authors

K H Neumann, F C Rector Jr

Persistence of thromboxane A2-like material and platelet release-inducing activity in plasma.

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Abstract

During the incubation of arachidonic acid with platelet-rich plasma, a persistent activity appeared that caused the release of [14C]5-hydroxytryptamine from indomethacin-treated platelets. By the time-course of its appearance and disappearance, this release-inducing activity could be dissociated from prostaglandin endoperoxides and associated with thromboxane A2-like material. This material persists in plasma because of its continued production and increased stability.

Authors

J B Smith, C Ingerman, M J Silver

Fluctuations in the affinity and concentration of insulin receptors on circulating monocytes of obese patients: effects of starvation, refeeding, and dieting.

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Abstract

The binding of 125I-insulin to insulin receptors on circulating monocytes of obese patients and normal volunteers has been determined under various dietary states. In the basal, fed state the monocytes of obese patients with clinical insulin resistance (n= 6) bound less insulin than normals (n =10) because of a decrease in insulin receptor concentration (obese = 6,000-13,000 sites per monocyte versus normals 15,000-28,000 sites per monocyte). The single obese patient without evidence of clinical insulin resistance demonstrated normal binding of insulin with 16,000 sites per monocyte. In all patients, the total receptor concentration was inversely related to the circulating levels of insulin measured at rest after an overnight fast. For the obese patients with basally depressed insulin binding, a 48-72-h fast lowered circulating insulin and increased binding to normal levels but only at low hormone concentrations; this limited normalization of 125I-insulin binding was associated with increased receptor affinity for insulin without change in receptor concentration. Refeeding after the acute fasting periods resulted in return to the elevated plasma insulin levels, the basal receptor affinity, and the depressed insulin binding observed in the basal, fed state. Chronic diet restored plasma insulin levels, insulin binding, and receptor concentration to normal without change in affinity. When the data from this study are coupled with previous in vivo and in vitro findings they suggest that the insulin receptor of human monocytes is more sensitive to regulation by ambient insulin than the receptors of obese mice and cultured human lymphocytes. The results further indicate than an insulin receptor undergoes in vivo modulation of its interaction with insulin by changing receptor concentration and by altering the affinity of existing receptors.

Authors

R S Bar, P Gorden, J Roth, C R Kahn, P De Meyts

The generation of fibrinopeptide A in clinical blood samples: evidence for thrombin activity.

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Abstract

Plasma fibrinopeptide A (FPA) concentrations were measured in clinical blood samples incubated in the collecting syringe for different time periods before addition to heparin and Trasylol, and the rate of in vitro generation of FPA was calculated as the mean increment in FPA concentration per minute over the linear portion of the generation curve. 36 normal individuals had a mean plasma FPA level of 0.64 +/- 0.56 pmol/ml and an FPA generation rate of less than 0.5 pmol/ml per min. Clinical samples with elevated plasma FPA levels manifested slow (less than 1 pmol/ml per min) (28 patients) or rapid FPA generation (greater than 1 pmol/ml per min) (33 patients). Slow FPA generation was found in 10/10 patients with venous thrombosis, in 4/4 with aortic aneurysm, and in several patients with acquired hypofibrinogenemia. In one such patient, addition of fibrinogen resulted in rapid FPA generation whereas thrombin addition was without effect. Rapid FPA generation was generally linear, was usually associated with slower fibrinopeptide B generation and was inhibited by parenteral or in vitro heparin. It is thought to reflect increased thrombin activity and was seen in patients with pulmonary embolism, active systemic lupus erythematosus, renal transplant rejection, and after infusion of prothrombin concentrates. The initial rate of FPA cleavage by thrombin at fibrinogen concentrations from 0.05 to 4 mg/ml showed little change between 2 and 4 mg/ml with a Km of 2.99 muM. At a fibrinogen concentration of 2.5 mg/ml the FPA cleavage rate was 49.2 +/- 1.6 nmol/ml per min per U of thrombin. Exogenous thrombin added to normal blood generated 21.7 nmol/ml per U of thrombin FPA in the first minute with a nonlinear pattern reflecting inactivation of thrombin and the presence of alternative substrates. Hence, the thrombin concentration in the blood cannot be calculated from the FPA generation rate. The FPA generation rates in clinical samples with rapid generation (1-28 pmol/ml per min) could be produced by 2 X 10(-5) to 5.6 X 10(-4) thrombin U/ml acting on purified fibrinogen at physiological conditions of pH, ionic strength, and temperature.

Authors

H L Nossel, M Ti, K L Kaplan, K Spanondis, T Soland, V P Butler Jr

Basophils in tuberculin and "Jones-Mote" delayed reactions of humans.

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Abstract

Jones-Mote reactions are delayed, erythematous, and mildly indurated cutaneous reactions originally described in humans sensitized by skin injection of heterologous proteins. Similar reactions in guinea pigs contain many basophils and are called cutaneous basophil hypersensitivity. In contrast, guinea pigs immunized with mycobacterial adjuvants have classical tuberculin-type delayed hypersensitivity reactions, which contain few basophils. This has led to a new classification of delayed responses, based largely on the presence or absence of basophils. We induced sensitization for Jones-Mote reactions in 20 normal humans by intradermal injections of keyhole limpet hemocyanin. Skin tests with KLH 1 wk later showed erythematous and indurated delyaed reactions in all subjects. Rebuck skin windows showed specific accumulations of basophils with a delayed time-course in 18 of 20 subjects. In 12 normals sensitized with oxazolone-keyhole limpet hemocyanin conjugates, skin reactions and in vitro lymphocyte stimulation showed carrier and not hapten specificity, suggesting that cutaneous responses were probably mediated by T cells. A comparative study of strongly positive PPD skin tests in patients with tuberculosis showed significant basophil accumulations in five of nine subjects. Thus, basophils occurred in human tuberculin and Jones-Mote reactions and were not a distinguishing feature of Jones-Mote reactions. We suggest that the occurrence of basophils at delayed reactions is under complex regulation and that basophil accumulations are an aspect of delayed hypersensitivity, rather than an indication of a distinctive and separate response.

Authors

P W Askenase, J E Atwood

Paraproteinemia: blood hyperviscosity and clinical manifestations.

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Abstract

Many of the clinical features of paraproteinemia result from impairment of blood flow through the vascular tree because of blood hyperviscosity. Studies were carried out in 65 patients with serum paraproteins (31 with IgG, 25 with IgM, and 9 with IgA) to examine the relationship between the blood viscosity and the frequency of selected clinical features. The blood and plasma viscosities were measured at low rates of shear. Blood hyperviscosity was present in 91% of the patients and plasma hyperviscosity in 75% of the patients. In each of the three immunoglobulin classes both the blood and plasma viscosities increased logarithmically with the paraprotein concentration being greatest in the case of IgM. In addition, the relationship between the hematocrit and the logarithm of blood viscosity tended to be linear at any given protein concentration. In patients with very high levels of paraprotein the blood viscosity was modified by low hematocrits; the latter was below 30 in 70% of patients in whom the concentration of paraprotein was above 4 g/100 ml. The prevalence of clinical complications involving the retinal circulation, the peripheral vascular system, and the central nervous system increased markedly with increasing blood viscosity, measured at 0.18 S-1. One or more of these regions was affected in greater than 80% of patients with blood viscosity above 60 centipoise and in less than 23% of patients with blood viscosity below 40 centipoise. These observations illustrate the complex relationship between blood viscosity, concentration of paraprotein, immunoglobulin class and hematocrit, and emphasize the importance of measuring the whole blood viscosity at low rates of shear in determining the risk of vascular complications.

Authors

M A McGrath, R Penny

Gonococci causing disseminated gonococcal infection are resistant to the bactericidal action of normal human sera.

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Abstract

The susceptibility of strains of Neisseria gonorrhoeae to the bactericidal action of normal human sera was determined for isolates from patients with disseminated gonococcal infection and uncomplicated gonorrhea. Serum susceptibility was correlated with penicillin susceptibility and auxotype. 38 of 39 strains (97%) of N. gonorrhoeae from Seattle patients with disseminated gonococcal infection were resistant to the complement-dependent bactericidal action of normal human sera. 36 of these were inhibited by less than or equal to mug/ml of penicillin G and required arginine, hypoxanthine, and uracil for growth on chemically defined medium (Arg-Hyx-Ura- auxotype). 12 of 43 isolates from patients with uncomplicated gonorrhea were also of the Arg-Hyx-Ura-auxotype, inhibited by less than or equal to 0.030 mug/ml of penicillin G, and serum resistant. Of the 31 remaining strains of other auxotypes isolated from patients with uncomplicated gonorrhea, 18 (58.1%) were sensitive to normal human sera in titers ranging from 2 to 2,048. The bactericidal action of normal human sera may prevent the dissemination of serum-sensitive gonococci. However, since only a small proportion of individuals infected by serum-resistant strains develop disseminated gonococcal infection, serum resistance appears to be a necessary but not a sufficient virulence factor for dissemination. Host factors such as menstruation and pharyngeal gonococcal infection may favor the dissemination of serum-resistant strains. Since serum-resistant Arg-Hyx-Ura strains are far more frequently isolated from patients with disseminated gonococcal infection than serum-resistant strains of other auxotypes, Arg-Hyx-Ura-strains may possess other virulence factors in addition to serum resistance.

Authors

G K Schoolnik, T M Buchanan, K K Holmes

The alteration of superoxide dismutase, catalase, glutathione peroxidase, and NAD(P)H cytochrome c reductase in guinea pig polymorphonuclear leukocytes and alveolar macrophages during hyperoxia.

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Abstract

Superoxide dismutase, catalase, glutathione peroxidase and NAD(P)H cytochrome c reductase were quantitated in polymorphonuclear leukocytes (PMN) and alveolar macrophages (AM) obtained from guinea pigs exposed up to 90 h to 85% oxygen. PMN and AM were sonicated and separated into a 16,000-g pellet, a 100,000-g pellet, and a 100,00-g supernate. Superoxide dismutase activity increased in both cells within 18 h, persisted for 66 h and decreased by 90 h. The highest rate of increase was in the 100,000-g pellet containing 3.4% of total enzyme activity in PMN but 28% in AM. The enzyme induction in PMN and AM was partially inhibited by daily intracardiac injections of 50 mg/kg actinomycin D. During oxygen exposure, catalase activity in PMN and AM decreased to 60% of its original activity, and gluthathione peroxidase was reduced in PMN to 60% and in AM to 20% of control values. Although NAD(P)H cytochrome c reductase decreased to 50% in PMN, no change was noted in AM. Upon exposure to superoxide anion, purified catalase, the glutathione peroxidase of the 100,000-g supernate, NADH, and NADPH cytochrome c reductases of the 16,000-g pellet decreased to 66+/-5%, 72+/-4%, 52+/-8%, and 40+/-9%, respectively, of their original activity. This inactivation was prevented by 0.1 mg superoxide dismutase. These in vitro observations could explain the decreased catalase and glutathione peroxidase activity demonstrated in vivo that may lead to an intracellular accumulation of hydrogen peroxide. Increased hydrogen peroxide concentrations have been found to inactivate superoxide dismutase thus impairing the first defense mechanism against superoxide anion.

Authors

M Rister, R L Baehner

Alterations of myocardial amino acid metabolism in chronic ischemic heart disease.

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Abstract

Arteriovenous differences (A-V) of all naturally occurring amino acids, lactate, and oxygen were measured simultaneously with coronary sinus blood flow (CSBF) in 8 normal subjects and 11 patients with coronary artery disease at rest and during pacing stress. Mean values for CSBF and myocardial oxygen consumptions (MVO2) for the two groups were similar at rest and during pacing, although mean CSBF and MVO2 increased significantly in both groups in the paced as compared to the rest state. Alanine (ala) was the only amino acid released by the myocardium, while only glutamic acid(glu) demonstrated uptake. Mean A-V ala was negative at rest in the control and coronary disease groups (-4.8+/-3.8 vs. -22.0+/-3.0 nmol/ml, respectively), but was significantly more negative in the coronary group (P less than 0.001) and not statistically different than zero in the normals. A-V ala became significantly negative with pacing in the normals (-10.0+/-4.3 nmol/ml), remained unchanged in the coronary group (-23.0+/-2.9 nmol/ml), and was significantly more negative in the coronary group (P less than 0.05). Calculation of data on the basis of net ala flux ([A-V] X [CSBF X hematocrit]) yielded similar results as that obtained with A-V differences. A-V glu was significantly positive in normals (27.7 +/- 8.9 nmol/ml, P less than 0.01) and coronary patients (59.9 +/- 8.9 nmol/ml, P less than 0.01) at rest but significantly greater in the latter group (P less than 0.001). With pacing, A-V glu remained significantly greater than zero in coronary patients (35.3 +/- 6.3 nmol/ml) and decreased to zero in the normals (4.3 +/- 11.8 nmol/ml). Calculation of net glu flux (nmol/min) at rest yielded data similar to that based on A-V difference. With pacing, net glu flux in the coronary patients did not decrease due to the augmentation of CSBF. No relation between A-V glu or ala and CSBF, MVO2 or A-V lactate was noted. The data demonstrate that specific alterations of myocardial amino acid metabolism characterize patients with chronic ischemic heart disease.

Authors

G H Mudge Jr, R M Mills Jr, H Taegtmeyer, R Gorlin, M Lesch

Glucose and ATP levels in pancreatic islet tissue of normal and diabetic rats.

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Abstract

It has been suggested that the hyperglucagonemia observed in diabetic animals and man may be due to an impairment of glucose uptake and metabolism by the alpha-cells resulting in a decreased production of ATP. To test this hypothesis glucose, ATP, glucagon, and insulin were measured in pancreatic islets of normal and alloxan or streptozotocin diabetic rats. Two experimental approaches were used. In the first, the pancreas was perfused in vitro for assessing insulin and glucagon release due to 10 mM amino acids with and without 5 mM glucose. These perfusions were performed in the presence and absence of insulin. After perfusion, the pancreas was frozen and processed for analysis of islet glucose, ATP, insulin, and glucagon content. The second approach was to investigate the islet sucrose, urea, and glucose spaces together with ATP, insulin, and glucagon content in vivo in normal and in insulin-treated and untreated streptozotocin diabetic rats. Perfusion of the pancreas in vitro with 5 mM glucose resulted in higher glucose content of normal islets than in alloxan and streptozotocin diabetic islets. Similarly in the in vivo studies, the intracellular glucose space of the streptozotocin diabetic islets was 30% the value found in normals. In the in vivo experiments, despite the relatively small intracellular glucose space of alpha-cell islets, the ATP content of these islets was only 15-20% lower than the ATP content of normal islets. In the in vitro experiments, perfusion with glucose resulted in ATP contents of alpha-cell islets and of normal mixed alpha-beta-cell islets which were indistinguishable. However, the ATP content of alpha-cell islets was maintained for prolonged periods in the absence of glucose in contrast to mixed islets, composed primarily of beta-cells, in which the ATP level decreased by 45% when glucose-free medium was perfused for sustained periods. Finally, insulin infused in high concentrations or administered to the diabetic animal had no effect on the glucose spaces or the ATP contents of normal or alpha-cell islets. It can be calculated that in vivo the intracellular glucose level of islets from streptozotocin treated rats is approximately 15 mM. Since in normals an extracellular glucose concentration of this magnitude inhibits stimulated glucagon release completely, it would seem unlikely that a lack of intracellular glucose is the cause of the apparent glucose "blindness" of the alpha-cells in diabetes. In fact, in perfusion studies as little as 2.5 mM free intracellular glucose was sufficient to suppress glucagon secretion from diabetic alpha-cells. The results of the ATP measurements clearly eliminate a possible energy deficit of diabetic alpha-cells as cause of the apparent glucose resistance of alpha-cells.

Authors

F M Matschinsky, A S Pagliara, S N Stillings, B A Hover

The critical role of the adrenal gland in the renal regulation of acid-base equilibrium during chronic hypotonic expansion. Evidence that chronic hyponatremia is a potent stimulus to aldosterone secretion.

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Abstract

Recent studies have shown that chronic hypotonic volume expansion (HVE) induced by administration of vasopressin and water stimulates distal hydrogen ion secretion and thereby (a) permits dogs with HCl-acidosis to restore acid-base equilibrium to normal despite continued acid feeding and (b) permits normal dogs to conserve filtered bicarbonate quantitatively despite the natriuresis induced by water retention. To examine whether these effects of chronic HVE are mediated by augmented mineralocorticoid secretion, urinary and plasma aldosterone levels were monitored during prolonged administration of vasopressin. In HCl-fed animals, the HVE-induced rise in plasma [HCO3] (from 13.8 to 21.3 meq/liter) was associated with a rise in aldosterone excretion from 0.45 to 0.88 mug/day (P less than 0.02). In normal animals, in which plasma [HCO3] remained stable during HVE (21.9 vs. 20.0 meq/liter), aldosterone excretion rose from 0.51 to 2.28 mug/day (P less than 0.02) and plasma aldosterone concentration rose from 8.1 to 39.8 ng/100 ml (P less than 0.01). Vasopressin and water were also administered to adrenalectomized animals maintained on glucocorticoids and a slightly subphysiologic replacement schedule of mineralocorticoids. In the HCl-fed adrenalectomized group, plasma [HCO3], instead of rising to normal, showed no significant change (16.9 vs. 15.0 meq/liter). In the non-HCl-fed adrenalectomized group, plasma [HCO3], rather than remaining stable, fell significantly (20.3 vs 16.5 meq/liter, P less than 0.1). Two conclusions can be drawn from this study: (a) the well-known inhibitory effect of volume expansion on aldosterone secretion can be overridden by a potent stimulatory effect on the adrenal produced by severe chronic hypotonicity, and (b) the response of plasma [HCO3] observed during severe chronic HVE is mediated by augmented mineralocorticoid secretion. These findings, furthermore, offer a possible explanation for the puzzling observation that plasma [HCO3] in patients with the syndrome of inappropriate antidiuretic hormone secretion is maintained at normal levels even in the face of severe hyponatremia.

Authors

J J Cohen, H N Hulter, N Smithline, J C Melby, W B Schwartz

Insulin release and cyclic AMP accumulation in response to glucose in pancreatic islets of fed and starved rats.

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Abstract

The dose as well as the time kinetics of insulin and adenosine-3', 5' -monophosphate (cyclic AMP) responses to glucose were compared in pancreatic islets of fed and starved rats. There was a preferential impairment of the early phase of glucose-induced insulin release in perifused islets of rats starved for 16 and 48 h. Similarly, the accumulation of 3H cyclic AMP in islets prelabeled with 3H-2-adenine was less in islets of 48 h starved than fed rats, during the first 10-min of stimulation with 26.7 mM glucose in the presence of 0.1 mM of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, whereas at 30 and 60 min 3H cyclic AMP responses to glucose were similar in fed and starved islets. Also, in 10-min incubations with glucose 3.3, 6.7, 10.0, 13.3, and 26.7 mM without and with 0.1 mM and 1.0 mM 3-isobutyl-1-methylxanthine, insulin release correlated strongly with the accumulation of 3H cyclic AMP in the islets of fed as well as starved rats. The thresholds for glucose-induced insulin and 3H cyclic AMP responses were higher and the maximal responses were lower in starved than fed islets. Preincubation of islets of 48-h starved rats with 16.7 mM glucose for 60 min corrected the impaired insulin and 3H cyclic AMP responses to glucose. Starvation-induced impairment of insulin secretory responses to glucose, and their restoration by preincubation with glucose in vitro, may represent acute regulatory effects of glucose on the adenylate cyclase-cyclic AMP system in the pancreatic beta cell.

Authors

A Rabinovitch, V Grill, A E Renold, E Cerasi

Radioimmunoassay of the binding protein for vitamin D and its metabolites in human serum: concentrations in normal subjects and patients with disorders of mineral homeostasis.

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Abstract

A radioimmunoassay for the binding protein for vitamin D and its metabolites (DBP) has been developed. Suitable rabbit anti-DBP antiserum was elicited after primary and one booster injection. Anti-DBP antisera, as well as antigroup-specific component antisera, produced a single, monospecific line of percipitation when reacted against purified DBP and human serum. DBP was iodinated with 125I and 125I-DBP was purified by gel filtration on Sephadex G-200. Binding of 125I-DBP by 20 nl of rabbit anti-DBP antisera was approximately 50% and was sharply competed for by 0.4-4.0 ng of DBP standard. Displacement of 125I-DBP by human serum dilutions or standard DBP gave identical curves, and only weak competition was observed with old and new world primate sera. Apo- and holo-DBP possessed indistinguishable immunoreactivity. The assay detects DBP in 1-10 nl of human serum with reasonable accuracy and with reasonable intra- and interassay precision. The mean serum concentration (+/- SEM) for a group of 40 normal adults was 525 +/- 24 mug/ml and no sex difference was observed. Higher levels were found in sera from pregnant women and women receiving oral contraceptives, and decreased concentrations were observed in premature cord and hypoproteinemic sera. No significant correlation between serum DBP levels and serum 25-hydroxycalciferol levels was found, and the DBP content of sera from vitamin D-deprived and vitamin D-treated subjects was indistinguishable from that of normal adults. DBP accounts for 6- of the alpha globulin in normal human serum. Considering the normal serum content of the parent vitamin and its metabolites to be approximately 0.1-0.2 mum, these immunoassay data confirm previous saturation analyses of human serum antiricketic sterol-binding capacity and suggest that greater than 95% of DBP circulates as the apoprotein under normal conditions.

Authors

J G Haddad Jr, J Walgate

Pathogenic mechanisms in pulmonary fibrosis: collagen-induced migration inhibition factor production and cytotoxicity mediated by lymphocytes.

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Abstract

The universal features of the histopathology of fibrotic lung disease are derangement of parenchymal collagen and infiltration of the parenchyma with chronic inflammatory cells. To determine if this cellular reaction might be associated with autoimmunity to a consitituent of the alveolar interstitium, peripheral blood lymphocytes were exposed to human type I collagen in vitro and evaluated for the production of migration inhibition factor and cytotoxicity. Data from 18 patients with idiopathic pulmonary fibrosis, 8 patients with pulmonary fibrosis other than idiopathic pulmonary fibrosis, 12 patients with nonfibrotic lung disease, and 9 normals demonstrated that circulating lymphocytes from more than 94% of patients with fibrotic lung disease take part in processes where the recognition of collagen results in migration inhibition factor production and lysis of collagen-coated sheep red blood cells. These collagen-induced cell-mediated phenomena are obviated with human T-lymphocyte antiserum. Collagen-induced migration inhibition factor production and cytotoxicity were found in less than 20% of patients with nonfibrotic disease and were not found in normals. Qualitatively, there was no organ (lung, skin) or species (human, rabbit) collagen specificity in these assays, but human lung alpha 2 chains were recognized more often than alpha 1(I) chains. Circulating lymphocytes from patients with fibrotic disease are present in a normal T to B ratio. These lymphocytes did not incorporate [3H]thymidine when exposed to collagen but did when exposed to T-cell mitogens. These in vitro observations suggest that circulating T-lymphocytes and lung collagen may be intimately associated in the pathogenesis of human fibrotic lung disease.

Authors

T C Kravis, A Ahmed, T E Brown, J D Fulmer, R G Crystal

Electrophysiological study of isolated perfused human collecting ducts: Ion dependency of the transepithelial potential difference.

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Abstract

Cortical and outer medullary collecting duct segments were dissected from human kidneys and perfused in vitro. The transepithelial potential difference was measured and found to be lumen positive +6.8 +/- 0.6 mV (n= 20). This lumen-positive potential difference was inhibited by ouabain and furosemide but not by acetazolamide. Replacement of chloride in bath and perfusion fluids caused a reversible decrease of the potential difference to near zero. We conclude from these studies: (a) the lumen-positive potential difference is dependent upon the presence of chloride ion suggesting the existence of an active electrogenic chloride reabsorptive process in the human collecting duct and (b) it is possible to examine human renal physiology directly using in vitro microperfusion of tubule segments.

Authors

H R Jacobson, J B Gross, S Kawamura, J D Waters, J P Kokko

The chromosomal order of genes controlling the major histocompatibility complex, properdin factor B, and deficiency of the second component of complement.

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Abstract

The relationship of the genes coding for HLA to those coding for properdin Factor B allotypes and for deficiency of the second component of complement (C2) was studied in families of patients with connective tissue disorders. Patients were selected because they were heterozygous or homozygous for C2 deficiency. 12 families with 15 matings informative for C2 deficiency were found. Of 57 informative meioses, two crossovers were noted between the C2 deficiency gene and the HLA-B gene, with a recombinant fraction of 0.035. A lod score of 13 was calculated for linkage between C2 deficiency and HLA-B at a maximum likelihood value of the recombinant fraction of 0.04. 18 families with 21 informative matings for both properdin Factor B allotype and HLA-B were found. Of 72 informative meioses, three recombinants were found, giving a recombinant fraction of 0.042. A lod score of 16 between HLA-B and Factor B allotypes was calculated at a maximum likelihood value of the recombinant fraction of 0.04. A crossover was shown to have occurred between genes for Factor B and HLA-D, in which HLA-D segregared with HLA-A and B. These studies suggest that the genes for Factor B and C2 deficiency are located outside those for HLA, that the order of genese is HLA-A, -B, -D, Factor B allotype, C2 deficiency, that the genes coding for C2 deficiency and Factor B allotypes are approximately 3--5 centimorgans from the HLA-A and HLA-B loci, and that the apparent lack of recombinants between the Factor B gene and C2 deficiency gene suggests that these two genes lie in close proximity to one another.

Authors

D Raum, D Glass, C B Carpenter, C A Alper, P H Schur

The measurement of thrombin in clotting blood by radioimmunoassay.

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Abstract

We have developed a radioimmunoassay for human thrombin using rabbit anti-human thrombin IgG. The assay can measure 2 ng thrombin/ml plasma, 500-fold more sensitive than clotting assays. Human prothrombin is less reactive in the assay than thrombin by at least four orders of magnitude, and there is no demonstrable cross-reactivity with human factor Xa, the clotting factor structurally most similar to thrombin. The assay does not detect thrombin bound to anthithrombin III. Using the assay, we have demonstrated that plasma from 20 normal subjects does not contain detectable thrombin. We measured thrombin generation in clotting blood in polypropylene tubes and observed that thrombin appears (approximately equal to 3 ng/ml) within 45 S-5 min after venipuncture. This material is thrombin, not intermediates of prothrombin activation, since it disappears after addition of heparin, which promotes thrombin antithrombin III complex formation. After a plateau of 2-10 min, there is further thrombin generation, which results in clotting after 15-27 min at a level of 40-50 ng thrombin/ml. The thrombin generated 9-25 min before clotting may activate factors V and VIII and stimulate platelet aggregation and release. In contrast, the cascade hypothesis assigns a role for thrombin only late in blood clotting. Radioimmunoassay of thrombin and other clotting factors will be useful for clinical and physiological studies of blood clotting especially since the assay seems specific for thrombin and is independent of other activities that affect bioassays.

Authors

M A Shuman, P W Majerus

Human myeloma IgA half-molecules.

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Abstract

A lambda, IgAl myeloma protein that formed two chain half-molecules was obtained from a patient who had typical multiple myeloma. His serum contained 1.3 g/100 ml of an IgA paraprotein of gamma-1 electrophoretic mobility, his urine predominantly lambda Bence Jones protein, and only small amounts of IgA paraprotein. Analytical ultracentrifugation of the isolated serum IgA protein showed 7.0S and 4.5S protein peaks but no IgA polymers. When the 7.0S and 4.5S protein peaks were tested with an antiserum specific for alpha chain, both fractions were antigenically deficient compared to control IgA myeloma proteins but showed a line of identity to their F(ab')2 fragments. The serum and 7.0S protein fraction showed double precipitin lines in IgA radial immunodiffusion plates and in immunoelectrophoretic analysis, one line being formed by the myeloma protein and the other by residual normal IgA. The myeloma protein did not form a precipitin line with antisera specific for the IgA Fc fragment. Sodium dodecylsulfate-urea-polyacrylamide gel electrophoresis demonstrated that both the 7.0S and 4.5S fractions of the myeloma protein consisted of covalently linked heavy and light chains, 4.5S fraction being apparently the half-molecule of the 7.0S protein. The heavy chain had a mol wt of 46,500 daltons compared to 55,000 daltons for normal alpha chains. Reduction and alkylation in aqueous solutions resulted in dissociation of the 7.0S myeloma protein fractions into smaller units, probably half-molecules, suggesting that the noncovalent interactions between the alpha chains were substantially weakened or absent, presumably as a result of a deletion in the Fc portion of the alpha chain. The catabolic rates of the radio-labeled 7.0S and 4.5S protein in rhesus monkeys were similar to those of control IgA myeloma proteins; the excretion of protein-bound radioactivity of the IgA half-molecules into the urine was no greater than that of the 7.0S or of control IgA myeloma proteins. It is suggested that the myeloma IgA half-molecule is probably derived from an IgAl mutant that is carried in the human genome and that it is unlikely a representative of a rare IgA subclass or an IgA l allotypic variant.

Authors

H L Spiegelberg, B G Fishkin

Pathogenesis and characterization of hyperglucagonemia in the uremic rat.

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Abstract

The pathogenesis of hyperglucagonemia and of the alterations in the pattern of circulating immunoreactive glucagon (IRG) associated with renal insufficiency was studied in rats in which a comparable degree of uremia was induced by three different methods, i.e., bilateral nephrectomy, bilateral ureteral ligation, and urine autoinfusion. Nephrectomized and ureteral-ligated rats were markedly hyperglucagonemic (575 +/- 95 pg/ml and 492 +/- 54 pg/ml, respectively), while IRG levels of urine autoinfused animals (208 +/- 35 pg/ml) were similar to those of control rats (180 +/- 26 pg/ml), indicating that uremia per se does not account for the hyperglucagonemia observed in renal failure. Similarly, plasma IRG composition in this group of animals was indistinguishable from that of controls, in which 88.2 +/- 5.9% of total IRG consisted of the 3,500-mol wt fraction. The same component was almost entirely responsible (82.6 +/- 4.1%) for the hyperglucagonemia observed in ligated rats, while it accounted for only 57.6 +/- 5.0% of the circulating IRG in nephrectomized animals. In the latter group, 36.8 +/- 6.6% of total IRG had a mol wt of approximately 9,000, consistent with a glucagon precursor. This peak was present in samples obtained as early as 2 h after renal ablation and its concentration continued to increase with time reaching maximal levels at 24 h. These results confirm that the kidney is a major site of glucagon metabolism and provide evidence that the renal handling of the various circulating IRG components may involve different mechanisms. Thus, the metabolism of the 3,500-mol wt fraction is dependent upon glomerular filtration, while the uptake of the 9,000-mol wt material can proceed in its absence, as long as renal tissue remains adequately perfused. This finding suggests that the 9,000-mol wt component may be handled by peritubular uptake.

Authors

D S Emmanouel, J B Jaspan, S F Kuku, A H Rubenstein, A I Katz, A H Huen

Inhibitory effects of hypermagnesemia on the renal action of parathyroid hormone.

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Abstract

Available evidence indicates that serum magnesium (Mg++) levels influence the secretion rate of parathyroid hormone (PTH). Whether serum Mg++ concentrations also modify the action of PTH on its target organs has not been definitively established. The present experiments were designed to study this possibility. The effect of infusing PTH on the urinary excretion of cyclic AMP (cAMP) and PO4= was examined in five normal dogs at two different levels of serum Mg++. At normal serum Mg++ concentrations (1.89 +/- 0.14 mg/100 ml), PTH infusion increased cAMP excretion from 1.76 +/- 0.27 to 4.87 +/- 1.00 nmol/min and fractional PO4= excretion (FEPO4) from 1.58 +/- 0.36% to 23.1 +/- 2.17%. When an identical amount of PTH was given to the same dogs at a serum Mg++ of 4.36 +/- 0.20 mg/100 ml, FEPO4 increased to only 6.02+/-1.89% and cAMP from 1.31 +/- 0.23 to 1.89 +/- 0.39 nmol/min. Identical results were obtained in thyroparathyroidectomized hypermagnesemic dogs. Increased serum Mg++ levels had no effect on the phosphaturia produced by the infusion of dibutyryl cAMP to thyroparathyroidectomized dogs. In vitro studies using rat renal cortical slices revealed a progressive decrease in cAMP production in response to PTH as the Mg++ concentrations were increased in the incubation medium. The overall results indicate that hypermagnesemia inhibits the phosphaturic response to PTH by decreasing the renal production of cAMP. Plasma magnesium, therefore, may participate in a double feedback mechanism, not only controlling the release of PTH, but also altering the biological activity of the hormone at the level of the target organ.

Authors

E Slatopolsky, A Mercado, A Morrison, J Yates, S Klahr

The effects of colcemid on hematopoiesis in the mouse.

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Abstract

Colcemid was found to induce a dose and schedule dependent marrow magakaryocytosis and peripheral thrombocytosis. The response could be divided into early and late components. The early component appears to have been due to a direct stimulatory effect, probably by enhancement of endoreduplication in metaphase arrested megakaryocyte precursors. The ealy stimulatory response was blunted on toxic drug schedules. In contrast, the late component of the thrombopoietic response was demonstrated best on the most toxic drug schedules. It coincided temporally with the reactive restoration of the mononuclear marrow and blood cell elements, respectively. Thus, the late component appears to be a nonspecific rebound phenomenon. On comparing the thrombopoietic properties of Colcemid with those of the vinca alkaloids in experimental systems, the former appears to have a more favorable therapeutic index. The data suggest that colchicine and its derivatives may be useful agents in the treatment of clinical thrombocytopenic states.

Authors

P A Bunn Jr, S E Shackney, S S Ford