An ASCI tradition. Presidential address.

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Authors

J S Fordtran

Selective release of archidonic acid from the phospholipids of human platelets in response to thrombin.

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Arachidonic acid is unique amongst human platelet fatty acids in that it is the precursor of prostaglandins and thromboxanes. Since a number of these oxygenated products of arachidonic acid have potent effects on platelet function, an understanding of the metabolsim of their precursor is important. Human platelets have a mechanism for incorporating arachidonic acid from plasma into their phospholipids and, in response to thrombin, they reveal mechanisms for hydrolyzing this arachidonic acid from platelet phosphatidylcholine and phosphatidylinositol. This report deals with the specificity of these mechanisms. The present studies show that human platelets contain phospholipase A2 activities that preferentially release arachidonic acid. One of these activities specifically utilizes 1-acyl-2-arachidonyl-phosphatidyl-choline. Another utilizes platelet phosphatidylinositol and/or phosphatidylserine, both of which are highly enriched with arachidonic acid.

Authors

T K Bills, J B Smith, M J Silver

Potentiation of the function of Hageman factor fragments by high molecular weight kininogen.

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Patients lacking high molecular weight (HMW) kininogen have profound abnormalities of the Hageman factor-dependent pathways of coagulation, kinin formation, and fibrinolysis. The ability of HMW kininogen to potentiate the Hageman factor fragments (HFf) activation of prekallikrein and Factor XI in plasma was studied. HFf only partially converted Factor XI to XIa and prekallikrein to kallikrein in plasma deficient in HMW kininogen (Williams trait), while enhanced activation of Factor XI and prekallikrein by HFf resulted after reconstitution with HMW kininogen. In a system using highly purified components, HMW kininogen increased the initial rate of prekallikrein activation whether the kallikrein formed was assayed by arginine esterase activity or kininforming ability. The potentiation of prekallikrein activation occurred over a 12-fold range of enzyme (HFf) concentration and was nonhyperbolic with respect to substrate (prekallikrein). HMW kininogen exerted its effect even in the absence of prekallikrein since the hydrolysis of acetylglycyl-lysine methyl ester by HFf was increased by HMW kininogen. These results suggest that one of the functions of HMW kininogen is to augment the catalytic action of HFf.

Authors

C Y Liu, C F Scott, A Bagdasarian, J V Pierce, A P Kaplan, R W Colman

Activation and function of human Hageman factor. The role of high molecular weight kininogen and prekallikrein.

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The activation and function of surface-bound Hageman factor in human plasma are dependent upon both high molecular weight (HMW) kininogen and prekallikrein. HMW kininogen does not affect the binding of Hageman factor to surfaces, but it enhances the function of surface-bound Hageman factor as assessed by its ability to activate prekallikrein and Factor XI. The initial conversion of prekallikrein to kallikrein by the surface-bound Hageman factor in the presence of HMW kininogen is followed by a rapid enzymatic activation of Hageman factor by kallikrein. The latter interaction is also facilitated by HMW kininogen. Kallikrein therefore functions as an activator of Hageman factor by a positive feedback mechanism and generates most of the activated Hageman factor during brief exposure of plasma to activating surfaces. HMW kininogen is a cofactor in the enzymatic activation of Hageman factor by kallikrein and it also augments the function of the activated Hageman factor generated. The stoichiometry of the Hagman factor interaction with HMW kininogen suggests that it enhances the activity of the active site of Hageman factor. Since HMW kininogen and prekallikrein circulate as a complex, HMW kininogen may also place the prekallikrein in an optimal position for its reciprocal interaction with Hageman factor to proceed. The surface appears to play a passive role upon which bound Hageman factor and the prekallikrein-HMW kininogen complex can interact.

Authors

H L Meier, J V Pierce, R W Colman, A P Kaplan

Salicylate Metabolism in Twins: EVIDENCE SUGGESTING A GENETIC INFLUENCE AND INDUCTION OF SALICYLURATE FORMATION

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To evaluate the contribution of genetic influences on the individual variation in plateau serum salicylate levels, salicylate metabolism was studied in seven pairs of identical and six pairs of fraternal twins.

Authors

Daniel E. Furst, Niroo Gupta, Harold E. Paulus

The Measurement of Apolipoprotein A-I and A-II Levels in Men and Women by Immunoassay

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To study apolipoprotein A-II, a simple, precise, and accurate immunodiffusion assay was developed and applied in a population sample of industrial employees. Apolipoprotein A-II (A-II) did not increase with age in men (r = −0.20, n = 172), but showed a slight increase with age in women (0.1 mg/dl per yr, r = 0.20, n = 188). A-II correlated significantly with apolipoprotein A-I (A-I) (r = 0.71) and high density lipoprotein (HDL) cholesterol (men, r = 0.64; women, r = 0.49). The A-I/A-II ratio was significantly related to HDL cholesterol (men, r = 0.29; women, r = 0.44). Women on no medication (n = 92) had A-II levels similar to men (34±5 and 33±5 mg/dl, mean±SD, respectively), whereas women on oral contraceptives or estrogens had significantly higher levels (39±6 mg/dl, n = 75, P < 0.01). The plasma A-I/A-II weight ratio was 3.6±0.4 for men and 3.8±0.5 for women. In the d = 1.10-1.21 subfraction, both males and females had similar A-I, A-II, and HDL cholesterol levels (men: mean, 97, 27, and 32 mg/dl, respectively; women: mean, 104, 28, and 36 mg/dl, respectively). Women had approximately twice the amount of A-I, A-II, and HDL cholesterol than men in the d = 1.063-1.10 fraction (men: mean, 10, 2, and 10 mg/dl, respectively; women: mean, 24, 4, and 19 mg/dl, respectively). The A-I/A-II weight ratio in the d = 1.063-1.10 fraction (men, 5.1±0.7; women, 6.1±1.3) was significantly greater (P < 0.01) than that in the d = 1.10-1.21 fraction (men, 3.7±0.2; women, 3.8±0.2). Furthermore, the weight ratio of cholesterol to total apoprotein A in the d = 1.063-1.10 fraction (men, 0.75±0.09; women, 0.67±0.05) was significantly higher (P < 0.01) than that found in the d = 1.10-1.21 fraction (men, 0.26±0.04, women, 0.28±0.05). Thus, the compositions of HDL hydrated density subclasses are significantly different from each other. These results suggest that the differences in HDL between men and women are due primarily to differences in the relative proportions of HDL subclasses rather than to the intrinsic differences in HDL structure.

Authors

Marian C. Cheung, John J. Albers

Stimulation of Gastrin Secretion and Synthesis in Antral Organ Culture

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The purpose of the present study was to examine stimulation of gastrin release and the synthesis of gastrin directly by measurement of incorporation of [3H]tryptophan into gastrin in rat antral mucosal explants maintained in organ culture. Gastrin synthesis and secretion were assessed simultaneously at intervals over the 24-h duration of explant culture. Antral mucosal explants from fed female Wistar rats (4-5 wk, 100-150 g) were cultured at 37°C (95% O2/5% CO2) in medium containing 70% Trowell-T8 and 10% NCTC-135 without unlabeled tryptophan, 10% dialyzed fetal calf serum and [3H]tryptophan (100 μCi/ml). Antral tissue was harvested at regular intervals during 24-h culture periods. Incorporation of [3H]tryptophan into immunoreactive gastrin was determined by techniques utilizing double-antibody immunoprecipitation. Antral tissue protein synthesis was assessed by measurements of incorporation of [3H]tryptophan into tissue protein of cultured antral explants. In paired experiments, gastrin synthesis and secretion in the presence of dibutyryl cAMP (DBCAMP) were compared to those observed under control conditions. Gastrin and protein specific activity progressively increased with time. Gastrin specific activity at 30 min increased from 3.3±0.5 (SEM) to 55.2±10.6 fmol [3H]tryptophan/pmol gastrin (or from 1.57±0.48 to 26.28±5.05 pmol [3H]tryptophan/μg gastrin) at 24 h: specific activity of antral tissue protein at 30 min increased from 33.6±8.4 to 1,660±236 fmol [3H]tryptophan/μg protein at 16 h. Culturing of explants for 4 h in the presence of cycloheximide (100 μg/ml) inhibited both gastrin synthesis and protein synthesis by greater than 90 and 95%, respectively. DBCAMP (10 mM) significantly increased both the synthesis and secretion of antral gastrin when compared with control cultured explants. Results of these experiments provide direct demonstration of gastrin synthesis by rat antral mucosal explants in organ culture, indicate that both gastrin and total antral protein synthesis are inhibited by cycloheximide, and demonstrate DBCAMP-induced stimulation of both gastrin synthesis and secretion, suggesting the potentially important role of cyclic AMP in gastrin cell function.

Authors

Richard F. Harty, Jan C. van der Vijver, James E. McGuigan

Metabolic control of circulation. Effects of iodoacetate and fluoroacetate.

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The circulatory effects of selective metabolic inhibition of glycolysis and of the tricarboxylic acid cycle by iodoacetate and fluoroacetate were studied in intact chloralose-anesthetized dogs. Pulmonary arterial blood pressure and vascular resistance increased after administration of both inhibitors, but neither systemic hemodynamics nor myocardial contractility changed significantly. Coronary blood flow did not change after iodoacetate administration but increased four- to five-fold after fluoroacetate. Administration of normal saline had no effect on any of the parameters. The changes in pulmonary arterial blood pressure and coronary blood flow after fluoroacetate were not mediated via the autonomic nerves or adrenergic neurohumors because they still occurred after autonomic nervous system inhibition. Neither myocardial oxygen consumption nor left ventricular work changed. A selective increase in myocardial blood flow also occurred in conscious dogs after fluoroacetate administration; hepatic artery flow was reduced, but other organ flows did not change significantly. These results indicate that pulmonary pressor and coronary dilator effects may be produced in intact dogs by selective metabolic blockade, in the absence of reduced oxygen supply or impairment in the electron transport system. These results also suggest that the increases in pulmonary arterial blood pressure, coronary blood flow, and cardiac output that occur during hypoxia probably are related to separate metabolic events in the tissue.

Authors

C S Liang

Modulation of in vitro erythropoiesis. The influence of beta-adrenergic agonists on erythroid colony formation.

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Canine marrow erythroid colony growth is enhanced by agents linked to the adenyl cyclase/cyclic AMP (cAMP) system, including cAMP, a phosphodieterase inhibitor (RO-20-1724), cholera enterotoxin, and beta-adrenergic agonists. The adrenergic effect is mediated by receptors having beta2-subspecificity. These receptors are distinct from putative receptors for erythropoietin and those acted upon by cholera enterotoxin. In addition, the population of cells most responsive to beta-agonists is distinct from the majority of erythropoientin-responsive cells, perhaps representing a subpopulation of this class of cell. This demonstration of an adenyl cyclase-linked mechanism regulating mammalian erythroid colony growth provides a model for the modulation by other hormones or small molecules of in vitro and, perhaps, in vivo erythropoiesis.

Authors

J E Brown, J W Adamson

Interaction between High Density and Low Density Lipoproteins during Uptake and Degradation by Cultured Human Fibroblasts

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High density lipoprotein (HDL) inhibited the binding (trypsin-releasable radioactivity), internalization (cell-associated radioactivity after trypsinization), and degradation (TCA-soluble non-iodide radioactivity) of 125I-low density lipoprotein (125I-LDL) by cultured normal human fibroblasts. At HDL:LDL molar ratios of 25:1 (protein ratios about 5:1), these parameters were reduced by about 25%. Unlabeled LDL was about 25 times more effective in reducing 125I-LDL binding, implying that if HDL and LDL bind at common sites the affinity of HDL for these sites is very low or that the interaction is on some other basis. The fractional reduction in 125I-LDL binding at a given HDL: 125I-LDL ratio was independent of 125I-LDL concentration and occurred equally with fibroblasts from a subject with homozygous familial hypercholesterolemia. Reciprocally, the binding, internalization, and degradation of 125I-HDL were reduced by LDL. Preincubation of fibroblasts with HDL (or LDL) reduced the subsequent binding of 125I-LDL (or 125I-HDL) during a second incubation. In other studies HDL reduced the net increase in cell cholesterol content induced by incubation with LDL. HDL alone had no net effect on cell cholesterol content.

Authors

N. E. Miller, D. B. Weinstein, T. E. Carew, T. Koschinsky, D. Steinberg

Dysfibrinogenemia Associated with Liver Disease

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To test the possibility that a functionally abnormal fibrinogen may exist in some patients with liver disease, we studied the plasma and purified fibrinogens of five patients whose plasma thrombin times were prolonged at least 40% over normal controls. In no patient was there evidence of disseminated intravascular coagulation and/or fibrinolysis. No abnormalities were detected by immunoelectrophoresis of plasmas or purified fibrinogens. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of reduced patient fibrinogens showed normal mobility and amount of Aα, Bβ, and γ chains. Alkaline polyacrylamide gel electrophoresis and gradient elution, DEAE-cellulose chromatography of admixtures of radio-iodinated patient 125I-fibrinogen and normal 131I-fibrinogen showed identical mobility in the gel and simultaneous elution from the column, respectively. Thrombin and Reptilase (Abbott Scientific Products Div., Abbott Laboratories, South Pasadena, Calif.) times of purified patient fibrinogens were prolonged, and calcium ions improved but did not completely correct these defects. Increasing amounts of thrombin progressively shortened the clotting times of patient fibrinogens but not to the level of normal. Addition of equal amounts of patient fibrinogen to normal fibrinogen resulted in a prolongation of the thrombin time of the normal protein. Thrombin-induced fibrinopeptide release was normal. Fibrin monomers prepared from patient plasmas and purified fibrinogens demonstrated impaired aggregation at low (0.12) and high (0.24) ionic strength. These studies demonstrate that some patients with liver disease and prolonged plasma thrombin times have a dysfibrinogenemia functionally characterized by an abnormality of fibrin monomer polymerization.

Authors

Joseph E. Palascak, Jose Martinez

Effect of ascorbic acid on arylsulfatase activities and sulfated proteoglycan metabolism in chondrocyte cultures.

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A correlation between increased arylsulfatase activities and decreased sulfated proteoglycan content in human osteoarthritic articular cartilage suggested a possible interrelationship between these parameters. Since we had previously shown that ascorbate caused a decrease in levels of arylsulfatase A and B activities in normal chondrocyte cultures, the validity of the above relationship was examined by measuring the effect of vitamin C on the biosynthesis and distribution of 35S-labeled proteoglycans and arylsulfatase A and B activities in cell extracts of chondrocytes derived from normal and osteoarthritic tissue. Arylsulfatase A and B activities were found to be reduced in the presence of ascorbic acid in all normal and osteoarthritic cell lines examined when measured 3, 6, 10, and 13 days after the introduction of the vitamin in the culture medium. Acid phosphatase activity, on the other hand, was found to be elevated in the presence of ascorbate. The inhibitory effect by ascorbic acid on arylsulfatase activities could be reversed by withdrawing the vitamin from the nutrient medium. Addition of EDTA to the cell extracts before assay also reversed the inhibiton. Sulfated proteoglycan biosynthesis as reflected in 35S-sulfate uptake per milligram of DNA was significantly increased in the presence of ascorbic acid. The distribution of the newly synthesized molecules between the cell layer and medium fractions was altered. In the presence of ascorbate, more deposition into the cell layer of newly synthesized macromolecules occurred. These data suggest an inverse relationship between arylsulfatase activities and the stability of the newly synthesized sulfated proteoglycans in the extracellular matrix.

Authors

E R Schwartz, L Adamy

The Rate of Synthesis of Glycosaminoglycans and Collagen by Fibroblasts Cultured from Adult Human Liver Biopsies

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Adult human liver biopsies were cultured from normal, alcoholic hepatitis, chronic active hepatitis, fibrosis plus alcoholic hepatitis (active cirrhosis), inactive cirrhosis, and drug hepatitis. The synthesis of collagen was estimated in cultures from 58 livers by measuring the conversion of [14C]proline to the [14C]hydroxyproline of collagen; that of glycosaminoglycans in cultures from 57 livers by the incorporation of [3H]acetate and 35SO4 into glycosaminoglycans (GAG). The synthesis of procollagen was increased only in cultures from alcoholic hepatitis, both in the pulse medium (P < 0.05) and in the chase medium (P < 0.02). The synthesis of insoluble collagen was increased in cultures from chronic (active) hepatitis (P < 0.01), fibrosis plus alcoholic hepatitis (active cirrhosis) (P < 0.001), and inactive cirrhosis (P < 0.05). Essentially all radioactive GAG was soluble in culture media. The predominant GAG were chondroitin-4 or -6-SO4. The synthesis of GAG was increased only in cultures from fibrosis plus alcoholic hepatitis (active cirrhosis) both in the pulse medium (P < 0.01) and chase medium (P < 0.001).

Authors

John T. Galambos, Martin A. Hollingsworth Jr., Arthur Falek, W. Dean Warren, J. R. McCain

Decreased Fluidity of Red Cell Membrane Lipids in Abetalipoproteinemia

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Acanthocytic red cells in patients with abetalipoproteinemia are morphologically similar to the red cells in spur cell anemia. Fluidity of membrane lipids is decreased in spur cells due to their excess cholesterol content. Acanthocyte membranes have an increased content of sphingomyelin and a decreased content of lecithin. To assess the effect of this abnormality of acanthocyte membrane lipid composition on membrane fluidity, we studied red cells from five patients with abetalipoproteinemia and four obligate heterozygote family members.

Authors

Richard A. Cooper, John R. Durocher, Mary H. Leslie

Big and Little Forms of Osteoclast Activating Factor

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We have further characterized osteoclast activating factor (OAF) using a bioassay for bone resorption which utilizes the release of previously incorporated 45Ca from fetal rat long bones in organ culture. When supernatant media from activated leukocyte cultures were concentrated on Amicon PM10 membranes (assigned molecular weight cutoff 10,000 daltons) and chromatographed on Sephadex G-50 columns, the bone-resorbing activity eluted between the molecular weight markers chymotrypsinogen (25,000 daltons) and cytochrome c (12,500 daltons). This peak of biological activity has been called big OAF. When filtrates from the PM10 membranes were concentrated on Amicon UM2 membranes (assigned molecular weight cutoff 1,000 daltons) and chromatographed on Sephadex G-50 columns, some of the biological activity eluted between the molecular weight markers chymotrypsinogen and cytochrome c (big OAF), but there was a separate peak of biological activity which eluted with [3H]proline (140 daltons). This second peak has been called little OAF. Little OAF was eluted from Bio-Gel P6 columns between the molecular weight markers calcitonin (approximately 3,500 daltons) and vitamin B12 (1,330 daltons), but was retained by Spectrapor dialysis tubing (nominal molecular weight cutoff 3,500 daltons). Big OAF was converted to little OAF by equilibration in 1 M NaCl or 2 M urea. Little OAF was self-associated back to big OAF by equilibration in buffers of low ionic strength (Tris-HCl 10-50 mM). Little OAF was extracted into the organic phase in ethyl acetate after acidification of the sample to pH 3.5. The biological activity remained in the aqueous phase after ethyl acetate extraction at pH 7.5-8.4. Little OAF has been purified more than 6,000-fold compared with the original material so that bone-resorbing activity is maximal in a sample with a protein concentration of 80 ng/ml.

Authors

Gregory R. Mundy, Lawrence G. Raisz, James L. Shapiro, Janet G. Bandelin, Robert J. Turcotte

Urinary kallikrein and plasma renin activity as determinants of renal blood flow. The influence of race and dietary sodium intake.

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We investigated the relationship of the kallikrein-kinin system and the renin-angiotensin system in the regulation of blood pressure, salt and water excretion, and renal blood flow. Normotensive and hypertensive black and white men were studied during unresticted sodium intake as well as on a 10-meq/day sodium intake; potassium intake was held constant throughout the study (80 meq/day). During unrestricted sodium intake, urinary kallikrein activity was greater in white normotensives than white hypertensives or black normotensives. There was no difference (P greater than 0.05) between white and black hypertensives or between black normotensives and black hypertensives. All groups had greater urinary kallikrein activity on low sodium vs. unrestricted sodium intake, but the increase in black hypertensives was small, and they excreted significantly less kallikrein than the ogher groups on the low sodium diet. Plasma renin activity showed similar increments after sodium restriction in all groups. Urinary kallikrein activity correlated with renal blood flow in all groups except the black normotensives on low sodium intake. Renal blood flow could be correlated uniformly with log (urinary kallikrein activity/supine plasma renin activity) in all groups on either diet. Urinary sodium and potassium excretion and urine volume were not different among the groups. We conclude: (a) important racial differences exist in urinary kallikrein activity that are unrelated to sodium or potassium excretion or urine volume; (b) dietary sodium restriction further delineates racial differences and suggests alternative pathophysiologic mechanisms for huma hypertension; (c) urinary kallikrein activity correlates with renal blood flow; and (d) our data support the concept that the kallikrein-kinin system and the renin-angiotensin system contribute to the regulation of renal blood flow and may account for racial differences in renal vascular resistance.

Authors

S B Levy, J J Lilley, R P Frigon, R A Stone

Peripheral airways as a determinant of ventilatory function in the human lung.

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We have investigated the morphological differences responsible for the variability in two tests of pulmonary function, maximal expiratory flow rates (MEF) and the frequency dependence of dynamic compliance (CDYN ratio). Functional measurements were obtained from 53 normal and minimally diseased postmortem human lungs. Morphological measurements performed on these same lungs included airway diameter at three levels in the bronchial tree, the amount of bronchial gland mass, and the alveolar surface to volume ratio. Multiple regression analysis suggests that the diameter of the peripheral conduction airways (membranous bronchioles) is the major morphological determinant for both MEF and the CDYN ratio in lungs at any particular age. Age-dependent changes in both functional tests were associated primarily with differences in the alveolar surface to volume ratio. Minimal emphysema and a lesion associated with cigarette smoking, respiratory bronchiolitis, have no demonstrable effect on either MEF or the CDYN ratio. These studies provide further evidence that the peripheral conducting airways are a major determinant of ventilatory function in the normal human lung.

Authors

D E Niewoehner, J D Knoke, J Kleinerman

Mechanisms of the Puromycin-Induced Defects in the Transglomerular Passage of Water and Macromolecules

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To investigate the mechanism(s) of increased filtration of serum proteins after glomerular injury, polydisperse samples of uncharged [3H]dextran (D) or anionic [3H]dextran sulfate (DS) were infused into 14 control and 16 puromycin aminonucleoside- (PAN) treated Munich-Wistar rats. Fractional clearances of D or DS ranging in radius from 18 to 42Å were determined in these rats, together with direct measurements of the forces governing the glomerular filtration rate of water. Whole kidney and single nephron glomerular filtration rates were ∼40% lower in PAN-treated rats, relative to controls, due mainly to a marked reduction in the glomerular capillary ultrafiltration coefficient and, to a lesser extent, to a small reduction in glomerular plasma flow rate as well. In PAN-treated rats, as in normal controls, inulin was found to permeate the glomerular capillary wall without measurable restriction, and both D and DS were shown to be neither secreted nor reabsorbed. Fractional clearances of uncharged D were reduced after PAN administration, falling significantly for effective D radii from 22 to 38Å. Utilizing a theory based on macromolecular transport through pores, these results indicate that in PAN-treated rats, effective pore radius is the same as in controls, ∼44Å. In PAN nephrosis, however, the ratio of total pore surface area/pore length, a measure of pore density, is reduced to approximately one-third that of control, due very likely to a reduction in filtration surface area. In contrast to the results with uncharged D, fractional clearances of DS were found to increase after PAN administration for all DS radii studied. These results with D and DS suggest that proteinuria in PAN nephrosis is due, not to an increase in effective pore radius or number of pores, but rather to a diminution of the electrostatic barrier function of the glomerular capillary wall, thereby allowing increased passage of polyanions such as DS and albumin.

Authors

Michael P. Bohrer, Christine Baylis, Channing R. Robertson, Barry M. Brenner, Julia L. Troy, Wayne T. Willis

Effects of Acetazolamide on Proximal Tubule Cl, Na, and HCO₃ Transport in Normal and Acidotic Dogs during Distal Blockade

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It has been suggested that the establishment of a tubular fluid to plasma chloride gradient in the late proximal tubule by the reabsorption of bicarbonate (and other anions) in the early proximal tubule is responsible for a significant part of sodium chloride and water reabsorption in the proximal tubule. In the present study the effects of acetazolamide on proximal tubule water and electrolyte excretion were examined in 6 normal dogs and 10 chronic ammonium chloride-loaded dogs during distal blockade produced by ethacrynic acid and chlorothiazide administration. During distal blockade control urine/plasma osmolality and urine/plasma sodium were close to unity in all experiments. Urine/plasma chloride and urine/plasma bicarbonate were 1.21±0.02 and 0.75±0.07 in normal and 1.24±0.01 and 0.04±0.01 in acidotic dogs, respectively. After the administration of acetazolamide (20 mg/kg i.v.), there was a significant increase in urine flow, absolute and fractional excretion of sodium, bicarbonate, and chloride in all animals. Associated with these effects, urine/plasma osmolality and urine/plasma sodium remained unchanged but urine/plasma chloride decreased significantly to 1.15±0.01 in normal and to 1.19±0.01 in acidotic dogs. In acidotic dogs there was a significant correlation between the increase in bicarbonate, sodium, or chloride excretion after acetazolamide and the plasma bicarbonate level (range 6.8-12.5 meq/liter). These data demonstrate a significant effect of acetazolamide on bicarbonate, sodium, and chloride reabsorption in the proximal tubule even in the face of severe acidosis. Moreover, the data suggest that the decrease in chloride reabsorption (and accompanying sodium) after acetazolamide is related to the decrease in bicarbonate reabsorption and the associated decrease in the transtubular chloride gradient.

Authors

Shyan-Yih Chou, Jerome G. Porush, Paul A. Slater, Carlos D. Flombaum, Tahir Shafi, Paul A. Fein

Radioimmunoassay of Human Apolipoprotein CII: A STUDY IN NORMAL AND HYPERTRIGLYCERIDEMIC SUBJECTS

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A specific, precise, and sensitive double-antibody radioimmunoassay for the measurement of human apolipoprotein CII (apoCII) was developed. ApoCII was labeled with 125I (chloramine-T) and monospecific antibody was raised in rabbits. No appreciable cross-reactivity with apolipoproteins CI, CIII, AI, AII, low density lipoproteins, and lipoprotein-free plasma was observed. Lipoproteins containing apoCII displaced the standard curve in parallel. ApoCII measurement was not affected by pretreatment of plasma with tetramethylurea, ethanol-diethyl ether, or heating.

Authors

M. L. Kashyap, L. S. Srivastava, C. Y. Chen, G. Perisutti, M. Campbell, R. F. Lutmer, C. J. Glueck

The Intestinal Brush Border Membrane in Diabetes: STUDIES OF SUCRASE-ISOMALTASE METABOLISM IN RATS WITH STREPTOZOTOCIN DIABETES

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Diabetes stimulates the functional activity of the intestinal brush border membrane with enhancement of both hydrolytic enzyme activity and membrane transport systems. To determine the mechanism of this effect, we studied the effects of streptozotocin diabetes on the metabolism of one membrane protein, sucrase-isomaltase, which increases its activity in diabetes. The protein was purified and an antiserum prepared. Sucrase-isomaltase from control and diabetic rats was immunologically identical as shown by Ouchterlony double-diffusion analysis of papain-solubilized mucosal proteins. The increase in sucrase enzyme activity in diabetic animals (31.0±1.4 U SEM 5 days after streptozotocin vs. 13.1±1.0 in controls) was the consequence of increased enzyme protein and not an alteration in catalytic efficiency as demonstrated by quantitative immunoprecipitin reactions.

Authors

Ward A. Olsen, Helen Korsmo

Cold Urticaria: RECOGNITION AND CHARACTERIZATION OF A NEUTROPHIL CHEMOTACTIC FACTOR WHICH APPEARS IN SERUM DURING EXPERIMENTAL COLD CHALLENGE

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Sera were obtained from the venous effluents of cold-challenged arms of patients with idiopathic cold urticaria without plasma or serum cryoproteins; these sera exhibited increased neutrophil chemotactic activity without alterations of the complement system. A two- to fourfold augmentation of the base-line neutrophil chemotactic activity of serum from the immersed extremity began within 1 min, peaked at 2 min, and returned to base-line levels within 15 min, whereas there was no change in the serum chemotactic activity in the control arm. The augmented chemotactic activity in the serum specimens from the challenged arm of each patient appeared in a high molecular-weight region, as assessed by the difference in activity recovered after Sephadex G-200 gel filtration of the paired lesional and control specimens. Sequential purification of this high molecular-weight activity by anion- and cation-exchange chromatography revealed a single peak of activity at both steps. The partially purified material continued to exhibit a high molecular weight, being excluded on Sepharose 4B, and had a neutral isoelectric point. The partially purified material showed a preferential chemotactic activity for neutrophilic polymorphonuclear leukocytes, required a gradient for expression of this function, and exhibited a capacity to deactivate this cell type. This active principle, termed high molecular-weight neutrophil chemotactic factor, exhibited a time-course of release that could be superimposed upon that of histamine and the low molecular-weight eosinophil chemotactic factor and may represent another mast cell-derived mediator.

Authors

Stephen I. Wasserman, Nicholas A. Soter, David M. Center, K. Frank Austen

Spatial and Nonspatial Influences on the TQ-ST Segment Deflection of Ischemia: THEORETICAL AND EXPERIMENTAL ANALYSIS IN THE PIG

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Spatial and nonspatial aspects of TQ-ST segment mapping were studied with the solid angle theorem and randomly coded data from 15,000 electrograms of 160 anterior descending artery occlusions each of 100-s duration performed in 18 pigs. Factors analyzed included electrode location, ischemic area and shape, wall thickness, and increases in plasma potassium (K+). Change from control in the TQ-ST recorded at 60 s (ΔTQ-ST) was measured at 22 ischemic (IS) and nonischemic (NIS) epicardial sites overlying right (RV) and left (LV) ventricles. In IS regions, ΔTQ-ST decreased according to LV > septum > RV and LV base > LV apex. In NIS regions, LV sites had negative (Neg) ΔTQ-ST which increased as LV IS border was approached. However, RV NIS had positive (Pos) ΔTQ-ST which again increased as RV IS border was approached. With large artery occlusion IS area increased 123±18%, ΔTQ-ST at IS sites decreased (−38.1±3.6%), and sum of ΔTQ-ST at IS sites increased by only 67.3±10.3%. In RV NIS Pos ΔTQ-ST became Neg. With increased K+, ΔTQ-ST decreased proportionately to log K+ (r = 0.97±0.01) at IS and NIS sites on the epicardium and precordium. TQ-ST at 60 s was obliterated when K+ = 8.7±0.2 mM. All findings were significant (P < 0.005) and agreed with the solid angle theorem. Thus, a transmembrane potential difference and current flow at the IS boundary alone are responsible for the TQ-ST. Nonspatial factors affect the magnitude of transmembrane potential difference, while spatial factors alter the position of the boundary to the electrode site.

Authors

Roger P. Holland, Harold Brooks, Barbara Lidl

Prostaglandin biosynthesis by rabbit renomedullary interstitial cells in tissue culture. Stimulation by angiotensin II, bradykinin, and arginine vasopressin.

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Abstract

Rabbit renomedullary interstitial cells were isolated and grown in tissue culture. These cells synthesize 0.8 ng of prostaglandin E2 (PGE2) per microgram cellular protein per hour in monolayer tissue culture; prostaglandins A2 and F2alpha (PGA2 and PGF2alpha) biosynthesis was 10 and 5% of PGE2 biosynthesis, respectively. Arachidonic acid markedly stimulated the production of PGE2 and PGF2alpha, with conversion rates of 0.24 and 0.02%/h, respectively. Angiotensin II, hyperosmolality, bradykinin, and arginine vasopressin each stimulated PGE2 biosynthesis; maximum stimulation was 20, 3.7, 3.6, and 3.2 times basal production, respectively. PGE2 biosynthesis by the renomedullary interstitial cells was inhibited by isoproterenol, potassium, nonsteroidal anti-inflammatory agents (indomethacin, naproxen, ibuprofen, suprofen, meclofenamate, and acetylsalicylic acid), mepacrine (a phospholipase inhibitor), hydrocortisone, and cortisone. The rabbit renomedullary interstitial cell in tissue culture is a model system for the study of hormonal regulation of PGE2 biosynthesis.

Authors

R M Zusman, H R Keiser

Glucagon and Insulin Binding to Liver Membranes in a Partially Nephrectomized Uremic Rat Model

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Abstract

To investigate the role of glucagon and insulin receptor binding in the glucagon hypersensitivity and insulin resistance which characterize the glucose intolerance of uremia, liver plasma membranes were prepared from control rats (blood urea nitrogen [BUN] 15±1 mg/100 ml, creatinine 0.7±0.2 mg/100 ml), and from 70% nephrectomized rats (BUN 30±2 mg/100 ml, creatinine 2.2±0.2 mg/100 ml), and from 90% nephrectomized rats (BUN 46±3 mg/100 ml, creatinine 4.20±0.7 mg/100 ml), 4 wk after surgery. As compared to controls, the 90% nephrectomized rats had significantly higher levels of plasma glucose (95±4 vs. 125±11 mg/100 ml), plasma insulin (28±9 vs. 52±11 μU/ml), and plasma glucagon (28±5 vs. 215±18 pg/ml). Similar, but less marked, elevations were observed in the 70% nephrectomized animals.

Authors

Vijay Soman, Philip Felig

The Occurrence and Effects of Human Vitamin E Deficiency: A STUDY IN PATIENTS WITH CYSTIC FIBROSIS

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Abstract

The role of vitamin E in human nutrition was studied by investigation of patients with cystic fibrosis (CF) and associated pancreatic insufficiency. Vitamin E status was assessed by measurement of the plasma concentration of the principal circulating isomer, α-tocopherol. Results of such determinations in 52 CF patients with pancreatogenic steatorrhea revealed that all were deficient in the vitamin. The extent of decreased plasma tocopherol varied markedly but correlated with indices of intestinal malabsorption, such as the serum carotene concentration and percentage of dietary fat absorbed. Supplementation with 5-10 times the recommended daily allowance of vitamin E in a water-miscible form increased the plasma α-tocopherol concentrations to normal in all 19 CF patients so evaluated.

Authors

Philip M. Farrell, John G. Bieri, Joseph F. Fratantoni, Robert E. Wood, Paul A. di Sant'Agnese

Isolation and Characterization of an Abnormal High Density Lipoprotein in Tangier Disease

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Abstract

The nature of the high density lipoproteins has been investigated in five patients homozygous for Tangier disease (familial high density lipoprotein deficiency). It has been established that Tangier high density lipoproteins, as isolated by ultracentrifugation, are morphologically heterogenous and contain several proteins (Apo B, albumin, and Apo A-II). An abnormal lipoprotein has been isolated from the d = 1.063-1.21 g/ml ultracentrifugal fraction by agarose-column chromatography which contains apoprotein A-II as the sole protein constituent. In negative-stain electron microscopy, these lipoproteins appeared as spherical particles 55-75 Å in diameter. By a variety of criteria (immunochemical, polyacrylamide electrophoresis, amino acid composition, and fluorescence measurements), apoprotein A-I the major apoprotein of normal high density lipoproteins and the C apoproteins were absent from this lipoprotein. As demonstrated by 125I very low density lipoprotein incubation experiments with Tangier plasma, C apoproteins did not associate with lipoproteins of d = 1.063-1.21 g/ml. Tangier apoprotein A-II, isolated to homogeneity by delipidation of the apoprotein A-II-containing lipoprotein or Sephadex G-200 guanidine-HCl chromatography of the d = 1.063-1.21 g/ml fraction, was indistinguishable from control apoprotein A-II with respect to amino acid composition and migration of tryptic peptides in urea-polyacrylamide electrophoresis. The ability of Tangier apoprotein A-II to bind phospholipid was demonstrated by in vitro reconstitution experiments and morphological and chemical analysis of lipid-protein complexes.

Authors

Gerd Assmann, Peter N. Herbert, Donald S. Fredrickson, Trudy Forte

Intestinal Calcium Absorption in Exogenous Hypercortisonism: ROLE OF 25-HYDROXYVITAMIN D AND CORTICOSTEROID DOSE

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Abstract

Pharmacologic doses of corticosteroids impair intestinal calcium absorption and contribute to negative calcium balance. However, the relationship between the impaired calcium absorption and a possible defect in the conversion of vitamin D to its physiologically active form, 1,25-dihydroxyvitamin D, is unknown. We compared fractional calcium absorption (double-isotope method, 100-mg carrier) and serum 25-hydroxyvitamin D (25-OH-D) (Haddad method) in 27 patients receiving pharmacologic doses of prednisone with 27 age-, sex-, and season-matched normal subjects. In patients receiving high daily doses of prednisone (15-100 mg/day), calcium absorption (P < 0.02) and serum 25-OH-D (P < 0.001) were decreased. However, in patients receiving low doses (8-10 mg/day) or high doses (30-100 mg) of prednisone on an alternate-day schedule, both of these parameters were normal. Calcium absorption in the patients treated with daily prednisone correlated inversely with the dose of corticosteroids (r = −0.52, P < 0.025) and, in all steroid-treated patients, correlated directly with serum 25-OH-D (r = 0.58, P < 0.01). In four patients who received high-dose corticosteroid therapy for an average of 4 wk, serum 25-OH-D decreased by 35.5% from pretreatment values. Administration of a physiologic or near-physiologic dose of synthetic 1,25-dihydroxyvitamin D3 (0.4 μg daily for 7 days) to patients receiving high-dose corticosteroids led to an increase in calcium absorption in all patients. These results suggest that calcium malabsorption in the corticosteroid-treated patients is due to a dose-related abnormality of vitamin D metabolism and not to a direct effect of corticosteroids on depressing transmucosal intestinal absorption of calcium.

Authors

Robert G. Klein, Sara B. Arnaud, J. C. Gallagher, Hector F. Deluca, B. Lawrence Riggs

Complement (C5-a)-induced granulocyte aggregation in vitro. A possible mechanism of complement-mediated leukostasis and leukopenia.

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Abstract

Activated plasma complement will induce biphasic aggregation of human granulocytes dectable by standard nephelometric techniques. The responsible active component was suggested to be C5a by molecular weight and heat-stability assays; moreover, aggragating activity was ablated by anti-C5 but not anti-C3 antibodies. C5a prepared by trypsinization of purified C5 reproduced the aggregating activity of whole activated plasma, whereas plasma from a C5-deficient donor did not support aggregation. Embolization of granulocyte aggregates might be a previously unsuspected cause of leukostasis and pulmonary damage in various clinical situations where intravascular complement activation occurs.

Authors

P R Craddock, D Hammerschmidt, J G White, A P Dalmosso, H S Jacob

A possible role for malonyl-CoA in the regulation of hepatic fatty acid oxidation and ketogenesis.

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Abstract

Studied on the oxidation of oleic and octanoic acids to ketone bodies were carried out in homogenates and in mitochondrial fractions of livers taken from fed and fasted rats. Malonyl-CoA inhibited ketogenesis from the former but not from the latter substrate. The site of inhibition appeared to be the carnitine acyltransferase I reaction. The effect was specific and easily reversible. Inhibitory concentrations were in the range of values obtained in livers from fed rats by others. It is proposed that malonyl-CoA functions as both precursor for fatty acid synthesis and suppressor of fatty acid oxidation. As such, it might be an important element in the carbohydrate-induced sparing of fatty acid oxidation.

Authors

J D McGarry, G P Mannaerts, D W Foster