This study was designed to investigate the pathogenesis of type I (distal) renal tubular acidosis.
M. L. Halperin, M. B. Goldstein, A. Haig, M. D. Johnson, B. J. Stinebaugh
To study limb vascular responses to K+ in man, paired intrabrachial arterial infusions of isosmolar NaCl (control) and isosmolar KCl (0.077, 0.154, and 0.307 meq K+/min) in isosmolar NaCl were made in 20 normotensive men and 20 men with essential hypertension of mild to moderate severity. Limb blood pressures were monitored, limb blood flow was measured by indicator-dilution, and limb vascular resistance was calculated as mm Hg/ml flow/min/100 cm3 limb volume. Measured concentrations of K+ in limb venous plasma during infusion of 0.307 meq K+/min ranged from 4.8 to 9.0 meq/liter. Changes in limb venous hematocrit, sodium, calcium, magnesium, and osmolality were similar during control and KCl infusions. The infusions did not significantly change systemic blood K+ concentration or blood pressures. Compared to NaCl, KCl decreased limb resistance (P < 0.05) in both normotensives and hypertensives, in a dose-related manner. Resting limb vascular resistances (IR) in hypertensives were greater (P < 0.05) than those in normotensives. Despite a positive correlation (P < 0.05) between IR and magnitude of response to K+, responses in hypertensives to K+ were not greater than those in normotensives. Further, analysis of covariance indicated that responses to 0.307 meq K+/min in hypertensives as a group were, in fact, less (P = 0.02) than those in normotensives. These results indicate that the vasodilator response to K+ may be attenuated in a significant proportion of essential hypertensive men, as it is in renal hypertensive animals. These abnormal responses to K+ in hypertensives may indicate an underlying defect in vascular K+ metabolism.
Henry W. Overbeck, Randall S. Derifield, Motilal B. Pamnani, Tümay Sözen
Ethacrynic acid (EA) has been reported to reduce cholera toxin-induced intestinal fluid secretion in the intact animal. We explored the nature of this inhibition in vitro by measuring unidirectional, transmural fluxes of 22Na and 36Cl across isolated rabbit ileal mucosa. Under control conditions (short-circuited mucosa bathed in bicarbonate-Ringer), there was net absorption of Na and Cl. Theophylline (10 mM), cyclic AMP (5 mM), and cholera toxin (added in vivo) abolished net Na flux and produced net Cl secretion. In the presence of either theophylline or cAMP, addition of 0.1 mM EA to the serosal bathing solution abolished net Cl secretion and restored net Na absorption. Cholera toxin-treated mucosa was exposed to 0.05 and 1.0 mM EA. The lower concentration restored net Na absorption but did not significantly reduce Cl secretion. The higher concentration abolished net transport of both Na and Cl. Short-circuit current and Na flux measurements in the presence and absence of glucose indicated that 0.1 mM EA does not inhibit glucose-coupled Na transport. Short-circuit current measurements in the presence of 1.0 mM EA suggested that even this concentration of EA does not inhibit glucose-coupled Na transport. Thus EA appears to specifically inhibit Cl (or NaCl) secretion without inhibiting the absorptive Na “pump.” The anti-secretory effect of 0.1 mM EA does not appear to result from inhibition of adenylate cyclase since secretion stimulated by addition of 5 mM cAMP was abolished. Furthermore, 0.1 mM EA did not significantly reduce theophylline-augmented and cholera toxin-augmented cAMP levels in ileal mucosa. We conclude that EA interacts specifically with the active Cl (or NaCl) secretory mechanism of the small intestine at a step beyond generation of cAMP.
Qais Al-Awqati, Michael Field, William B. Greenough III
The plasma pyridoxal-5′-phosphate (PLP) level of alcoholic subjects has been compared with that of non-alcoholic individuals in order to ascertain the incidence of abnormal vitamin B6 metabolism in chronic alcohol abuse. 66 alcoholic subjects were selected on the basis that they did not exhibit abnormal liver function tests and hematologic findings. 35 of them had plasma PLP concentrations less than 5 ng/ml, the lowest value encountered in 94 control subjects, indicating a high incidence of deranged PLP metabolism in alcoholic patients even when hepatic and hematologic abnormalities are absent. The biochemical basis for the altered PLP metabolism in chronic alcohol abuse was examined. Low plasma PLP levels in alcoholics were not accompanied by decreased pyridoxal kinase and pyridoxine phosphate oxidase activities in erythrocytes. Further studies with erythrocytes demonstrated that the cellular content of PLP is determined not only by the activities of these PLP-synthesizing enzymes but also by the activity of a phosphate-sensitive, membrane-associated, neutral phosphatase, which hydrolyzes phosphorylated B6 compounds.
Lawrence Lumeng, Ting-Kai Li
We studied the influence of prolonged exposure to hyperoxia (O2 > 98%) on protein synthesis and on the ultrastructure of the granular pneumocyte. To study protein synthesis, as indicated by l-[U-14C]-leucine incorporation into protein, lung slices were incubated with radioactive leucine and a surface-active fraction was obtained by ultracentrifugation of lung homogenates. We found that, following an initial depression in protein synthesis after 48 h of hyperoxia, protein synthesis in rats exposed to oxygen for 96 h rose to greater than control levels. This increase in protein synthesis was noted in whole lung protein and in protein present in the surface-active fraction.
Gloria D. Massaro, Donald Massaro
An in vitro system for perifusion of rat pancreatic islets has been utilized to define the effects of cholinergic agents on the dynamics of insulin release. In the absence of glucose the effects of either acetylcholine or acetyl-β-methylcholine were minimal at concentrations up to 10−5 mM. In the presence of low glucose concentration (2.4 mM), both of the muscarinic agents produced dose-dependent biphasic insulin release. Under these conditions significant insulin release was observed over both phases at concentrations of the muscarinic agents as low as 10−8 mM. Further, the dose response curves relating muscarinic concentration to the total amount of insulin released in each of the two phases showed lack of parallelism between the curves. Nicotinic acid in concentrations up to 10−5 mM had no effect on insulin release in the presence of 2.4 mM glucose. When the glucose concentration was increased to 16.4 mM, the effects of the muscarinic agents were significantly less than those observed in the presence of 2.4 mM glucose. This held true whether the effect was defined as absolute increment due to the muscarinic agent or as percentage of enhancement. Atropine inhibited insulin release induced by both acetylcholine and by 16.4 mM glucose. These data indicate that cholinergic stimulation can play a significant role in modifying insulin release patterns.
R. Sharp, S. Culbert, J. Cook, A. Jennings, I. M. Burr
The effects of calcium on the renal actions of parathyroid hormone (PTH) were studied in vivo and in vitro. In parathyroidectomized rats, variable levels of blood calcium concentration were induced by intravenous infusion of calcium. The renal responses to the injected PTH, i.e. phosphate and cyclic AMP excretion, were compared in these animals. After PTH injection, the increases of both phosphate and cyclic AMP excretion were less in the calcium-infused animals than in the control group without calcium infusion. There was an inverse correlation between the renal responses to PTH and plasma calcium concentration of 4.2-13.5 mg/100 ml. But calcium had no effect on phosphate excretion induced by infusion of dibutyryl cyclic AMP. In the in vitro experiments, the increase of cyclic AMP concentration in response to PTH was less in renal cortical slices taken from the calcium-infused animals than in ones from the control group without calcium infusion. Calcium also inhibited the activation of renal cortical adenylate cyclase in response to PTH, but calcium had no effect on phosphodiesterase. The data indicate that calcium directly inhibits renal actions of PTH both in vivo and in vitro. Such inhibitory mechanism is probably at or before the step of PTH-dependent cyclic AMP generation in the kidney.
Nama Beck, Harbans Singh, Sarah W. Reed, Bernard B. Davis
During phagocytosis, new phospholipid is synthesized from triglyceride fatty acid and may be utilized to form the membranes of phagocytic vesicles. In addition, hydrogen peroxide, which can peroxidize unsaturated fatty acids, is generated. Since both of these processes could change membrane fatty acid composition during the conversion of cytoplasmic granules and plasma membranes to phagosomes, the lipid compositions of these structures were examined. Phagocytic vesicles were prepared by density gradient centrifugation of polystyrene latex particles after phagocytosis. Granule and plasma membrane fractions were isolated by density gradient and differential centrifugation. Phospholipids and fatty acids were analyzed by thin-layer chromatography and gas-liquid chromatography.
James E. Smolen, Stephen B. Shohet
The in vivo and in vitro responses to ragweed antigen E were evaluated in 28 untreated atopic patients with ragweed hayfever. The methods employed included direct skin testing, measurement of total serum IgE, measurement of specific IgE anti-ragweed antibodies, leukocyte histamine release, lymphocyte transformation, and release of lymphocyte mediators (migration inhibitory factor and mitogenic factor). The patients could be divided into sensitive and insensitive groups on the basis of their in vitro reactivity to antigen E. 20 patients in the sensitive group had statistically higher levels of total serum IgE, higher levels of specific IgE anti-ragweed antibodies, and greater leukocyte sensitivity as measured by antigen-induced histamine release than did eight patients in the insensitive group. Lymphocytes from sensitive patients produced greater amounts of migration inhibitory factor and mitogenic factor when challenged by antigen E than did lymphocytes from insensitive patients. A possible role for the lymphocyte in this allergic disease is discussed. The results of this study indicate that the immune response to ragweed antigen is complex and involves components of both immediate and delayed hypersensitivity.
Ross E. Rocklin, Hobert Pence, Hyman Kaplan, Richard Evans
The influxes of Na+ and K+ into the human red cell appear to be interrelated. This relationship was investigated under conditions in which either Na+ or K+ concentration outside the cell was varied or one cation was replaced by Mg2+, choline+, or Li+. The effects of furosemide on Na+ and K+ movements were studied in the presence of ouabain.
James S. Wiley, Richard A. Cooper
Acute clearance studies were performed in thyroparathyroidectomized animals to determine the actions and interactions of thyrocalcitonin (TCT), cyclic adenosine 3′5′-monophosphate (cAMP), 25-hydroxycholecalciferol (25HCC), and calcium ion on the reabsorption of phosphate, calcium, sodium, and potassium by the kidney. The infusion of 25HCC in a dosage of 60 U/h to moderately saline-expanded animals (2.5% body weight) induced a fall in the excretion of all of the ions under study after 90-120 min similar to that observed in previous experiments from this laboratory. Mean decrements in fractional excretion were: phosphate, 42.0% (P < 0.005); calcium, 25.0% (P < 0.005); sodium, 23.4% (P < 0.001); and potassium, 14.7% (P < 0.005). The superimposition of either porcine or salmon TCT (1-100 MRC U/h for 2 h) resulted in no further alterations in electrolyte excretion. However, the infusion of TCT during steady-state saline expansion, before the administration of 25HCC, obviated the renal transport effects of the vitamin D metabolite. Both in the latter studies, as well as those in which similar doses of TCT were given to hydropenic animals, the hormone itself failed to induce any consistent alteration in electrolyte excretion. Cyclic AMP (50 mg/h) caused an increase in the excretion of phosphate, sodium, and potassium and no change in calcium excretion. Like TCT, the nucleotide blocked the action of 25HCC on the kidney. Raising the mean level of serum ultrafilterable calcium to 3.02±0.25 mEq/liter from 1.62±0.17 mEq/liter likewise prevented enhanced ionic reabsorption due to 25HCC.
Jules B. Puschett, William S. Beck Jr., Adam Jelonek, Pedro C. Fernandez
Further studies have been performed to define the kinetic characteristics of nuclear triiodothyronine (T3) binding sites in rat liver (J. Clin. Endocrinol. Metab. 1972. 35: 330). Sequential determination of labeled T3 associated with nuclei and cytoplasm over a 4-h period allowed analysis of the relationship of T3 in nuclear and cytoplasmic compartments. A rapid interchange of hormone between nuclei and cytoplasm was demonstrated, and in vitro incubation experiments with nuclei yielded no evidence favoring metabolic transformation of T3 by the nuclei. In vivo displacement experiments were performed by subcellular fractionation of liver ½ h after injection of [125I]T3 with increasing quantities of unlabeled T3. The nuclear binding capacity for T3 could be defined (0.52 ng/mg DNA). Analysis of these experiments also allowed an estimation of the association constant of nuclear sites for T3 (4.7 × 1011M−1). The affinity of these sites for T3 was estimated to be 20-40 fold greater than for thyroxine (T4). Chromatographic analysis of the nuclear radioactivity after injection of labeled T4 indicated that the binding of T4 by the nucleus could not be attributed to in vivo conversion of T4 to T3 but reflected intrinsic cross-reactivity of the two iodothyronines at the nuclear binding sites.
Jack H. Oppenheimer, Harold L. Schwartz, Diona Koerner, Martin I. Surks
Substances such as bilirubin that bind tightly to plasma proteins cannot readily be removed from blood. We describe here the use of affinity chromatography as a new approach to the removal of proteinbound metabolites and toxins from blood. Agarose beads were coupled via cyanogen bromide to human serum albumin so as to contain 30-50 mg of albumin/g wet wt. Such beads, when exposed to plasma from a patient with congenital nonhemolytic jaundice labeled with [14C]-bilirubin, bound more than 150 μg bilirubin/g of beads. The binding was saturable, concentration-dependent, relatively independent of flow rate, and reversible by elution with plasma, albumin, or 50% (vol/vol) ethanol. The beads could be repeatedly reused without loss of efficiency after ethanol elution and long storage in the cold. Salicylate, cortisol, and taurocholate, which bind weakly to albumin, were retarded by the beads but eluted with neutral buffer. Thyroxine, taurolithocholate, chenodeoxycholate, and digitoxin bound tightly but were eluted with 50% ethanol. Digoxin did not bind at all. When whole blood was passed over agarose-albumin beads, bilirubin was removed, calcium and magnesium fell slightly, but red cells, white cells, platelets, clotting factors, and a variety of electrolytes and proteins were substantially unchanged. Agarose-albumin beads may be useful for removing protein-bound substances from the blood of patients with liver failure, intoxication with protein-bound drugs, or specific metabolic deficits. Furthermore, it may be possible to make useful adsorbents by attaching other proteins to agarose or other polymer beads.
Paul H. Plotz, Paul D. Berk, Bruce F. Scharschmidt, Joyce Kay Gordon, John Vergalla
In vitro studies indicate that bilirubin and other albumin-bound substances can be efficiently removed from plasma by filtration over albumin-conjugated agarose beads. The effectiveness of this technique in vivo was investigated in rats by using a closed extracorporeal hemoperfusion system. Five Gunn rats whose endogenous bilirubin pool had been labeled with [3H]bilirubin and five Sprague Dawley rats with surgically created biliary obstruction were chosen as models of unconjugated and conjugated hyperbilirubinemia. Indocyanine green was injected into rats and its removal also studied. In the Gunn rats, 98% of the bilirubin was removed from plasma during the initial pass over the column as determined isotopically and chemically. Plasma bilirubin levels fell more than 70% from 8.2±1.6 mg/100 ml (mean±SD) to 2.6±0.5 mg/100 ml during a 1-h hemoperfusion. An average of 1,061 μg of bilirubin was recovered from the columns, representing 22.5±4.2% of the total exchangeable bilirubin pool and 96±36.4% of the plasma pool. Results were similar in the rats with biliary obstruction and in those given indocyanine green. Normal Sprague Dawley rats experience minimal changes in formed blood elements, electrolytes, and proteins as the result of hemoperfusion. When the total volume of the column did not exceed 51% of the estimated blood volume of the animal, the survival rate was 100% in 20 studies, and the procedure was without observable ill effect. Extrapolation of both in vitro and in vivo data to man suggests that extracorporeal hemoperfusion over albumin-agarose columns may be a practical means of assisting hepatic excretory function.
Bruce F. Scharschmidt, Paul H. Plotz, Paul D. Berk, Jeanne G. Waggoner, John Vergalla
Effects of the diuretic ethacrynic acid on osmotic water permeability were investigated in the isolated perfused collecting tubule of the rabbit kidney.
Hemolytic anemia caused by oxidative drugs is thought to result from the oxidation of intracellular and membrane sulfhydryl groups of the erythrocyte. This process is more likely to occur in those erythrocytes in which the intracellular mechanism for reduction of disulfides is abnormal (e.g., glucose-6-phosphate dehydrogenase deficiency). If a membrane sulfhydryl group is critical in the pathogenesis of druginduced hemolytic anemia, it follows that this specific group must be dependent on intracellular reductive mechanisms for maintenance of the reduced state.
Alvin Zipursky, Marlene Stephens, Elizabeth J. Brown, Per Larsen
Incubation of human leukocytes with ascorbic acid at neutral pH and at concentrations 10-50 times that of normal blood levels augmented both the in vitro random migration and chemotaxis of the cells by 100-300% without influencing their phagocytic capacity. Enhancement of mobility by ascorbate was evident for isolated neutrophils, eosinophils, and mono-nuclear leukocytes and was independent of the specific chemotactic stimulus. Stimulation by ascorbate of the hexose monophosphate shunt of adherent neutrophils and augmentation by ascorbate of neutrophil mobility had comparable dose-response relationships, could be reversed by washing the cells, and were both suppressed by preincubation of the neutrophils with 6-aminonicotinamide, but not with the neutrophil-immobilizing factor. Glutathione, the proposed intermediate for ascorbate action, similarly stimulated hexose monophosphate shunt activity and enhanced migration. The enhancement in vitro of leukocyte mobility by ascorbate at concentrations found in some normal tissues, therefore, appears to be dependent upon stimulation of the leukocyte hexose monophosphate shunt.
Edward J. Goetzl, Stephen I. Wasserman, Irma Gigli, K. Frank Austen
Total and unbound testosterone and Δ4-androstenedione have been determined in 104 cord blood samples. The same sexual steroids and pituitary gonadotropins have been measured in 46 normal male infants aged 27-348 days and 34 normal female infants aged 19-332 days.
Maguelone G. Forest, Pierre C. Sizonenko, Anne M. Cathiard, Jean Bertrand
Bacterial endocarditis was produced by intravenous injection of Streptococcus viridans into rabbits with preexisting sterile endocardial vegetations. After 6 h had elapsed, bacteria in the vegetations could not be eradicated by brief treatment with antimicrobials to which the streptococci were sensitive. However, when treatment with penicillin was continued for 4 days, the animals were cured. The 6-h infection therefore offered a model in which treatments could be conveniently compared over a short period. Synergism was demonstrated between penicillin and streptomycin in endocarditis due to a fully penicillin-sensitive streptococcus, a point which had not been previously proved in vivo. The clinical implications are discussed.
David T. Durack, Lawrence L. Pelletier, Robert G. Petersdorf
Peripheral blood lymphocytes from 15 patients with variable immunodeficiency and severe panhypogammaglobulinemia were evaluated for B and T cell surface markers. B cells were enumerated by immunofluorescent detection of both surface immunoglobulin (Ig) and the ability to bind aggregated Ig complexes. T cells were identified by their ability to form nonimmune rosettes with sheep red blood cells. Four distinct patterns were observed which were designated types I-IV. Type I: six patients had normal percentages (8.5-19.0%) of Ig-bearing B lymphocytes. Type II: four patients were observed to have B lymphocytes (4.5-15.0%) which lacked fluorescence-detectable surface Ig. Type III: the peripheral blood of these four patients contained a subpopulation (11.3-20.0%) of lymphocytes which apparently lacked both B and T cell markers (“null” cells). Type IV: one patient's blood was characterized by a subpopulation (18.0-22.0%) of lymphocytes which bore both B and T cell markers. Patients of each type had some clinical features in common. It is concluded that evaluation of lymphocyte surface markers provides a means of separating patients with variable immunodeficiency and panhypogammaglobulinemia into distinct groups which appear to differ in the nature of their fundamental defect.
H. B. Dickler, N. F. Adkinson Jr., R. I. Fisher, W. D. Terry
Elevation of plasma glucagon concentration has been observed in starvation and illnesses associated with increased catabolism such as diabetes mellitus and severe infections. Thus, we examined plasma glucose, immunoreactive insulin (IRI, microunits per milliliter) and glucagon (IRG, picograms per milliliter) responses to a beef meal (1 g/kg body wt) and intravenous glucose (1.5 g/min for 45 min) in patients with chronic renal failure (CRF).
Gordon L. Bilbrey, Gerald R. Faloona, Martin G. White, James P. Knochel, Julio Borroto
[1-14C]glucose oxidation to CO2 and conversion into glyceride by adipose tissue from nonobese and obese subjects has been studied in vitro in the presence of varying medium glucose and insulin concentrations as functions of adipose cell size, the composition of the diet, and antecedent weight gain or loss.
L. B. Salans, G. A. Bray, S. W. Cushman, E. Danforth Jr., J. A. Glennon, E. S. Horton, E. A. H. Sims
Recent studies have demonstrated that the antidiuresis associated with intravenous (i.v.) infusion of the beta adrenergic agonist, isoproterenol (ISO), is mediated by release of endogenous vasopressin. To examine whether beta-adrenergic stimulation causes vasopressin release by a direct cerebral action, ISO was infused into the carotid artery in a dose estimated to equal the amount of catecholamine reaching the cerebral circulation in the i.v. studies. This intracarotid infusion did not alter renal or systemic hemodynamics, urinary osmolality (Uosm) or free-water clearance (CH2O). Although renal perfusion pressure was maintained constant in all experiments i.v. ISO was consistently associated with a decrease in total peripheral resistance and systemic arterial pressure as cardiac output increased. To investigate whether the decrease in cerebral perfusion pressure with i.v. ISO might be responsible for vasopressin release, the carotid arteries were bilaterally constricted both above and below the carotid sinus to lower carotid perfusion pressure by a mean of 25 mmHg, a decrement comparable to that observed during i.v. ISO. Constriction of the carotid arteries above the carotid sinus did not affect Uosm or CH2O, while constriction below the sinus was associated with an antidiuresis as Uosm increased from 155±25 to 385±58 mosmol/kg (P < 0.001) and CH2O decreased from 1.20 to −0.44 ml/min (P < 0.001). This antidiuresis was not significantly different from that observed during i.v. ISO. Since these results suggested that changes in autonomic neural tone from arterial baroreceptors are responsible for vasopressin release with i.v. ISO, studies were performed in animals with denervated baroreceptors. While sham-operated animals and animals with bilateral cervical vagotomy showed a reversible antidiuresis with i.v. ISO infusion, dogs with complete denervation of arterial baroreceptors did not show a significant alteration in renal water excretion (Uosm, 187 to 182 mosmol/kg and CH2O, 0.59 to 0.74 ml/min). The results therefore indicate that ISO stimulates vasopressin release by altering baroreceptor tone rather than by a direct central or depressor effect of the catecholamine. These same baroreceptor pathways have been recently shown to be involved in the suppression of vasopressin release with norepinephrine and may well be the common pathway whereby nonosmotic stimuli control vasopressin release.
Tomas Berl, Pravit Cadnapaphornchai, Judith A. Harbottle, Robert W. Schrier
Patient B. J. with chronic myelocytic leukemia excreted 0.5-1.1 g protein per day in the urine. Gel filtration on Sephadex G-75 showed about one-third of this protein to be in molecular weight range 20,000-40,000 (fraction BJC). BJC, prepared from 9 liters of urine by gel filtration, was chromatographed on carboxymethylcellulose. Two proteins were eluted from the resin in pure form (as shown by zone and immunoelectrophoresis) in yields representing 8 and 3 mg/liter of urine: BJC1 and BJC2. Their amino acid compositions were identical. BJC1 contained 61% carbohydrate (33% hexose, 11% sialic acid, 13% glucosamine, 5% galactosamine). BJC2 contained one-fourth to one-half as much of each carbohydrate. Molecular weight of BJC1 was estimated at 29,000 by gel filtration. Neither glycoprotein reacted with rabbit antiserum to normal human serum.
Daniel Rudman, Rajender K. Chawla, Alejandro E. Del Rio, Bettye Hollins
This study investigates whether soluble collagen can initiate platelet aggregation or whether a higher degree of polymerization is required. Purified rat skin collagen was prepared in four states. Soluble monomeric collagen, containing 2 μM calcium chloride, was maintained at 4°C until use. A previously uncharacterized form of collagen, soluble microfibrillar collagen, was prepared from monomeric collagen containing calcium chloride by allowing it to polymerize at 23°C. Viscometric and electron microscopic characterization of microfibrillar collagen indicated polymerization to ordered native filaments. Particulate native macrofibrillar collagen was prepared from monomeric collagen by allowing it to polymerize at 37°C in the absence of calcium. Particulate collagen, in which the fibers were randomly associated, was prepared by salt precipitation of calcium-free monomeric collagen. Microfibrillar and native macrofibrillar collagen initiated platelet aggregation, with a lag phase of approximately 60 s. Monomeric collagen initiated aggregation with a lag phase of approximately 180 s. The duration of the lag phase for platelet aggregation initiated by monomeric collagen was independent of the dose. Salt-precipitated particulate collagen did not initiate platelet aggregation. Agents which prolong the transition from monomeric collagen to fibrillar collagen (urea, arginine) retarded or prevented the aggregation of platelets by monomeric collagen. Sodium borohydride, which stabilizes the intraand intermolecular cross-links of collagen did not affect platelet aggregation. Penicillamine, which displaces the intermolecular cross-links and binds the intramolecular cross-links of collagen, did not prevent platelet aggregation. The data suggest that an architectural requirement exists for the initiation of self-perpetuating platelet aggregation; that tropocollagen units do not fulfill this requirement; that a soluble collagen preparation, microfibrillar collagen, contains the minimal structural unit; and that cross-linkages within collagen do not play a critical role in platelet aggregation.
Russell Jaffe, Daniel Deykin
Renal production of ammonia by the left kidney was studied in 31 acidotic dogs (NH4Cl) after acute constriction of the renal artery. Renal ammoniagenesis fell in direct proportion with the reduction in glomerular filtration rate and renal plasma flow. The renal extraction of glutamine by the experimental kidney fell in direct proportion with the reduction in renal hemodynamics. Extracted glutamine remained greater than filtered glutamine indicating that both the luminal and antiluminal transport sites were operative. The relationship between renal extraction of glutamine and ammoniagenesis observed during control was maintained after renal artery constriction (1.7 μmol NH3 produced for each μmol of glutamine extracted). Systemic venous or renal intra-arterial infusion of glutamine during arterial constriction increased renal production of ammonia to or above control values. These observations indicate that the mechanisms responsible for glutamine extraction and ammonia production were operating normally despite reduced hemodynamics. When measured immediately after arterial clamping, the renal venous pNH3 was found to rise significantly decreasing progressively thereafter towards control values. The extracted fraction of total glutamine delivered to the kidney (31%) did not change after acute reduction of the glutamine load. Thus, the antiluminal extraction site was incapable of lowering renal venous plasma glutamine concentration below 0.33 μM/ml. In a second series of experiments, the properties of the antiluminal site of transport for glutamine were studied after complete occlusion of the left ureter in acidotic and nonacidotic animals. Under these circumstances, it was demonstrated that the antiluminal site is capable of extracting sufficient glutamine to maintain total ammonia production at 60% or more of control. In acidotic animals, changes in cellular pNH3 appeared to play a key role on the antiluminal extraction of glutamine since the significant rise in renal blood flow often observed after ureteral occlusion prevented the rise in pNH3 noted when blood flow remained constant. Thus, when renal blood flow rose glutamine extraction and ammonia production were maintained at control values. In these acidotic animals, glutamine infusion failed to influence ammonia production until luminal transport was restored by release of ureteral clamp and resumption of glomerular filtration. The latter observation establishes that reabsorbed glutamine is utilized at least in part for ammonia production.
Guy Lemieux, Patrick Vinay, Pierre Cartier
Metabolic clearance (MCR) and production rates (PR) of human thyrotropin (hTSH) were determined by the constant infusion to equilibrium method 57 times in 55 patients. 16 control patients had a mean hTSH MCR of 50.7 ml/min. The mean hTSH MCR was significantly (P < 0.02) higher in 19 euthyroid men (51.6 ml/min) than in 12 euthyroid women (43.0 ml/min), but this apparent sex difference disappeared when the MCR were corrected for surface area, 25.8 (men) versus 25.2 ml/min per m2 (women). Hypothyroid patients had significantly (P < 0.005) lower hTSH MCR (30.9 ml/min), and hyperthyroid patients had significantly (P < 0.05) higher hTSH MCR (60.9 ml/min) than controls. The hTSH MCR in patients with “decreased thyroid reserve” (40.9 ml/min), hyperfunctioning thyroid nodule (53.8 ml/min), and “empty sella syndrome” (46.6 ml/min) were not significantly different from controls. The mean hTSH PR in controls (104.3 mU/day) was significantly (P < 0.005) different from that in patients with “decreased thyroid reserve” (956 mU/day), hypothyroidism (4,440 mU/day), hyperthyroidism (< 43.9 mU/day) and a hyperfunctioning thyroid nodule (< 38.7 mU/day). In primary hypothyroidism intravenous triiodothyronine therapy (50 μg/day) for 10 days decreased the hTSH PR (from 4,244 to 2,461 mU/day) before changes in the hTSH MCR (from 33.1 to 33.7 mU/day) were observed.
E. Chester Ridgway, Bruce D. Weintraub, Farahe Maloof
This study was undertaken to assess the efficacy of an amino acid mixture formulated for intravenous use from estimates of requirements for essential amino acids of human adults, and from data previously derived from a study using casein hydrolysate as the amino acid source. This mixture contained 39.4% essential amino acids, with glycine, alanine, arginine, histidine, and proline selected to supply the nonessential nitrogen.
G. H. Anderson, D. G. Patel, K. N. Jeejeebhoy
The acute effect of i.v. and direct intrarenal arterial infusion of 25-hydroxycholecalciferol (25HCC) and 1,25-dihydroxycholecalciferol (1,25-DHCC) on renal handling of phosphorus was evaluated in the following groups of rats: (a) intact animals, (b) parathyroidectomized (PTX) hypocalcemic rats, (c) PTX rats in which normocalcemia was maintained with calcium supplements and (d) PTX animals in which urinary phosphorus was augmented by (i) i.v. sodium phosphate, (ii) expansion of the extracellular fluid volume with normal saline, and (iii) i.v. parathyroid hormone (PTH). Clearances of inulin (CIn), phosphorus (CP), and fractional clearances of phosphorus (CP/CIn) of the experimental groups were compared with those of the corresponding control groups, and the clearances of the infused kidneys with those of the contralateral kidneys.
Mordecai M. Popovtzer, John B. Robinette, Hector F. Deluca, Michael F. Holick
Cytidine deaminase, an enzyme that catalyses the deamination of both cytidine and its nucleoside analogues including the antineoplastic agents cytosine arabinoside (ara-C) and 5-azacytidine (5-azaC), has been partially purified from normal and leukemic human granulocytes. The purification procedure included heat precipitation at 70°C, ammonium sulfate precipitation, calcium phosphate gel ion exchange, and Sephadex G-150 gel filtration. The enzyme has mol wt 51,000, isoelectric pH of 4.8, and maximum activity over a broad pH range of 5-9.5. The enzyme is stabilized by the presence of the sulfhydryl reagent, dithiothreitol.
Bruce A. Chabner, David G. Johns, C. Norman Coleman, James C. Drake, Warren H. Evans
The serum of a patient (L'ec) with an IgM lambda monoclonal protein was noted to bind albumin on immunoelectrophoresis. Analytical ultracentrifugation of the L'ec serum demonstrated 23S and 12S peaks, but no 4S (albumin) boundary. Immunologically identical 20S and 9S IgM proteins were isolated from the serum and the addition in vitro of either the patient's albumin or albumin isolated from normal serum was shown to reconstitute the 23S and 12S boundaries. The binding of high molecular weight IgM to albumin was demonstated by Sephadex G200 chromatography with 125I-labeled albumin and isolated IgM. Immunoelectrophoresis of the L'ec IgM developed with aggregated albumin (reverse immunoelectrophoresis) also demonstrated the binding of albumin to IgM. That all of the patient's IgM complexed with albumin was shown by affinity chromatography employing an aggregated albumin-immunoadsorbent column. Binding was shown to be of the noncovalent type by polyacrylamide gel electrophoresis in 8 M urea. With hot trypsin proteolysis, Fabμ and Fcμ5 fragments were isolated, and monomer albumin was shown to complex only with the Fabμ fragment by both analytical ultracentrifugation and molecular sieve chromatogaphy employing 125I-labeled Fab fragments. 1 mol of Fabμ fragment bound 1 mol of monomer albumin.
Stephen Hauptman, Thomas B. Tomasi Jr.
Both cholera enterotoxin and certain prostaglandins have been shown to stimulate intestinal fluid secretion in vivo, to cause ion flux changes in vitro similar to those caused by addition of cyclic 3′,5′-adenosine monophosphate (cyclic AMP), and to activate intestinal mucosal adenyl cyclase. It has been suggested that the effects of the enterotoxin on intestinal cyclic AMP metabolism may be indirect, and that locally synthesized prostaglandins may serve as required intermediates for the effects of the enterotoxin in activating intestinal mucosal adenyl cyclase. In order to clarify certain aspects of the mechanisms by which these two agents alter intestinal mucosal cyclic AMP metabolism and ion transport, their effects on cyclic AMP accumulation in rabbit ileal mucosa were examined in vitro. Addition of 5 μg per ml (75 μg per 150 mg mucosa) of purified cholera enterotoxin produced a peak increase in cyclic AMP level in 3 h but there was a time delay of at least 30 min before any effect was observed. Inhibition of cyclic nucleotide phosphodiesterase with theophylline failed to reduce this time delay. In contrast, addition of prostaglandin E1 (PGE1) increased the cyclic AMP level rapidly, a peak effect being observed in 2 min. The time of the peak prostaglandin-induced changes in cyclic AMP level and short-circuit current correlated closely. A maximal increment in cyclic AMP level was achieved with 5 × 10−5 M PGE1. When 10−4 M PGE1 was added to mucosa already maximally stimulated with cholera toxin, the resulting cyclic AMP level was equal to the sum of the levels reached when each agent was added alone. Furthermore, the effects of the enterotoxin on mucosal cyclic AMP levels were not influenced by indomethacin under conditions where mucosal prostaglandins synthesis was inhibited. The results suggest that endogenous prostaglandins do not provide an essential link in the activation of intestinal mucosal adenyl cyclase by cholera enterotoxin. The present study also indicates that the effect of cholera enterotoxin on intestinal mucosal cyclic AMP metabolism involves a definite time delay which is not due to cyclic nucleotide phosphodiesterase activity.
Daniel V. Kimberg, Michael Field, Elaine Gershon, Antonia Henderson
Two normal collie dogs were given 1,200 R total body irradiation followed by successful marrow grafts from their grey collie littermates with cyclic hematopoiesis. During observation periods of 97 and 41 days after grafting, both previously normal recipients showed regular cyclic fluctuations of their granulocyte and reticulocyte counts similar to those observed in their donors. These findings suggest that canine cyclic neutropenia is due to a defect in the marrow stem cell.
Paul L. Weiden, Barbra Robinett, Theodore C. Graham, John Adamson, Rainer Storb