Issue published September 1, 1974

T he interrelationships of arterial oxygen flow rate index, oxygen binding by hemoglobin, and oxygen consumption have been examined in patients with acute myocardial infarction. Proportional extraction of oxygen increased in close association with decreasing oxygen flow rate, and hence, whole body oxygen consumption was constant over nearly a three-fold variation in arterial oxygen flow rate. A reduction in hemoglobin-oxygen affinity at in vivo conditions of pH. Pco₂ and temperature also occurred in proportion to the reduction in arterial oxygen flow rate. Therefore, the increased proportional removal of oxygen from arterial blood at low oxygen flow rates, required to maintain oxygen consumption, may have been facilitated by the reduced affinity of hemoglobin for oxygen at in vivo conditions. However, the decrease in affinity did not appear to explain more than 30-40% of the increased extraction.Respiratory alkalosis was a frequent occurrence in these patients and 2,3-diphosphoglycerate was positively associated with blood pH as well as with the time-averaged proportion of deoxyhemoglobin in arterial and venous blood.Hemoglobin-oxygen affinity measured at standard conditions and the mixed venous oxygen saturation were equally good indicators of reduced arterial oxygen flow rate in patients without shock. However, S̄vo₂ is more easily measured and is a more useful indicator of reduced oxygen flow rate, since its relationship to oxygen flow appears to be independent of affinity changes and time.

T he resting membrane potential and intracellular potassium and sodium concentration of three guinea pig hind limb muscles were studied. These properties are related to the gross color, the speed of contraction, and the biochemically defined fiber type composing the muscle. The resting membrane potential and intracellular content were: white vastus (grossly white, fast-twitch glycolytic) -85.3 mV. potassium 171.9 meq/liter; soleus (grossly red, slow-twitch oxidative) -69.7 mV, potassium 137.5 meq/liter; and red vastus lateralis (grossly red, fast-twitch oxidative glycolytic) -71.7 mV, potassium 139.6 meq/liter. In soleus and red vastus lateralis, the relative permeability of sodium to potassium was 0.041 and 0.036, while in white vastus it was 0.015. These results give us the first exception to the hypothesis that fast-twitch fibers have higher intracellular potassium and higher resting membrane potential than slow-twitch fibers.

T he rate of ingestion of inhaled bacteria by pulmonary alveolar macrophages is an important determinant of host defense. This parameter was investigated by infecting rats with finely dispersed aerosols bearing Staphylococcus aureus in high concentrations (about 10^s bacteria/ft³/min). These aerosols deposited more than 10⁶ bacteria/murine lung. At 0, 2½, and 5 h after infection, bacterial clearance rates were measured in the left lung, and the intracellular or extracellular location of 100 bacteria was determined histologically in the right lung (perfused in situ). The clearance rates at 2½ and 5 h were 44.5% and 76.9%, respectively. The percentages of intracellular bacteria were: 0 h, 54.8%; 2½ h, 87.1%: 5 h, 91.9%. When rats were exposed for 4 h to 2.5 ppm of ozone (O₃), bacterial clearance did not occur — 15.3%, although 78.7% of the bacteria were intracellular. Clumps of more than 10 bacteria—usually intracellular—were also present. These experiments demonstrate that phagocytic ingestion is an exceedingly rapid process, that in this experimental model the inactivation of inhaled staphylococci results almost entirely from phagocytosis, and that ozone-induced reductions in bacterial clearance are due to severe impairment of intrapulmonary killing mechanisms and minor impairment of bacterial ingestion.

T ransverse muscle strips, 2-mm wide, were cut serially from the gastroduodenal junction in opossums, cats, dogs, and man. Electrical field stimulation with trains of rectangular current pulses of 0.5 ms in all opossums, all cats, some dogs, and the one human specimen induced relaxation in strips from the thickened circular muscle proximal to the mucosal junction. In some opossums weak relaxations also occurred in the first few strips below the mucosal junction. All other strips contracted or showed no response. This relaxation in opossums was abolished by tetrodotoxin but was not affected by antagonists to adrenergic and cholinergic transmission, nor by tripelennamine, methysergide, pentagastrin, secretin, cerulein, or cholecystokinin. Optimal frequency for stimulus-relaxation was 12 Hz. Chronaxie was 0.85 ms. The junctional strips also showed greater resistances to stretch than those remote from the junction. With apparent species variations, the junctional muscle possesses a nonadrenergic inhibitory innervation which is either absent or unexpressed in adjacent muscle of stomach and duodenum. This suggests the existence of a distinctive inhibitory neural control mechanism for pyloric muscle.

V asoactive intestinal peptide (VIP), originally isolated from hog small intestinal mucosa, has been shown to cause small intestinal secretion. More recently, this peptide has been identified in the plasma and tumors of patients with the so-called “pancreatic cholera” syndrome. In order to explore the possible role of VIP in the pathogenesis of this syndrome, we examined the effects of this peptide and other hormones on the cyclic AMP levels, adenylate cyclase activity, and ion transport in in vitro preparations of ileal mucosa. In rabbit ileal mucosa, VIP (20 μg/ml) caused a prompt fivefold increase in cyclic AMP level, whereas nine other hormones, which have been postulated to cause intestinal secretion, failed to exert such an effect. Pentagastrin and glucagon also failed to increase cyclic AMP levels in canine ileal mucosa. An increase in mucosal cyclic AMP levels was observed at a VIP concentration of 0.1 μg/ml and appeared to be nearly maximal at 2.0 μg/ml. VIP (100 μg/ml) stimulated adenylate cyclase activity in a membrane preparation from rabbit ileal mucosa. Secretin (6.0 × 10^-5 M) failed to do so. When added to the serosal side of isolated rabbit ileal mucosa clamped in an Ussing chamber, VIP (2 μg/ml) increased short-circuit current (SCC) and caused net secretion of both Cl and Na. Net Cl secretion exceeded net Na secretion. These effects of VIP on mucosal cyclic AMP metabolism and ion transport are similar to those observed with cholera enterotoxin and certain prostaglandins. VIP was also tested with normal human ileal mucosa. At a concentration of 2 μg/ml it caused a fivefold increase in cyclic AMP level and an increase in SCC of the same magnitude as that caused by 5 mM theophylline. Addition of a second 2-μg/ml dose of VIP and addition of theophylline after VIP produced no further change in SCC. We conclude the VIP stimulates adenylate cyclase and active ion secretion in both rabbit and human ileal mucosa. This may be related to the pathogenesis of diarrhea in patients with the pancreatic cholera syndrome.

E lucidation of mechanisms involved in the control of colonic production of mucus requires direct examination of glycoprotein synthesis and secretion by colonic mucosa. In the past, the limited viability of intestinal mucosa in vitro has hampered such investigations. When maintained in an organ culture system, mucosal biopsies of rabbit colon and human rectum remained viable for 24 h as documented by morphologic appearance and a steady rate of protein synthesis and secretion. These biopsies also incorporated ¹⁴C-labeled glucosamine into tissue glycoproteins and secreted labeled glycoproteins at a steady rate for 24 h. Glucosamine was predominantly incorporated into macromolecules that were ultimately secreted, in contrast to leucine, which was predominantly incorporated into tissue macromolecules. When studied by autoradiography, cultured rabbit colonic biopsies synthesized and secreted glycoproteins in vitro at cellular sites and over a time-course similar to those previously described for the intestine of intact animals. Acetylcholine consistently stimulated secretion of labeled glycoproteins but did not alter glycoprotein synthesis. In contrast, cycloheximide inhibited glycoprotein synthesis but had no effect on the secretion of newly synthesized glycoproteins. Rectal biopsies from patients with active ulcerative colitis incorporated increased amounts of [¹⁴C]glucosamine into glycoproteins during organ culture and secreted labeled glycoproteins more rapidly into the incubation medium when compared to biopsies obtained from healthy volunteers These findings indicate that organ culture provides a useful means of directly examining the synthesis and secretion of glycoproteins by healthy and diseased colonic mucosa.

A ddition of increasing amounts of ¹²⁵I-labeled desialylated thyroxine-binding globulin (DTBG) to hepatic cell membranes resulted in a progressive increase in binding. Saturability of membrane sites was indicated by a concentration beyond which further increases in [¹²⁵I]DTBG resulted in no further binding. The binding curve for [¹²⁵I]DTBG was similar to binding curves of desialylated orosomucoid, fetuin, and ceruloplasmin.An inhibition assay system using hepatic cell membranes showed that desialylated orosomucoid had a greater affinity for membrane binding sites than did DTBG but desialylated fetuin and ceruloplasmin bound less avidly than DTBG.Serum from normal persons and patients with a variety of illnesses was tested for its ability to inhibit [¹²⁵I]DTBG binding. The inhibitory activity of 1 ml of normal serum was equivalent to that of 0.2-2 μg DTBG. Patients with Laënnec's cirrhosis, biliary cirrhosis, and hepatic metastases had greatly increased inhibitory activity in their serum. Patients with jaundice due to extrahepatic obstruction had inhibitory activity not significantly different from that found in normal serum.Column chromatography of normal serum on Sephadex G-200 resulted in inhibitory activity throughout the range of protein molecular weight. Desialylation of normal serum with neuraminidase enhanced the inhibitory activity but did not change the distribution of the activity. Gel chromatography of cirrhotic serum showed markedly increased inhibitory activity associated with the macroglobulins and the 4.5S peak and a new peak of inhibitory activity in the low molecular weight area was also seen.Inhibition of desialylated glycoprotein binding to liver cell membranes by serum from patients with hepatocellular disease raises the possibility that desialylated serum glycoproteins accumulate in the circulation and that patients with compromised hepatocellular function may no longer be able to clear them from the circulation. Alternatively, accumulation of desialylated glycoproteins in the circulation could result from defective protein synthesis by the diseased liver.

T he effects of isoproterenol, norepinephrine, dobutamine, exercise, and nitroglycerin on left ventricular diameter, pressure, velocity of shortening, dP/dt, dP/dt/P, arterial pressure, left circumflex coronary blood flow, and coronary vascular resistance were examined in healthy conscious dogs with normal coronary perfusion and in the same animals after moderate global ischemia had been induced by partial occlusion of the left main coronary artery. In the normal nonischemic heart, all interventions improved left ventricular performance, as evidenced by increases in dP/dt/P and velocity at the same or lower left ventricular end-diastolic diameter. Interventions, which in the normal heart caused large increases in heart rate and myocardial contractility, e.g. isoproterenol and exercise, or which decreased coronary perfusion pressure, e.g. nitroglycerin or isoproterenol, elicited paradoxical responses in moderate global ischemia, i.e., left ventricular enddiastolic diameter and pressure rose, and dP/dt/P and velocity fell substantially. On the other hand, norepinephrine, which increased coronary perfusion pressure along with myocardial contractility but did not increase heart rate, improved left ventricular function. Dobutamine, which did not alter heart rate or arterial pressure substantially while improving myocardial contractility, produced an intermediate response between that of norepinephrine and isoproterenol in the presence of moderate global myocardial ischemia. Thus, interventions that increase myocardial O₂ requirements, by increasing heart rate and myocardial contractility without augmenting coronary perfusion pressure, can produce a paradoxical depression of ventricular function in the presence of global myocardial ischemia.

S tudies of four pregnancy-associated plasma proteins (PAPP's) were made on 206 plasma samples obtained from 175 pregnant women between 12 and 44 wk of gestation. The concentration of three PAPP's (A, C, and D) were assayed by quantitative crossed immunoelectrophoresis. They showed a gradual but small rate of increase during the 2nd trimester, which became more rapid in the 3rd trimester. PAPP-A continued to rise steadily during this period, while PAPP-C and PAPP-D (recently identified as human placental lactogen) tended to reach a plateau. Although PAPP-B could not be quantitated because of technical problems, it was detected in over 50% of the samples from the last 2 mo of gestation, but was almost never seen in those obtained during the 12th-28th wk of gestation. Various parameters were analyzed to determine their possible correlation with the PAPP levels during the last month of gestation. The race and age of the mother showed no influence on any of the PAPP's, while parity, sex of fetus, and infant birth weights appeared to affect the plasma concentration of some of the PAPP's. In the two instances studied, mothers of twins showed abnormally high PAPP levels.

A highly specific antiserum to 3,3′,5′-triiodothyronine (reverse T₃, rT₃) was prepared by immunization of rabbits with D,L-rT₃-human serum albumin conjugate. Of the various thyroid hormone derivatives tested, only 3,3′-diiodothyronine (3,3′-T₂) cross-reacted significantly (10%) with rT₃-binding sites on the antiserum, while thyroxine (T₄) and triiodothyronine (T₃) cross-reacted by less than 0.1%. The antiserum was used in a simple, sensitive, precise, and reproducible radioimmunoassay (RIA) for measurement of rT₃ in ethanolic extracts of serum. The dose-response curves of inhibition of the binding of [¹²⁵I]rT₃ to antibody obtained by serial dilutions of serum extracts were essentially parallel to the standard assay curve. Recovery of nonradioactive rT₃ added to serum before extraction averaged 93%.Serum rT₃ concentrations were found to be (mean±SD) 41±10 ng/100 ml in 27 normal subjects, 103±49 ng/100 ml in 22 hyperthyroid patients, 19±9 ng/100 ml in 12 hypothyroid patients, and 54±7 ng/100 ml in five subjects with elevated serum thyroxine-binding globulin: the values in each of the latter three groups of individuals were significantly different from normal. Reverse T₃ was detected regularly in normal or supranormal concentrations in serum of 12 hypothyroid patients rendered euthyroid or mildly hyperthyroid by treatment with synthetic T₄. It is suggested that serum rT₃ values noted here should be taken to reflect the relative changes in serum rT₃ rather than its absolute values in health and thyroid disease. True serum rT₃ may be somewhat different because: (a) D.L-rT₃ employed in the standard curve and L-rT₃ present in human serum may react differently with anti-D,L-rT₂. (b) Even though 3,3′-T₂, which cross-reacted 10% in rT₃ RIA, has been considered unlikely to be present in human serum, it may circulate in low levels. (c) Cross-reaction of T₄ in rT₃ RIA of 0.06% although small, could contribute to RIA estimates of rT₂; the effect of T₄ would be particularly important in case of serum of hyperthyroid patients. Thus, serum rT₃ concentration in hyperthyroid patients averaged 89±48 μg/100 ml after correction for cross-reaction effects of T₄: this value was about 14% lower than that before correction (see above).Serum rT₃ concentration in cord sera of seven newborns averaged 136±19 ng/100 ml; it was clearly elevated and within the range of values seen in hyperthyroid patients. This was the case when the mean T₄ concentration in the newborn cord sera was moderately higher than normal and about one-half that in hyperthyroid patients, whereas serum T₃ was markedly below the normal adult level. A Pronase hydrolysate of thyroglobulin prepared from pooled normal thyroid glands contained 0.042, 3.0, and 0.16 μg/mg protein of rT₃, T₄, and T₃, respectively. The various data suggest that: (a) rT₃ is a normal component of human serum and thyroglobulin: (b) peripheral metabolism of T₄ is an important source of the rT₃ present in serum: (c) peripheral conversion of T₄ to T₃ and rT₃ may not necessarily be a random process.

T he action of intravenous atropine on meal-and pentagastrin-induced gastric acid secretion was studied in six duodenal ulcer patients.A test meal of 10% peptone solution adjusted to pH 5.0 was maintained in the stomach at at distention presure of 15 cm H₂O, and a modification of the intragastric titration method of Fordtran and Walsh was used to measure gastric acid output by monitoring the rate at which a solution of 0.5 M sodium bicarbonate had to be added to keep the pH of the gastric content constant at the initial (pH 5.0) value. Serum gastrin concentrations were measured simultaneously by radioimmunoassy. The dose of 25 μg/kg-h atropine inhibited meal-induced acid secretion by about 70% and that evoked by pentagastrin by about 30%. The serum gastrin response to the test meal was not significantly altered by atropine.We conclude that atropine is a very strong inhibitor of meal-induced gastric acid secretion and does not significantly change serum gastrin response to feeding in duodenal ulcer patients when postprandial gastric acidity (pH 5.0) and intragastric pressure (15 cm H₂O) are kept constant.

T o determine whether the molecular configuration of vitamin B₁₂ influences the attachment of intrinsic factor-vitamin B₁₂ complex to ileal microvillous membrane receptor sites, we have examined the kinetics of uptake of intrinsic factor-bound cyanocobalamin by brush borders and microvillous membranes isolated from guinea pig ileum, and have compared this uptake with that of intrinsic factor alone and with that of intrinsic factor complexed with various analogs of cyanocobalamin.We first studied the kinetics of binding of cyanocobalamin and other cobamides to human gastric intrinsic factor. The binding of cyanocobalamin showed saturation kinetics and, at relatively high concentrations of cyanocobalamin, a Scatchard plot of binding was linear. The dissociation constant for the intrinsic factor-cyanocobalamin complex was 0.066 nM. When the binding of various vitamin B₁₂ analogs to intrinsic factor was determined by competition experiments, the analogs could be separated into two categories: those with affinities similar to that of cyanocobalamin and those with affinities much lower than that of cyanocobalamin. The affinity of cyanocobalamin for intrinsic factor was not altered by various substitutions at the -CN position, while removal of a single amido group on the corrin ring of substitution of the dimethylbenzimidazole base greatly reduced affinity. Removal of the base totally abolished binding. These findings, confirming those reported by others, are consistent with the concept that the cyanocobalamin molecule fits into a “pocket” in the intrinsic factor molecule, with the nucleotide base facing inward and the -CN side of the planar corrin ring facing outward.We then investigated the attachment of intrinsic factor-bound cyanocobalamin to ileal receptor. Attachment to microvillous membranes showed saturation kinetics with a dissociation constant of 0.25 nM. Attachment was rapid and was 70% complete within 5 min; the second-order rate constant for attachment was 1.3 × 10⁶ M^-1 s^-1. The half-time for dissociation of intrinsic factor-bound cyanocobalamin from the ileal receptor was approximately 35 min. Free intrinsic factor inhibited the attachment of intrinsic factor-bound cyanocobalamin, but the rate of attachment of free intrinsic factor was slower than that of intrinsic factor bound to cyanocobalamin. When intrinsic factor was complexed with various analogs of cyanocobalamin, the affinities of these complexes for ileal microvillous membranes were similar to that of intrinsic factor-bound cyanocobalamin. These findings suggest that the molecular configuration of vitamin B₁₂ is not a major determinant in the interaction between intrinsic factor-bound vitamin B₁₂ and its ileal receptor site.

P lasma luteinizing hormone (LH) and testosterone (T) were measured by radioimmunoassay in nine pubertal boys and three sexually mature young men at 20-min intervals for 24 h. Plasma LH and T were also measured in one boy during a delayed sleep onset study. Polygraphic monitoring was carried out to identify precisely sleep onset. Wakefulness, and specific sleep stages. In all nine pubertal boys the plasma T concentration fluctuated and was significantly higher during normal nocturnal sleep as compared to daytime waking. This increased T secretion during sleep was temporally linked to the characteristic pubertal sleep augmentation of LH secretion. To define further the relationship of this increased T secretion to sleep, plasma LH and T were also measured in three of the pubertal boys after acute (1-day) reversal of the sleep-wake cycle. One of these boys was also studied after 3 days of sleep-wake cycle reversal. The results of these studies showed that plasma T was now augmented during the reversed daytime sleep period; the mean T concentrations during this period were significantly higher (P < 0.001) than during nocturnal waking in all four studies. Measurement of plasma LH and T in the three sexually mature young men showed episodic secretion of LH and T during both waking and sleep periods; there was no consistent significant augmentation of LH or T secretion during sleep. This study demonstrates that (a) in normal pubertal boys and sexually mature young men plasma T fluctuates episodically; (b) there is marked augmentation of T secretion during sleep in pubertal boys, which is dependent on increased LH secretion; (c) this pubertal LH-T secretory “program” is dependent on sleep, since it shifts with delayed sleep onset and reversal of the sleep-wake cycle; and (d) this demonstrable tropic effect of LH on T is evident only during puberty, since sexually mature young men fail to show any consistent relationship between LH and T secretion either awake or asleep.

T he structure of Hageman factor, isolated from human plasma, was analyzed before and after enzymatic activation. The purified molecule is a single polypeptide chain of 80,000 molecular weight (mol wt) sedimenting at 4.5S. An amino acid analysis has been performed. The concentration of Hageman factor in normal human plasma was found to be 29 μg/ml with variation between individuals ranging from 15 to 47 μg/ml. Treatment of the molecule with kallikrein, plasmin, or trypsin resulted in cleavage at two primary sites, yielding fragments of 52,000, 40,000, and 28,000 mol wt. No further changes occurred in the fragments with subsequent reduction. Prekallikrein-activating ability was associated exclusively with the 28,000 moiety.

C learance experiments were performed in female mongrel dogs, either intact or thyro-parathy-roidectomized (T-PTX), under pentobarbital anesthesia, to examine the unusual hypocalciuric property of thiazide diuretics. The relationship between calcium clearance (C_Ca) and sodium clearance (C_Na) was determined in normal dogs, C_Ca = 0.79 C_Na; constant infusion of chlorothiazide (CTZ) to provide drug concentrations in plasma of approximately 40 μg/ml modified this relationship; C_Ca = 0.30 C_Na (P < 0.001). The magnitude of the dissociating effect of CTZ on the urinary Ca/Na relationship was found to be most highly correlated with urinary drug concentration. Infusion of CTZ (1 mg/min) into one renal artery caused a unilateral decrease (25%) in C_Ca/GFR while producing a unilateral increase (80%) in C_Na/GFR. The same dose of CTZ in T-PTX dogs produced an increase in C_Na/GFR without causing a change in C_Ca/GFR. The defective response in T-PTX dogs could be ascribed to poor tubular secretion of the drug; when urinary drug concentrations were elevated in T-PTX dogs to the levels found in intact dogs (by infusing more drug), C_Ca/GFR fell to an equivalent extent. T-PTX dogs showed substantially lower renal extraction of CTZ (42%) than intact dogs (57%); PTH administration to T-PTX dogs increased extraction toward normal (49%). The defective secretion of CTZ could not be attributed to either a decreased tubular maximum or a decreased renal blood flow.

C ell suspensions enriched in human blood monocytes, obtained from normal peripheral blood by sedimentation on sodium diatrizoate-Ficoll gradients or from the blood of patients with neutropenia and monocytosis, accumulated malonyldialdehyde, a labile catabolite of lipid peroxidation, during incubations with polystyrene beads or heat-killed Staphylococcus epidermidis. Mixed blood leukocytes principally composed of granulocytes or granulocytes purified by density gradient sedimentation did not accumulate malonyldialdehyde during incubations with these particles, but did when ingesting particles containing linolenate. The phospholipid fatty acid composition of monocyte-enriched and purified granulocyte preparations from the same donors were compared. The molar fraction of arachidonate (20:4) in phospholipids from monocyte-rich preparations was 62% greater than that of purified granulocytes. The findings indicate that human monocytes, possibly because of a greater content of polyunsaturated fatty acids in their membranes, peroxidize a greater quantity of endogenous lipids than granulocytes during endocytosis. Normal human granulocytes have the capacity to peroxidize ingested lipids. However, mixed leukocytes from two patients with chronic granulomatous disease produced little malonyldialdehyde when engulfing linolenate-containing particles. Therefore the capacity to peroxidize lipid is related to cellular oxygen metabolism, a function in which chronic granulomatous disease granulocytes are dificient.Malonyldialdehyde chemically prepared by hydrolysis of tetramethoxypropane, by extraction from peroxidized linolenic acid, or purified from extracts of phagocytizing rabbit alveolar macrophages had bactericidal activity against Escherichia coli and S. epidermidis. Therefore, toxic catabolites of lipid hydroperoxides may potentiate the bactericidal activity of hydrogen peroxide in mononuclear phagocytes.

K inetics of 5α-androstane-3α, 17β-diol (3α-diol) were studied in man. Clearance rates were determined by both the constant infusion and single injection techniques. Production rates were calculated as the product of clearance rate data and plasma values in the a.m. obtained by a radioimmunoassay specific for 3α-diol. Mean metabolic clearance rates were 1,776±492 (SD) liters/day in males and 1,297±219 (SD) liters/day in females. Metabolic clearance rates by single injection were similar. Calculated production rates are 208±26 (SD) μg/day in males and 35±11 μg/day in females, which are significantly different. Hepatic extraction of 3α-diol determined by hepatic vein catheterization during constant infusion was 76% which was greater than expected from information on in vitro binding in plasma. The kinetic data is of interest since 3α-diol has a calculated inner pool (V₁) volume of 12-14 liters, similar to 17β-hydroxyandrost-4-en-3-one (testosterone) and 5α-androstan-17β-ol-3-one (dihydrotestosterone), but the calculated outer pool (V₂) of 33.5 liters is very large as are the metabolic rate and transfer constant. In contrast to testosterone and dihydrotestosterone, 3α-diol, although bound to sex hormone binding globulin, has a high metabolic clearance of which a large fraction represents extrahepatic (splanchnic) metabolism. A production rate of 3α-diol similar to dihydrotestosterone together with rather unique kinetic characteristics encourages further investigation of the biological role of this potent androgen.

T he mechanisms involved in the production of hypoglycemic coma were studied in rabbits. Measurements were made in brain, cerebrospinal fluid (CSF), and plasma of osmolality, Na⁺, K⁺, Cl^-, water content, exogenous insulin, glucose, lactate, and glutamate, while pH, Pco₂, Po₂, and bicarbonate were evaluated in arterial blood, 35 min after i.v. injection of insulin (50 U/kg), plasma glucose did not change, but brain K⁺ content increased significantly. Grand mal seizures were observed in unanesthetized animals (±SD) 133±37 min after administration of insulin, at a time when brain glucose was normal, but brain tissue content of Na⁺, K⁺, osmoles, and water was significantly greater than normal. Coma supervened 212±54 min after insulin injection, at which time brain glucose, lactate, and glutamate were significantly decreased. At both 35 and 146 min after insulin administration, exogenous insulin was present in brain, but not in the CSF. After 208 min of insulin administration, animals were given i.v. glucose and sacrificed 35 min later. Most changes in the brain produced by hypoglycemia were reversed by the administration of glucose. Hypoxia (Po₂ = 23 mm Hg) was produced and maintained for 35 min in another group of animals. Hypoxia caused brain edema but did not affect brain electrolyte content. However, brain lactate concentration was significantly greater than normal. The data indicate that the seizures noted early in the course of insulin-induced hypoglycemia are temporally related to a rise in brain osmolality secondary to an increased net transport into brain of Na⁺ and K⁺, probably caused by insulin, per se. As hypoglycemia persists, there is also depletion of energy-supplying substrates (glucose, lactate, glutamate) in the brain, an event which coincides with the onset of coma. The brain edema observed during hypoxia is largely due to an increase in brain osmolality secondary to accumulation of lactate.

P seudomonas pneumonia was produced in dogs with radiation-induced leukopenia. Treatment of this infection with either gentamicin alone or gentamicin plus daily granulocyte transfusion was compared in a randomized controlled trail. The dogs receiving granulocytes plus gentamicin survived significantly longer than those treated with gentamicin alone (P < 0.05). The Pseudomonas immunotype which was inoculated into the dogs were recovered at autopsy from none of the granulocyte-transfused dogs, whereas seven or eight of the dogs treated with gentamicin alone had the inoculated Pseudomonas immunotype in the area of induced pneumonia at autopsy. As measured by the limulus test, the granulocyte-transfused dogs also did not have endotoxemia as frequently as the dogs given only gentamicin (P < 0.05). This controlled study establishes that transfused granulocytes can favorably alter the course of experimental Pseudomonas pneumonia and suggests that granulocyte transfusion may be a useful therapy in serious bacterial infections of leukopenic subjects.

T he diabetogenic activity of streptozotocin has been correlated with a reduction in pyridine nucleotide synthesis in the mouse pancreatic islet. To determine the specificity of this reduction for diabetogenicity, a comparative study of streptozotocin, its cytotoxic moiety, 1-methyl-1-nitrosourea, and alloxan was performed. Streptozotocin administered intraperitoneally (i.p.) producd a dose-related reduction in islet NAD which was proportional to the degree of diabetogenicity. A diabetogenic dose, 200 mg/kg, attained a peak plasma N-nitroso intact streptozotocin concentration of 0.224 μmol/ml and reduced the mean islet NAD from a control of 0.78 to 0.15 pmol. At borderline, 150 mg/kg, and nondiabetogenic, 100 mg/kg, doses, plasma concentrations reached 0.161 and 0.136 μmol/ml, and NAD was 0.36 and 0.86 pmol/islet, respectively. 1-Methyl-1-nitrosourea, 100 mg/kg, attained a maximum N-nitroso intact 1-methyl-1-nitrosourea concentration of 0.162 μmol/ml and reduced the mean NAD to 0.58 pmol/islet, and was nondiabetogenic; 200 mg/kg attained a peak plasma concentration of 0.344 μmol/ml and depressed NAD to 0.38 pmol/islet, and was inconsistently diabetogenic. Islet NAD of 0.4 pmol/islet or greater is required for integrity of the beta cell. A diabetogenic dose of alloxan, 500 mg/kg, did not depress NAD, 0.85 pmol/islet, therefore confirming that its mechanism of diabetogenicity differs from that of streptozotocin.In vivo uptake of [methyl-¹⁴C]streptozotocin by islets was 3.8 times that of [methyl-¹⁴C]-1-methyl-1-nitrosourea, whereas uptake by the exocrine pancreas favored 1-methyl-1-nitrosourea over streptozotocin 2.4:1. The decreased islet uptake of 1-methyl-1-nitrosourea correlates with the 3.5 times increased molar dosage required to produce islet NAD depression comparable to that of streptozotocin, 150 mg/kg. These studies indicate that the glucose carrier of streptozotocin facilitates uptake of its cytotoxic group, 1-methyl-1-nitrosourea, into islets.

T he sequential changes that occur during the precipitation on mild heating of the unstable hemoglobins, Hb Christchurch, Hb Sydney, Hb Köln, and Hb A, were examined with particular attention to the possibility of an accompanying oxidative process. Hb Christchurch, Hb Sydney, and Hb A precipitated with equal amounts of α- and β-chains and full heme complement. Hb Köln, however, was one-half hemedepleted and showed a slight excess of precipitated β-chains. In all cases the spectrum of the precipitated material was typical of a hemichrome. There was no evidence that sulfhydryl oxidation contributed to the precipitation process. Reduced glutathione was unable to protect the hemoglobin against precipitation, and mixed disulfide formation between the precipitating hemoglobin and glutathione was insignificant, even in the presence of excess glutathione. No blockade of β93 cysteines could be demonstrated in the unstable hemoglobins.Precipitation of oxyhemoglobin and carboxyhemoglobin in all cases gave nonspecific oxidation of approximately two of the six hemoglobin sulfhydryl groups to give intra- and intermolecular disulfide bonds. Single α- and β-chains, plus polymers of up to five or six chains linked by disulfide bridges, were demonstrated by polyacrylamide gel electrophoresis. This disulfide oxidation was not observed with deoxy- or methemoglobin and did not appear to influence the rate of precipitation. These findings fit the theoretical prediction that autoxidation of oxy- and carboxyhemoglobin is accompanied by formation of a free radical, with the reactions of this free radical being confined intramolecularly.Together, these results are in keeping with predictions based on the known structural abnormalities of the unstable hemoglobins, all of which result in greater molecular flexibility. Our findings support the conclusion that the usual precipitating event is altered bonding at the heme to give the formation of hemichromes. There is no evidence of an accompanying oxidative process that could pose a threat to the integrity of the red cell.

T ransfer factor (TF) derived from donors with strong delayed hypersensitivity to coccidioidin (CDN) was administered to four patients with active disseminated or progressive pulmonary coccidioidomycosis. The clinical and immunologic response to TF was studied.Before the administration of TF, all four patients had defective thymus-derived lymphocyte (T-cell) function. In no case were lymphocytes in culture stimulated to incorporate [³H]thymidine when exposed to CDN. Cases 1 and 2 had no skin test response to CDN or other antigen, nor was antigen-induced migration inhibition factor (MIF) release detected. Cases 3 and 4 had skin reactivity to CDN as well as MIF release. Lymphocyte reactivity to phytohemagglutinin (PHA), as measured by the incorporation of [³H]thymidine, was low or absent in all.After the administration of TF, patients with negative skin tests became reactive to CDN, MIF release was present in all but case 1, and lymphocyte stimulation was present in response to CDN in all.Lymphocyte reactivity to PHA was also increased after the administration of TF in all cases. All responses to single doses of TF were transient, lasting no more than 10 days. Subsequent doses were less effective at restoring lymphocyte stimulation once it had waned. Multiple doses of TF administered at frequent intervals appear to be the most effective way to maintain lymphocyte reactivity.Clinical response to the administration of TF correlated closely with specific transfer as measured by response to CDN in skin test, lymphocyte stimulation, and MIF release. After TF administration, all patients mounted a more effective host response against the infecting fungus. In each patient, smears and cultures became negative. Fistulas, when present, diminished in extent or closed; and pulmonary infiltrates cleared. Nonspecific signs of infection such as fever, weight loss, and anorexia also improved. Clinical improvement paralleled immunologic improvement. When immunologic improvement was transient so was clinical improvement. Multiple doses of TF at frequent intervals may maintain transferred T-cell reactivity. TF may prove to be a useful adjunct in the management of patients with coccidioidomycosis. Whether TF from CDN-negative donors may have similar effects is not known and requires exploration.

T he uptake and release of ⁴⁵Ca from the intestinal mucosal epithelium were investigated under a variety of conditions. The initial rate of uptake characterized a calcium pool with a half-time of saturation of less than 2 min. The entry of ⁴⁵Ca into this pool was inhibited by NaCN and ethacrynic acid and was stimulated by the removal of Cl^- from the incubation. The initial rate of ⁴⁵Ca release was also inhibited by NaCN and removal of Na⁺ from the incubation. Parathyroid hormone administration enhanced the release of ⁴⁵Ca from cells prepared from parathyroid-ectomized animals. These observations suggest that calcium transport across the brush border and basallateral membranes are identifiable components of the kinetics of ⁴⁵Ca uptake and release and that parathyroid hormone stimulates a sodium-dependent mechanism of calcium transport across the basal-lateral membranes.

P revious reports suggest that the site of the energy-dependent intestinal calcium transport against an electropotential and concentration gradient is located along the basal-lateral membrane of the mucosal cell. Accordingly, basal-lateral membranes were prepared from rat intestinal homogenates in order to identify the enzyme mediating this step in the transport process. An alkaline phosphatase was delineated which utilized ATP as a substrate and was dependent on both Na^- and Ca⁺⁺ with optimum enzyme activity at 200 mM and 0.04 mM, respectively. Furthermore, the activity of the enzyme was demonstrated to decrease with the advance in age of the animal and to decrease with removal of the parathyroid glands, consistent with a decreased rate of ⁴⁵Ca release from mucosal cells under the same experimental conditions. Calcium binding to basal-lateral membrane fragments was also sodium dependent and enhanced by the prior administration of parathyroid extract. The consistent correlation between the rate of calcium transport across the basal-lateral membrane of the mucosal cell and the activity of this Na, Ca-dependent phosphatase under a variety of experimental conditions suggest that this enzyme may mediate the parathyroid hormone-sensitive active transport of calcium across the intestine.

T he rate of passive absorption into the intestinal mucosal cell is determined by at least two major diffusion barriers: an unstirred water layer and the cell membrane. This study defines the morphology and permeability characteristics of these two limiting structures. The unstirred water layer was resolved into two compartments: one behaves like a layer of water overlying the upper villi while the other probably consists of solution between villi. The superficial layer is physiologically most important during uptake of highly permeant compounds and varies in thickness from 115 to 334 μm as the rate of mixing of the bulk mucosal solution is varied. From data derived from a probe molecule whose uptake was limited by the unstirred layer, the effective surface area of this diffusion barrier also was determined to vary with stirring rate and equaled only 2.4 cm²·100 mg^-1 in the unstirred condition but increased to 11.3 cm²·100 mg^-1 with vigorous mixing. This latter value, however, was still only 1/170 of the anatomical area of the microvillus membrane. With these values, uptake rates for a number of passively absorbed probe molecules were corrected for unstirred layer resistance, and these data were used to calculate the incremental free energy changes associated with uptake of the -CH₂- (-258 cal·mol^-1), -OH (+564), and taurine (+1,463) groups. These studies, then, have defined the thickness and area of the unstirred layer in the intestine and have shown that this barrier is rate-limiting for the mucosal uptake of compounds such as fatty acids and cholesterol; in addition, the lipid membrane of the microvillus surface has been shown to be a relatively polar structure.

t he absorption of vitamin B₁₂ in many animals requires its prior association with intrinsic factor (IF) and attachment to a specific receptor in the intestine. Employing Triton X-100, we have solubilized from guinea pig ileum a factor that binds intrinsic factor-vitamin B₁₂ complex (IF-B₁₂). This binding factor was soluble to the extent that it was not sedimented by centrifugation at 100,000 g for 1 h and was small enough to enter the included volume of a Sepharose 4-B column. Furthermore, the ileal extract contained no microfine particles of membrane upon electron microscopic search. When a portion of the extract was incubated with a mixture of gastric juice and ⁵⁷Co-labeled vitamin B₁₂, a portion of the radioactivity was excluded from a Sephadex G-200 column. When gastric juice from a patient with a congenital abnormality of IF that prevented its binding to intestine was substituted for normal human gastric juice, radioactivity was not excluded from the gel, indicating failure of this abnormal IF-B₁₂ to bind to the intestinal extract. These data suggested the presence of a specific binder of IF-B₁₂ in the ileal mucosal extract. The reactions of normal IF-B₁₂ with the solubilized binding factor and with the membrane-bound “receptor” had several characteristics in common, including calcium dependence, temperature independence, and pH optimum near neutral. Extracts from the distal intestine showed more activity than did those from the proximal. The solubilized binding facter seemed specific for IF-B₁₂ in that it was not blocked by prior incubation with excesses of either free vitamin B₁₂ or IF. Binding activity of the extract was decreased by incubation at pH 2.0, by heating to 56°C, and by incubation with chymotrypsin and dithiothretiol. Incubation with trypsin, neuraminidase, and sulphydryl blockers did not affect it.The Triton X-100 extract of guinea pig ileal mucosa contains a specific binding factor that probably is the receptor for IF-B₁₂. This appears to be a protein with function dependent on peptide and disulphide linkages.

I n view of the variables that obscure the pathogenesis of cardiomyopathy, a study was undertaken in mongrel dogs fed ethanol as 36% of calories for up to 22 mo. Both the experimental and control groups maintained body weight, hematocrit, plasma vitamin, and protein levels. Left ventricular function was evaluated in the intact anesthetized dog using indicator dilution for end-diastolic and stroke volume determinations. During increased afterload with angiotensin, the ethanol group exhibited a larger rise of end-diastolic pressure (P<0.01), whereas end-diastolic and stroke volume responses were significantly less than in controls. Preload increments with saline elicited a significantly higher end-diastolic pressure rise in the ethanol group (P<0.01). No hypertrophy, inflammation, or fibrosis was present and it was postulated that the enhanced diastolic stiffness was related to accumulation of Alcian Blue-positive material in the ventricular interstitium.To evaluate myocardial lipid metabolism, [1-¹⁴C]oleic acid was infused systemically. Plasma specific activity and myocardial lipid uptake were similar in both groups. There was a significantly increased incorporation of label into triglyceride, associated with a reduced ¹⁴CO₂ production, considered the basis for a twofold increment of triglyceride content. In addition, diminished incorporation of [¹⁴C]oleic acid into phospholipid was observed accompanied by morphologic abnormalities of cardiac cell membranes. Potassium loss and sodium gain, like the lipid alteration, was more prominent in the subendocardium. Thus, chronic ethanol ingestion in this animal model is associated with abnormalities of ventricular function without evident malnutrition, analogous to the preclinical malfunction described in the human alcoholic.

P revious work has suggested that resistance to vasopressin in two strains of mice with nephrogenic deficiency of urinary concentration may entail a defect in the action of vasopressin at the cellular level. Several components involved in this action were therefore examined in vitro in renal medullary tissues from control mice (genotype VII +/+) and two genotypes with mild diabetes insipidus (DI +/+ nonsevere) and marked (DI +/+ severe) vasopressin-resistant concentrating defects. No significant differences were found in the affinity of adenylate cyclase for [8-arginine]-vasopressin (AVP), tested over a range of hormone concentration from 10^-10 to 10^-5 M. However, maximal stimulation of adenylate cyclase by saturating concentrations of AVP (intrinsic activity) was markedly decreased from control values in DI +/+ severe mice, and decreased to a lesser extent in DI +/+ nonsevere animals. A significant correlation was found between the activity of adenylate cyclase maximally stimulated by AVP in a given genotype, and the urine osmolality in the same animals. There were no significant differences in maximal stimulation of renal medullary adenylate cyclase in control experiments: not when stimulated nonspecifically by sodium fluoride, nor when stimulated by AVP in tissues from rats with induced water diuresis as compared to antidiuretic rats. Nor were there significant differences between VII +/+ and DI +/+ severe mice in the activity of renal cortical adenylate cyclase, either basal or when stimulated by parathyroid hormone. Furthermore, the abnormal genotypes did not differ significantly from control mice in the renal medullary activities of cyclic AMP phosphodiesterase or cyclic AMP-dependent protein kinase, nor in the content of microtubular subunits (assessed as colchicinebinding protein). The results are compatible with the view that impaired stimulation of renal medullary adenylate cyclase by vasopressin might be the sole or contributing cause of the vasopressin-resistant concentrating defect in the diseased mice; however, a causal relationship has not yet been proved.

E ight patients with coronary heart disease and exertional angina pectoris successfully completed an 11-15 wk program of endurance exercise conditioning. Angina threshold was determined by upright bicycle ergometer exercise and by atrial pacing. The product of heart rate and arterial systolic blood pressure at the exercise angina threshold was higher after conditioning. suggesting that conditioning increased the maximum myocardial O₂ supply during exercise. However, when angina was induced by atrial pacing, heart rate, arterial blood pressure, coronary blood flow, and myocardial O₂ consumption at the angina threshold were the same before and after conditioning. Myocardial lactate extraction during atrial pacing was abnormal in the same five patients before and after conditioning. Conditioning caused no detectable changes in coronary collaterals as judged by coronary arteriograms.The increase in exercise angina threshold appeared to be due to a functional adaptation in either myocardial O₂ supply or the relationship between hemodynamic work and myocardial O₂ consumption. The adaptation was limited to exercise, and did not occur during a different stress to myocardial O₂ supply, atrial pacing.