Issue published November 1, 1973

T he specific activity of sodium-potassium-activated adenosine triphosphatase (Na-K-ATPase) in homogenates of rat kidneys increases when the dietary intake of potassium is chronically increased. The effect is seen first and is most prominent in the outer medulla, but large loads of potassium elicit an increase in the cortex as well. Levels of Na-K-ATPase in brian, liver, and muscle, by contrast, are unaffected by potassium loading. Although the changes in enzyme activity in the kidney resemble those reportedly produced by aldosterone, they are not induced by experimental sodium deprivation, and they can be evoked by potassium loading in the absence of the adrenal glands. The results suggest that Na-K-ATPase of renal tubular cells, presumably in the distal tubules and collecting ducts, plays an important role in the phenomenon of potassium adaptation and in the process by which potassium is excreted into the urine.

J ejunal calcium absorption was measured from test solutions containing 1.0, 2.5, 5, and 10 mM calcium (as calcium gluconate). Absorption rates increased progressively as luminal calcium concentration was increased, although there was a tendency toward saturation of the absorptive process at the higher concentrations. Calcium absorption was higher in normal young adults than in normal subjects over age 60. In both groups a 300 mg calcium diet for 4-8 wk enhanced calcium absorption relative to absorption rates after 4-8 wk on a 2,000 mg calcium diet. This adaptation was more definite and dramatic in the young than in the old subjects. Indirect estimates suggest that adaptation to a low calcium diet and the higher absorption in young than old normal subjects are mediated by an increased V_max rather than a decreased K_m.

T he present study was designed to ascertain sequentially the pressor response to angiotensin II in young primigravid patients throughout pregnancy in order a) to define when in pregnancy resistance to the pressor effects of angiotensin II develops; b) to define the physiologic sequence of events leading to this resistance; and c) to ascertain whether sensitivity to infused angiotensin II could be detected before the onset of clinical signs of pregnancy-induced hypertension.With this prospective approach, two separate groups of patients were defined. The first group of patients remained normal throughout pregnancy. The second group consisted of those patients who, while clinically normotensive during the initial phase of the study, ultimately developed hypertension of pregnancy.192 patients were studied; of these, 120 patients remained normotensive and 72 developed pregnancy-induced hypertension. In both groups, vascular resistance to infused angiotensin II (more than 8 ng/kg/min required to elicit a pressor response of 20 mm Hg in diastolic pressure) was demonstrated as early as the 10th wk of pregnancy. In the group that remained normotensive, maximum mean vascular resistance occurred at 18-30 wk of pregnancy, (mean pressor dose required being 13.5 to 14.9 ng/kg/min). In those subjects who developed pregnancy-induced hypertension, the mean maximum dose required was 12.9 ng/kg/min, which was observed at the 18th wk of pregnancy. By the 22nd wk there was a clear separation of the two groups, with the mean dose requirement of the subjects destined to develop hypertension being progressively less than that of those who remained normal. The difference between the two groups became significant (P < 0.01) by 23-26 wk of pregnancy.Among patients requiring more than 8 ng/kg/min on one or more tests done between wk 28-32, 91% remained normotensive. Conversely, during the same time period among patients requiring less than 8 ng/kg/min, on at least one occasion, 90% developed pregnancy-induced hypertension.

T he effect of prolonged administration of alcohol on mitochondrial function and high-energy phosphate (ATP) of heart muscle was investigated in dogs. Animals were divided into two groups, a control group and a group that received alcohol. In the experimental series, dogs received 400 ml of a 25% solution of alcohol added to the food and drinking water. Measurements were carried out after ethanol had been withheld for 2 days. Total myocardial blood flow, cardiac output, and myocardial O₂ consumption remained at control levels. Measurement of cardiac contractility using the maximal rate of left ventricular pressure rise (dP/dt_max) showed no change in animals exposed to alcohol. When the afterload of the heart was increased with angiotensin, a slight but not significant decline in cardiac contractility was observed. Activities of various intramitochondrial and extramitochondrial enzymes were measured in both groups. After alcohol administration, the primarily intramitochondrial isocitrate dehydrogenase diminished. ATP in heart muscle of dogs exposed to alcohol declined, and mitochondrial oxygen consumption and respiratory control indices diminished. These observations suggest that the primary lesion leading to alteration of myocardial performance is a biochemical malfunction of the mitochondria, which at this early stage is not reflected in changes in myocardial contractility.

T he antibiotic ristocetin, in concentrations of 1.0-1.5 mg/ml, aggregated normal platelets in citrated platelet-rich plasma by a mechanism in which the release reaction played only a minor role. Platelet aggregation by ristocetin in a concentration of 1.2 mg/ml was absent or markedly decreased in 10 patients with von Willebrand's disease. Lesser degrees of abnormality were obtained with a concentration of 1.5 mg/ml. The magnitude of the defect in ristocetin-induced platelet aggregation correlated well with the degree of abnormality of the bleeding time and the levels of antihemophilic factor (AHF, VIII_AHF) procoagulant activity. In all patients, the defect in ristocetin-induced platelet aggregation was corrected in vitro by normal plasma. Correction was also obtained with a fraction of normal cryoprecipitate that eluted in the void volume with VIII_AHF after chromatography on a gel that excludes molecules larger than 5 × 10⁶. A similar fraction, devoid of VIII_AHF activity, obtained from patients with von Willebrand's disease had no corrective effect, but fractions obtained from patients with hemophilia were just as effective as those obtained from normal subjects. The correction activity of plasma and partially purified factor VIII was inhibited by a rabbit antibody to human factor VIII but not by a human antibody against VIII_AHF procoagulant activity. The studies provide further evidence that patients with von Willebrand's disease are deficient in a plasma factor that is necessary for normal platelet function. The activity of this factor appears to be associated with factor VIII but is unrelated to VIII_AHF procoagulant activity.

I n a previous paper, we showed that the abnormality of ristocetin-induced platelet aggregation in platelet-rich plasma in 10 patients with von Willebrand's disease could be corrected by a factor in normal plasma that was present in the same fractions as factor VIII procoagulant activity (antihemophilic factor, AHF, VIII_AHF) when prepared by chromatography on Bio-Gel 5 M (Bio-Rad Laboratories, Richmond, Calif.). This observation suggests that patients with this disorder are deficient in a plasma factor, associated with the factor VIII molecule, that is necessary for normal platelet function. In the present paper, we describe, an assay for this factor, the von Willebrand factor (VIII_VWF), based on the observation that a log-log relationship exists between the amount of ristocetin-induced aggregation of washed, normal platelets and the concentration of normal plasma present in the test system. We assayed the activity of VIII_VWF as well as antihemophilic factor procoagulant activity (VIII_AHF) and factor VIII antigen (VIII_AGN) in 15 patients with von Willebrand's disease and 20 normal subjects. A highly significant correlation (r ∼ 0.80) between VIII_VWF and both VIII_AHF was found in normal subjects and in patients with von Willebrand's disease. This finding, in addition to the observation that agarose gel chromatography fractions that have VIII_AHF procoagulant activity also have VIII_VWF activity, strongly suggests that the von Willebrand factor is associated with the factor VIII molecule. VIII_VWF in normal plasma was not inhibited by human anti-VIII, and VIII_VWF levels were normal in hemophilic plasma. Thus, the VIII_VWF site on the factor VIII molecule appears to be different from that determining VIII_AHF. Finally, the activity of VIII_VWF appeared to correlate better with the bleeding time than either VIII_AHF or VIII_AGN. This suggests that VIII_VWF assayed in this study may be the “anti-bleeding factor” that is deficient in von Willebrand's disease. These findings are consistent with a decreased synthesis of the factor VIII molecule in von Willebrand's disease and suggest the possibility of additional abnormalities of the site on the molecule that determines the activity of VIII_VWF.

I nfluence of altered blood oxygen affinity on maximal performance ability was evaluated in trained rats exercising to exhaustion in a graded treadmill test. Modification of blood oxygen affinity was achieved both by 2,3-diphosphoglycerate depletion, accomplished by exposure of animals to CO₂ and by exchange transfusion with blood exposed to bisulfite or stored in acid citrate dextrose, and by carbamylation of hemoglobin, produced by exchange transfusion of blood incubated with potassium cyanate. A decrease in oxygen tension at half-saturation of hemoglobin (P₅₀) from 36 to 23 mm Hg produced a decrease in resting central venous oxygen pressure of about 12 mm Hg. During exercise it caused an average decrease in work performance of about 10%, which was equivalent to that performance decrement caused by a decrease in hemoglobin concentration of approximately 10%. When superimposed on anemia, this change in blood oxygen affinity again caused a similar decrease in performance over and above that due to anemia alone. A marked rightward shift of the in vivo oxygen dissociation curve during severe exercise may have compensated for the reduced in vitro P₅₀.

T he effect of feeding diets containing 75% glucose or fructose on liver triglyceride formation in the rat was studied by both in vivo and in vitro techniques. The results were compared with those from control rats fed laboratory chow.Both high-sugar diets increased the capacity for triglyceride formation from sn-glycerol-3-P by rat liver homogenates and correspondingly increased incorporation of [1,3-¹⁴C]glycerol into hepatic triglyceride by the intact animal.These independent measures of hepatic triglyceride production changed with a similar time-course characteristic for each diet. The 75% fructose diet produced a greater increase in both determinations, reaching a maximum after 11 days.Despite the increase in hepatic triglyceride formation by both high-sugar diets, only the 75% fructose diet resulted in a consistent and sustained increase in serum triglyceride. This results most probably from differences in the fractional rate of serum triglyceride removal between the two groups.When serum triglyceride removal was inhibited by administration of Triton WR-1339, both high-sugar diets increased incorporation of [1,3-¹⁴C]glycerol in serum triglyceride in vivo and increased serum triglyceride level above that in control rats.

A n inhibitor of ciliary activity is present in the serum of cystic fibrosis (CF) patients and heterozygotes. Fractionation of CF serum has indicated that the inhibitor is associated with the serum IgG fraction. This study compared the activity of the CF inhibitor to that of rabbit antibody preparations directed against oyster cilia. The aim of this investigation was to determine whether the ciliary inhibitory mechanism in cystic fibrosis is related to a typical immunological reaction. Results from experiments utilizing fluorescent antibody techniques demonstrated that an antiserum directed against oyster ciliated epithelium binds immunologically with both human bronchial and oyster epithelial tissue. Results from experiments utilizing immunofluorescent tracing, passive hemagglutination, double immunodiffusion, and papain digestion, however, indicated that the interaction between the oyster cilia and the CF serum inhibitor was not a typical antigen-antibody reaction.

T he tissue localization of antihemophilic factor (AHF, Factor VIII) has been determined by immunofluorescent studies using monospecific rabbit antibody to human AHF. Specific staining demonstrating AHF antigens has been identified in endothelial cells of a wide range of human tissues. The staining pattern was observed in endothelial cells of arteries, capillaries, and veins as well as the cells lining hepatic and splenic sinusoids. Specific fluorescence was limited to these endothelial cells in sections of kidney, liver, spleen, lymph node, cardiac and smooth muscle, thyroid, umbilical cord, and skin. Absorption studies established that the staining was specific for cells in which there were proteins that had AHF antigens. The demonstration of fluorescence within the cytoplasm of endothelial cells suggests that these cells synthesize proteins that have AHF antigens.

E ndothelial cells were isolated from freshly obtained human umbilical cords by collagenase digestion of the interior of the umbilical vein. The cells were grown in tissue culture as a homogeneous population for periods up to 5 mo and some lines were subcultured for 10 serial passages. During the logarithmic phase of cell growth, cell-doubling time was 92 h. Light, phase contrast, and scanning electron microscopy demonstrated that cultured human endothelial cells grew as monolayers of closely opposed, polygonal large cells whereas both cultured human fibroblasts and human smooth muscle cells grew as overlapping layers of parallel arrays of slender, spindle-shaped cells. By transmission electron microscopy, cultured endothelial cells were seen to contain cytoplasmic inclusions (Weibel-Palade bodies) characteristic of in situ endothelial cells. These inclusions were also found in endothelial cells lining umbilical veins but were not seen in smooth muscle cells or fibroblasts in culture or in situ. Cultured endothelial cells contained abundant quantities of smooth muscle actomyosin. Cultured endothelial cells also contained ABH antigens appropriate to the tissue donor's blood type; these antigens were not detectable on cultured smooth muscle cells or fibroblasts. These studies demonstrate that it is possible to culture morphologically and immunologically identifiable human endothelial cells for periods up to 5 mo.

A ntihemophilic factor (AHF, Factor VIII) antigen has been demonstrated in cultured human endothelial cells by immunofluorescence studies using monospecific rabbit antibody to human AHF. Control studies with cultured human smooth muscle cells and human fibroblasts were negative. By radioimmunoassay it was demonstrated that cultured human endothelial cells contain AHF antigen which is released into the culture medium. Cultured smooth muscle cells and fibroblasts did not have this property. Cultured endothelial cells incorporated radioactive amino acids into high molecular weight, AHF antigen-rich protein fractions prepared from the culture media, 7% of the radioactive amino acid counts incorporated into this material were precipitated by globulin prepared from rabbit anti-AHF whereas normal rabbit globulin precipitated only 1.5% of the counts. Although cultured endothelial cells actively synthesize AHF antigen, AHF procoagulant activity was not detected in the culture medium. Studies seeking a basis for the lack of procoagulant activity have not clarified this deficiency, but they have established that exogenous AHF procoagulant activity is not inactivated by the tissue culture system.

I ntestinal brush borders prepared from vitamin D-deficient rats demonstrate increased susceptibility in vitro to fragmentation by shear forces or to loss of microvillus enzymes on treatment with EDTA. These effects are relatively nonspecific and are also observed in normal rats starved for 48 h. They may underlie prior observations that purport to demonstrate a vitamin D-dependent increase in brush border Ca-dependent ATPase. In addition, however, vitamin D increases ATPase activity dependent on certain divalent cations, including Ca and Zn, in whole-particulate suspensions pelleted by high-speed centrifugation of mucosal homogenates. This action is independent of changes in other microvillus enzymes, i.e. disaccharidases, and tissue distribution and cation specificity studies support the hypothesis that the mucosal whole-particulate ATPase is related to transport of Ca, Zn, and possibly other divalent cations.

P lasma concentrations of glucagon, insulin, glucose, and individual plasma amino acids were measured in normal nonobese and obese subjects before and after 3 days of dexamethasone treatment (2 mg/day) and in patients with Cushing's syndrome. The subjects were studied in the basal postabsorptive state and following the infusion of alanine (0.15 g/kg) or ingestion of a protein meal.In nonobese subjects dexamethasone treatment resulted in a 55% increment in basal glucagon levels and in a 60-100% increase in the maximal glucagon response to alanine infusion or protein ingestion. In obese subjects, basal glucagon rose by 110% following dexamethasone, while the response to alanine increased fourfold. In patients with Cushing's syndrome basal glucagon levels were 100% higher and the glucagon response to alanine infusion was 170% greater than in normal controls.Dexamethasone treatment in normal subjects resulted in a 40% rise in plasma alanine concentration which was directly proportional to the rise in basal glucagon. The remaining 14 amino acids were unchanged. In the patients with Cushing's syndrome alanine levels were 40% higher than in normal controls and were directly proportional to basal glucagon concentrations. No other plasma amino acids were significantly altered in the group with Cushing's syndrome.It is concluded that (a) glucocorticoids increase plasma glucagon concentration in the basal state and in response to protein ingestion or aminogenic stimulation; (b) this effect of glucocorticoids occurs in the face of obesity and persists in chronic hypercorticism; (c) hyperalaninemia is a characteristic of acute and chronic glucocorticoid excess, and may in turn contribute to steroid-induced hyperglucagonemia; and (d) increased alpha cell secretion may be a contributory factor in the gluconeogenic and diabetogenic effects of glucocorticoids.

1 0 patients with a single hyperfunctioning thyroid nodule each were studied for pituitary thyrotropin (TSH) suppression. They were judged to be euthyroid on clinical grounds. The total thyroxine (T₄D), free thyroxine (FT₄), total triiodothyronine (T₃D), and free triiodothyronine (FT₃) were normal in most of the patients. Incorporation of ¹³¹I into the hyperfunctioning thyroid nodules was not suppressed by the administration of physiological doses of T₃. Basal serum TSH concentrations were undetectable (<0.5 - 1.0 μU/ml) in all patients. The metabolic clearance of TSH in one patient before and after excision of the thyroid nodule was unchanged (40 vs. 42 ml/min) whereas the calculated production rate was undetectable before the operation (<29 mU/day) and normal after (103 mU/day). These data, in one patient, suggest that the undetectable concentration of TSH in these patients is a result of suppressed TSH secretion rather than accelerated TSH clearance.In eight patients, basal serum TSH concentrations failed to increase after the intravenous administration of 200 μg of thyrotropin-releasing hormone (TRH); minimal increases in serum TSH concentrations were observed in two patients. The suppression of TSH was evident despite “normal” concentrations of circulating thyroid hormones. The observation that normal serum concentrations of T₄D, FT₄, T₃D, and FT₃ may be associated with undetectable basal serum TSH concentrations and suppressed TSH response to TRH was also found in four hypothyroid patients given increasing doses of L-thyroxine and sequential TRH stimulation tests.

A lveolar macrophages lavaged from human lungs contain protease activity at an optimum pH of 3.0 and possibly a lesser peak of activity at pH 5.5. Protease activity measured at pH 4.1 is inhibited by purified alpha-1-antitrypsin.Fluorescent antibody studies of human alveolar macrophages showed that alpha-1-antitrypsin is present in normal alveolar macrophages. In addition, macrophages from a patient with a homozygous deficiency of alpha-1-antitrypsin exhibited less fluorescence when incubated in autologous serum than the same macrophages incubated in normal serum. Macrophages from normal subjects showed maximal fluorescence when removed from the lung and additional incubation with serum did not increase fluorescence.These results implicate the human alveolar macrophage as a possible source of an enzyme that may cause emphysema in patients deficient in alpha-1-antitrypsin. They also show that alpha-1-antitrypsin has access to the alveolus in normal subjects.

T his investigation was undertaken to evaluate possible genetic determinants of bone mass with the premise that inheritance of bone mass could be of etiologic importance in osteoporosis.Bone mass and width measurements were made with the photon absorption technique on the right radius of 71 juvenile and 80 adult twin paris. The variance of intrapair differences of bone mass in monozygotic (MZ) juvenile twins was 0.0013 g²/cm² compared to 0.0052 g²/cm² in the dizygotic (DZ) twins. For the adult twins the variance of intrapair differences in bone mass was 0.0069 for MZ and 0.0137 for DZ twins. Similar results were obtained for bone width. The significantly larger variation in intrapair differences in DZ twins indicates that these traits have significant genetic determinants. These intrapair differences were found to increase with age, suggesting that genetic-environmental interaction also contributes to the observed variation in bone mass.These data provide evidence that bone mass does have significant genetic factors, which alone or in conjunction with environmental factors may predispose persons to the development of osteoporosis.

B ile acid kinetics and biliary lipid composition were characterized in six women with gallstones before and after 6 mo of oral therapy with chenodeoxycholic acid, an agent that induces dissolution of cholesterol gallstones in man. Over a dosage range of 1-4 g/day, absorption varied from 0.8 to 2.3 g/day. The chenodeoxycholic acid pool expanded two-to sixfold, and bile became composed predominantly (> 90%) of chenodeoxycholic acid conjugated chiefly with glycine. Cholic acid and deoxycholic acid pools decreased markedly, so that the total bile acid pool expanded much less, about twofold on the average. Cholic acid synthesis decreased in five of the six patients, consistent with negative feedback inhibition of cholic acid synthesis by chenodeoxycholic acid. In four patients whose bile was above or close to saturation with cholesterol, the bile became unsaturated; in two patients, whose bile was unsaturated, it remained so. In five patients with radiolucent gallstones, chenodeoxycholic acid therapy was continued after completion of kinetic and composition measurements; the stones decreased in size or dissolved entirely during the subsequent 6 to 18 mo. Similar measurements of bile acid kinetics and biliary lipid composition were made before and after a 6-mo period without medication in a control group of six healthy women; no changes occurred.

T he metabolism of cholesterol and its 5-dihydro derivative, cholestanol, was investigated by means of sterol balance and isotope kinetic techniques in 3 subjects with cerebrotendinous xanthomatosis (CTX) and 11 other individuals. All subjects were hospitalized on a metabolic ward and were fed diets practically free of cholesterol and cholestanol. After the intravenous administration of [1,2-³H]cholestanol, the radioactive sterol was transported and esterified in plasma lipoproteins in an identical manner to cholesterol. In these short-term experiments, the specific activity-time curves of plasma cholestanol conformed to two-pool models in both the CTX and control groups. However, cholestanol plasma concentrations, total body miscible pools, and daily synthesis rates were two to five times greater in the CTX than control individuals. The short-term specific activity decay curves of plasma [4-¹⁴C]cholesterol also conformed to two-pool models in both groups. However, in the CTX subjects the decay was more rapid, and daily cholesterol synthesis was nearly double that of the control subjects. Plasma concentrations and the sizes of the rapidly turning over pool of exchangeable cholesterol were apparently small in the CTX subjects, and these measurements did not correlate with the large cholesterol deposits found in tendon and tuberous xanthomas.Despite active cholesterol synthesis, bile acid formation was subnormal in the CTX subjects. However, bile acid sequestration was accompanied by a rise in plasma cholestanol levels and greatly augmented fecal cholestanol outputs. In contrast, the administration of clofibrate lowered plasma cholesterol levels 50% and presumably reduced synthesis in the CTX subjects. Plasma cholesterol concentrations and fecal steroid excretion did not change significantly during this therapy.These findings indicate that the excessive tissue deposits of cholesterol and cholestanol that characterize CTX were associated with hyperactive neutral sterol synthesis. The demonstration of subnormal bile acid formation suggests that defective bile acid synthesis may predispose to the neutral sterol abnormalities.

N itroglycerin (TNG) causes a prolonged dilatation of coronary collaterals. To demonstrate a functional significance of this dilatation we measured the effect of TNG on myocardial contractile force in dogs 2½-4 wk after the left anterior descending coronary artery (LAD) had been embolized in closed-chest animals. Development of collaterals was documented by angiography. Via a left thoracotomy the main left coronary artery (LCA) and LAD distal to the embolized plug were cannulated. Coronary flow and perfusion pressure were recorded. Contractile force was measured with gauges sutured to epicardial areas supplied by the left circumflex coronary artery (LCf) and occluded LAD. Coronary perfusion pressure in the LCA was gradually decreased until the contractile force recorded by the LAD gauge diminished while the LCf gauge was unaffected. Under these conditions, with coronary perfusion pressure held constant with the aid of a Starling resistance, TNG (18 μg) injected into the LCA increased peripheral LAD pressure by 3-12 mm Hg and contractile force in the LAD region by 36% (range 20-90%), returning it to near-normal levels, while having minimal effect in the LCf area. These changes persisted for 5 min. When LCf and LAD areas were both ischemic, intracoronary TNG had minimal effect on peripheral LAD pressure and contractile force. Thus, TNG causes prolonged dilatation of coronary collaterals and presumed increased collateral flow with subsequent enhancement of myocardial contractile force in ischemic areas. This effect is seen only when ischemia is limited to an area supplied by the collaterals. When the whole heart is ischemic, collaterals are unresponsive to TNG, suggesting that these collaterals dilate fully when the regions from which they originate become ischemic.

X -ray diffraction analysis of bone from chronically uremic but nonacidotic rats with normocalcemia and hyperphosphatemia revealed smaller apatite crystals and an increase in the X-ray amorphous mineral fraction when compared to age-matched, pair-fed control animals, indicating less advanced mineral maturation in the uremic animals. Studies in animals with varied degrees of chronic renal insufficiency revealed a progression of the bone crystal maturational defect with advancing uremia.

T he immune response of C57BL/6 mice to allogeneic (DBA/2) mastocytoma cell suspensions was profoundly suppressed by intraperitoneal administration of 1 μg cholera enterotoxin 4 days after antigenic stimulation. The immune response assayed 11 days after antigen showed decreased cytolytically active thymusderived (T) lymphocytes and markedly depressed serumagglutinating titers. A comparable suppression of the immune response to skin allografts (DBA/2→C57BL/6) was also effected by cholera toxin administration, although there was no prolongation of allograft survival.The mechanism of the immune suppression is apparently related to the known adenylate cyclase stimulatory activities of choleragen.

A slow-moving hemoglobin with electrophoretic mobility similar to that of hemoglobin S was discovered in a white laboratory technologist. She had an elevated reticulocyte count, as did several members of her family. Her red cell survival was shortened. Amino acid analysis indicated that leucine at position β48 (CD7) had been replaced by arginine. The abnormal hemoglobin, called Okaloosa, was heat-precipitable and had decreased oxygen affinity. It exhibited a greater change in oxygen affinity than hemoglobin A when 2,3 DPG was added to “stripped” hemolysates. These findings cannot be readily explained by current views of structure-function relationships in the hemoglobin molecule. However, it is of interest that the amino acid in position CD7 is normally leucine in the α, β, δ, and γ-hemoglobin chains and in the myoglobin chain of man and a wide variety of other vertebrates.

M ost essential amino acids can be replaced by their α-keto-analogues in the diet. These ketoacids have therefore been proposed as substitutes for dietary protein. In order to determine their fate in tissues of normal animals, isolated rat liver and hindquarter (muscle) preparations were perfused with keto-analogues of valine, leucine, isoleucine, methionine, or phenylalanine. When perfused at 1.5-2.0 mM, all five compounds were utilized rapidly by the liver of 48-h starved rats, at rates varying from 49 to 155 μmol/h per 200g rat. The corresponding amino acids appeared in the medium in significantly increased concentrations. Perfusion with phenylpyruvate also led to the appearance of tyrosine. Urea release was unaltered. Measurement of metabolite concentrations in freeze-clamped liver revealed two abnormalities, particularly at ketoacid concentrations of 5 mM or above: a large increase in α-ketoglutarate, and a moderate to marked decrease in tissue glutamine. This decrease was quantitatively sufficient to account for nitrogen appearing in newly synthesized amino acids.Isolated hindquarters of fed rats were perfused with the same ketoacids at concentrations of 1.3-8.0 mM. All were utilized at rates varying from 1.4 to 7.0 μmol/h per g muscle perfused. The corresponding amino acids were released at greatly increased rates. Alanine and glutamate levels fell in some perfusions, but the principal nitrogen donor in muscle was not identified; the content of glutamine in tissue, and its rate of release into the perfusate remained constant.

B acterial multiplication in the lung associated with murine Sendai virus pneumonia is caused by virus-induced defects in pulmonary bactericidal mechanisms. The nature of this effect has been studied in animals immunized against the challenge bacteria. Mice were immunized against Proteus mirabilis by intraperitoneal inoculation and by aerosol inhalation. After the development of immunity, mice were infected aerogenically with 10⁴ TCID₅₀ of Sendai virus. 7 days later, during the height of the bronchial inflammation and pulmonary consolidation, the mice were challenged with an aerosol of viable ³⁵S-labeled Proteus mirabilis or ³²P-labeled Staphylococcus aureus.Nonimmunized virus-infected animals showed marked impairment of pulmonary bactericidal activity with subsequent multiplication of the bacterial strain in the case of Proteus mirabilis. Immunized nonvirus-infected animals showed enhancement of pulmonary bactericidal activity for the homologous and heterologous strains in comparison with nonimmunized animals. Virus-infected animals immunized by aerosol showed enhanced bactericidal activity against the homologous but not the heterologous bacterial strain. Neither virus infection nor immunization had a significant effect on the transport of particles in the lung. The data demonstrated that the bacterial multiplication associated with the virus pneumonia was prevented by preceding immunization against the homologous challenge organism. The data suggest a mechanism for controlling bacterial multiplication associated with virus pneumonias.

T he intrarenal distribution of radionuclide microspheres injected into the thoracic aorta was used to examine glomerular blood flow distribution (GBFD) in 26 healthy, unanesthetized puppies, ranging in age from 5 h to 42 days, and in 5 adult dogs. For analysis, the cortex was divided into four equally thick zones designated zone I (subcapsular) to zone IV (juxtamedullary).During the first 36 h of life, the highest flow rate was in zone II, which received 35.5±2.0%/g, compared with 26.8±1.4% to zone I, 23.7±1.4% to zone III, and 13.4±1.4% to zone IV. At age 6 wk, zone I had the highest rate of perfusion (48.6±2.1%, compared with 28.8±1.4% in zone II, 15.8±0.8%, in zone III, and 6.8±0.6% in zone IV). The 6-wk old animals resembled the adult animals, except for relatively greater perfusion per gram of zone I in the former group. Changes in relative GBFD did not correlate with those in arterial pressure or peripheral hematocrit.The distribution of glomeruli among the four zones of the cortex followed its own pattern of development. At birth and at 6 wk, the greatest density of glomeruli was in zone I (50.6±5.4 and 42.7±3.9%/g respectively, as compared with 24.1±2.9% in adults); in adults zone II contained the greatest density (39.1±1.6%).At birth the relative perfusion of glomeruli in zone I was only one-fifth that of glomeruli in zone IV, with intermediate values in zones II and III. By 6 wk of age, increased perfusion of the outer cortical glomeruli resulted in rates of flow in the four zones that did not differ significantly from each other. Relative perfusion in zone I continued to increase, so that in the adult animals perfusion in that zone was significantly greater than in the three deeper zones.These data demonstrate the marked hemodynamic changes that take place within the kidney during the first few weeks of life. The relatively greater blood flow of the most deeply situated nephrons in the early postnatal period suggests ascendancy of this population of nephrons and may have important functional implications.

G onococcal pili, pure by the criteria of electron microscopic examination and polyacrylamide gel electrophoresis in sodium dodecyl sulfate, have been prepared by repeated cycles of precipitation with 0.1 M MgCl₂, followed by dissolution in 0.01 M Tris pH 8, 0.01 M NaN₃. Using a fluorescein-conjugated antibody prepared to pili from a single strain, pilar antigen(s) was found to be present in each of 18 strains of gonococci tested, and absent from strains of pilated meningococci, nonpathogenic Neisseria sp., and Escherichia coli. Purified pili, labeled with ¹²⁵I were used in an antigen binding assay to quantitatively measure antibody to pili in rabbit sera and in 561 human sera. The range of antibody activity for 133 persons unlikely to have experienced gonorrhea was 0.1-1.6 μg/ml with a geometric mean of 0.5 μg/ml. This geometric mean antibody activity was significantly lower than the geometric mean for asymptomatically infected males (1.0 μg/ml, P < 0.002), males with symptomatic gonococcal anterior urethritis (1.6 μg/ml, P < 0.001), or asymptomatically infected females (4.2 μg/ml, P < 0.001). Antibody appeared in elevated levels (> 1.6 μg/ml) 2-3 wk after infection and returned toward control levels 1 or more months after treatment. Antibody levels higher than 1.6 μg/ml were found in 26 (50%) of 52 males with gonococcal anterior urethritis, in 10 (33%) of 30 males with asymptomatic urethral infection and in 50 (89%) of 56 asymptomatically infected females. In a high-risk group of 103 females for whom culture results and antibody to pili were compared, 58 (57%) had elevated antibody levels to pili and 86% of the infected females were within this seropositive group. The antigen binding assay may provide a means to detect asymptomatic gonococcal infection in women.

T he effect of impaired intestinal protein synthesis on chylomicron apoprotein composition was studied in mesenteric lymph fistula rats. Lymph was obtained from animals with impaired protein synthesis given intraperitoneal acetoxycycloheximide (ACH), a potent inhibitor of protein synthesis. Lymph chylomicrons were then isolated by ultracentrifugation and purified on agarose columns. Purified chylomicrons from control and ACH-treated animals were delipidated, and their apoprotein pattern was examined on sodium dodecyl sulfate (SDS) polyacrylamide gels.Because we had previously demonstrated a markedly increased lymph chylomicron size during the inhibition of protein synthesis, it was first necessary to determine whether chylomicron apoprotein composition normally varied with chylomicron size. Chylomicrons of varying sizes were prepared by differential ultracentrifugation, and their apoprotein composition was determined densitometrically on SDS polyacrylamide gels. No significant difference in apoprotein composition was found normally with varying chylomicron size.In contrast, however, chylomicrons from ACH-treated animals showed a 50% decrease in a major apoprotein band with R₁ 0.67. Other chylomicron apoproteins were not decreased as a result of impaired protein synthesis, suggesting differing rates of synthesis of the various chylomicron apoproteins. In vivo incorporation studies of [³H]leucine into the various apoproteins of lymph chylomicrons demonstrated that this apoprotein (R₁ 0.67) had the most rapid synthesis rate and suggested that it seemed most affected by impaired intestinal protein synthesis. Immunologic studies indicated that this apoprotein was immunologically related to high-density lipoproteins (HDL) and was present in chylomicrons isolated directly from small intestinal mucosa.These studies demonstrate that impaired intestimal protein synthesis is associated with a deficiency in one of the major chylomicron apoproteins and may in part explain the impaired lipid absorption seen during states of impaired protein synthesis.

I ntrapulmonary deposition of the proteolytic enzyme papain produces a lesion resembling emphysema in experimental animals. The natural history of this lesion has not been well defined. The present study was performed to evaluate changes in lung structure and function with aging in normal rats and rats exposed to an aerosol of papain at 2 mo of age. Groups of control and papain-exposed animals were studied at 4, 8, and 18 mo of age. The parameters of lung function studied were specific airways' conductance (G_aw/TGV), diffusing capacity per unit of alveolar volume (D_Lco/V_A), diffusing capacity (D_Lco), and functional residual capacity (FRC). Morphometric parameters were the postfixation lung volume (V_L) and mean chord length (L_M); internal surface area (ISA) and ISA extrapolated to both the mean V_L of the corresponding papain group and a V_L of 10 ml (ISA₁₀) were calculated.At 4 mo of age L_M and FRC were significantly increased and ISA, D_Lco/V_A, and D_Lco were significantly reduced in the papain group. At 8 mo of age L_M was significantly increased and ISA was significantly decreased in the papain group: physiologic studies were not performed in this group. At 18 mo of age L_M was significantly increased and D_Lco/V_A, D_Lco, and ISA were significantly decreased. Neither progression nor healing of the lesion was observed despite similar lung growth in both groups.This study demonstrates that a single proteolytic lung injury produces a fixed deficit of lung parenchyma. Progressive lung destruction may require repeated or continuous lung injury.

T he present studies were undertaken to determine the role, if any, of cyclic 3′,5′-adenosine monophosphate (cyclic AMP) as a chemical inducer of rat liver alkaline phosphatase. Cholera enterotoxin, given intravenously to rats, led to a rapid rise in the activity of hepatic adenyl cyclase that was 7½ times greater than control values in 6 h. Cyclic AMP levels were also significantly increased above control values while the activity of cyclic nucleotide phosphodiesterase was unchanged. Hepatic alkaline phosphatase activity was increased 5½ times above control in 12 h, but its rise followed that of adenyl cyclase and cyclic AMP by several hours. Cycloheximide inhibited the rise of hepatic alkaline phosphatase but not that of adenyl cyclase. The administration of glucagon, a known stimulator of hepatic adenyl cyclase, and of dibutyryl cyclic AMP, led to similar striking increases in hepatic alkaline phosphatase activity. This alkaline phosphatase increase was blocked by the prior administration of cycloheximide. Bile duct ligation, a known stimulator of hepatic alkaline phosphatase activity, failed to produce any significant changes in adenyl cyclase or cyclic AMP. Concomitant treatment of rats with bile duct ligation and cholera enterotoxin or bile duct ligation and glucagon, had no additive effect on the increase in hepatic alkaline phosphatase activity, although the increase occurred earlier. These results suggest that: (a) cyclic AMP may act as an inducer of hepatic alkaline phosphatase: (b) the stimulation of hepatic alkaline phosphatase by cholera enterotoxin is mediated by cyclic AMP; (c) the rise in hepatic alkaline phosphatase following bile duct ligation is not mediated by cyclic AMP; (d) the same alkaline phosphatase in rat liver may be induced by two (or more) mechanisms, only one of which requires cyclic AMP.

A prospective study used polymyxin B by aerosol to reduce colonization of the upper respiratory tract with nosocomial gram-negative bacilli. 58 high-risk patients from the Respiratory-Surgical Intensive Care Unit entered the trial. 33 were randomly selected to receive 2.5 mg/kg/day of polymyxin B by hand atomizer into the pharynx, and tracheal tube if present. 17 of 25 control patients became colonized with gram-negative bacilli as compared with 7 of 33 polymyxin-treated patients (p < 0.01). Control patients became colonized with a total of 33 gram-negative bacilli: 3 were Pseudomonas aeruginosa, 21 were species of Enterobacteriaceae. The polymyxin-treated patients became colonized with a total of 11 gram-negative bacilli: no P. aeruginosa and only 3 species of Enterobacteriaceae were recovered. Colonization increased with duration in Respiratory-Surgical Intensive Care Unit and with time of required controlled ventilation. Polymyxin most effectively prevented the increase in colonization in treated patients who stayed in the Respiratory-Surgical Intensive Care Unit for longer than 1 wk and who required controlled ventilation for at least 72 h.

A s a first step in our study of structure-function relationships among primate and non-primate growth hormones, human growth hormone (hGH) was subjected to the limited digestive activity of human plasmin. The lyophilized whole digest, containing less than 2% of unchanged hormone, had an average of 2.3 new amino-terminal groups per mole. The digest had the same potency as the native hormone (a) in causing weight gain in hypophysectomized rats; (b) in stimulating somatomedin production in hypophysectomized rats; (c) in stimulating upake of [³H]leucine into isolated diaphragm of hypophysectomized rats; (d) in accelerating transport of [¹⁴C]α-aminoisobutyric acid into isolated diaphragm of hypophysectomized rats; (e) in stimulating uptake of [3-0-methyl-¹⁴C]glucose by isolated adipose tissue of hypophysectomized rats; (f) in accelerating conversion of [¹⁴C]glucose to ¹⁴CO₂ by isolated epididymal adipose tissue of hypophysectomized rats. The digest also caused glucosuria in partially pancreatectomized rats treated with dexamethasone.These metabolic actions of plasmin-digested hGH in the array of animal tests were confirmed by comparable effects elicited in 11 human subjects (nine pituitary-deficient children and adolescents and two nondeficient adults). A single injection of the plasmin digest caused an increase in plasma free fatty acids and a fall in plasma amino acids. Seven daily injections caused positive balances of nitrogen, phosphorous, sodium, and potassium, gain in body weight, and in two of three subjects impairment of glucose tolerance. The potency of the plasmin digest in producing these metabolic effects in man was comparable to that of native hGH.Thus, 2-3 bonds in the hGH molecule can be cleaved by plasmin without impairing the hormone's growthpromoting, anabolic, diabetogenic, and adipokinetic actions for rat and man.

T he extractable and nonextractable collagen and glycosaminoglycuronans (GAG) were estimated and characterized in 32 dried, defatted human livers obtained at necropsy. 10 had normal livers. 22 of the 32 livers were from patients who drank in excess: 5 had fatty livers, 7 had alcholic hepatitis, and 10 had cirrhosis. Livers with alcoholic hepatitis or cirrhosis had significantly increased total and 1 N NaCl-extractable collagen. Only alcoholic hepatitis livers had significantly increased Tris-buffer-extractable GAG, but the amino acid composition of these GAG (proteoglycans) was no different from that of normal livers. The major fraction of these GAG had isoelectric pH (pI) ≤ 3.1 in all livers. Livers with alcoholic hepatitis or cirrhosis had significantly increased nonextractable GAG. The major GAG fraction of all livers was chondroitin-4 or -6-SO₄. Alcoholic hepatitis livers had a significant increase of hyaluronic acid and an unidentified hyaluronidase-resistant GAG. Fatty livers showed no differences from normal ones.The data indicates that alcoholic hepatitis is associated with a significantly increased fibroblast activity, but fatty livers of alcoholics are not. The changes in histologically “inactive” micronodular cirrhosis of alcoholic patients indicate continued activity of fibroblasts in the connective tissue of these cirrhotic livers.

T he mammalian antidiuretic hormone, 8-arginine-vasopressin, was found to increase net mucosal-to-serosal urea flux across the isolated toad urinary bladder 13-fold. This urea flux was accompanied by a 24-fold increase in solute-linked water movement across the membrane. Net urea flux and urea-linked volume flux were inhibited by 50% or more when thiourea was added to the mucosal medium at concentrations equal to those of urea. In contrast, thiourea did not inhibit osmotic water flux across the bladder in the presence of vasopressin. These observations are consistant with the view that thiourea and urea compete for a common site on a membrane carrier molecule.When bladders were exposed to vasopressin on the serosa and subsequently fixed with 1% glutaraldehyde on the mucosa, they were found to retain 74% of their prefixation permeability to urea. Net urea flux across these fixed bladders (in the absence of vasopressin) was markedly inhibited by thiourea, whereas osmotic water flux was not inhibited. These studies suggest that vasopressin induces the formation of “urea-channels” in the membrane that can be preserved by glutaraldehyde and blocked by thiourea.