Issue published January 1, 1969

T o determine the quantitative relationship of urinary hydroxyproline peptide excretion to collagen breakdown, known quantities of radioactive hydroxyproline peptides were administered to unlabeled animals and excertion of radioactivity in respiratory carbon dioxide, urine, and feces was measured. The major routes of excretion of collagen peptide metabolites were respiratory carbon dioxide (75%) and urine, as hydroxyproline-containing peptides (25%).Since the predominant urine hydroxyproline peptide linkage is proly-hydroxyproline, L-prolyl-L-hydroxyproline-³H was administered to unlabeled animals. Greater than 80% of the administered dipeptide was excreted in urine, suggesting that this peptide linkage is not hydrolyzed to a significant extent in vivo.These data suggest that urinary hydroxyproline excretion is a “fairly” sensitive indicator of collagen breakdown and can be used at the clinical level to quantitate changes in collagen breakdown.

B y measurement of its arginine esterase activity, plasma kallikrein was purified from fresh frozen ACD plasma. The steps involved alcohol fractionation, isoelectric precipitation, and carboxymethyl (CM) Sephadex and DEAE cellulose chromatography. Three enzymatically active fractions were finally isolated and termed plasma kallikreins I, II, and III; they represented purifications of 970,320- and 590-fold, respectively. All three kallikreins were active biologically; they increased vascular permeability in the guinea pig and released a kinin from human plasma, as measured in the rat uterus bioassay. Bradykinin and/or closely related kinins were identified in the kallikrein I plasma digest by radioimmunoassay.Kallikreins I, II, and III had similar ratios of hydrolytic activity on a variety of arginine and lysine esters and were immunochemically related. However, differences were present on physicochemical characterization: kallikrein I had S_20,[unk] of 5.7, a mol wt of 99,800, and migrated as a slow gamma globulin; kallikrein II migrated as a fast gamma globulin with a mol wt of 163,000, but the evidence suggested that it was closely related, if not interconvertible, with kallikrein I. Kallikrein III, on the other hand, migrated as an alpha globulin and reacted quite differently with inhibitors.

E vidence is presented in this paper that the kaolin-activated arginine esterase of plasma is related to plasma kallikrein activity. Such a relationship is based on studies that (1) establish a constant ratio of esterase activity on various synthetic substrates for the kaolin-activated arginine esterase, purified kallikrein(s), and preparations obtained during the fractionation procedure; (2) exclude other known plasma and tissue arginine esterases; (3) confirm the requirement for factor XII in the activation of the enzyme precursor; and (4) show similarities in behavior between the plasma esterase and purified kallikrein(s) toward a variety of inhibitors.Based on this probable identification, evidence is provided that the concentration of active factor XII determines the rate of activation of plasma kallikreinogen, and that the activation may be blocked by polybrene. Once activated, plasma kallikrein is rapidly inactivated by the naturally occurring plasma inhibitor, but the inhibition is incomplete. Acid or chloroform treatment of plasma rapidly inactivates the plasma inhibitor without affecting the concentration of plasma kallikreinogen.Another plasma arginine esterase with properties suggestive of permeability factor is activated by factor XII in the presence of synthetic substrates, but only at low ionic strength. The data suggest that this enzyme is closely related to plasma kallikrein and that it arises from a common precursor.

C ertain aspects of the metabolism of centrifuged young and old erythrocytes in hemoglobin H disease have been examined and compared with similar studies of beta thalassemia and normal cells. Glycolysis, hexose monophosphate shunt activity (HMPS), potassium flux, and glutathione (GSH) content were measured. The distributions of hemoglobins H and F, as well as the activities of erythrocyte glucose-6-phosphate dehydrogenase (G6PD) and glutamic oxalacetic transaminase (GOT), were utilized for estimations of the relative ages of the cell samples. The young erythrocytes in hemoglobin H disease differed in several respects from older hemoglobin H cells. They contained more soluble hemoglobin H and GSH and, after splenectomy, fewer inclusions. HMPS activity was subnormal in hemoglobin H young cells and rose to normal activity in old cells. Potassium flux tended to increase in old cells when inclusions were present.Beta thalassemia young cells contained less hemoglobin F and, after splenectomy, more inclusions than old cells. In addition, they had markedly increased glycolysis and HMPS activity. GSH was randomly distributed. Potassium flux was increased in younger cells and particularly increased when inclusions appeared in younger cells after splenectomy.The results are interpreted to indicate that inclusion formation is associated with increased erythrocyte cation permeability in the thalassemia syndromes. This is not related to the level of intracellular GSH.The decreased HMPS activity in young hemoglobin H cells may be due to the presence of the extra thiols of soluble hemoglobin H which can act as a reducing agent. The substitution of hemoglobin H for glutathione in this capacity would then spare the NADPH-requiring glutathione reductase system. As a consequence, HMPS activity would decline. However, in older cells the oxidized hemoglobin H precipitates; these must rely upon GSH and glutathione reductase activity for thiol reduction capacity. Accordingly, HMPS activity increases to normal in the old cell population.

S odium phenobarbital and various hormones, compounds capable of hepatic enzyme induction, were given to an infant boy with congenital, nonhemolytic, unconjugated, hyperbilirubinemia and severe kernicterus for prolonged periods between the ages of 2 and 25 months to determine their effect on serum bilirubin concentrations. Phenobarbital, 5 mg/day orally, on two occasions decreased serum bilirubin concentrations approximately threefold over a period of 30 days. Withdrawal of phenobarbital after the first study resulted in a gradual (30 days) return of serum bilirubin to pretreatment levels. The lower serum bilirubin concentrations observed when phenobarbital therapy was reinstituted were maintained for 61 days on 2.5 mg/kg per day of the drug. Orally administered L-triiodothyronine, 0.05-0.1 mg/day for 71 days, intramuscular human growth hormone, 1 mg/day for 21 days, and testosterone propionate, 0.1 mg/day for 9 days, did not decrease serum bilirubin levels below lowest control values of 18 mg/100 ml.Bilirubin-³H was administered twice before and once with bilirubin-¹⁴C during phenobarbital therapy to study the kinetics of bilirubin metabolism. Results of the first and second control studies and of the bilirubin-³H and bilirubin-¹⁴C phenobarbital studies, respectively, were as follows: total body bilirubin pools, 200, 184, 73, and 72 mg; half-lives, 111, 84, 37, and 39 hr; and turnover, 30, 37, 33, and 31 mg/day. The data show that the approximate threefold decrease in serum bilirubin concentration and total body pool resulted from a comparable decrease in bilirubin half-life without a significant change in turnover.In vitro histological (electron microscopy) and enzymological studies of liver obtained by surgical biopsies before and during phenobaribtal administration showed that both the hepatocyte content of agranular endoplasmic reticulum (AER) and the ability of liver homogenate to conjugate p-nitrophenol were significantly increased during phenobarbital treatment.The observations suggest that phenobarbital affects bilirubin metabolism by the induction of an enzyme(s) with a slow rate(s) of degradation (or rapid rate of degradation with limited capacity).

R adiosulfate, ³⁵SO₄, and radiobromide, ⁸²Br, were administered simultaneously to rats and dogs. In rats, the apparent volume of distribution of ⁸²Br averaged 30% of body weight and was constant between 0.5 and 35 hr after injection. The apparent volume of distribution of ³⁵SO₄, corrected for urinary loss, increased by 6% body weight/hr: the extrapolated volume at zero time was 88% of bromide space. Analysis of individual tissues and carcasses for ⁸²Br and inorganic ³⁵SO₄ showed that equilibration of both isotopes in several organs and in the whole carcass was rapidly achieved within 1 to 2 hr: no further increase in measured spaces occurred in 24 hr. The carcass inorganic sulfate space was 92%±2% of the bromide space in intact rats, and showed no increase with time. However, a progressively greater fraction of the injected ³⁵SO₄ was not recovered, owing to metabolic alteration. In eviscerated rats, the inorganic sulfate space was a smaller and much more constant fraction (79.8% ±0.4%) of the bromide space, showing that at least 20% of body bromide (and hence chloride) is nonextracellular. The viscera chiefly responsible for the higher ratio of spaces in the intact animal were the liver, small bowel, and kidney. In the last two organs, excess inorganic ³⁵SO₄ (beyond the bromide space) was attributable to trapped transcellular fluid in which sulfate had been concentrated more than chloride (or bromide). Excess sulfate in liver and cartilage could not be explained in this manner: the results suggest passive binding of sulfate, but could reflect active cell uptake in these tissues. No excess sulfate was found in skin or tail. The implications of these observations with respect to the distribution of body chloride and the measurement of extracellular space are discussed. The extracellular volume of the rat is estimated to be 24% of body weight.

T he effect of dietary calcium intake on calcium metabolism was studied in eight normal volunteers by multicompartmental analysis of radiocalcium and balance data. In paired studies of six normal subjects on normal and high or low calcium intakes, necessary and sufficient criteria were used to determine changes in calcium metabolic parameters produced by alterations in dietary calcium. These changes involved gastrointestinal calcium absorption rate, renal and endogenous fecal rate constants, and bone resorption rate. Bone accretion rate and compartment sizes need not change between the paired studies. The changes of parameters involving kidney, gut, and bone were in a direction to support calcium homeostasis and were compatible with the pattern of changes produced by parathyroid hormone. However, the source of the stimulus for hormone secretion was not apparent since plasma calcium concentrations showed no significant difference between paired studies. The implications of these findings relative to control of hormone secretion, calcium regulatory mechanisms, and metabolic bone disease are discussed.

S everal aspects of the effects of dietary fat on plasma lipids and lipoproteins were investigated in 12 subjects during the long-term feeding of formulas containing 40% of their calories as either saturated or unsaturated fats. The changes in fatty acid composition of plasma lipids, shown previously to occur after prolonged feedings of a dietary fat, required 10-14 days to be complete and were synchronous with the effect of the fat on plasma lipid concentrations. The change in lipid concentration occurred in low but not in high density lipoproteins. The effects on lipid levels of the low density lipoproteins were found to occur with little or no effect on the concentration of the protein moiety of these lipoproteins; as a result, cholesterol- and phospholipid to protein ratios in low density lipoproteins fell during unsaturated fat feeding. The effects of dietary fat on plasma phospholipids were studied in detail: the relative amounts of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and lysophosphatidylcholine were unaffected by the type of dietary fat. However, the molecular species of phosphatidylcholine were markedly affected. More than 90% of the fatty acids at the α-position were saturated during both saturated and unsaturated feedings. In contrast, during unsaturated feedings, linoleate at the β-position outnumbered oleate by approximately 4:1, whereas during saturated feedings these two types of fatty acids were present in nearly equal amounts.This paper also presents the following hypothesis for the lipid-lowering effect of unsaturated dietary fat: since unsaturated fatty acids occupy a greater area than saturated acids, they alter the spatial configuration of the lipids into which they are incorporated; as a result, fewer lipid molecules can be accommodated by the apoprotein of the low-density lipoproteins (LDL), and thus the lipid content of the lipoprotein is lowered. The experimental findings of this study, while not proving this hypothesis, are consistent with it.

T he absorption of endogenous cholesterol, labeled with tracer doses of cholesterol ¹⁴C or cholesterol-³H and of near physiological doses of vitamin D₃-³H was studied in rats with cannulated intestinal lymphatics. The effects of administering mixed micellar solutions of fatty acid, monoglyceride, and bile salt on the absorption of these labeled sterols was determined. It was observed that the specific activity of free cholesterol and the amounts of vitamin D₃ appearing in lymph were significantly increased during the intraduodenal administration of mixed micellar solutions of either linoleic or palmitic acid, in contrast to control rats receiving a micellar solution of taurocholate. These increases were related linearly to the lymph triglyceride level. In addition it was observed that when the linoleic acid solution was administered there was a more marked increase in the ratio of the specific activities of free and esterified cholesterol in lymph than with either the palmitic acid or taurocholate solutions.Additional studies in rats with intact lymphatics showed that the uptake of labeled cholesterol and vitamin D₃ from the intestinal lumen into the wall was similar whether the sterols were administered in taurocholate or in mixed micellar solution.These findings suggest that mixed micellar lipid increased the rate of appearance of labeled free cholesterol and vitamin D₃ in lymph by enhancing their transport out of the intestinal mucosa, rather than by an effect on uptake.

A ngiotensin infusion evokes marked increases in aldosterone secretion in primary aldosteronism and little change in secondary aldosteronism. The low plasma renin activity of primary aldosteronism and the elevated plasma renin activity of secondary aldosteronism are thought to account for this differential response. The effect of angiotensin on aldosterone and 18-hydroxycorticosterone secretion was studied during adrenal vein catheterization in seven patients with primary aldosteronism (whose plasma renin activity had been elevated following spironolactone therapy), one hypertensive patient with normal plasma renin activity and normal aldosterone secretion, two patients with secondary aldosteronism who had elevated plasma renin activity, and one anephric patient whose plasma renin activity was 0. Adrenal venous aldosterone and 18-hydroxycorticosterone were measured before and after a ten min sub-pressor angiotensin infusion.The cells of the aldosterone-producing adenoma (APA) respond to small increases in plasma angiotensin with large increases in secretion of aldosterone and 18-hydroxycorticosterone. The dose of angiotensin capable of evoking this response from the aldosterone-producing adenoma produces little or no change in the secretion of the steroids from nontumorous glands. The augmentation of aldosterone secretion, induced by angiotensin, in primary aldosteronism is due solely to increased secretion by the adenoma and not by the contralateral zona glomerulosa. The increased sensitivity of the aldosterone-producing adenoma is characteristic of the tumor. This response is independent of fluctuations in endogenous plasma renin activity. This sensitivity is not blunted by high plasma renin activity, nor is it a function of tumor mass for the effect is observed in aldosterone-producing adenomas regardless of size. ACTH injection after angiotensin infusion resulted in a marked increase in aldosterone concentration in the effluent from the nontumorous adrenal, but was not capable of producing further increases in aldosterone concentration in the effluent from the APA. In view of this exquisite sensitivity to infused angiotensin, it may be that the small variations in endogenous plasma renin activity that have been observed in primary aldosteronism may be capable of evoking large changes in aldosterone secretion in patients with aldosterone-producing adenomas.

( 1) Synthesis of deoxythymidine by either direct transfer of deoxyribosyl to thymine (pyrimidine deoxyribosyltransferase) or by a coupled deoxynucleoside phosphorylase mechanism is approximately twofold greater with normal leukocyte extracts (55 to 88% granulocytes) than with extracts prepared from leukocytes obtained from patients with chronic myelogenous leukemia. Activities in lymphocytes (normal or leukemic) are one-fifth the activity of normal granulocytes.(2) The lower activity in chronic myelogenous leukemia remains at 50% of normal even when patients are in hematologic remission with a normal per cent mature granulocytes in the peripheral blood.(3) The leukemic enzyme could not be distinguished from the normal by pH optima, thermal stability, or kinetic properties. The Km's for the deoxyribosyl acceptor and deoxyribosyl donors were identical for both enzymes. Both are subject to substrate inhibition by thymine and to inhibition by purine bases with similar Ki's. In addition, the transferase component of both the leukemic and the normal cell enzyme is activated by phosphate and arsenate. It appears, therefore, that there is no qualitative difference between the enzyme obtained from leukocytes of patients with chronic myelogenous leukemia and the enzyme obtained from normal leukocytes, suggesting that the difference in total cell activity is due to an actual decrease in amount of enzyme in chronic myelogenous leukemia or to a mixed cell population, one with a normal quantity of enzyme and the other with little or no active enzyme.(4) In both the normal cell and the leukemic cell extracts, transferase and phosphorylase activities could not be separated. The ratio of the two activities remained constant over a 140- and a 230-fold purification in normal and leukemic cell extracts, respectively. These and other observations indicate that transferase and phosphorylase activities are associated with the same protein.(5) The metabolism of pyrimidine and purine deoxynucleosides is similar for normal and leukemic cells. Catabolism of all deoxynucleosides tested was by direct phosphorolysis, except for deoxyadenosine which required initial deamination to deoxyinosine before phosphorolysis. In contrast to the greater rates of pyrimidine deoxynucleoside synthesis and cleavage with normal leukocyte extracts, the rates of purine deoxynucleoside synthesis and cleavage were approximately twofold greater with extracts prepared from cells of patients with chronic myelogenous leukemia. There was no significant difference in the rate of phosphorolytic cleavage of pyrimidine nucleosides (uridine) between the CML and normal leukocyte extracts.

I t is known that bisulfite ions can selectively deplete red blood cells of 2,3-diphosphoglycerate (2,3-DPG). Studies of the effects of bisulfite on sodium-potassium permeability and metabolism were undertaken to clarify the physiologic role of the abundant quantities of 2,3-DPG in human erythrocytes. Treatment of cells with bisulfite results in a reversible increase in the passive permeability to Na and K ions. Metabolism of glucose to lactate is increased, with a rise in the intracellular ratio of fructose diphosphate to hexose monophosphate. Cell 2,3-DPG is quantitatively converted to pyruvate and inorganic phosphate. The permeability effects of bisulfite are countered by ethacrynic acid and by such oxidizing agents as pyruvate and methylene blue. Taken together, the results suggest that the effects on Na-K flux of bisulfite are related more to the reducing potential of this anion than to its capacity to deplete cells of 2,3-DPG.

T he extent of dissociation of various hemoglobins into subunits was estimated from their elution volumes (V_e) on G-100 Sephadex. Under the same controlled conditions carboxyhemoglobins A, A3 (A₁), F, S, and C all had the same elution volumes. The carboxy and cyanmet derivatives of hemoglobin Kansas (a variant with very low oxygen affinity) had a relatively high V_e, indicating a decreased mean molecular weight and therefore an increased tendency to form dimers and even monomers. Conversely, the liganded derivatives of hemoglobin Chesapeake (a variant with high oxygen affinity) had a relatively low V_e, suggestive of an impaired degree of subunit dissociation. Deoxyhemoglobin Chesapeake had a V_e identical with that of deoxyhemoglobin A. Cat hemoglobin, known to have an unusually low oxygen affinity, was found to have a higher V_e than human, dog, rabbit, rat, or guinea pig hemoglobins.Haptoglobin is thought to bind αβ dimers in preference to the α²β₂-tetramer. The comparative haptoglobin affinities of the human hemoglobins were measured by competition between the test hemoglobin and radioactive reference hemoglobin for haptoglobin binding sites. Hemoglobins A, F, S, and C all seemed to bind equally readily, but hemoglobin Kansas and cat hemoglobin showed a higher affinity, and hemoglobin Chesapeake a lower affinity.These results are in accord with recently proposed models which predict that hemoglobins which have an increased degree of subunit dissociation will have a low oxygen affinity, and vice versa.

A method is described to determine the fatty acid composition of small samples of subcutaneous adipose tissue, and of fasting plasma free fatty acids (FFA) and triglycerides. These analyses were carried out on samples from five normal children, six diabetic children consuming a standard diabetic diet, 17 diabetic children prescribed a diet rich in corn oil since diagnosis 4-7 years ago, and 2 brothers with familial hypercholesterolemia on a corn oil diet for 3 yr. The results obtained showed that: (1) The composition of adipose tissue triglycerides in the diabetic children on a standard diet was similar to that in the normal children. (2) The 17 diabetic children were consuming different quantities of corn oil. (3) There was a highly significant correlation between the percentage of linoleic acid present in adipose tissue and in the fasting plasma FFA fraction. It is therefore concluded that future assessments of the adherence of these diabetic children to their corn oil diet will be possible by examination of the fasting plasma FFA fraction, obviating the need for repeated adipose tissue biopsies. (4) The sum of the concentration of saturated and monounsaturated fatty acids of the same chain length in adipose tissue was similar to that in the fasting plasma FFA fraction, even though the proportions of individual acids were different in the two fractions.

I n 12 dogs anesthetized with chloralose, angiotensin (angiotensin II amide) given intravenously increased the glomerular filtration rate (GFR) of an ischemic kidney while simultaneously having little effect on the GFR of the contralateral kidney. In the ischemic kidney, in 14 of 30 observations, increments of GFR greater than 100% of mean control GFR (9 ml/min) occurred in response to angiotensin. The magnitude of the increase in GFR produced by angiotensin was independent of dose (range 0.005-0.050 μg/kg per min), the degree of accompanying pressor response, and alterations in renal blood flow (RBF) (electromagnetic flow-meter). In the ischemic kidney, increments of GFR could be produced by sub-pressor doses of angiotensin.Dissociations between increments of GFR and sodium excretion occurred. Equivalent increments of GFR in the ischemic kidney in dogs receiving either 5% glucose in water or 10% mannitol in 0.3% saline were associated with natriuresis only in the latter group: a) as an initial response of the contralateral kidney to renal arterial constriction (RAC) in spite of a concomitant reduction in RBF and an unchanged GFR; b) in the ischemic kidney on giving angiotensin. The natriuresis produced by angiotensin was independent of the magnitude of elevations in blood pressure, altered filtration fraction, and was associated with a further reduction in RBF. After release of RAC in the dogs receiving mannitol, an antinatriuresis was again observed in response to angiotensin.The presence of unilateral renal ischemia allowed the demonstration of a differential action of angiotensin on the GFR of an ischemic and nonischemic kidney. The natriuresis in response to angiotensin requires, in addition to mannitol, the participation of undefined factors invoked by unilateral renal ischemia.

E xtracts from human platelets contain the enzymes of de novo fatty acid biosynthesis. The pattern of incorporation of acetate-1-¹⁴C into fatty acids by intact platelets indicates that these enzymes function in platelets. The level of acetyl-coenzyme A (CoA) carboxylase activity in extracts of platelets from normal subjects is 0.036 ±0.01 mμmole of malonyl-CoA formed per min per mg of protein and that of fatty acid synthetase is 0.075 ±0.016 mμmole of malonyl-CoA utilized per min per mg of protein. Thus, platelets are the only formed elements of the blood capable of de novo fatty acid synthesis. The capacity of platelets to synthesize fatty acids is similar to human liver based on enzyme activity per milligram of soluble protein.Acetyl-CoA carboxylase was purified 16-fold from platelet extracts, and this partially purified enzyme was compared to enzyme from rat liver. The two enzymes were similar with respect to requirements, substrate affinities, pH profile of activity, inhibition by malonyl-CoA, and aggregation in the presence of citrate. Thus, while fatty acid synthesis may serve a different function in platelets than in liver, the properties of acetyl-CoA carboxylase from these tissues are alike.The levels of the enzymes of fatty acid synthesis were significantly higher in platelets from splenectomized subjects than in controls. Acetyl-CoA carboxylase levels were 0.086 ±0.027 mμmole of malonyl-CoA formed per min per mg of protein, and fatty acid synthetase levels were 0.151 ±0.039 mμmole of malonyl-CoA utilized per min per mg of protein. These changes in the enzymes of fatty acid synthesis occurred promptly after splenectomy with peak values being reached within 7-10 days.

H uman blood platelets were subjected to osmotic shock, brief sonication, pressure homogenization, or treatment with adenosine diphosphate (ADP). These procedures demonstrated an abundance of cytoplasmic microfibrils. The fibrils resembled those found on electron microscopy of partially purified thrombosthenin, the actomyosin-like protein isolated from platelets, and they also appeared to resemble the myofilaments of smooth muscle. Similar fibrils were not found in leukocytes studied under identical conditions. Treatment with colchicine (2 × 10^-5 mole/liter) resulted in the disappearance of microtubules but did not affect the morphology of the microfibrils or interfere with platelet-dependent clot retraction. Thus, microfibrils rather than microtubules may represent the morphologic counterpart of the contractile protein. Brief osmotic shock at low temperature or treatment with 10^-4 M ADP caused the marginal band of microtubules to be replaced by a bundle of intertwining microfibrils. The apparent inter-conversion of microtubules and microfibrils under a variety of conditions led to the hypothesis that fibrils and tubules consist of similar subunits whose degree of polymerization might be dependent on local cytoplasmic forces. Furthermore, on the basis of these observations, it is postulated that the contractile properties of the cells may be vested in the microfibrils, whereas the tubules may serve to maintain the highly asymmetric shape characteristic of circulating and irreversibly aggregated platelets.

T he concentrations of plasma glucose, insulin, growth hormone, and immunoreactive growth hormone-like substance in subhuman primate fetal and maternal plasma were examined after the intravascular administration of glucose, arginine, or tolbutamide to the fetus. Cannulation of interplacental vessels permitted studies on the fetus in utero without disruption of fetal-placental-maternal anatomic integrity. Single glucose injections, glucose infusions, and arginine infusions into the fetus did not alter fetal plasma insulin concentrations. In contrast, tolbutamide injections elicited an immediate 3-4-fold increase in fetal plasma insulin concentrations. A bidirectional placental transfer of insulin was demonstrated with the use of simultaneously injected insulin-¹²⁵I to the mother and insulin-¹³¹I to the fetus.Simian fetal plasma contained a substance which cross-reacted with immunologic identity to human growth hormone. In contrast, simian maternal plasma and amniotic fluid reacted with immunologic nonidentity to human growth hormone. Although glucose administration to the fetus did not suppress nor did arginine infusion consistently augment fetal plasma growth hormone levels, the latter were observed to vary in individual experiments.The plasma responses to the same stimuli in the neonate were also examined. In contrast to the fetal experiments, glucose injection in the neonate elicited a delayed rise in the concentration of plasma insulin. Similar to the fetus, the plasma concentration of insulin increased after tolbutamide injection and did not change in response to arginine infusion. The initial concentrations of neonatal plasma growth hormone were significantly lower when contrasted with the initial fetal plasma levels. There was no difference in the responsiveness of the fetal and neonatal growth hormone-releasing mechanisms when challenged by glucose or arginine infusion.The data indicate that the fetal plasma concentration of growth hormone is labile, that fetal growth hormone metabolism may differ from that in the neonate, and that pancreatic islet cell responsiveness rapidly changes after delivery.

Q uantitative chemical analyses of the subcellular distribution patterns for acid and alkaline phosphatase, beta glucuronidase and peroxidase were obtained for human peripheral blood leukocytes of four patients with chronic granulomatous disease (CGD). Five young adults with acute infections served as controls. The observations were made on fractions obtained by homogenization and centrifugation of leukocytes previously incubated with or without particles for ingestion. Distributions in resting CGD and normal cells were very similar for acid and alkaline phosphatase and peroxidase, but the proportion of beta glucuronidase in the granule fraction of CGD cells was depressed, with an increased proportion in the soluble fraction. Release of granule-bound enzymes during phagocytosis of a variety of particles was the same for CGD and control cells, except that release of beta glucuronidase was less marked in CGD cells. Total enzymatic activity of CGD cells for the hydrolases studied was normal. The data indicated that granular enzymes are released in a normal fashion in phagocytizing CGD cells. Supportive evidence of release of enzymes into the phagocytic vacuole of CGD cells was obtained by an electron microscopic study of myeloperoxidase.

T he interaction of the anticoagulant drug warfarin and its metabolites with human plasma albumin was studied by equilibrium dialysis. A 20-fold variation of buffer ionic strength (0.017-0.340) caused no significant change in the warfarin association constant. But the binding strength rose significantly as the pH was increased from 6.0 to 9.0 and then declined at pH 10.0. The 6-, 7-, and 8-hydroxywarfarin metabolites showed a 7- to 23-fold reduction in binding strength at pH 10.0. These data indicate that the molecular basis of the interaction is nonelectrostatic and that the introduction of polar hydroxyl groups on the coumarin nucleus by metabolism reduces its hydrophobic binding surface. The interaction was markedly exothermic and showed a positive entropy (increased molecular disorder), which suggests cooperative hydrogen and hydrophobic bonding as the molecular basis for the binding of warfarin to albumin.The marked albumin binding and nonpolar character of warfarin explains the respective absence and presence of the unchanged drug in urine and plasma of warfarintreated patients, while the more polar character and lesser albumin binding of the metabolites probably determines their absence in plasma and presence in urine. The relatively marked binding to albumin of the 4′-hydroxywarfarin metabolite suggests that it may occur in the plasma of warfarin-treated patients. The data suggest that a direct correlation exists between the interaction of warfarin with plasma albumin and the interaction with the warfarin receptor site.

5 min after intravenous injection into rats of ¹⁴C- or ³⁶Cl-carbon tetrachloride, liver lipids were found labeled. Most of the radioactivity was found in the phospholipid fraction. The metabolites were shown to comprise a heterogeneous group of branched long-chain chlorinated fatty acids, probably containing the trichloromethyl side chain. Surviving liver slices also formed these metabolites. In a simple chemical system which generates trichloromethyl free radicals, carbon tetrachloride added to methyl oleate to form esters which behaved like the metabolites during counter-current distribution and urea adduction. The evidence strongly suggests the formation of these metabolites by free radical attack on unsaturated lipids. The relation of these observations to current theories of carbon tetrachloride intoxication is discussed.

F ew studies have been published on peptide hydrolase activities of human small intestine mucosa. We developed methods to screen tissue extracts for such enzymes and to quantitate hydrolase activities for dipeptides containing the aromatic amino acid L-phenylalanine. The screening procedure indicated glycyl-L-proline hydrolase activity was reduced in biopsy specimens from patients with flattened intestinal mucosa. To explore this further, we established optimal assay conditions for hydrolase activities (a) glycyl-L-proline, (b) L-phenylalanyl-L-proline, (c) L-alanyl-L-phenylalanine, and (d) L-phenylalanylglycine. Biopsy specimens from patients with various intestinal disorders, but without flattened mucosa, and from three patients with flattened mucosa, showed a disproportionate reduction in activities (a) and (b), with the reduction being significantly more marked in the latter patients. We suggest that intestinal imidopeptide hydrolase activities, such as (a) and (b), are sensitive to changes in intestinal disease generally, particularly to the altered physiology associated with flattening of the mucosa, and are secondary to, rather than a cause of, the intestinal pathology.Our finding that intestinal alkaline phosphatase activity tended to parallel imidopeptide hydrolase activity, and that activity (a) was partially localized to the particulate fraction of mucosal homogenate, suggested that imidopeptide hydrolase activities may be located in the microvilli of the intestinal epithelium and that, like alkaline phosphatase activity, they may be reduced in flattened mucosae, in part at least because of the pathologic changes in the microvilli.In our studies of control subjects we did not detect peptide hydrolase activity deficiency analogous to asymptomatic disaccharidase deficiency.