Issue published December 1, 1970

A technique was devised for a more accurate measurement than has been heretofore possible of one of the factors responsible for hyperglycemia in the complex syndrome of diabetes. This factor is termed impedance and represents the tissues' insensitivity or resistance to insulin-mediated glucose uptake. It was measured by use of steady-state exogenous insulin and glucose infusions during a period of pharmacological suppression of endogenous insulin secretion. Endogenous new glucose production was also inhibited. Impedance as calculated is a direct function of steady-state glucose concentrations, since exogenous insulin concentrations were similar in all studies. Two groups of normal weight subjects were studied. One had maturity onset latent diabetes, and the other (matched for age, weight, and per cent adiposity) was normal. Impedance was closely reproducible in the same individual and remained relatively constant during prolonged infusions. The diabetics had average infusion glucose concentrations (and thus impedance) 68% higher than the normal group, and it is of note that their previously measured glucose intolerance differed by a similar degree; that is, the diabetic's intolerance (as defined by mean weighted plasma glucose response after oral glucose) was 52% greater than that of the normal individuals.

A group of 32 sexual ateliotic dwarfs with an isolated deficiency of human growth hormone (HGH) were shown previously to resemble subjects with genetic diabetes mellitus in terms of hyperlipemia, carbohydrate intolerance, and patterns of insulin secretion. 11 of these dwarfs had needle biopsies of the quadriceps femoris carried out and tissue fixed for electron microscopy. Capillary basement membrane thickness was measured and compared with measurements previously obtained in diabetics and normal controls. Measurements were similar in controls and dwarfs (1080 ±27 A and 1086 ±90 A, respectively) and significantly less than in diabetics (2403 ±119 A). Placed in juxtaposition with the absence of retinopathy in dwarfs and the high incidence in the diabetic group (41%), the data support the thesis that these anatomical abnormalities are largely independent of serum lipid and carbohydrate abnormalities. The data are consistent with a supportive, if not causative role of growth hormone in the pathogenesis of these lesions.

C ell surface receptors for human γG antibodies directed against bacterial antigens were demonstrated on human neutrophils using an in vitro bacteriocidal-phagocytic assay. These results were confirmed by adherence of sensitized erythrocytes to monolayers of neutrophils or monocytes. Erythrocytes sensitized indirectly with antibacterial γG antibodies after passive sensitization with bacterial antigens adhered to both neutrophils and monocytes. Erythrocytes sensitized directly with conventional anti-D γG antibodies adhered only to monocytes, while those sensitized with the hyperimmune anti-CD γG antibody Ripley adhered to both monocytes and neutrophils. Adherence of anti-Rh or antibacterial γG antibodies to monocytes and neutrophils could be inhibited by whole γG, myeloma globulins of the γ₁ or γ₃ subclasses, or Fc fragments, but not by Fab fragment.These results indicate that receptors for the Fc portion of human γG antibodies exist on both neutrophils and monocytes, and that γG antibodies differ in their ability to attach to these two cell types. Differences in the behavior of the γG antibodies studied may be related to differences in the density of antibodies on the erythrocyte surface and receptors on the phagocytic cells.

P lasma lipoprotein alterations in nine insulin-dependent diabetics with hyperlipemia have been related to the lipid accumulating in eruptive xanthomas evolving in these patients. Histochemical and electron microscopic examination of xanthomas have been correlated with the lipid analyses in order to obtain additional evidence regarding the lipoprotein origin of lipids accumulating in the lesions. Both analytical and morphologic evidence suggested that circulating chylomicrons significantly contribute to the xanthoma lipids. All the patients had large quantities of circulating triglyceriderich chylomicrons which carried approximately 70% of the triglyceride found in the plasma. The fatty acid pattern of chylomicron and xanthoma triglycerides were similar. Triglyceride constituted the major lipid found in the xanthomas when they were sampled during their eruption. These findings, take in conjunction with histochemical and electron microscopic evidence of chylomicron particles in the dermal capillary walls, support the theory that blood lipoproteins, and particularly chylomicrons, permeated the vascular walls and the triglycerides carried by these lipoproteins apparently accumulated in tissue macrophages and perithelial cells which evolved into foam cells. Initiation of appropriate therapy resulted in clearance of the chylomicronemia and a concomitant resolution of the xanthomas as reflected by a decrease in total xanthoma lipid. Sequential studies of resolving xanthomas in five patients revealed that xanthoma triglyceride was mobilized more rapidly than cholesterol, resulting in a redistribution of the xanthoma lipids, so that the resolving lesions were cholesterol rich. Consistent with this change in lipid composition, correlative electron microscopy revealed loss of amorphous material from many of the foam cell vacuoles.

O ur previous studies have indicated the presence of a macromolecular inhibitor of in vitro mineral growth, as well as a mineral nucleational agent in extracellular matrix fluid aspirated by micropuncture methods from epiphyseal hypertrophic cell cartilage. In this report, new miniaturized methods were used to extract proteinpolysaccharide complexes (PPC) from cartilage, to isolate a light fraction (PPL-C), and further, to separate it into R1, R2, and SR2 subfractions. These methods were applied to PPL-C complexes separated from microdissected epiphyseal cartilages and to cetylpyridinium chloride (CPC) precipitates of extracellular matrix fluid aspirated from similar cartilages. Most of all of the inhibitory action on an in vitro system of mineral growth shown by whole cartilage PPL-C and by cartilage fluid PPC obtained from noncalcifying sites was contained in the R2 fraction which represented ¼-[unk] of the total hexuronate.The R2 fraction was diminished or absent from calcified cartilage fluids and from whole calcified epiphyseal septa. The ratio R1 + R2: SR2 ranged from 0.37 to 0.71 in the fluids and whole tissue samples of noncalcified cartilages. The R2 fraction was distinguished from SR2 by a 2- to 3-fold higher protein: hexuronate ratio. These data are interpreted to indicate that the inhibitory R2 fraction was degraded or otherwise inactivated at the zone of provisional calcification and that this inhibitor participates in the physiological mechanism that regulates endochondral calcification.

S imultaneous studies of albumin and fibrinogen metabolism have been conducted using the carbonate-¹⁴C method before and after a 13 day course of prednisolone in eight patients with hepatocellular disease. Initially six patients were hypoalbuminemic. The mean plasma albumin and fibrinogen concentrations and albumin and fibrinogen synthetic rates were all lower than the corresponding values in a group of control subjects. Prednisolone therapy was associated with significant increases in the plasma concentration and synthetic rate of albumin but changes in the intravascular albumin pools were not significant. It is inferred that a low synthetic rate of albumin in a patient with liver disease does not necessarily represent the maximum capacity of the diseased liver to synthesize this protein. Changes in the plasma concentration, intravascular pool, and synthetic rate of fibrinogen were small and inconsistent. The data are compatible with a selective action of corticosteroids on hepatic protein metabolism and with the existence of different mechanisms for the control of albumin and fibrinogen synthesis.

H emorrhagic hypotension in anesthetized dogs produces a marked decrease of the cortical blood flow, whereas the medullary blood flow is well preserved. These animals were maintained at blood pressures of 50 mm Hg during a 3 hr period after which their blood pressures were restored by the reinfusion of blood or dextran, or both. In the first group of animals, the reinfusion of blood reestablished the blood pressure to control values, but the cortical blood flow was still nonuniformly decreased whereas the medullary blood flow appeared to be increased. In the second group of animals, phenoxybenzamine failed to protect the kidney completely since after blood reinfusion, the same anomalies described for the preceding group were found in 7 out of 10 dogs. The animals of the third group were reinfused with 50% of the shed blood and 10 ml/kg of a 10 g/100 ml solution of low molecular weight dextran. The modifications of the intrarenal distribution of the blood flow were less marked in this group although the blood flow rate of the inner cortex and the outer medulla was always elevated under these conditions. The reinfusion of low molecular weight dextran alone (20 ml/kg of a 10 g/100 ml solution) restored the blood pressure to levels slightly lower than those observed under control conditions but reestablished a normal pattern of intrarenal blood flow. The reinfusion of high molecular weight dextran was inefficient in correcting completely the anomalies of the renal blood flow. Mechanisms such as the increased sympathetic tone, the liberation of angiotensin, and the intravascular cellular aggregation could possibly account for the persisting anomalies of the renal circulation after reinfusion and are discussed.

I n patients heterozygous for abnormal hemoglobins there is usually less than 50% of the mutant hemoglobin present in peripheral blood. The synthetic rates of α-chain mutants compared to α^A have not been reported to date. In this study the production of α^A- and α^I-chains has been measured in peripheral blood and bone marrow of two patients with approximately 30% hemoglobin I, an α-chain abnormality (α^{16 lys → glu}). The results suggest that the decreased amount of α^I compared to α^A is due solely to diminished biosynthesis of the α^I-chains. The relative rates of synthesis of α^I- and α^A-chains are similar in both nucleated red cells and reticulocytes indicating that no change occurs during erythroid cell maturation which preferentially affects either α^I or α^A production.

K inetic parameters and the renal clearances of plasma adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP) were evaluated in normal subjects using tritium-labeled cyclic nucleotides. Each tracer was administered both by single, rapid intravenous injection and by constant intravenous infusion, and the specific activities of the cyclic nucleotides in plasma and urine were determined.Both cyclic AMP and cyclic GMP were cleared from plasma by glomerular filtration. The kidney was found to add a variable quantity of endogenous cyclic AMP to the tubular urine, amounting to an average of approximately one-third of the total level of cyclic AMP excreted. Plasma was the source of virtually all of the cyclic GMP excreted.Plasma levels of the cyclic nucleotides appeared to be in dynamic steady state. The apparent volumes of distribution of both nucleotides exceeded extracellular fluid volume, averaging 27 and 38% of body weight for cyclic AMP and cyclic GMP, respectively. Plasma production rates ranged from 9 to 17 nmoles/min for cyclic AMP and from 7 to 13 nmoles/min for cyclic GMP. Plasma clearance rates averaged 668 ml/min for cyclic AMP and 855 ml/min for cyclic GMP. Approximately 85% of the elimination of the cyclic nucleotides from the circulation was due to extrarenal clearance.

G lucagon, infused intravenously into fasting, well-hydrated, normal men in doses of 25-200 ng/kg per min, induced up to 30-fold increases in both plasma and urinary cyclic AMP. Cyclic GMP levels were unaffected by glucagon. Simultaneous cyclic AMP and inulin clearance studies demonstrated that the glucagon-induced increase in urinary cyclic AMP was entirely due to glomerular filtration of the elevated plasma levels of the nucleotide.The cyclic AMP response to glucagon was not mediated by parathyroid hormone or epinephrine, and trypsintreated glucagon was completely inactive.The perfused rat liver released cyclic AMP into the perfusate in response to glucagon, indicating that the liver is a possible source of the cyclic AMP entering the extracellular fluids in response to glucagon in vivo.

T he free proline, free glutamic acid, and total collagen contents of the livers of cirrhotic and noncirrhotic patients were determined. The amounts of free proline in the sera of the patients were also determined. The results indicated that certain metabolic changes occurred in cirrhotic livers of humans that were similar to the metabolic changes observed previously in CCl₄-induced cirrhosis in the rat. The amount of free proline was coordinate with the increase in total collagen, and both were inversely related to the amount of free glutamic acid. The average proline concentration in sera of cirrhotic patients was not higher than that of non cirrhotic patients, suggesting that the metabolic alteration noted above is a local event in the liver related to fibrogenesis. These and other results suggest that the pool size of free proline may play a prime role in regulation of collagen biosynthesis in liver cirrhosis.

H uman granulocytes (G) contain a vitamin B₁₂-binding protein (B₁₂BP). There is evidence that chronic myelogenous leukemia leukocytes (CMLL) may synthesize B₁₂BP. Our prior studies suggested that intact, living intravascular G synthesize and release such protein into extracellular compartments in vivo.In the present study, CMLL were incubated in Trisbuffered Hank's basal salt solution (pH 7.2) containing 0.1% human serum albumin to study release of B₁₂BP into the medium. B₁₂BP was released continuously and in increasing amounts over a 5 hr period at 37°C; this release was inhibited almost completely when the cells were incubated at 4°C and by about half as much in the presence of N-ethylmaleimide (1 mmole/liter). Cycloheximide (50 μg/ml) had no effect on the release of B₁₂BP but significantly inhibited incorporation of leucine-³H into leukocyte protein. G incubated with 20 mg/ml of compound 48/80, an experimental histamine-releasing agent, had a 6-fold increase in release of B₁₂BP over a 2 hr period.Subcellular fractionation studies of human granulocytes demonstrate that most of the B₁₂BP is associated with the granular (20,000 g) layer with an excellent correlation observed between its subcellular distribution and that of acid phosphatase.These findings suggest that the release of B₁₂BP from G is mediated by an active process and provide further evidence that granulocytes are secretory as well as phagocytic cells.

T he role of glucagon in the metabolic adaptation to prolonged fasting in man has been examined. Plasma immunoreactive glucagon was determined during 6-wk fasts and during infusion of exogenous glucagon using an assay which minimized nonpancreatic immunoreactivity.Plasma glucagon concentrations rose twofold to a peak on the 3rd day of fasting and then declined thereafter to a level maintained at or above postabsorptive. Insulin concentration declined to a plateau by the 3rd day. Thus a persisting altered relationship of glucagon and insulin concentrations characterized the fasted state. A synergism of low insulin and relative or absolute elevation of glucagon levels is viewed as a hormonal mechanism controlling the rate of hepatic substrate extraction for gluconeogenesis.Glucagon was infused systemically into 4-6 wk fasted subjects at three dose levels. A marked sensitivity of individual plasma free amino acids to the induced elevations of plasma glucagon within the physiologic range was demonstrated. At higher concentrations, equivalent to those present in the portal vein, stimulation of hepatic gluconeogenesis occurred, and the effects on glucose, insulin, and growth hormone levels and on ketone metabolism were induced.

T his report describes a search for tumor-specific transplantation antigens in man by testing four sets of identical siblings using the mixed leukocyte transformation reaction. In each case, one member of the set had acute leukemia in relapse. In no instance did the leukemic cells of the patient stimulate the cells of the normal twin, nor did cells from the normal twin stimulate cells from the leukemic patient. Possible explanations for the failure to detect a leukemia-associated antigen in these studies are discussed.

S ecretin-stimulated insulin release was studied in normal subjects. In response to rapid intravenous injections (pulses) of secretin, insulin levels reached a peak between 2 and 5 min and returned to basal levels with 15 min. In contrast to large glucose pulses, increasing secretin pulses did not elicit sustained or prolonged insulin responses. In addition, insulin responses to a pulse and infusion were essentially identical with that of a pulse alone. Increasing secretin pulses given in 1 day were associated with decreasing insulin responses but not when the same pulses of secretin were given over a 2 day period. When time was the sole variable, insulin responses progressively decreased after identical 15-U secretin pulses given every 30 min, but were unchanged when the interval was 105 min. These observations indicate that secretin in contrast to glucose stimulates insulin release which is uniphasic. They suggest that release occurs only from a stored, readily available pool. This insulin pool appears to be relatively small and can be discharged faster than it refills.

T ransport of plasma-free fatty acids (FFA) and of fatty acids in triglycerides of plasma very low density lipoproteins (VLDL-TGFA) was studied in two normal subjects, five patients with type IV hyperlipoproteinemia, and two patients with type I hyperlipoproteinemia. After intravenous pulse-labeling with albumin-bound 1-palmitate-¹⁴C, specific radioactivity of plasma FFA and VLDL-TGFA were determined at intervals up to 24 hr. The results were analyzed using several different multicompartmental models each compatible with the experimental data. Fractional transport of VLDL-TGFA was distinctly lower (no overlap) in the type IV patients than in the control subjects, both on a usual balanced diet (40% of calories from carbohydrate) and on a high-carbohydrate diet (80% of calories). However, net or total transport of VLDL-TGFA in the type IV patients was not clearly distinguishable from that in the control subjects, there being considerable overlap on either diet. The results suggest that in this group of type IV patients the underlying defect leading to the increased pool size of VLDL-TGFA is not overproduction but a relative defect in mechanisms for removal of VLDL-TGFA. Since some of these type IV patients had only a moderate degree of hypertriglyceridemia at the time they were studied, and since it is not established that patients with the type IV phenotype constitute a biochemically homogeneous population, the present results should not be generalized.Four studies were done (in two control subjects and two type IV patients) in which the kinetic parameters in the same individual were determined on the balanced diet and on the high-carbohydrate diet. All subjects showed an increase in VLDL-TGFA pool size. Using two of the models for analysis, all showed an increase in net transport of VLDL-TGFA; using the third model, three of the four studies showed an increase in VLDL-TGFA transport. The results are compatible with the interpretation that the carbohydrate-induced increase in VLDL-TGFA, both in controls and type IV patients, is at least in part due to an increased rate of production of VLDL-TGFA. The magnitude of the increase was approximately the same in controls and patients. Thus, metabolic adjustment to a high-carbohydrate regimen in these type IV patients may not be basically different from that in normal controls; the higher levels of VLDL-TGFA reached may simply be another reflection of a defective removal mechanism. An alternative interpretation, compatible with the data, would involve both a carbohydrate-induced increase in fractional rate of release of VLDL-TGFA from liver to plasma and a decrease in fractional removal of VLDL-TGFA from plasma without increase in net production rate. The simpler hypothesis of a single primary effect on net VLDL-TGFA production from FFA seems more likely.

T hree different multicompartmental models of free fatty acid (FFA) and very low density lipoprotein triglyceride fatty acid (VLDL-TGFA) transport in man are formulated from plasma FFA and VLDL-TGFA tracee and tracer data collected over a 24 hr interval after the injection of palmitate-¹⁴C. All modeling and data fitting were performed on a digital computer using the SAAM program. Structural differences in the three models relate to the position of the slowly turning over compartment required to generate the late portion of the plasma VLDL-TGFA tracer data. The positions of this slow compartment are along the hepatic pathway from FFA to VLDL-TGFA (model A) or in the distribution system of VLDL-TGFA (model B) or in the distribution system of FFA (model C). Although all three models are equally consistent with our experimental data and are supported by observations of others, each reveals inconsistency with some data obtained from the literature. Consequently, a combination model of FFA-TGFA transport, incorporating properties of models A, B, and C would be more consistent with all available data. Experiments that would help to determine the quantitative significance of each of the slow compartments in the combination model are suggested.Several other models suggesting recycling of plasma VLDL-TGFA through the plasma FFA pool, kinetic heterogencity of the plasma VLDL-TGFA pool, and contamination of plasma VLDL-TGFA radioactivity with low density lipoprotein (LDL) TGFA radioactivity were tested. The first model does not explain the late portion of the plasma VLDL-TGFA tracer data. The second and third models, while consistent with our tracee and tracer data, have steady-state implications with respect to the extent of kinetic heterogeneity and size of the LDL-TGFA contaminant that make them unlikely.Assumptions underlying other investigator's models of FFA and TGFA transport in man are reviewed within the logical framework of our models. Quantitative differences among the various models are shown by evaluating all of the models with respect to a common set of plasma FFA and VLDL-TGFA data.

M oderate increases of ureteral back pressure usually cause decreases of glomerular filtration rate and even greater decreases of sodium excretion. It has been assumed previously that increased ureteral back pressure does not enhance renal tubular sodium reabsorption directly and that the decreases of sodium excretion are caused by the decreases of glomerular filtration rate. In the experiments reported here, the effect of increased ureteral back pressure on urinary sodium excretion was studied in dogs in which changes of filtration rate were minimized by infusing saline while ureteral back-pressure was increased.When ureteral back pressure was increased on one side by 10-23 cm of water, the inulin clearance of the experimental kidney decreased by only 3-12% in 21 experiments, did not change significantly (±2%) in eight experiments, and increased by 3-8% in seven experiments. The sodium excretion of the experimental kidney decreased in all experiments regardless of whether its inulin clearance increased, decreased, or was unchanged from control values.When the inulin clearance of the experimental kidney increased or remained unchanged during increased ureteral back pressure, its reabsorption of sodium increased more than could be accounted for by the increase of filtered sodium. When the inulin clearance of the experimental kidney decreased during increased ureteral back pressure, its reabsorption of sodium decreased less than could be accounted for by the decrease of filtered sodium.Therefore, the effect of increased ureteral back pressure to decrease urinary sodium excretion is caused in part by increased tubular reabsorption of sodium.

I n order to study the splanchnic metabolism of blood-borne estrogens, a constant infusion of estrone-6,7-³H was made in a series of dogs, and arteriovenous (A-V) differences at equilibrium were determined for estrone-6,7-³H and for its products estradiol-17β, estrone sulfate, estrone glucosiduronate, and estradiol-17β glucosiduronate across the splanchnic bed (artery-hepatic vein), the small intestine (artery-superior mesenteric vein), and the spleen (artery-splenic vein). Per cent extractions (100 - [V/A] 100) were calculated. The plasma metabolic clearance rate (MCR) for estrone was measured. Principal findings were as follows: mean MCR was 731 liters/day per m², SEM 50. By comparison with estimated hepatic plasma flow and using the observed splanchnic extraction of estrone, 45-71% of estrone metabolism was calculated to be extrasplanchnic. The significant mean per cent extractions were as follows (SEM in parentheses): splanchnic bedestrone 85.9 (1.92), estradiol-17β 88.11 (3.36), estrone sulfate 27.9 (5.22), estrone glucosiduronate -48.5 (9.33), estradiol-17β glucosiduronate -33.3 (80.3); small intestine—estrone 45.3 (2.60), estradiol-17β 46.1 (12.9), estrone glucosiduronate - 30.8 (7.9); spleen—estrone 35 (3.8), estrone glucosiduronate 12 (3.7). These results lead to the following conclusions. Both estrone and estradiol-17β are nearly completely extracted in one passage through the splanchnic bed. There is net uptake of estrone sulfate and net production of estrone glucosiduronate and of estradiol-17β glucosiduronate by the splanchnic bed. There is net uptake of estrone and of estradiol-17β by the intestine, associated with substantial net production of estrone glucosiduronate. There is net uptake of estrone by the spleen and a small but significant net uptake of estrone glucosiduronate.

T he separate effects of volume expansion and of increased delivery of sodium on sodium reabsorption in the diluting segment of the distal nephron were studied in man. In six normal subjects during a sustained water diuresis, sodium delivery to the distal nephron was increased without volume expansion by the administration of acetazolamide. In these subjects, free water clearance rose linearly as a function of urine flow. In five patients with complete, central diabetes insipidus, distal sodium delivery was increased by the infusion of hypertonic saline during a sustained water diuresis. In four of these five patients, changes in free water clearance were also observed during hypertonic saline diuresis in the presence of distal blockade of sodium reabsorption with chlorothiazide. At high rates of distal delivery the following observations were made: (a) free water clearance was lower and fractional sodium excretion higher during saline diuresis compared to acetazolamide diuresis; (b) although free water clearance was moderately reduced by chlorothiazide at low rates of urine flow, there was no difference in free water clearance between saline loading alone and saline plus chlorothiazide at high rates of urine flow; and (c) during saline loading free water clearance rose without evidence of a limit when increased distal delivery was accompanied by spontaneous increases in glomerular filtration rate, but tended toward a limit when glomerular filtration rate remained constant.The data indicate that during acute volume expansion with saline, there is a decrease in the fraction of delivered sodium reabsorbed in the distal nephron when compared to the response of the distal nephron to comparable increases in distal sodium delivery in the absence of volume expansion.

T he characteristics of the disappearance of radioiodinated synthetic human calcitonin from plasma have been studied in man. After single injection the disappearance curve was multiexponential. The number of exponentials of the theoretical curve fitting the best with the experimental data varied individually. Metabolic clearance rate was determined both from single injection and constant infusion studies, and fast initial distribution volume from the former. Metabolic clearance rate values in normal man calculated from constant infusion studies were 82.3 ±3.4 ml/min per m². Values derived from single injection studies were similar, 77.0 ±4.7 ml/min per m². These results were compared to those obtained in end stage, renal failure patients. Metabolic clearance rate was considerably lower and volume of fast initial distribution slightly larger in that group. This fact emphasizes the important role of kidneys in the utilization and/or the degradation of human calcitonin.

A state of supersaturation of urine with respect to brushite is considered to be important in the formation of renal stones composed of calcium phosphate. 56 supersaturated urine specimens and 44 undersaturated specimens were incubated with collagen (Sigma collagen). Most of the supersaturated specimens calcified the collagen, whereas none of the undersaturated ones did so. Among samples which calcified the collagen, whereas none of the undersaturated ones did so. Among samples which calcified the collagen, the activity product of Ca⁺⁺ and HPO₄⁼ after incubation with collagen was essentially the same as that after incubation of the same specimen with brushite; it usually differed from that obtained after incubation with octacalcium phosphate or hydroxyapatite. The molar calcium-to-phosphorus ratio of the solid phase in collagen was approximately 1. These results suggested that the solid phase formed in collagen is brushite. This conclusion was confirmed by the direct identification of brushite in collagen by X-ray diffraction.

A lterations in human cerebral blood flow and related blood constituents were studied during exposure to acute hypoxia. Observations were made during serial inhalation of decreasing O₂ concentrations with and without maintenance of normocarbia, during 8 min inhalation of 10% O₂, and after hyperventilation at an arterial P_O2 of about 40 mm Hg. In the range of hypoxemia studied, from normal down to arterial P_O2 of about 40 mm Hg, the magnitude of the cerebral vasodilator response to hypoxia appeared to be largely dependent upon the coexisting arterial CO₂ tension. The mean slope of the increase in cerebral blood flow with decreasing arterial O₂ tension rose more quickly (P < 0.05) when eucapnia was maintained when compared with the slope derived under similar hypoxic conditions without maintenance of eucapnia. When 12 subjects inhaled 10% oxygen, cerebral blood flow rose to more than 135% of control in four whose mean decrease in arterial CO₂ tension was - 2.0 mm Hg. The remaining eight had flows ranging from 97 to 120% of control, and their mean decrease in CO₂ tension was - 5.1 mm Hg. When mean arterial P_O2 was 37 mm Hg, hyperventilation was carried out in 10 subjects. Arterial P_O2 increased insignificantly, arterial P_CO2 declined from 34 to 27 mm Hg (P < 0.05), and cerebral blood flow which had been 143% of control decreased to 109%, a figure not significantly different from control.These data demonstrate the powerful counterbalancing constrictor effects of modest reductions in CO₂ tension on the vasodilator influence of hypoxia represented by arterial P_O2 reductions to about 40 mm Hg. Indeed, mild hyperventilation completely overcame the vasodilator effect provided by an arterial O₂ tension as low as 40 mm Hg. The effects of hypoxia on the control of the cerebral circulation must be analyzed in terms of the effects of any associated changes in CO₂ tension.

S everal unstable mutant hemoglobins have alterations which affect areas of the molecule involved in the attachment of heme to globin. Loss of heme from globin has been demonstrated during the denaturation of some of these unstable mutants. The importance of heme ligands for the stability of hemoglobin was illustrated in the present experiments on the denaturation of several hemoglobins and hemoglobin derivatives by heat, oxidative dyes, and alkali. Heating of normal hemolysates diluted to 4 g of hemoglobin per 100 ml at 50°C for 20 hr in 0.05 M sodium phosphate, pH 7.4, caused precipitation of 23-54% of the hemoglobin. Dialysis against water or dilution of the sample decreased denaturation to 12-20%. Precipitation was decreased to less than 3.5% by the presence of 0.015 M potassium cyanide. Increasing the ionic strength of the medium increased precipitation. Cyanide prevented the formation of inclusion bodies when red cells containing unstable hemoglobin Philly, β35 tyr → phe, were incubated with the redox dye new methylene blue. Conversion to methemoglobin increased the rate of alkali denaturation of hemoglobin but the presence of potassium cyanide returned the denaturation rate to that of ferrohemoglobin. The ability of cyanide to decrease heat precipitation of hemoglobin may depend on a dimeric or tetrameric state of the hemoglobin molecule. Purified β-chains, which exist as tetramers, were stabilized but purified monomeric α-chains were not rendered more heat resistant by the ligand. Stabilization of hemoglobin by cyanide required binding of the ligand to only one heme of an αβ-dimer. Hemoglobin Gun Hill, an unstable molecule with heme groups present only on the α-chains was quite heat stable in the presence of cyanide. The binding of cyanide to the iron atom in methemoglobin is thought to be associated with increased planarity of the heme group and increased stability of the heme-globin complex. The stabilizing effect of cyanide in the above experiments suggests that Heinz body formation, heat precipitation of hemoglobin, and the increased alkali denaturation of methemoglobin depend on changes of heme-globin binding.

A radioimmunoassay for serum digoxin concentration has been used to study the interrelationships of circulating levels of the drug and various factors in the clinical setting in 48 hospitalized patients with cardiac rhythm disturbances due to digoxin intoxication. 131 patients on maintenance doses of digoxin without toxicity and 48 patients with equivocal evidence of digoxin excess were also studied and compared with the toxic group.Patients with cardiac rhythm disturbances due to digoxin intoxication tended to be older and to have diminished renal function compared with the nontoxic group; body weight, serum potassium concentration, underlying cardiac rhythm, and nature of cardiac disease were not significantly different for the groups as a whole. Despite comparable mean daily digoxin dosages, digoxin intoxicated patients had a mean serum digoxin concentration of 3.7 ±1.0 (SD) ng/ml, while nontoxic patients had a mean level of 1.4 ±0.7 ng/ml (P < 0.001), 90% of patients without evidence of toxicity had serum digoxin concentrations of 2.0 ng/ml or less, while 87% of the toxic group had levels above 2.0; the range of overlap between the two groups extended from 1.6 to 3.0 ng/ml. Patients with atrioventricular block as their principal toxic manifestation had a significantly lower mean serum digoxin concentration than those in whom ectopic impulse formation was the chief rhythm disturbance.Patients with equivocal evidence of digoxin excess had received comparable daily maintenance doses of digoxin but had a mean serum concentration of 1.9 ±0.8 ng/ml, intermediate between those of the nontoxic (P < 0.005) and toxic (P < 0.001) groups. Renal function as judged by mean blood urea nitrogen concentration was also intermediate.The data indicate that knowledge of the serum digoxin concentration, weighed in the clinical context, is useful in the management of patients receiving this drug.

T he effects of parathyroid hormone (PTH) on plasma and urinary adenosine 3′,5′-monophosphate (cyclic AMP) levels were studied in normal subjects. Under basal conditions normal adults have plasma concentrations of cyclic AMP ranging from 10 to 25 nmoles/liter and excrete from 1.5 to 5 μmoles of cyclic AMP per g of urinary creatinine. About one-half to two-thirds of the cyclic AMP excreted in the urine is derived from the plasma by glomerular filtration, and the remainder is produced by the kidney. Renal production of cyclic AMP is partly under the control of PTH. It can be suppressed by infusions of calcium and stimulated by infusions of the calcium chelating agent, EDTA. Infusions of PTH in doses up to 10 mU/kg per min were associated with dose-related increases both in urinary cyclic AMP and phosphate. Infusions of PTH in doses ranging from 20 to 80 mU/kg per min did not lead to any further increase in phosphaturia but did lead to further marked increases in urinary cyclic AMP. A modest increase in plasma cyclic AMP was noted when PTH was infused at 40 mU/kg per min. Anephric patients failed to show appreciable increases in plasma cyclic AMP in response to large doses of PTH but did show expected increases in response to glucagon. Surgical removal of parathyroid adenomas from nine patients with primary hyperparathyroidism was invariably followed by a decrease in urinary cyclic AMP, PTH, in large doses, and calcium infusion produced up to 2-fold increases in the other known naturally occurring cyclic nucleotide, guanosine 3′,5′-monophosphate (cyclic GMP).

T he plasma low density lipoproteins (LDL) in biliary obstruction are characterized almost exclusively by the presence of the immunochemically distinct lipoprotein families, lipoprotein B (LP-B) and lipoprotein X (LP-X). It is suggested that LP-X, with its uniquely high content of unesterified cholesterol and phospholipid, is primarily responsible for the unusual lipid composition of LDL and the abnormal plasma lipid composition in obstructive jaundice.To show their protein moieties, we isolated LP-X and LP-B from the LDL in plasma obtained from patients with obstructive jaundice. A separation procedure was employed which combines ultracentrifugation, heparin precipitation, and ethanol fractionation. Whereas LP-B was characterized by the presence of apolipoprotein B (ApoB), intact LP-X contained a protein moiety of unique composition consisting of a mixture of albumin (approximately 40%) and the specific apolipoprotein, ApoX (60%). These two protein moieties were separated by preparative ultracentrifugation at d 1.21 g/ml of a solution of partially delipidized LP-X. LP-X thus comprises an albumin-lipoprotein complex in which the masked antigenic site of albumin can be revealed by partial or total delipidization.Apolipoprotein X, the characteristic nonalbumin protein moiety of intact or partially delipidized LP-X, was immunochemically different from ApoA, ApoB, albumin, γ-globulins, and other serum proteins. The results of analytical ultracentrifugation and the immunochemical and electrophoretic properties of ApoX indicated it to be a complex protein consisting possibly of several nonidentical polypeptides. ApoX was characterized by its amino acid composition, and by serine and threonine as the major N-terminal and alanine as the major C-terminal amino acids. It has been suggested that ApoX is similar to, if not identical with, apolipoprotein C.

T he importance of ceruloplasmin in iron metabolism was studied in swine made hypoceruloplasminemic by copper deprivation. When the plasma ceruloplasmin level fell below 1% of normal, cell-to-plasma iron flow became sufficiently impaired to cause hypoferremia, even though total body iron stores were normal. When ceruloplasmin was administered to such animals, plasma iron increased immediately and continued to rise at a rate proportional to the logarithm of the ceruloplasmin dose. The administration of inorganic copper induced increases in plasma iron only after ceruloplasmin appeared in the circulation. Thus, ceruloplasmin appeared to be essential to the normal movement of iron from cells to plasma.Studies designed to define the mechanism of action of ceruloplasmin were based on the in vitro observation that ceruloplasmin behaves as an enzyme (ferroxidase) that catalyzes oxidation of ferrous iron. Retention of injected ferrous iron in the plasma of ceruloplasmin-deficient swine was significantly less than that of ferric iron, reflecting impaired transferrin iron binding. Rat ceruloplasmin, which has little ferroxidase activity, was much less effective than porcine or human ceruloplasmin in inducing increases in plasma iron. These observations suggest that ceruloplasmin acts by virtue of its ferroxidase activity.Eight patients with Wilson's disease were evaluated in order to investigate iron metabolism in a disorder characterized by reduced ceruloplasmin levels. Evidence of iron deficiency was found in six of these, and in five of the six, plasma ceruloplasmin was less than 5% of normal. In comparison, the two patients without evidence of iron deficiency had ceruloplasmin levels of 11 and 18% of normal. It is suggested that iron deficiency tends to occur in those patients with Wilson's disease who have the severest degrees of hypoceruloplasminemia, possibly because of defective transfer of iron from intestinal mucosal cells to plasma.

T he pyrogenic properties of some C-19 and C-21 steroids were examined by in vitro incubation of human blood leukocytes with serum-buffer solutions of the steroids and injection of the 18-hr supernatants into rabbits. In previous studies this method demonstrated release of leukocyte endogenous pyrogen by etiocholanolone. With two exceptions, steroids known to cause fever in man, such as 11β-OH etiocholanolone and 3α-hydroxy-5β-pregnane-20-one were also pyrogenic in vitro. All steroids tested which are nonpyrogenic in man, such as androsterone, 3β-OH etiocholanolone, and 3α, 17α-dihydroxy-5β-pregnan-20-one were also nonpyrogenic in vitro. Solubility in aqueous solution did not correlate with pyrogenic capacity.Inhibition of pyrogen release from human leukocytes in vitro by hydrocortisone and estradiol was demonstrated. Hydrocortisone-treated leukocytes released less pyrogen than did normal leukocytes when stimulated either by etiocholanolone or by phagocytosis of heat-killed staphylococci. On the other hand, estradiol-treated blood leukocytes and mononuclear cells showed significant suppression of pyrogen release when phagocytosis, but not etiocholanolone, was used as the stimulus. When blood cells were incubated with progesterone, greater than normal amounts of pyrogen were released following phagocytosis, and the inhibiting effect of estradiol could be partially reversed. Neither estradiol nor hydrocortisone appeared to act on rabbit leukocytes.These studies indicate that a variety of naturally-occurring steroids may alter pyrogen release from leukocytes. Alterations in steroid balance in man may influence normal temperature regulation and contribute to clinical fevers.

T he failure of human serum to give rise to anaphylatoxin activity could be attributed to the presence of a potent inactivator of anaphylatoxin in human serum. The inactivator was isolated and characterized as an α-globulin with a molecular weight of approximately 310,000. It was found to abolish the activity of both anaphylatoxins, which are derived respectively from the third and the fifth component of complement, and of bradykinin. Inactivation of C3-derived anaphylatoxin and of bradykinin was accompanied by release of C-terminal arginine from these peptides. The anaphylatoxin inactivator was shown to hydrolyze the synthetic substrates hippuryl-L-arginine and hippuryl-L-lysine and to be inhibited by ethylenediaminetetraacetate (EDTA) or phenanthroline. These observations indicate that the anaphylatoxin inactivator constitutes a metal-dependent enzyme resembling in specificity pancreatic carboxypeptidase B.