The intestinal fate of two tripeptides (triglycine and trileucine), which differ markedly in solubility and molecular weight, have been investigated by jejunal perfusion in healthy human volunteers. Rates of glycine or leucine uptake from test solutions containing triglycine or trileucine were greater than from test solutions containing corresponding amounts of free glycine or free leucine, respectively. The rate of glycine uptake from a 100 mM triglycine solution was greater than that from a 150 mM diglycine solution. At each infused load of triglycine (e.g., 1,000 mumol/min) the rates (micromoles/minutes per 30 cm) of either triglycine disappearance (810 +/- 40) or glycine absorption (2,208 +/- 122) were markedly greater than the luminal accumulation rates of either diglycine (56 +/- 10) or free glycine (110 +/- 18). The luminal accumulation rate of free leucine during infusion of a 5 mM trileucine solution was over threefold greater than that of free glycine during the infusion of a 5 mM triglycine solution. Luminal fluid exhibited no hydrolytic activity against triglycine, but contained some activity against trileucine. Saturation of free amino acid carrier system with a large load of leucine did not affect glycine absorption rate from a triglycine test solution, but isoleucine markedly inhibited the uptake from a trileucine solution. When the carrier system for dipeptides was saturated with a large amount of glycylleucine, the disappearance rate of triglycine was considerably reduced while that of trileucine remained unaffected. After addition of glycylleucine to tripeptide solutions, there was a minimal increase in the luminal accumulation of diglycine, while dileucine accumulation was incresed by 62-fold.
S A Adibi, E L Morse, S S Masilamani, P M Amin
The abilities of 14 strains of aerobic gram-positive cocci and gram-negative bacilli to adhere in vitro to human or canine aortic valve leaflets were compared. 2-mm sections of excised valve leaflets were obtained by punch biopsy and were incubated under standardized conditions in suspensions of bacteria. Valve sections were subsequently washed and homogenized, and quantitative techniques were used to determine the proportions of bacteria from the initial suspensions that had adhered to the valve sections. Comparable results were obtained when these adherence ratios were determined by two independent methods based either on measurements of bacterial viability or of radioactivity in 51Cr-labeled bacteria. For each bacterial strain, the adherence ratio was constant over a wide range of concentrations of bacteria in the incubation medium. Strains of enterococci, viridans streptococci, coagulase-positive and coagulase-negative staphylococci and Pseudomonas aeruginosa (adherence ratios 0.003-0.017) were found to adhere more readily to valve sections than strains of Escherichia coli and Klebsiella pneumoniae (adherence ratios 0.00002-0.00004). The organisms that most frequently cause bacterial endocarditis were found to adhere best to heart valves in vitro, suggesting that the ability to adhere to valvular endothelium may be an important or essential charcteristic of bacteria that cause endocarditis in man.
K Gould, C H Ramirez-Ronda, R K Holmes, J P Sanford
We have tested the hypothesis that severe lypoxia causes apnea, regardless of the arterial CO2 and pH, and that extreme hypoxia causes gasping. Acute experiments with airway occlusion and with low inspired oxygen (FIo2) were performed on anesthetized adult dogs and monkeys. Arterial oxygen saturation was recorded continuously with fiberoptic oximetry, and Pco2 by an electrode catheter. In addition, blood samples were obtained for Po2, Pco2, and pH. Apnea was induced regularly when the Pao2 fell below 10 torr, whether the Paco2 was high with asphyxia (63 torr) or low (26 torr) with low FIo2. Similarly, the Pao2 at apnea was the same whether the pH was 7.17 with asphyxic hypoxia or 7.46 with hypoxic hypoxia. Gasping occurred at even lower Pao2 (below 5 torr) after 1 or 2 min of apnea. Gasping promptly restored the Pao2 to levels of moderate hypoxia (over 30 torr) which permitted resumption of regular respiration, with gradual elimination of the gasping. Fetal monkeys at term were studied in a similar manner from the moment of cord clamping. Their blood gases with apnea were quite similar to adult values in the narrow range of Pao2 and the wide range of Paco2 and pH. In the fetus, gasping was less immediately effective in improving arterial oxygen, but more persistent than in the adult. Regular respirations would not develop in the absence of oxygen in either the fetus or adult animal.
W G Guntheroth, I Kawabori
Antigen-ginding lymphocytes capable of binding native DNA (DNA-ABC) were identified in the peripheral blood of normal controls and patients with systemic lupus erythematosus (SLE) by autoradiography with 125I-nDNA. 12 patients with active SLE had 404 +/- 273 (mean +/- SD) DNA-ABC/105 lymphocytes, while 7 inactive SLE patients and 13 normals had 120 +/- 48 and 48 +/- 36, respectively. All three groups were significantly different from one another (p less than 0.01). No significant correlation was detected between the quantity of anti-native DNA (nDNA) antibody and number of DNA-ABC; however, most patients with large amounts of anti-nDNA antibody had both active disease and large numbers of DNA-ABC. Numbers of DNA-ABC and lymphocytes with surface immunoglobulin (Ig) did not change significantly after an 18-h incubation at 37degreeC. After depletion of B-lymphocytes by passage over bead columns coated with a complex of IgG and anti-IgG, the great majority of DNA-ABC were removed in both normal subjects and SLE patients. Labeling lymphocytes sequentially with 125I-nDNA, followed by an indirect fluorescence technique for identification of surface Ig, indicated that the great mahority of radiolabeled cells had surface Ig by fluorescence microscopy in four normals (average 93%) and five patients with active SLF (average 82%). The predominance of nDNA-sensitive B-lymphocytes in the peripheral blood of both normals and SLE patients is consistent with the concept that the induction of the anti-nDNA antibody response is due to the stimulation of preexisting nDNA-specific B lymphocytes by mechanisms other than those necessarily involving participation of nDNA-specific T lymphocytes.
A D Bankhurst, R C Williams Jr
Experiments were carried out in normal dogs to characterize the mechanisms by which sodium bicarbonate administration results in increased excretion of phosphate. Infusion of sodium bicarbonate alone increased fractional phosphate excretion from 0.8 to 29.3%. During bicarbonate administration, ionized calcium fell and mean parathyroid hormone values increased from 59.6 to 230.4 muleq/ml. In the same group of dogs, administration of sodium bicarbonate plus calcium prevented the fall in ionized calcium, and parathyroid hormone levels remained unchanged. In these dogs fractional phosphate excretion increased from 2.4 to only 4.9%. Similar results were obtained in thyroparathyroidectomized dogs receiving sodium bicarbonate. In these dogs fractional excretion of phosphate increased from 0.6 to 4.5%. Under all three experimental conditions no differences were observed in sodium or bicarbonate excretion or in urinary or plasma pH. Administration of hydrochloric acid, after phosphaturia had been induced by the infusion of bicarbonate, resulted in a decrease in plasma bicarbonate and an acid urine; however, the phosphaturia persisted even in the presence of an acid urine pH. In five thyroparathyroidectomized dogs infused with parathyroid hormone throughout, administration of identical amounts of sodium as either NaCl or NaHCO3 resulted in a similar degree of phosphaturia despite significant differences in urine pH. These experiments suggest that a rise in parathyroid hormone levels, resulting from a fall in ionized calcium, is the major mechanism by which bicarbonate administration produces phosphaturia. An increased natriuresis per nephron, as a consequence of extracellular fluid volume expansion, contributes to the phosphaturia. On the other hand, alkalinization of the urine does not play a significant role in the phosphaturia seen after bicarbonate administration.
A Mercado, E Slatopolsky, S Klahr
As an extension of metabolic studies of the cholesteryl ester component of rat very low density lipoproteins, we have studied the metabolism of the B apoprotein component labeled by intravenous injection of [3H]lysine. The B apoprotein separated from other apoproteins by delipidation and selective precipitation with tetramethylurea could not be distinguished from B apoprotein prepared by the conventional gel filtration technique. After injection of [3H]lysine, specific activity of B apoprotein was maximal in very low density and low density lipoproteins 1 and 11/2-h later, respectively, in a manner consistent with a precursor-product relationship. When protein-labeled very low density lipoproteins were injected into rats, the relationships of specific activity again indicated that B apoprotein of very low density lipoproteins may be the sole precursor of that of low density lipoproteins. However, less than 10% of the B apoprotein that disappeared from very low density lipoproteins appeared in density lipoproteins. To evaluate the sites of removal of B aproprotein of very low density lipoproteins from plasma, protein-labeled very low density lipoproteins were incubated with unlabeled high density lipoproteins to reduce radioactivity in non-B apoproteins selectively by molecular exchange. Most of the B apoprotein was rapidly removed by the liver. The extensive hepatic uptake of both the cholesteryl ester and B apoprotein components of rat very low density lipoproteins may explain the characteristically low concentrations of plasma low density lipoproteins in the rat.
O Faergeman, T Sata, J P Kane, R J Havel
To investigate the in vivo whole blood metabolic clearance rates and sites of metabolism of prostaglandins A1 and E1 in man, constant infusions of the tritiated compounds were administered to normal subjects and to patients undergoing cardiac catheterization. The whole blood metabolic clearance rate of [3H]prostaglandin A1 in eight men was 5,003 +/- 864 liters/day (SD) or 2,546 +/- 513 liters/day per m2 (SD). Nonradioactive prostaglandin A1 was similarly infused in two subjects, and the metabolic clearance rates were determined, utilizing a specific radioimmunoassay. The clearance rates with this method correlated closely with those determined by the isotope infusions. Extraction studies of prostaglandin A1 showed that pulmonary, splanchnic, renal, and extremity perfusions resulted in 8.1 +/- 4.1, 56.1 +/- 10.1, 50.3 +/- 3.4, and 34.4 +/- 5.9% (SEM) removal, respectively. With [3H]=prostaglandin E1, the whole blood metabolic clearance rate was determined from the pulmonary artery concentration in three patients and averaged 4,832 +/- 1,518 liters/day (SD) or 2,686 +/- 654 liters/day per m2 (SD). Pulmonary extraction was 67.8 +/- 6.8% (SEM) and extremity removal averaged 6.6 +/- 4.9% (SEM). These results indicate that A prostaglandins are metabolized by several organs, such as the liver and kidney, and possibly by intravascular pathways as well. In man, the E prostaglandins are primarily metabolized by the lung, but extraction is not complete and approximately one-third may escape lung metabolism. Thus, these findings suggest that both E and A prostaglandins in the venous circulation may reach the systemic circulation in man.
M Golub, P Zia, M Matsuno, R Horton
The dose dependence of the acute effects of ethanol upon liver intermediary metabolism in vivo has been demonstrated in rats. Ethanol was given i.p. in doses of 0.69, 1.7, and 3.0 g/kg in equal volumes (20 ml/kg). The liver was freeze-clamped 120 min after injection, and multiple metabolites were measured in the perchloric acid extract of the tissue. Each group showed a significantly different pattern of metabolites, redox states, and phosphorylation potentials although the rate of ethanol disappearance, at least between the two highest dose groups, was not significantly different. The mitochondrial free [NAD+]/[NADH] ratios and the cytoplasmic free [NADP+]/[NADPH] ratio were paradoxically most reduced with the lowest dose of ethanol and became progressively more oxidized with increasing dose. Once established, the differences in these ratios between the groups tended to persist with time, relatively independent of the concentration of ethanol. In a somewhat different pattern, the phosphorylation potential ([ATP]/[ADP][P1]) remained at the control level in the low-dose group but was significantly elevated in the two higher-dose groups. The results, therefore, show distinct and complicated dose-dependent patterns of intermediary metabolism that cannot be explained completely by any one hypothesis but that imply significant dose-dependent effects of ethanol upon intermediary metabolism not directly related to NADH production.
R W Guynn, J R Pieklik
The turnover of 125I-labeled low density lipoprotein (LDL) and the total body balance of cholestrol were studied in a 6-yr-old girl with the homozygous form of familial hypercholesterolemia (FH) before and after the surgical creation of an end-to-side portacaval shunt. The results were compared with those of similar studies simultaneously performed in untreated patients with the heterozygous form of FH and with the results of earlier studies performed on normolipidemic subjects. Before shunt surgery, the rate of synthesis of LDL in the FH homozygote (mg/kg per day) was fourfold higher than in normolipidemic subjects and twofold higher than in her heterozygous mother. The fractional catabolic rate for LDL in the homozygote was decreased to 33% of normal control values. The rate of cholesterol synthesis, estimated by chemical sterol balance, was higher in the FH homozygote than in two FH heterozygotes of similar age studied simultaneously. When considered in relation to the markedly elevated level of plasma cholesterol, the observed rate of cholesterol synthesis in the FH homozygote was inappropriately elevated. Bile acid production was normal in all three children. 5 mo after shunt surgery, the rate of LDL synthesis in the homozygote had declined by 48% as compared with the preoperative value, and this caused a 39% drop in the plasma LDL cholesterol level despite a 17% reduction in the fractional catabolic rate of the lipoprotein. The rate of cholesterol synthesis fell by 62% as compared with the preoperative value. The findings of an inappropriately elevated rate of production of both cholesterol and LDL as well as a reduced fractional catabolic rate for the lipoprotein in the untreated FH homozygote are consistent with results of studies in cultured fibroblasts indicating that the primary genetic defect in FH involves a deficiency in a cell-surface receptor for LDL that regulates both cholesterol synthesis and LDL degradation. Although the mechanism for the decline in production of cholesterol and LDL after portacaval shunt surgery is unknown, it was observed that these changes were associated with marked increases in the plasma concentrations of bile acids and glucagon.
D W Bilheimer, J L Goldstein, S M Grundy, M S Brown
Bile salts play a major role in bile formation and biliary lipid secretion. Sodium taurodihydrofusidate (TDHF), a derivative of the antibiotic fusidic acid, closely resembles bile salts in terms of structure, micellar characteristics, and capacity ot solubilize otherwise insolbule lipids. We have therefore studied the biliary secretion of this bile salt analogue and its influence on bile formation and biliary lipid secretion in primates. Alert, unanesthetized female rhesus monkeys prepared with a total biliary fistula were allowed to reach a steady bile salt secretion rate before each study. In three animals (group I),[14C]TDHF was infused intravenously. Most of the compound was secreted rapidly in bile chemically unchanged. The biliary secretion of this drug produced a twofold increase in bile flow; however, the bile salt output was markedly reduced during the infusion. In spite of this reduction, the phospholipid output remained essentially unchanged whereas the cholesterol output increased almost twofold. In five other animals (group II), the effect of TDHF on the bile salt secretion was further investigated by an intravenous infusion of [14C]taurocholate followed by a combined infusion of [14C]taurocholate and TDHF. When TDHF was added to the infusate, a reduction in the [14C]taurocholate output and a progressive rise in the plasma [14C]taurocholate concentration were observed in each animal. An analysis of the data in both groups indicates that (a) the most likely explanation to account for the decreased bile salt output is that the bile salt analogue, TDHF, interfered with bile salt secretion into the biliary canaliculi; (b) TDHF induces a greater secretion of biliary water than was observed with bile salts, an effect consistent with a stimulation of the bile salt-independent canalicular flow; (c) at similar 3alpha-hydroxysteroid secretion rates TDHF caused a significant increase in cholesterol secretion compared to that induced by bile salt. This finding suggests that TDHF affects cholesterol metabolism or secretion in a way distinct from bile salts. Thus, the solubilization of biliary lipids in mixed micelles, although essential, is only one of the factors which determine their secretion into bile.
M Beaudoin, M C Carey, D M Small
Measurements of mean left ventricular (LV) and regional myocardial blood flow rates were made at rest in 161 patients with 133Xe and a multiplecrystal scintillation camera. Myocardial perfusion rates were correlated with assessments of the degree of coronary artery disease made from the arteriograms obtained during the same studies. In patients with normal coronary arteries without heart failure, the presence of hypertension, aortic stenosis, or aortic insufficiency was not associated with changes in mean LV perfusion from the control value of 61+/-7 ml/100 g-min. However, mean LV perfusion was significantly reduced in patients with normal coronary arteries who had cariomyopathy and impaired ventricular performance. Mean LV perfusion was not significantly different from control values in patients with "mild" coronary artery disease (less than 50% obstruction) or in patients with significant isolated disease (greater than 50% obstruction) of the left anterior descending (lad) artery. Significant reductions in mean LV perfusion were found in patients with greater than 50% obstruction of two coronary arteries (LAD + right or LAD + circumflex) and in patients with triple-vessel disease. The average perfusion rate for regions distal to LAD obstructions in patients with isolated LAD disease was not lower than the LAD perfusion in control patients, but was significantly reduced in patients with LAD + right coronary artery disease (43+/-14 ml/100 g-min). In the latter group average perfusion distal to the LAD lesion was significantly lower than the average regional perfusion rate for the remainder of the LV. However, the mean blood flow rate for the remainder of the LV was also significantly lower than control values despite the lack of significant circumflex disease. The data demonstrate that the presence of radiographically "mild" or significant isolated LAD coronary disease is not associated with reductions in mean LV perfusion at rest, but that mean LV perfusion is reduced in the presence of significant disease of two or three coronary artieries. None of the patients experienced angina during the resting studies and most had clinical evidence of ventricular failure. The observation of depressed LV perfusion in this group, as in the patients with cardiomyopathy, raises the possibility that a lowered resting blood supply may be adequate for a reduced level of performance of a diseased ventricle. The lack of selective reductions of regional perfusion at rest in the majority of the patients with LAD lesions suggests that regional myocardial blood flow must be measured during an intervention which increases myocardial oxygen consumption in order to assess the physiological significance of lesions which are observed at coronary arteriography.
P J Cannon, D H Schmidt, M B Weiss, D L Fowler, R R Sciacca, K Ellis, W J Casarella
A preparative scheme has been developed for the purification of a trace protein in human serum exhibiting nonsuppressible insulin-like activity (NSILA). This scheme consisted of (a) adsorption chromatography of serum utilizing the sulfonic acid polystyrene resin, Dowex 50, at pH 6.8; (b) (200 gel filtration at pH 8.9; and (c) acrylamide gel electrophoresis in a discontinuous preparative system. Throughout all procedures, NSILA fractionated as a single molecular species approximating 90,000 mol wt. The purified protein exhibited a single band by disk gel electrophoresis, an isoelectric pH approximating 6.2, doublet bands of 90,000 mol mt by analytical sodium dodecyl sulfate gel electrophoresis, and a biologic specific activity approximating 50 mU/mg. Serum somatomedin (sulfation factor) activtiy did not fractionate with NSILA in this scheme, and partially purified NSILA did not stimulate radiosulfate uptake into hypophysectomized rat costal cartilage. This protein appears to represent the major constituent of serum NSILA: its purification and partial characterization provides the first step towards elucidation of its metabolic role.
P L Poffenbarger
Two separate lymphocyte populations, each bearing easily detectable surface immunoglobulin, have been detected in human peripheral blood. The first, B cells, has surface determinants that are stable at 37degreeC, but are removed by pronase and regenerate in culture. The cells are nylon adherent and have a receptor for C3, and studies with unit gravity sedimentation indicate they are mostly small lymphocytes. B cells comprise 9.5% of the total lymphocytes, with the normal range from 3-16%. As many or more lymphocytes lack membrane-incorporated Ig determinants but have an Fc receptor that binds IgG1 and IgG3 in normal serum maximally at 4degreeC. This receptor for cytophilic IgG is removed by pronase but not by trypsin. The second population has been named L lymphocytes because of membrane-labile IgG determinants. L cells do not adhere to nylon, do not form rosettes with sheep erythrocytes sensitized with antibody and mouse complement, and are larger than small lymphocytes. These lymphocytes with cold-reactive Fc receptors for serum IgG do not form E-rosettes or respond to phytohemaggutinin. Since L cells do not have surface markers of T and B lymphocytes, it is likely that they comprise a separate population.
D A Horwitz, P I Lobo
The distribution of calcium pyrophosphate mineral phase, almost exclusively confined to articular cartilage in chondrocalcinosis, and the high level of pyrophosphate (PPi) ion relative to serum in synovial fluid in patients with either chondrocalcinosis or advanced osteoarthritis led to an investigation of whether cartilage cells elaborate PPi ions. Incubates of articular cartilage from young rabbits but not from mature rabbits, as well as growth plates cartilage, released PPi into incubation media during a 4h period. Control rabbit ear cartilage and synovial membrane elaborated negligible amounts of PPi. The PPi was shown to be undialyzable but could be dissociated from the alkaline phosphatase by ultracentrifugation. In 16 patients with osteoarthritis, a substantial output of PPi by samples of articular cartilage from the knee was demonstrated. It is postulated that either rapid cell division and matrix synthesis found in the base of ulcerating osteoarthritic cartilage or remodeling calcified sites are the source of the PPi in such osteoarthritic cartilage. It is further hypothesized that this PPi output accounts at least in part for the elevated PPi levels found in synovial fluid of patients with osteoarthritis.
D S Howell, O Muniz, J C Pita, J E Enis
The interrelationship between apolipoprotein B in very low density lipoprotein (VLDL-B) and in low density lipoprotein (LDL-B) was studied in seven normal and hyperlipidemic men and women, with purified radioiodinated VLDL. The time-course of the appearance of radioactivity in LDL was followed. As the specific activity curves intersected at the masimal height of the LDL-B curve, it was inferred that all or most LDL-B peptide is derived from VLDL-B peptide. This transfer was further quantitated in seven normotriglyceridemic subjects by simultaneous i.v. injection of purified 131I-VLDL and 125I-LDL. By a deconvolution method, a quantitative description of the rate of entry of 131Ivldl-b into 131I-LDL-B was derived by analysis of 131I-LDL-B and 125I-ldl-b radioactivity in plasma. The results indicate that approximately 90% of VLDL-B mass is converted into LDL-B in subjects with normal serum triglyceride concentrations. The synthetic rates of VLDL-B and LDL-B peptide were simultaneously measured in six normal subjects, and two patients with heterozygous familial hypercholesterolemia (type IIa). The turnover rates for VLDL-B and LDL-B peptide were similar in these subjects. The findings in the three parts of this study were consistent with the view that most if not all VLDL-B is converted into LDL-B peptide, and most if not all LDL-B is derived from VLDL-B peptide in normotriglyceridemic subjects.
G Sigurdsson, A Nicoll, B Lewis
Human bone marrow is known to contain significant numbers of bursa-dependent lymphocytes. The presence of thymus-dependent (T) cells is controversial. Bone marrow cells obtained from healthy volunteers was fractionated by density centrifugation. A lymphocte-enriched subpopulation was shown to be reactive to alloantigens in mixed lymphocyte culture and to contain substantial numbers of T lymphocytes. The T lymphocytes were detected by cell surface markers (rosette formation with sheep RBC) and by response to the mitogens phytohemagglutinin and concanavalin A. Bone marrow T cells exhibited functional characteristics quantitatively different from peripheral blood T cells, suggesting that they may represent a particular subpopulation of T cells. The lymphocyte-enriched fraction additionally contained committed granulopoietic stem cells capable of colony formation in semisolid gel. The presence of T cells in human bone marrow is consistent with findings in other mammals and may explain the high incidence of graft versus host disease in bone marrow transplant recipients.
R P Gale, G Opelz, O M Kiuchi, D W Golde
We have studied the in vitro effects of dexamethasone on isolated rat adipocytes at concentrations of dexamethasone therapeutically achieved in man. Glucose oxidation, glucose transport, and insulin binding were assessed. In dexamethasone-treated cells, glucose oxidation was decreased by 30-40% both in the absence of insulin (basal state) and at low insulin levels (less than 25 mu/ML). At maximally effective insulin levels (over 100 muU/ml) no differences existed between control and treated cells. If glucose transport were the rate-limiting step for glucose oxidation in the basal state and at low (submaximal) insulin levels, but not at maximally effective insulin concentrations, then these data could be explained by postulating that dexamethasone has a direct affect on glucose transport and does not affect intracellular oxidative pathways. We tested this hypothesis by directly assessing glucose transport in dexamethasone-treated cells. Glucose transport was assessed by measuring the uptake of [14C]2-deoxy glucose. These studies demonstrated a 30-40% decrease in 2-deoxy glucose uptake by treated cells both in the basal state and at all insulin concentrations. Thus, a direct glucocorticoid effect on the glucose transport system seems to account for the decreased ability of dexamethasone-treated cells to oxidize glucose. Since dexamethasone treatment leads to decreased insulin binding to adipocytes in vivo, we examined the possibility that the in vitro decreases in insulin-mediated glucose transport could be due to decreased insulin receptors. Insulin binding to control and treated adipocytes was measured, and no differences were found. Therefore, in cntrast to previously reported in vivo studies, adipocytes treated in vitro with dexamethasone retain a normal ability to bind insulin. Thus, these studies suggest that all of the in vitro effects of dexamethasone on glucose oxidation are due to direct inhibition of the glucose transport system.
J M Olefsky
Cleavage of human fibrinogen and fibrin by plasmin is associated with modification of native antigenic expression and the exposure of cleavage-associated neoantigenic sites on the derivative molecular fragments. In this study, the presence of humoral antibodies in man to cleavage-associated neoantigens has been demonstrated by primary antigen binding radioimmunochemical assays. Specific binding of radioiodinated human fibrinogen D fragment by serum immunoglobulins was demonstrated in 52 of 59 random normal human sera and was independent of immunoglobulin concentration. Binding was mediated by F (ab)2 fragments of IgG, and specificity for neoantigens was indicated by the capacity of the D fragment but not native fibrinogen to competitively inhibit the antibody. The population distribution of antibody to these cleavage-associated neoantigens indicated the presence of a major group of individuals (77%) with a mean antigen binding capacity of 11.8 pmol/ml serum. Two minor populations with : (a) low or undetectable binding capacities (less than 6.0 pmol/ml serum) and (b) exhibiting markedly elevated binding capacities (less 18.0 pmol/ml serum) were delineated. Independent of these features, sera could also be readily separated into two groups that differed with respect to relative antibody affinity. The antibodies in most sera exhibited marked heterogeneity of binding affinity, whereas a small group of sera contained antibodies exhibiting relative homogeneity of binding affinity. Specific antibody was rather equally distributed between the major immunoglobulin classes, and in no serum was the antibody restricted to a single immunoglobulin class. Antibodies capable of binding fibrinogen fragments X, Y, and D and fibrin D fragment were detected in most sera. The quantity of antibody differed for different fragments with X greater than Y congruent to D greater than fibrin D. The presence of antibody capable of binding any single fragment was statistically correlated with the presence of antibody capable of binding other cleavage fragments. No antibody to the E fragment was detected. Antibody to cleavage fragments was not demonstrable in sera containing fibrinogen or fibrin cleavage fragments. Demonstration of this humoral immune response to the products of the fibrinolytic systems provides a new interface between the coagulation and immune system.
E F Plow, T S Edgington
In lead intoxication photosensitivity is usually absent, despite concentrations of protoporphyrin in the erythrocytes equal to or greater than in erythropoietic protoporphyria. Profound differences in the distribution of protoporphyrin in aging erythrocytes were demonstrated by age-dependent fractionation of cells on discontinuous density gradients. In erythropoietic protoporphyria the concentration of protoporphyrin declined extremely rapidly with erythrocyte age; the bulk of the protoporphyrin was lost in less than 3 days and the concentration of fluorescent erythrocytes in the gradient paralleled the decline of protoporphyrin. In lead intoxication the protoporphyrin concentration declined only slightly with cell aging and erythrocytes of all ages fluoresced. In the bone marrow from a patient with erythropoietic protoporphyria all reticulocytes, but only occasional late normoblasts, fluoresced, suggesting a single population. Sterile incubation in plasma (pH 7.5) demonstrated rapid diffusion of protoporphyrin from the erythrocytes in erythropoietic protoporphyria, but not in lead intoxication. Plasma protoporphyrin was elevated in erythropoietic protoporphyria, but not in lead intoxication. Estimates of the daily loss of protoporphyrin from erythropoietic tissue in erythropoietic proporphyria suggested an order of magnitude similar to the total blood protoporphyrin. Therefore, it is not necessary to postulate a preponderant extraerythropoietic source to explain the amount of fecal excretion. A significant amount of the diffused protoporphyrin probably reaches the skin with resulting photosensitivity. In contrast, in lead intoxication protoporphyrin remains within the erythrocyte throughout its life span ; there is no diffusion into the plasma and hence no photosensitivity.
S Piomelli, A A Lamola, M F Poh-Fitzpatrick, C Seaman, L C Harber
Acidic solvents extract the same porphyrin-protoporphyrin-from the erythrocytes of patients with either erythropoietic protoporphyria or lead intoxication. However, extractable protoporphyrin disappears rapidly, both in vivo and in vitro, from erythrocytes in erythropoietic protoporphyria but slowly, if at all, in lead intoxication. Consistent with these observations, fluorescence spectroscopy revealed that the intracellular state of the erythrocyte protoporphyrin is different in the two diseases. Spectrofluorometric measurements coupled with fractionations and biochemical syntheses showed that in erythropoietic protoporphyria the protoporphyrin is bound as the free base to hemoglobin molecules at sites other than the heme binding sites. In lead intoxication the fluorescent porphyrin is also bound to hemoglobin but is present as zinc protoporphyrin. The data suggest that the zinc protoporphyrin is bound at heme binding sites. Acidic extraction solvents remove the chelated zinc, but zinc protoporphyrin may be extracted intact from erythrocytes with acetone, ethanol, or the detergent Ammonyx-LO.
A A Lamola, S Piomelli, M G Poh-Fitzpatrick, T Yamane, L C Harber
Group A and group C meningoccal polysaccharide vaccines were evaluated in infants. No significant local or systemic reactions were observed with 908 doses of vaccine given to 396 infants between 3 and 12 mo of age. The antibody response varied with the age of the infant, vaccine dose, molecular weight of vaccine, prior immunization with vaccine, and prior exposure to naturally occurring cross-reactive antigens. Only 7% of 3-mo-old infants had detectable antibody responses to primary immunization with 5-200 mug of A vaccine, presumably because of suppressive effects of high concentrations of maternal anti-A. More than 90% of 7- and 12-mo-old infants responded to A vaccine, achieving geometric mean anti-A concentrations of 0.38 and 0.98 mug/ml, respectively. The dose-response curve was flat between 10 and 200 mug of A vaccine. Geometric mean anti-A concentrations of 2.51 and 4.00 mug/ml were induced in 7- and 12-mo-old infants by booster injections of A vaccine. Approximately 90% of 3-mo-old infants had detectable antibody responses to primary immunization with C vaccine. The 100-mug dose appeared to be optimal, resulting in geometric mean anti-C concentrations of 0.49, 1.55, and 2.64 mug/ml in 3-, 7-, and 12-mo-old infants, respectively. Significant booster responses were not observed with C vaccine. Indeed, except for the 10-mug dose, booster injections of C vaccine in 7- and 12-mo-old infants resulted in lower anti-C concentrations than did primary immunizations.
R Gold, M L Lepow, I Goldschneider, T L Draper, E C Gotschlich
Serum glucocorticoid levels were determined in 20 mothers and 43 premature infants who received prenatal betamethasone therapy for prevention of respiratory distress syndrome (RDS). Maternal betamethasone peaked at 75 microg cortisol equivalents per 100 ml 1 h after injection of 12 mg steroid and declined to half by 6 h. Betamethasone in cord blood was 14.3 microg cortisol equivalents per 100 ml at 1 h, decreased to a level of 4.7 at 20 h, and was not detected 2 days after a second dose at 24 h. After the second dose, the mean level of cortisol in cord blood was 5.9 microg per 100 ml compared with 13.05 microg per 100 ml (p less than 0.001) in untreated premature infants. The unbound glucocorticoid activity in treated infants delivered 1-10 h after the second dose (mean, 8.4 microg per 100 ml) is similar to the unbound cortisol level after birth in untreated premature infants who develop RDS. These findings indicate that (a) serum glucocorticoid levels in the physiologic stress range can induce lung maturation in the human and (b) antenatal treatment with this dose of betamethasone does not expose the human fetus to potentially harmful pharmacologic levels of steroid.
P L Ballard, P Granberg, R A Ballard
A variety of studies in man and animals demonstrate that testosterone (T) is aromatized to estradiol (E) in the hypothalamus and limbic system. These observations suggested the possibility that conversion to E is an absolute requirement for the biologic activity of T on the hypothalamic-pituitary axis. Since this hypothesis implies a common mechanism of action of these two steroids, the demonstration of divergent effects of T and E on luteinizing hormone (LH) secretion would exclude this possibility. To test this hypothesis, the actions of T and E on three separate aspects of LH release (mean LH, pulsatile LH secretion, and responsiveness to LH-releasing hormone [LH-RH]) were contrasted. T and E, infused at two times their respective production rates into normal men, reduced mean LH levels similarly during 6 h of steroid infusion and for 6 h thereafter. However, these steroids exerted different effects on pulsatile secretion. E reduced the amplitude of spontaneous LH pulse from pre- and postinfusion control levels of 75+/-14 and 68+/-5.6% (SEM) to 39+/-5.7%. In contrast, T increased pulse amplited to 96+/-14% and decreased pulse frequency from basal levels of 3.4+/-0.31 to 1.8+/-0.31 pulses/6h. The site of suppressive action was determined by administering 25 microgms of LH-RH to the same men during T and E infusions and during three additional control periods without steroid administration. LH-RH produced similar 170-190% increments in serum LH during the three control periods and during T infusion. In contrast, E markedly blunted (76+/-31%, p less than 0.005) the LH response to LH-RH. Under the conditions of acute steroid infusion at doses (utilized in these experiments) producing similar inhibition of mean LH, E but not T acted directly on the pituitary to diminish LH-RH responsiveness. As further support that androgens can act without conversion to estrogens, the effects of a nonaromatizable androgen, dihydrotestosterone (DHT), on mean LH levels were studied. DHT, infused at the same rate as T, suppressed mean LH to a similar but somewhat greater extent than T. Since T and E produced divergent effects on LH secretion and a nonaromatizable androgen, DHT, suppressed mean LH, aromatization is not a necessary prerequisite for the action of androgens on the hypothalamic-pituitary axis.
R J Santen
To investigate the physiology of thyrotropin-releasing hormone (TRH) secretion from hypothalamus and brain, a method for measurement of peripheral plasma TRH concentrations in rats was developed. Blood was collected in heparin and dimercaptopropanol containing [3H]TRH to determine recovery. The plasma was extracted with methanol and the redissolved dried methanol extracts applied to anti-TRH Sepharose columns. These columns bound greater than 80% of 125I-TRH applied and had a capacity in excess of 20 ng TRH. TRH was eluted from the anti-TRH Sepharose with acetic acid and quantitated by radioimmunoassay of the lyophilized acetic acid eluate. Mean recovery of unlabeled TRH was 44.7+/-6.1% (SD) and mean recovery of [3H]TRH was 44.0+/-4.0%. Mean plasma TRH concentrations, corrected for recovery, in plasma pools from eight groups of normal male rats (four to seven pools/experiment, five to seven rats/pool) ranged from 7 to 30 pg/ml (mean, 16). In experiments in which rats were given 5, 10, 15, 0r 50 mug thyroxine daily for 1 wk or in thyroidectomized rats, mean plasma TRH concentrations did not differ significantly from those of control animals sacrificed at the same time. In each experiment, four to seven plasma pools, each from five to seven rats, were processed from both control and experimental groups. No changes in plasma TRH concentrations were found in rats exposed to cold (4degreeC) for 30, 60, and 90-180 min. Signigicant increases in plasma thyrotropin (TSH) concentrations were found in all cold-exposed animals. These results provide no evidence that thyroid hormone excess of deficiency affects TRH secretion. If TRH secretion is responsible for cold-induced increases in plasma TSH concentrations, the increase in TRH secretion is of insufficient magnitude to alter periperal plasma TRH concentrations.
C H Emerson, R D Utiger
The solubility of triclinic calcium pyrophosphate dihydrate (CPPD) crystals was measured under varying conditions using 45Ca-labeled crystals, expressing solubility as micromoles per liter of 45Ca in solution. In a 0.1-M Tris-HC1 buffer pH 7.4, the solubility of accurately sized CPPD crystals (37-20mum) was 60muM with maximal solubility being attained after about 8 h incubation at 37degreeC. Reduction in crystal size, decrease in pH, increase in ionic strength, Mg++, citrate, and albumin all increased solubility. The most marked effects on solubility occurred when changing the calcium concentration or by enzymatic hydrolysis of inoganic pyrophosphate to orthophosphate. It was found that decreasing the ionized calcium level below 5 mg/100 ml resulted in a progressive enhancement of solubility. The observed solubility-enhancing effects of albumin could be explained solely on its calcium-binding ability and thereby, altered ionized calcium level. Diffusible calcium in synovial fluid was only 40% of the total calcium concentration, which means most joint fluids are normally near the critical concentration of 5 mg/100 ml of ionized calcium, below which solubility is enhanced. During surgery, especially parathyroidectomy, calcium levels fall, favoring dissolution of CPPD crystals. We speculate that the slight decrease in crystal size during dissolution frees them from their cartilaginous mold, resulting in a dose-dependent inflammatory reaction as they are "shed" into the joint space. Crystal shedding may be reinforced by the modest fall in joint fluid pH accompanying the inflammatory response.
R M Bennett, J R Lehr, D J McCarty
Antibody-dependent cellular cytotoxicity (ADCC), has been shown to be independent in vitro of thymus-derived lymphocytes, but the precise nature of the effector lymphocyte has not been fully clarified. To further study the identity of the ADCC effector cell type(s), peripheral blood leukocytes were purified by Ficoll-Hypaque density centrifugation and fractionated into surface immunoglobulin-positive [Ig(+)] and surface immunoglobulin-negative [Ig(-)] populations by chromatographic separation on Sephadex G-200 anti-human immunoglobulin columns. After column fractionations, the ADCC effector activity against antibody-coated autologous lymphocytes was predominantly and consistently found in the Ig(-) fraction. This latter population was then further fractionated, by rosetting techniques, into two subpopulations, The first was depleted by lymphocytes with surface receptors for sheep red blood cells [E(+)]and the second was depleted of lymphocytes with receptors for sheep red blood cell-antibody-complement [EAC-(+)]. Analysis of these populations showed that ADCC effector activity was predominantly a property of the Ig(-) lmyphocytes which are E(-) but EAC(+). These lymphocytes have been referred to as "null lymphocytes" and probably represent a subset of bone marrow-derived (B) cells. In addition, variable and low levels of ADCC activity were observed in some Ig(+) populations (B cells). Further purification of the null cell population by filtration over nylon wool columns to reduce the number of contaminating latex ingesting monocytes did not reduce ADCC effector activity. Isolated null cell ADCC effector activity was inhibited by either rabbit anti-human F(ab)2 or normal pooled rabbit gamma globulin, but not by rabbit F(ab)2 anti-human F)ab)2 or media. This supports the contention previously suggested in studies using unfractionated lymphocyte populations that the ADCC effector cell recognizes the Fc portion of the antibody molecule. The variable and low level of activity noted in the Ig(+) populations is unexplained but possibly due to a variable population of null cell-derived Ig(+) lymphocytes within the whole Ig(+) population. In conclusion, these experiments demonstrate that, in vitro, the major ADCC effector activity of circulating human peripheral blood lymphocytes resides in the Ig(-), E(-), EAC-(+) subpopulation termed "null cells." Since it has been noted that in certain disease states, such as immunodeficiency syndromes, autoimmune disorders, and neoplasms, the percentage of this population of lymphocytes in the peripheral blood is elevated, it is speculated that these cells, perhaps through their ADCC function, may play an important pathophysiologic role in these diseases.
A M Brier, L Chess, S F Schlossman
The immunosuppressive activities of two phase-specific drugs, 6-mercaptopurine (6-MP) and methotrexate, and a cycle-specific agent, cyclophosphamide, were evaluated on the lymphocytic component of established tuberculin hypersensitivity in guinea pigs. In these animals, purified protein derivative (PPD)-sensitive lymphocytes are in an intermitotic phase of their proliferative cycle. Neither phase-specific drug significantly altered either the number or functional activities of these lymphocytes. By two in vitro criteria, PPD-induced lymphoproliferation and elaboration of migration inhibition factor (MIF), the responses of lymph node cells were equivalent to sensitized controls. In addition, these agents did not deplete pools of T lymphocytes, impair responses to phytohemagglutinin (PHA), nor inhibit cutaneous reactivity if employed before sensitization. In contrast, cyclophosphamide showed broader immunosuppressive effects including significant toxicities for intermitotic lymphocytes. This drug depleted pools of T cells and markedly impaired the in vitro proliferative responses of residual lymphocytes. The latter occurred with both PHA and PPD. Suppression of PHA reactivity was a dose-dependent phenomenon but was evident even with small quantities of this alkylating agent. The suppression of antigen-induced responses was independent of the proliferative status of target lymphocytes in vivo, after a single large dose, it persisted for more than 3 wk. In total, these results indicate that the effective use of cytotoxic drugs as immunosuppressants must include consideration of both the cycle specificities of the agent and the proliferative activities of the target lymphoid population.
Splenectomy markedly impaired the production of circulating anti-endotoxin antibodies during the initial 10 days after .v. administration of a Boivin preparation of Escherichia coli endotoxin (ET) in both rabbit and man. Increase in antibodies with secondary (flocculating and bactericidal) activities were virtually abolished, whereas increases in antibodies with primary (binding) activity were significantly reduced. On the basis of these findings, splenectomized rabbit and man were employed to test the hypothesis that the early phase (less than 72 h) of pyrogenic tolerance to endotoxin is independent of anti-endotoxin antibody but that such antibody contributes significantly to the later phase (less than or equal to 72 h) of tolerance. In the splenectomized rabbit, the initial pyrogenic reponses to ET and the subsequent tolerant responses at 24 and 48 h were comparable to sham-operated controls...
S E Greisman, E J Young, J B Workman, R M Ollodart, R B Hornick
To clarify alterations in carbohydrate metabolism which occur in pregnancy, metabolic clearance rates of insulin, proinsulin, and C-peptide were measured by the constant infusion technique in term-pregnant rats and in virgin littermates. In addition, placental permeability to these peptides was evaluated by simultaneous determination of their concentration in fetal blood, amniotic fluid, and maternal arterial blood and the renal extraction and excretion of insulin and C-peptide were determined during simultaneous studies of renal hemodynamics. The metabolic clearance rate (MCR) of insulin was higher (P less than 0.005) in pregnant animals (61.5+/-1.7 ml/min per kg nonconceptus body weight) than in virgin littermates (51.5+/-2.2 ml/min per kg). Insulin disappearance from the circulation after both single injection and discontinuance of a constant infusion was also faster in gravid animals. In contrast, the MCR of proinsulin and C-peptide, and the disappearance of C-peptide from the circulation were similar in pregnant and control rats. The placenta was virtually impermeable to each of the three polypeptides since their mean levels in both fetal blood and amniotic fluid did not exceed 2.5 ng/ml and were only minimally influenced by pharmacological concentrations as high as 60 ng/ml in the maternal circulation. The renal clearance of insulin (renal arteriovenous insulin difference X renal plasma flow) was lower, and its contribution to insulin MCR was less in pregnant animals than in controls (19.4+/-1.5% vs. 28.7+/-3.7%, P less than 0.05), whereas the renal clearance and renal clearance/MCR of C-peptide were similar in pregnant rats and virgin littermates. These results indicate that the peripheral metabolism of insulin is accelerated in pregnancy, while that of pro-insulin and C-peptide is unaffected. Since transplacental passage of insulin is negligible and its renal clearance is not increased, the enhanced MCR of insulin in pregnancy is due to increased metabolism at an extrarenal site probably within the placenta itself.
A I Katz, M D Lindheimer, M E Mako, A H Rubenstein
The effect of magnesium chloride or magnesium sulfate infusion on circulating levels of immunoreactive calcitonin (iCT) was evaluated on nine occasions in three patients with metastatic medullary carcinoma of the thyroid. One patient was normocalcemic and had normal circulating levels of immunoreactive parathyroid hormone (iPTH), one patient was hypocalcemic and had surgical hypoparathyroidism, and one patient had mild to moderate hypercalcemia associated with bone metastases. The basal serum iPTH levels were undetectable in the latter two patients. In every instance magnesium administration produced a rapid and striking fall in circulating iCT and usually a detectable fall in serum calcium. During the hypermagnesemic state, serum iPTH fell from normal to undetectable in the patient with normal parathyroid function, while serum iPTH levels remained undetectable in the hypoparathyroid patient and in the patient with hypercalcemia associated with bone metastases. The results of these studies indicate that: (a) contrary to what has been reported in normal experimental animals, magnesium administration lowers circulating iCT in human subjects with thyroid medullary carcinoma and (b) the calcium-lowering effect produced by magnesium in patients with medullary carcinoma may, in part at least, be due to a redistribution of body calcium that is not mediated by the actions of either parathyroid hormone or clacitonin.
C Anast, L David, J Winnacker, R Glass, W Baskin, L Brubaker, T Burns
Methods for quantitation of the major apoproteins of human serum very low density lipoprotein have been developed employing tetramethylurea, which delipidates the lipoprotein and selectively precipitates apolipoprotein B. Six soluble apoproteins are separated by electrophoresis in polyacrylamide gel. One of these is a previously unrecognized species of R-alanine (R4-alanine), more anionic than the R3-alanine polypeptide. Conditions of staining have been found which yield reproducibly linear chromogenic response with native lipoprotein and with each purified apoprotein. Recovery of protein in the seven species measured accounts for over 97% of the total in the very low density lipoprotein of normolipidemic individuals and in most samples from individuals with endogenous hyperlipemia. The mean content of apolipoprotein B in 43 samples from normolipidemic subjects was 36.9(+/-1.2 SEM)% of total protein, The distribution of the major soluble apoproteins as mean (+/-SEM) percentage of the soluble fraction was : R-serine, 5.3+/-o.5; arginine-rich, 20.6+/-1.0; R-glutamic, 10.6+/-0.4; R2-alanine, 28.3+/-0.7; R3-alanine, 26.9+/-0.5; and R4-alanine, 8.0+/-0.5. Distribution of the apoproteins was a function of particle diameter of very low density lipoprotein in fractions separated by gel permeation chromatography and by density gradient ultracentrifugation. In fractions below 700-800 A, apolipoprotein B comprised an increasing percentage of the total protein with decreasing particle diameter. Among the soluble proteins the percentage of the arginine-rich and R-serine polypeptides increased and that of the R-glutamic polypeptide declined progressively with decreasing particle size. Apoprotein distribution was similar in fractions of similar particle size from normolipidemic and hyperlipemic subjects with the exception that all fractions from the hyperlipemic subjects contained more R-serine and some, more arginine rich polypeptide. Even in the absence of chylomicrons, the distribution of soluble apoproteins in particles of diameters greater than 700-800 A was usually similar to that of the smallest particles. This suggests that the largest particles may include products of the partial catabolism of chylomicrons.
J P Kane, T Sata, R L Hamilton, R J Havel
Platelets secrete lysosmal enzymes during the "platelet release reaction" early in clot formation. This study was undertaken to identify primary lysosomes of platelets and to detemine their origin in megakaryocytes. Using electron microscopy and cytochemistry, we localized two lysosomal enzymes, arylsulfatase and acid phosphatase, in megakaryocytes and platelets of normal and thrombocytopenic rats. In platelets and mature megakaryocytes, reaction product for both enzymes is confined to vesicles measuring 175-250 nm. These vesicles, which are primary lysosmes, first appear in the earliest recognizable megakaryocytes and increase in number during cellular maturation. In immature and maturing megakaryocytes, arylsulfatase and acid phosphatase can also be demonstrated in an organell similar to GERL (Golgi-endoplasmic reticulumlysosome), i.e., single smooth-surfaced cisternal with associated vesicles near the stacked Golgi cisternae. Scant reaction product for acid phosphatase is also sometimes seen in Golgi cisternae and endoplasmic reticulum. No reaction product was found in alpha-granules at any stage of megakaryocyte maturation, nor in alpha- or serotonin granules of platelets. Thus, our findings indicate that the primay lysosomes of megakaryocytes and platelets are small vesicles derived from GERL early in megakaryocyte differentiation. They can be indentified only after cytochemical staining and are distinct from both alpha- and serotonin granules.
M E Bentfeld, D F Bainton
An asymptomatic woman (Ms. Williams) was found to have a severe abnormality in the surface-activated intrinsic coagulation, fibrinolytic, and kinin-generating pathways. Assays for known coagulation factors were nromal while Fletcher factor (pre-kallikrein) was 45%, insufficient to account for the observed markedly prolonged partial thromboplastin time. Plasminogen proactivator was present at 20% of normal levels and addition of highly purified plasminogen proactivator containing 10% plasminogen activator partially corrected the coagulation and fibrinolytic abnormalities but not the kinin-generating defect. This effect was due to its plasminogen activator content. In addition, Williams trait plasma failed to convert prekallilrein to lakkilrein or release kinin upon incubation with kaolin. Kininogen antigen was undetectable. When normal plasma was fractionated to identify the factor that corrects all the abnormalities in Williams trait plasma, the Williams factor was identified as a form of kininogen by its behavior on ion exchange chromatography, gel filtration, disc gel electrophoresis, and elution from an anti-low molecular weight kininogen immunoadsorbent. High molecular weight kininogen as well as a subfraction of low molecular weight kininogen, possessed this corrective activity while the bulk of low molecular weight kininogen functioned only as a kallikrein substrate. Kininogen therefore is a critical factor required for the functioning of Hageman factor-dependent coagulation and fibrinolysis and for the activation of prekallikrein.
R W Colman, A Bagdasarian, R C Talamo, C F Scott, M Seavey, J A Guimaraes, J V Pierce, A P Kaplan
Flaujeac trait plasma resembled Hageman trait or Fletcher trait, in that the intrinsic coagulation pathway, plasma fibinolytic pathway, kinin-forming system, permeability factor of dilution (PF/dil) phenomenon were abnormal. The defect in each assay was reconstituted by afactor separable from Hageman factor or Fletcher factor. This substance was an alpha-globulin with an approximate mol wt of 170,000. Flaujeac plasma did not release a kinin upon incubation with kallikrein and was deficient in total kininogen antigen. Antiserum to kininogen inhibited the activity of the factor in solution. Flaufeac factor was identified as a kininogen of high molecular weight (HMW-kininogen). The mean total kininogen antigen in four children of the proposita was 51% (range 34-62%) of normal. A functional coagulation assay of HMW-kininogen in the children was 34% (range 23-55%). The results were consistent with autosomal recessive inheritance. The plasma pathways of intrinsic coagulation, fibrinolysis, kinin formation, and PF/dil generation are dependent upon HMW-kininogen. We believe this is the first demonstration of biological function for a kininogen apart from its role as a substrate for kallikreins.
K D Wuepper, D R Miller, M J Lacombe