The distribution, blood transport, and metabolic clearance of physiological concentrations of antidiuretic hormone were studied in 10 hydrated normal subjects with radioiodinated arginine vasopressin (125I-AVP). At 37 degrees C no binding of 125I-AVP to plasma proteins could be demonstrated, but some metabolites were associated with plasma proteins. 125I-AVP was rapidly distributed into a space approximating the extracellular fluid volume. Metabolic breakdown products became demonstrable within minutes after injection. The mean metabolic clearance rate of 125I-AVP was 4.1 ml/min/kg and the mean plasma half-life 24.1 min. Renal clearance had a mean value of 80 ml/min and accounted for 27% of the total metabolic clearance. It is concluded that in man antidiuretic hormone circulates as a free (non-protein bound) peptide, diffuses readily into the extracellular fluid space, and is metabolized within minutes. A plasma half-life of 24 min is consistent with the duration of antidiuresis after hormone administration or release.
G Baumann, J F Dingman
Obese-hyperglycemic (ob/ob) mice have the interesting feature of being hyperinsulinemic, thus having some characteristics in common with human maturity-onset diabetics. As the cause of hyperinsulinemia in these mice is not established, and as the liver is known to play a role in determining the amount of hormone that reaches the periphery, it was hypothesized that an anomaly in the hepatic handling of insulin might prevail in obese-hyperglycemic mice. Immunoreactive insulin was therefore measured in the perfusate before and after a single passage through perfused livers of lean and ob/ob mice, permitting. It was found that the removal of insulin by livers of lean mice increased with increasing concentrations of the hormone in the portal vein. The removal process had a limited capacity, however, and as a consequence the percentage of hormone removed by the liver actually decreased when portal insulin concentrations increased. Insulin removal by livers of ob/ob mice had qualitatively the same characteristics but was considerably less efficient than in normal livers. Due to this, more insulin was found in the perfusate leaving the liver of ob/9b mice than in that of controls, at any insulin concentration tested. These observations suggest that in obese-hyperglycemic mice more of the hormone may reach the periphery and thus contribute to hyperinsulinemia. The present study further suggests that the anomaly of insulin removal observed in perfused livers of ob/ob mice might be secondary to hyperinsulinemia, since it was partly corrected upon artificially decreasing the circulating levels of insulin (e.g. via a fast, anti-insulin serum, or streptozotocin treatment) before perfusion. The characteristics of hepatic insulin removal reported in this study, as well as the differences observed between livers of lean and ob/ob mice, may reflect changes in membrane insulin receptors and/or in processes responsible for the degradation of the horomone.
C Karakash, F Assimacopoulos-Jeannet, B Jeanrenaud
Acid-stimulating action and clearance of pure natural human big gastriin (HG-34-I) and little gastrin (HG-17-I) were assessed in four male subjects with inactive duodenal ulcer (DU) disease. Disappearance half-times for HG-17-I after intravenous infusion (5.2 min) or rapid intravenous injection (6.4 min) were six to eight times shorter than those for HG-34-I (41.5 and 37.8 min, respectively). Studies of clearance of synthetic human little gastrin (HG-17-I) were performed in three of these same four DU subjects, eight additional male DU subjects, and eleven normal male subjects. The disappearance halftime of synthetic HG-17-I averaged 6.2 min in both the DU subjects and the normal subjects. These data suggest that clearance of exogenous gastrin is not altered in patients with DU. Acid secretion in response to rapid intravenous injection of HG-34-I reached a higher peak and lasted longer than in response to an equimolar dose of HG-17-I; the total response to HG-34-I was about three times that to HG-17-I. During constant intravenous infusion, acid responses to equimolar exogenous doses of the two peptides were similar but the increment in molar concentration of circulating gastrin was six to eight times greater with HG-34-I than with HG-17-I. Chromatography of serum obtained during infusions of HG-34-I revealed no evidence of conversion to HG-17-I, nor was there any increase in circulating G-34 activity during infusions of HG-17-I. The increment in serum gastrin concentration required to produce half-maximal stimulation of gastric acid secretion (D50) was estimated in each subject for each gastrin from curves relating acid secretion to change in serum gastrin concentration produced by infusion of these peptides. After instilling peptone solution into the stomach, acid secretion was measured by intragastric titration, and increases in circulating G-17 and G-34 were determined by chromatography and radioimmunoassay of serum. Increases in circulating G-17 and G-34 in response to the peptone meal, taken together, were equivalent to 1.5 times the D50 determined from infusions of G-34 and G-17. Acid secretion during the same time period averaged 55% of maximal rates. Although G-34 comprised approximately three-fourths of the total molar concentration of circulating gastrin after stimulation, it was estimated to contribute less than half of the acid-stimulating activity.
J H Walsh, J I Isenberg, J Ansfield, V Maxwell
These studies evaluated the influence of an initial exposure of thyroid slices to thyroid-stimulating hormone (TSH) on the subsequent responsiveness to the hormone. Bovine thyroid slices were incubated with or without 50 mU/ml TSH for varying periods and then incubated in hormone-free medium for varying periods. Subsequently, slices were incubated for 20 min with 10 mM theophylline and with or without TSH. Cylic AMP was measured after the third incubation. Phosphodiesterase and adenylate cylase were assayed in homogenates prepared from slices after the second incubation. In some experiments prostaglandin E1, puromycin, thyroxine, and triiodothyronine and propylthiouracil were included in the media. In other experiments, low does of TSH (1 AND 10 mU/ml) were used instead of 50 mU/ml. Slices previously exposed to TSH have decreased responsiveness of the adenylate cyclase-cylic AMP system. Such refractoriness is hormone specific since initial exposure to prostaglandin E1 decreases the subsequent response to this substance but not to TSH. Refractoriness to TSH develops only when the first incubation is at least 30 min. It is not reversed by 5 h of incubation without hormone. Incubation of thyroid slices with puromycin does not eliminate refractoriness. The decreased response to TSH cannot be explained by release of thyroxine, triiodothyronine, or iodide from the slices. Phosphodiesterase activity is not increased during the refractory period. The decreased cyclic AMP response to TSH is associated with diminished response of adenylate cyclase activity to the hormone. Guanosine triphosphate (1 mM) increased adenylate cyclase activity in both control and TSH treated tissue, but the effect was significantly less in the latter. Although with guanosine triphosphate, TSH increased adenylate cyclase activity in TSH treated tissue, the enzyme activity was still less than that present in control tissue incubated with guanosine triphosphate and TSH. NaF caused an equivalent stimulation of adenylate cyclase in both control and TSH treated tissue. These results suggest that the refractoriness represents an alteration in hormone binding or the coupling of the bound hormone to the adenylate cyclase activity rather than any modification of the catalytic site of the enzyme.
S J Shuman, U Zor, R Chayoth, J B Field
In order to assess the renal tubular site(s) at which sodium reabsorption is enhanced in chronic sodium-depletion, seven normal dogs, six salt-depleted dogs, and three normal dogs receiving aldosterone were studied during a steady-state water diuresis under Pentothal anesthesia and during progressive hypotonic saline diuresis. For both maintenance of the water diuresis and progressive hypotonic saline diuresis 0.45% NaCl was used. During the steady state water diuresis delivery of sodium to the diluting segment of the nephron as approximated by solute-free water clearance + sodium clearance/glomerular filtration rate (CH2O + CNa/GFR) was significantly lower in salt-depleted dogs compared to normal dogs with or without aldosterone. During progressive hypotonic saline infusion fractional free water excretion (CH2O/GFR) was similar in all three groups as CH2O + CNa/GFR increased up to 12-14 ml/min-100 ml GFR. Thereafter, CH2O/GFR continued to rise in virtually a straight line in salt-depleted dogs but leveled off in normal dogs with or without aldosterone. These data demonstrate that enhanced sodium reabsorption in the diluting segment of the nephron is an important determinant of the renal sodium retention in chronic extracellular volume contraction in dogs in addition to confirming the presence of increased proximal tubule sodium reabsorption in these animals.
S Y Chou, L F Ferder, D L Levin, J G Porush
Random one-way microtiter mixed lymphocyte cultures between 43 rheumatoid arthritis (RA) patients and 45 controls consisting of 26 normal subjects and 19 miscellaneous non-RA patients were performed and results were evaluated as relative responses. Low responses (consisting of relative response less than 38%) were found in 31 out of 43 RA patients in cultures against eight of the RA stimulators. The remaining 35 RA stimulators tested yielded only normal mixed lymphocyte culture reactions. The same RA patients used as responders never produced low responses when stimulated by non-RA lymphocytes. But six of the control subjects gave low responses to two RA stimulators. The low responses did not appear to correlate with intake of aspirin, prednisolone, or gold salts, nor could they be reproduced by addition of RA serum of 7S or 19S fractions thereof containing either polyclonal or monoclonal rheumatoid factors. Short-term culture and washing before mixing with the allogeneic cells did not change the low responses suggesting that in vivo bound autoantibodies against lymphocyte receptors were not involved. Study of the inheritance of HLA and mixed lymphocyte culture determinants in the family of patient A. C. who most frequently elicited low responses indicated she was homozygous for a lymphocyte-defined determinant which has been called R. The low responses to A. C. could be interpreted as typing responses based on sharing of the same or of a similar lymphocyte-defined determinant. This gene appears to be increased in patients with RA with respect to non-RA controls and may reflect an association of genes within the HLA chromosomal region leading to predisposition for the development of RA.
The fate of glucose in the colon of rats and man was investigated by measuring breath 14CO2 and fecal 14C after direct instillation of 14C-labeled glucose, acetate, and lactate into the cecum. For the 6 h after administration of as much as 400 mg of [U-14C]-glucose to the rat and 12.5 g to man, 14CO2 excretion was as rapid after intracecal as after intragastric instillation. Less than 20% of 14C instilled into the cecum as glucose was recovered in feces and only about 15% of this fecal 14C was in a dialyzable form. The conversion of intracecally administered glucose to CO2 was dependent upon the presence of the colonic flora, as evidenced by the minimal excretion of 14CO2 after administration of [14C]glucose to germ-free rats. In contrast, acetate and lactate, fermentation products of glucose, were converted to CO2 as rapidly in germ-free rats as in their conventional counterparts. Measurement of O2 availability in the colonic lumen indicated that insufficient O2 was available for the aerobic metabolism of glucose by the colonic bacteria. These experiments suggest that the colon bacteria anaerobically metabolize most of the glucose to short-chain fatty acids, which are absorbed and oxidized by the host. Most of the remaining fecal glucose is converted to a larger molecular form that has limited osmotic activity. Thus, the colonic flora benefits the host by reducing the osmotic load of nonabsorbed carbohydrate and by making possible the salvage of a large percentage of the calories of carbohydrate, which is not absorbed in the small bowel.
J H Bond Jr, M D Levitt
Insulin binding to isolated adipocytes from 16 normal and 14 obese patients was studied. The data indicated that, as a group, adipocytes from the obese patients bound significantly less insulin than normal. However, of the 14 obese patients, 5 were not hyperinsulinemic and 4 of these 5 subjects had normal insulin binding. These subjects were also younger, and had the onset of obesity in childhood. When these five patients were separated from the original 14 obese patients, enhanced differences in insulin binding to adipocytes were observed when normals and the remaining 9 obese subjects were compared. Similar findings were obtained with isolated circulating mononuclear cells from these same patients. Presumably the five normoinsulinemic obese patients were not insulin-resistant, and, thus, the data indicate that insulin binding to adipocytes was decreased only in insulin-resistant obese patients. This conclusion was strengthened by finding a highly significant correlation (r=-0.71, p less than 0.001) between insulin binding to adipocytes and fasting plasma insulin level, while a weaker correlation (r=-0.49,p less than 0.01) existed between insulin binding and degree of obesity. Finally, when insulin binding to adipocytes and mononuclear cells from the same individual was compared, a significant positive correlation was found (r=0.53,p less than 0.01). In conclusion: (a) insulin binding to adipocytes and mononuclear cells is decreased in cells from insulin-resistant obese patients; (b) a significant inverse relationship exists between fasting plasma insulin level and insulin binding to adipocytes; and (c) in obesity, events that affect insulin receptors on adipocytes similarly affect insulin receptors on mononuclear cells.
J M Olefsky
Phospholipase D preferentially contained in human eosinophil polymorphonuclear leukocytes as compared to other leukocytes was isolated by sequential asion and cation exchange chromatography and gel filtration. The purified eosinophil enzyme specifically liberated choline from I-alpha-phosphatidyl choline with a pH optimum of 4.5-6.0 and exhibited a pI of 5.8-6.2 on polyacrylamide-gel isoelectric focusing, which are properties shared by phospholipase D from plant sources; however, its apparent mol wt of 60,000 is approximately one-half that of the plant enzymes. Eosinophil and cabbage phospholipase D inactivated a partially purified rat platelet-activating factor (PAF) in a time- and dose-dependent reaction. The cleavage of this PAF activity was attributed to the inherent phospholipase D activity of the eosinophil enzyme since the two activities chromatographed together at each purification step, and there was apparent reciprocal inhibition of choline-generating activity by PAF and of PAF-inactivating activity by phosphatidyl choline. Thus, possible regulatory functions of the eosinophil in immediate hypersensitivity reactions include inactivation of a PAF by phospholipase D as well as degradation of slow-reacting substance of anaphylaxis by arylsulfatase B.
L A Kater, E J Goetzl, K F Austen
The injection of sodium maleate (200-400 mg/kg) into rats produces aminoaciduria along with glycosuria and phosphaturia, resembling the Fanconi syndrome. This experimental model was studied by means of microinjections into proximal convoluted tubules of the kidney, stop-flow diuresis, and microperfusion of single nephrons. Our results show that, in maleate-treated rats, competition between amino acids or related structures (L-proline, L-OH-proline, and glycine) possesses the same characteristics, and net influx of amino acids appear normal at the proximal nephron. Data obtained by classical stop-flow techniques and single nephron microperfusions also indicate a normal entry of labeled amino acids (L-lysine, glycine, L-valine, L-proline, L-cystine), and 3-0-methyl-D-[3H]glucose and [32P]phosphate from the luminal side of the proximal tubule cell. However, the efflux of molecules from the cell appears enhanced throughout the proximal and distal tubule; molecules that exit at this site are excreted directly into the urine. Our results suggest that the phosphaturia, aminoaciduria, and glycosuria of the experimental Fanconi syndrome can be explained by a modification of the cell membrane permeability (increased efflux) at distal sites of the nephron rather than by a modification of the membrane transport (decreased influx) at the proximal sites, as is currently accepted. Our data also stress the importance of efflux phenomena in membrane transport.
M Bergeron, L Dubord, C Hausser, C Schwab
Previous investigations have suggested that significant hypotension during hemodialysis may result from abnormalities of sympathetic nervous system activity. To further evaluate these phenomena, plasma dopamine beta-hydroxylase (D beta H) and cold pressor test (proposed indexes of efferent sympathetic nervous system activity) and amyl nitrite inhalation (an index of the entire baroreceptor reflex arc) were studied in two groups of patients: group I, patients exhibiting a mean arterial pressure decrease to less than 70 mm Hg during less than 10% of dialyses; group II (hemodialysis hypotension), patients with a mean arterial pressure decrease to less than 70 mm Hg during more than 90% of dialyses. The groups were similar with respect to plasma renin activity, renin response to ultrafiltration, age, duration of dialysis, nerve conduction velocity, plasma protein concentration, hematocrit, dialysis weight change, resting heart rate, sex, race, blood pressure and heart rate response to cold pressor test, and 125I-albumin plasma volume. Supine mean arterial pressure was higher in patients with hemodialysis hypotension than in patients without hemodialysis hypotension (group I) both before and after dialysis. Plasma D beta H activity was significantly higher in patients with hemodialysis hypotension (group II) than in group I both before and after dialysis. Amyl nitrite inhalation, expressed as change in delta R-R interval/mean arterial pressure decrease, was less in hemodialysis hypotension patients. These results suggest that hemodialysis hypotension may result from a lesion in the baroreceptors, cardiopulmonary receptors, or visceral afferent nerves. Furthermore, elevated mean arterial pressure in patients with hemodialysis hypotension may be neurogenic in origin, as reflected by plasma D beta H activity, and appears similar to the hypertension that follows baroreceptor deafferentation of experimental animals.
J J Lilley, J Golden, R A Stone
Studies were undertaken to determine whether deoxyribonuclease I, (DNase I) once immobilized on activated nylon microspheres, would be capable of degrading circulating DNA in vitro and in vivo in an extracorporeal circulation system in dogs. Nylon microspheres were prepared and after gentle hydrolysis and glutaraldehyde treatment, demonstrated a retention of up to 4.73 mg of Dnase I. In vitro studies showed that DNase I immobilized on microspheres degreded a significant percentage of 125I-native DNA (nDNA) within 15 min. Mongrel dogs were injected with 125I-nDNA and a variation in initial t 1/2 in individual animals was observed. Therefore, for experimental studies, 125I-nDNA was injected and decay was recorded during a control period in which untreated microcapsules were utilized in the extracorporeal system. DNase I microspheres were then introduced into the extracorporeal circuit which resulted in an acceleration of degradation of acid precipitable 125I-nDNA. When 200 mug of unlabeled DNA with 125I-nDNA was injected, a similar augmentation of DNA degradation was noted after extracorporeal circulation over DNase I microcapsules. This effect could not be attributed to release of DNase I from the microspheres since no 131I-DNase was detected in the serum or organs of the dogs at the conclusion of the experiments. 125I-nDNA:anti-DNA complexes were passively injected into dogs and after a similar control period of circulation over untreated microcapsules. DNase I microspheres were introduced. Results showed a rapid acceleration in the degradation rate of 125I-nDNA:anti-DNA complexes precipitable with (NH4)2SO4. Extracorporeal circulation over nylon microspheres resulted in no significant alteration of the host's hematocrit or platelet count, and little residual cellular debris on the microcapsules. These data suggest that DNAase immobilized on nylon microspheres may have a potential role in the specific therapy of systemic lupus erythematosus, when it is desirable to hydrolyze DNA circulating free or in combination with antibody.
D S Terman, A Tavel, T Tavel, D Petty, R Harbeck, G Buffaloe, R Carr
The metabolism of [3H]vitamin D3 in hepatectomized vitamin D-deficient rats has been studied. Hepatectomy drastically disrupts vitamin D3 metabolism as revealed by prolonged high levels of [3H] vitamin D3 in the plasma compartment even 12 h after dose in contrast to sham-operated controls. Some conversion of [3H] vitamin D3 to [3H]25-hydroxyvitamin D3 was detected in hepatectomized rats, but the amount was small in spite of the high circulating levels of [3H]vitamin D3. Since the liver initially takes up much of an administered dose in normal animals and the conversion of [3H]vitamin D3 to [3H]25-hydroxyvitamin D3 is small in hepatectomized rats in spite of high circulating [3H]-vitamin D3, it is concluded that the liver plays a major role in the metabolism of vitamin D3 to 25-hydroxy-vitamin D3.
E B Olson Jr, J C Knutson, M H Bhattacharyya, H F DeLuca
The location of phospholipids in the human platelet plasma membrane was probed with 2, 4, 6-trinitrobenzenesulfonate (TNBS). TNBS does not penetrate inintact cells and can label phosphatidylethanolamine (PE) and phosphatidylserine (PS). In tact platelets, PE is not accessible to TNBS during the initial 15 min. However, 6.9% PE reacts with TNBS after 30 min and 17.9% PE is labeled after 90 min. In intact platelets, PS is not labeled even after 2 h. In contrast, in phospholipids extracted from platelets 71% PE and 26.5% PS react with TNBS within 5 min. This indicates that PS is inaccessible and PE is relatively inaccessible to TNBS in intact platelets. After incubation of platelets with thrombin, there is increased labeling of PE but no labeling of PS. The incubation of platelets with thrombin (0.05 U/ml) for 5 min results in 16.2% increase of PE labeling during subsequent 30-min incubation with TNBS. PS does not appear to be a component of the functional surface of platelets. However, exposure of PE may have a critical role in platelet hemostatic function. The implication of the study is that there is asymmetry of phopholipids in the platelet plasma membrane which has considerable physiological significance.
P K Schick, K B Kurica, G K Chacko
The expression of selected lymphocyte surface-membrane markers was evaluated in 37 patients with acute viral hepatitis B, 10 of whom were studied serially through the resolving and convalescent phases of disease. Bone marrow-derived (B) lymphocytes were identified by reference to surface immunoglobulin, whereas normal thymus-derived (T) lymphocytes were assayed by their capacity to form spontaneous nonimmune rosettes with sheep erythrocytes (E rosettes, ER). During the acute and resolving phases of viral hepatitis B, the relative and absolute number of ER-positive lymphocytes was significantly reduced, whereas the number of surface immunoglobulin-positive lymphocytes and the absolute lymphocyte count remained normal. This resulted in the appearance of a third population of cells, deficient in respect to both surface immunoglobulin and ER markers. Such cells accounted for nearly 25% of peripheral blood lymphocytes, approximately 5 x 105ml blood. Depression of the number of ER-positive lymphocytes occurred at least once during the course of disease in every patient studied serially, and was observed in 55 of 67 individual assays of the 37 cases of acute viral hepatitis B. Lymphocytes from some patients reacquired ER function when cultured in fetal calf serum but not in the presence of autologous serum. Such autologous serum was capable of suppressing ER function of lymphocytes from normal donors. The extrinsic suppression of er function by a serum factor (designated as the Rosette Inhibitory Factor), was found to be time dependent, characterized by a 4-h latent period and requiring approximately 18 h for maximum attenuation of ER function. The Serum Rosette Inhibitory Factor was: (a) heat and freeze-thaw stable, (b) nondialyzable, (c) physically separable from hepatitis B surface antigen, (d) not a lymphocytotoxic antibody, and (e) had the buoyant density of a lipoprotein. This extrinsic mechanism was observed in 41.8% of patients with reduced numbers of ER-positive lymphocytes. The Rosette Inhibitory Factor was not detectable in the serum of the remaining 58.2% of the cases of acute and resolving viral hepatitis B despite the presence of reduced numbers of ER-positive lymphocytes. The lymphocytes from these cases did not reacquire ER function when cultured in the absence of autologous serum. The mechanisms responsible for the suppression of normal ER function in these cases appears to be intrinsic to the lymphocytes and not the result of a humoral factor. The T lymphocyte lineage of cells deficient in respect to ER function, whether of intrinsic or extrinsic type, was demonstrated by their capacity to form spontaneous rosettes with neuraminidase-treated sheep erythrocytes. Both intrinsic and extrinsic suppression of T lymphocyte ER function commonly occurred during the first 4 wk of acute viral hepatitis B.9 of the 10 patients followed serially continued to manifest defective ER function at 12 wk...
F V Chisari, J A Routenberg, T S Edgington
Chediak-Higashi (CH) syndrome is a genetic disorder of children and certain animal species including the beige mouse. We have previously described a membrane abnormality in CH mouse polymorphonuclear leukocytes (PMH). Whereas normal mouse PMN do not form surface caps with concanavalin A except after treatment with agents such as colchicine that inhibit microtubule assembly, CH mouse PMN show spontaneous cap formation. This capping is inhibited by 3',5 cyclic guanosine monophosphate and by the cholinergic agonists carbamylcholine and carbamyl beta-methylcholine that increase 3',5' cyclic guanosine monophosphate generation. These data suggested that microtubule function may be impaired in CH syndrome perhaps secondary to an abnormality in 3',5' cyclic guanosine monophosphate generation. The cholinergic agonists were also shown to prevent development of the giant granules that are pathognomonic of CH syndrome in embryonic fibroblasts isolated from CH mice and cultured in vitro. In this report it is shown that an extreme degree of spontaneous concanavalin A cap formation is also characteristic of peripheral blood PMN from two patients with CH syndrome. This indicates an abnormality of microtubule function in CH syndrome in man. 3',5' cyclic guanosine monophasphate, carbamylcholine, and carbamyl beta-methylcholine reduce spontaneous capping in CH cells. In addition, it is shown that monocytes isolated from the patients' blood and incubated in tissue culture generate a large complement of abnormal granules. When the same cells mature in vitro in the presence of carbamylcholine or carbamyl beta-methylcholine, the proportion of cells containing morphologically normal granules is significantly increased. These responses can be reproduced in vivo in the beige (CH) mouse. Animals treated for 3 wk and longer with carbamylcholine or carbamyl beta-methylcholline show normal granule morphology and a normal degree of concanavalin A cap formation in peripheral blood PMN leukocytes.
J M Oliver, R B Zurier
In this study we have demonstrated that in native bile, lipids are organized in the form of a lipoprotein (bile LP) carrying albumin as apoprotein. The lipid composition of bile LP is almost identical to lipoprotein-X (LP-X, the characteristic lipoprotein of cholestasis). However, it differs from LP-X inits protein/lipid ratio and immunological and electrophoretic characteristics. Bile lipoprotein can be converted into "LP-X-like" material in vitro by adding albumin or serum to native bile. The LP-X-like material formed in vitro has physicochemical and chemical characteristics similar or identical to LP-X isolated from serum. As bile lipoprotein can be converted into LP-X-like material by the addition of albumin to bile, LP-X can be converted into bile-LP-like particles by adding bile salts to a LP-X-positive serum. Furthermore, experimental connection of the common bile duct to the vena cava is followed after a few hours by the appearance of LP-X-like material in the plasma. These facts taken together strongly suggest that bile LP is a precursor lipoprotein for LP-X and that it refluxes into the plasma pool under cholestatic conditions.
E Manzato, R Fellin, G Baggio, S Walch, W Neubeck, D Seidel
The effects of coronary occlusion and of subsequent isoproterenol infusion were examined in conscious dogs. Left ventricular (LV) function was assessed by measurements of LV diameter, pressure, velocity and dP/dt/P, and regional myocardial function was assessed by measurements of segment length (SL) and velocity of SL shortening in normal, border, and ischemic zones. Regional myocardial function was measured from the same sites, along with intramyocardial electrograms and regional myocardial blood flow as determined by radioactive microspheres. Coronary occlusion resulted in graded loss of function from the normal to severely ischemic zones with graded flow reductions and graded increases in ST segment elevation. Isoproterenol improved overall LV function, and function in the normal zone. Isoproterenol also improved function in 19 of 21 border-zone segments and in all moderately ischemic segments, while elevating further the ST segments. These changes were accompanied by increases in myocardial blood flow. In contrast, in severely ischemic segments, isoproterenol resulted in a deterioration of function, in that paradoxical motion occurred in segments previously akinetic during systole, while paradoxical motion was intensified in those segments in which it was already present. These changes were accompanied by further ST segment elevation but not by concurrent increases in blood flow. In addition, in 2 of 21 border zone segments, myocardial blood flow fell and these segments responded to isoproterenol with complete loss of function; paradoxical motion developed. Thus, in the conscious dog, a strong inotropic agent can improve function, even in the ischemic myocardium, as long as the required additional blood flow can be provided wither through primary or collateral channels.
S F Vatner, R W Millard, T A Patrick, G R Heyndrickx
Polydisperse [3h] dextran was infused into eight Munich-Wistar rats in the early autologous phase of nephrotoxic serum nephritis (NSN), thereby permitting direct measurements of pressures and flows in surface glomeruli and fractional clearances for dextrans [(U/P) dextran/(U/P) inulin] ranging in radius from 18 to 42 A. Despite glomerular injury, evidenced morphologically and by a marked reduction in the glomerular capillary ultrafiltration coefficient, the glomerular filtration rate remained normal because of a compensating increase in the mean net ultrafiltration pressure. In NSN rats, as in normal controls, inulin was found to permeate the glomerular capillary wall without measurable restriction, and dextrans were shown to be neither secreted nor reabsorbed. For dextran radii of 18, 22, 26, 30, 34, 38, and 42 A, (U/P) dextran/(U/P) inulin in NSN and control rats, respectively, averaged 0.90 vs. 0.99, 0.81 vs. 0.97, 0.63 vs. 0.83, 0.38 vs 0.55, 0.20 vs. 0.30, 0.08 vs. 0.11, and 0.02 vs. 0.03. Using a theory based on macromolecular transport through pores, the results indicate that in NSN rats, effective pore radius is the same as in controls, approximately 50 A. In NSN, however, the ratio of total pore surface area to pore length, a measure of the number of pores, is reduced to approximately 1/3 that of control, probably due to a reduction in capillary surface area. These results suggest that proteinuria in glomerular disease is not due simply to increases in effective pore radius or number of pores, as previously believed. Using a second theoretical approach, based on the Kedem-Katchalsky flux equations, dextran permeability across glomerular capillaries was found to be slightly lower, and reflection coefficient slightly higher in NSN than in control rats.
R L Chang, W M Deen, C R Robertson, C M Bennett, R J Glassock, B M Brenner, J L Troy, I F Ueki, B Rasmussen
To determine whether the increased filtration of serum proteins after glomerular injury is the consequence of altered electrostatic properties of the glomerular capillary wall, we measured fractional clearances of the anionic polymer, dextran sulfate, in nine Munich-Wistar rats in the early autologous phase of nephrotoxic serum nephritis (NSN). In agreement with previous studied from this laboratory, whole kidney and single nephron glomerular filtration rates were normal in NSN rats despite histological evidence of glomerular injury, and despite a marked reduction in the glomerular capillary ultrafiltration coefficient to approximately one-third of normal. In the companion study (9), it was shown that in NSN rats the mean fractional clearances of neutral dextrans over the range of effective molecular radii from 18 to 42 A were reduced, compared to normla. In contrast, in the present study the mean fractional clearances for dextran sulfate over the same range of molecular radii were significantly greater than those found previously for normal Munich-Wistar rats. The fractional clearance of dextran sulfate molecules of the same molecular radius as serum albumin (approximately 36 A) was increased markedly, from 0.015 +/- 0.005 (SEM) in nonnephritic controls to 0.24 +/- 0.03 in NSN (P less than 0.001). The sialoprotein content of glomeruli, estimated by the colloidal iron reaction, was reduced in NSN rats as compared to normal controls. It is concluded that the abnormal filtration of anionic serum proteins, such as albumin, seen in glomerulopathies is, at least in part, the consequence of loss of fixed negative charges from the glomerular capillary wall.
C M Bennett, R J Glassock, R L Chang, W M Deen, C R Robertson, B M Brenner, j l Troy, I R ueki, B Rasmussen
Transplantation of human pancreatic islets to diabetic patients may require that donor islets be kept viable in vitro for extended time periods before transfer to the recipient. We have maintained isolated pancreatic islets obtained from the human cadaveric pancreas in tissue culture for 1-3 wk, after which we studied the structure and function of the islets. Electron micrographs of the cultured islets showed a satisfactory preservation of both beta-cells and alpha 2-cells. After culture for 1 wk, the islet oxygen uptake proceeded at a constant rate at a low glucose concentration (3.3 mM) and was significantly enhanced by raising the glucose concentration to 16.7 mM. Likewise, after culture for 1 wk, the islets responded with an increased insulin release when exposed to 16.7 mM glucose with or without added theophylline (10 mM). Islets cultured for 1-3 wk were able to incorporate [3H]leucine into proinsulin, as judged by gel filtration of acid-alcohol extracts. Glucagon release from the cultured islets was reduced significantly by 16.7 mM glucose alone, but stimulated by glucose (16.7 mM) plus theophylline (10 MM). It is concluded that viable pancreatic islets can be isolated from the pancreas of adult human donors and maintained in tissue culture for at least 1 wk without loss of the specific functions of the alpha 2- and beta-cells. It remains to be established whether such islets will survive and remain functionally competent after transplantation to human recipients.
A Andersson, H Borg, C G Groth, R Gunnarsson, C Hellerström, G Lundgren, J Westman, J Ostman
In the absence of vitamin E deficiency, red cell lipid peroxidation has not been clearly demonstrated in freshly drawn blood obtained from patients with various hemolytic anemias despite indirect evidence that oxidative decomposition of cell membrane unsaturated fatty acids occurs in these particular hemolytic states. Recent studies have indicated that malonaldehyde, a decomposition product of oxidized polyunsaturated fatty acids, is able to covalently cross-link the amino groups of protein or lipid resulting in a fluorescent compound. In the present study we have utilized spectrofluorescent technique to assess whether such fluorescence is present in red cell lipid extracts in association with lipid peroxidation. In vitro red cell lipid peroxidation produced by ultraviolet radiation or the oxidant gas ozone was associated with the development of a fluorescent peak (excitation maximum 360 nm; emission maximum 440 nm) in lipid-containing red cell extracts Similar fluorescence was observed after incubation of red cells with malonaldehyde or with malonaldehyde-containing extracts of peroxidized red cell lipid. Spectrofluorescent evaluation of chloroform: isopropanol extracts obtained from the freshly drawn red cells of six patients receiving the oxidant hemolytic drug diaminodiphenylsulfone also revealed a peak at 440 nm which ranged from 39 to 78 U. In contrast, the levels in samples obtained from 11 hematologically normal subjects were 17-27 fluorescence U. No evidence for an increase in blood levels of free malomaldehyde was observed using the 2-thiobarbituric acid test which is the most commonly performed assay of lipid peroxidation. Serum vitamin E levels were within the normal range. Density separation indicated that the bulk of the fluorescence was present in older red cells. A similar fluorescent peak was also observed in lipid-containing extracts of red cells obtained from rabbits repeatedly injected with phenylhydrazine. The finding of fluorescent spectra consistent with the cross-linking of aminolid by malonaldehyde in the red cells of patients receiving diaminodiphenylsulfone indicates that in vivo red cell lipid peroxidation does occur in the absence of vitamin E deficiency.
B D Goldstein, E M McDonagh
The correlation between the incidence and level of immune complexes in serum and synovial fluid and the various clinical and biological manifestations of rheumatoid arthritis has been studied. Immune complexes were quantitated using a sensitive radioimmunoassay, the 125I-Clq binding test, in unheated native sera and synovial fluids from 50 patients with seropositive (RA +) and 45 with seronegative (RA -) rheumatoid arthritis, 17 with other inflammatory arthritis, and 37 with degenerative and post-traumatic joint disease. The following observations were made: (a) when compared to the results from patients with degenerative and post-traumatic joint diseases, the 125I-Clq binding activity (Clq-BA) in synovial fluid was found to be increased (by more than 2 SD) in most of the patients with RA + (80%) and RA - (71%) and in 29% of patients with other inflammatory arthritis; the serum Clq-BA was also frequently increased in both RA + (76%) and RA - (49%) patients, but only exceptionally in patients with other inflammatory arthritis (6%); (b) a significant negative correlation existed between the Clq-BA and the immunochemical C4 level in synovial fluids from patients with RA + and RA -; (c) neither the serum nor the synovial fluid Clq-BA in rheumatoid arthritis significantly correlated with the erythrocyte sedimentation rate, the clinical stage of the disease, or the IgM rheumatoid factor titer; and (d) the serum Clq-BA in patients with rheumatoid arthritis and extra-articular disease manifestations (40 +/- 34% in those with RA +,32 +/- 29% in those with RA -) was significantly increased as compared to the serum Clq-BA in patients with joint disease alone (24 +/- 30% in those with RA +, 10 +/- 13% in those with RA -). Experimental studies were carried out in order to characterize the Clq binding material in rheumatoid arthritis. This material had properties similar to immune complexes: it sedimented in a high molecular weight range on sucrose density gradients (10-30S) and lost the ability to bind Clq after reduction and alkylation, or after acid dissociation at pH 3.8, or after passage through an anti-IgG immunoabsorbant. DNase did not affect the Clq BA. These results support the hypothesis that circulating as well as intra-articular immune complexes may play an important role in some pathogenetic aspects of rheumatoid arthritis. The 125I-Clq binding test may also be of some practical clinical value in detecting patients who have a higher risk of developing vasculitis.
R H Zubler, U Nydegger, L H Perrin, k Fehr, J McCormick, P H Lambert, P A Miescher
To evaluate gonadotropin release in polycystic ovary syndrome (PCO), one or more of the following hypothalamic-pituitary function tests were performed on 24 patients with the syndrome. These tests included (a) the pulsatile pattern and day-to-day fluctuation of gonadotropin release; (b) effects of exogenous estrogen and antiestrogen (clomiphene) administration on gonadotropin release; and (c) pituitary responsiveness to maximal (150 mug) and submaximal (10 mug) luteinizing hormone-releasing factor (LRF) injections. In 10 of the 14 patients sampled frequently (15 min) for 6 h, luteinizing hormone (LH) levels were elevated above the concentration seen in normal cycling women (except the LH surge). These high LH concentrations appeared to be maintained by and temporally related to the presence of exaggerated pulsatile LH release, either in the form of enhanced amplitude or increased frequency. In all subjects, levels of follicle-stimulating hormone (FSH) were low or low normal, and a pulsatile pattern was not discernible. In four patients, daily sampling revealed marked day-to-day fluctuation of LH but not FSH. That the elevated LH levels were not related to a defect in the negative-feedback effect of estrogen was suggested by the appropriate fall of LH in four patients given an acute intravenous infusion of 17beta-estradiol. This infusion had no effect on FSH levels. In addition, clomiphene elicited rises of both LH and FSH that were comparable to the ones observed in normal women given the same treatment. The clomiphene study also suggested that the positive-feed-back mechanism of estrogen on LH release was intact when the preovulatory rises of 17beta-estradiol induced appropriate LH surges. The elevated LH levels appeared to be related to a heightened pituitary responsiveness to the LRF. This was found in the 11 and 2 patients given maximal (150 mug) and submaximal (10 mug) doses of LRF, respectively. The augmented pituitary sensitivity for LH release correlated with the basal levels of both estrone (P less than 0.025) and 17beta-estradiol (P less than 0.02). The net increase in FSH was significantly greater (P less than 0.001) in the PCO patients than the normal women with maximal doses of LRF. With the smaller dose study none of the injections had a discernible effect on FSH concentrations in either subject. The disparity between LH and FSH secretion could be explained by the preferential inhibitory action of estrogen on FSH release, coupled with a relative insensitivity of FSH release. These data indicate that in these PCO patients the abnormalities of the hypothalamic-pituitary regulation of gonadotropin secretion was not an inherent defect but represented a functional derangement consequent to inappropriate estrogen feedback, which led to a vicious cycle of chronic anovulation and inappropriate gonadotropin secretion.
R Rebar, H L Judd, S S Yen, J Rakoff, G Vandenberg, F Naftolin
Virtually all preparations of DNA used to detect antibody to native DNA (nDNA) by binding assays have been found to be subtly contaminated by single stranded DNA. Because recent DNA binding data have directly challenged the unique role previously attributed to these antibodies in systemic lupus erythematosus (SLE), resolution of the consequent ambiguity is of theoretical and practical importance. It is proposed that a synthetic nDNA molecule (dAT) might circumvent this difficulty by being antigenically equivalent to nDNA while, on theoretical grounds, lacking significant contamination with single stranded DNA or other cellular antigens. These expectations were generally confirmed by biochemical and immunological analyses. In clinical studies, sera from 124 pateints with SLE and from controls were examined for their ability to bind dAT. In contrast to results with KB binding, patients with non-SLE rheumatologic disorders were indistinguishable from normals by dAT binding. dAT binding was elevated in 85% of sera from SLE patients with clinically-judged active nephritis but in only 9% of those with inactive renal disease. Active non-renal disease, including cerebritis, was not associated with increased dAT binding. Individual non-lupus sera which bound increased amounts of KB DNA, failed to bind dAT. It is suggested that such binding resulted from contaminating non-nDNA antigens. When elevated, dAT binding, like KB binding, varied with disease activity and might thus be useful as a parameter thereof. In several patients elevated dAT binding led to the finding, on biopsy, of clinically silent, active, diffuse proliferative nephritis. It is concluded that use of synthetic nDNA antigens such as dAT may offer theoretical and practical advantages over naturally-derived preparations in detecting anti-nDNA, both clinically and for investigational purposes. Also, caution is urged in interpreting DNA binding data derived from incompletely characterized systems, particularly with regard to the occurrence of anti-nDNA antibodies in serum.
C R Steinman, U Deesomchok, H Spiera
Dihydrotestosterone binding was measured in culture fibroblasts from 14 control subjects and from 12 patients with five different types of hereditary male pseudohermaphroditism. Two assays of binding were used--an intact monolayer assay and density gradient centrifugation of cell extracts. In the intact monolayer assay of normal cells the uptake of [3H]dihydrotestosterone consisted of two components. The first was a high affinity component that exhibited saturation at approximately 1 nM dihydrotestosterone. The second was a low affinity component that was not saturable with concentrations of steroid up to 5 nM. Twice the number of high affinity binding sites were present in fibroblasts grown from genital skin (foreskin, labia majora, and scrotum) as from nongenital sites (37 vs. 14 fmol/mg protein). In the density gradient assay in 5-10% sucrose, the major peak of dihydrotestosterone binding was in the 8S region in low molarity buffer and in the 4S region in 0.5 M KCl. High affinity binding was normal in cells from two patients with familial incomplete male pseudohermaphroditism, type 2, an autosomal recessive defect in which dihydrotestosterone formation is deficient, and in cells from a patient with male pseudohermaphroditism due to 17 beta-hydroxysteroid dehydrogenase deficiency, an autosomal recessive defect of testosterone synthesis. High affinity binding was low by both methods in fibroblasts from five patients with complete testicular feminization. Furthermore, binding by both methods was also low in cells from three subjects with familial incomplete male pseudohermaphroditism, type 1, a presumed X-linked recessive disorder of androgen resistance, and in fibroblasts grown from a subject with the incomplete form of testicular feminization. The finding that dihydrotestosterone binding is abnormal in two forms of hereditary androgen resistance in addition to complete testicular feminization suggests either that these disorders are the result of allelic mutations affecting the function of the androgen-binding protein or that normal dihydrotestosterone binding requires the participation of more than one gene product.
J E Griffin, K Punyashthiti, J D Wilson
Recent studies indicate that oxygen radicals such as superoxide or singlet oxygen may be important in the functional activity of human granulocytes. We have examined the possible importance of these radicals in the functional capacity of human blood monocytes. Monocytes, like granulocytes, generate chemiluminescence during phagocytosis. Chemiluminescence is impaired 50-90% by superoxide dismutase, an enzyme which enhances the dismutation of superoxide to hydrogen peroxide. These results indicate that superoxide is related to the chemiluminescence generated by monocytes. Superoxide dismutase in a concentration which impaired chemiluminescence also impaired the staphylococcal killing by monocytes. Hexose monophosphate shunt activity and hydrogen peroxide production by granulocytes and monocytes were also evaluated. The oxidation of [1-14C]glucose was used as a measure of hexose monophosphate shunt activity and the oxidation of [14C]formate as an estimation of hydrogen peroxide production. The oxidation of both substrates by monocytes was increased during phagocytosis but, in contrast to results in granulocytes, was not further increased by the addition of superoxide dismutase. These data indicate that superoxide may be important in bactericidal activity of human monocytes. Our results also suggest that the metabolism of oxygen radicals in monocytes and granulocytes may be different.
A L Sagone Jr, G W King, E N Metz
This study was designed to examine local effects of acute cellular injury on regional myocardial blood flow. Studies were carried out in awake dogs chronically prepared with indwelling catheters in the aorta and left atrium and an occluder on the left circumflex coronary artery. Regional myocardial blood flow was measured by using 7-10-mum radioisotope-labeled microspheres after reestablishing inflow to a region subjected to a 2-h complete coronary occlusion. Microspheres were injected 15 s, 15 min, 4 h, and 3 days after reperfusion to assess effects of cell injury at varying intervals after reperfusion. Effects of acute cellular injury on blood flow were assessed by determining the relationship between regional blood flow and the extent of subsequent cellular necrosis measured in multiple tissue samples, weight 1-2 g, from the entire ischemic zone. The extent of cellular necrosis was determined from histological sections of each tissue sample. Prolonged ischemia effected local tissue responses which altered perfusion as a function of the interval after reperfusion and the subsequent extent of myocardial necrosis. Although the net response in each region immediately after reperfusion was vasodilation, the hyperemia in regions which subsequently suffered cellular necrosis was attenuated in direct proportion to the extent of subsequent infarction. Blood flow to acutely injured regions remained equal to, or in excess of, flow to nonischemic regions 15 min after reperfusion, but at 4 h and 3 days after reperfusion, flow was significantly decreased in regions with greater than 50% infarction. Thus, these data indicate that prolonged ischemia initiates tissue responses which progressively reduce myocardial perfusion after reperfusion. These effects on tissue perfusion may result from normal responses to irreversible injury and (or) abnormal responses to reversible and thus, potentially alterable, ischemic injury.
F R Cobb, R J Bache, F Rivas, J C Greenfield Jr
Histamine release from peripheral blood leukocytes challenged with anti-human IgE was studied in patients with chronic urticaria and nonatopic controls. 19 of 23 controls, but only 6 of 20 patients, released over 20% of the total available leukocyte histamine. The response to anti-IgE concentrations of 1.66, 0.33, 0.066, and 0.013 mug antibody N/ml was significantly lower in patients than in controls. Serum IgE levels were significantly higher in the patients but total histamine content of about 10(7) leukocytes was not. Deuterium oxide (D2O) greatly increased histamine release (in both groups), indicating that the anti-IgE interacted with the basophils of urticaria patients. Passive sensitization of leukocytes with biologically active IgE was achieved in both patients and control subjects whose cells responded to anti-IgE, but was not achieved in either patients or control subjects whose cells were nonresponsive to anti-IgE challenge. 125I-anti-IgE autoradiographic studies revealed no obvious quantitative abnormality in the amount of basophil-bound IgE in chronic urticaria patients. Ionophore stimulation of aliquots of the same leukocytes used for anti-IgE challenge demonstrated that the urticaria patients' basophils were capable of releasing normal amounts of histamine. Leukocyte cyclic AMP levels in the two groups were not significantly different either in base-line levels or in responsiveness to stimulation with isoproterenol. These data indicate that chronic urticaria patients have a (acquired?) defect in leukocyte histamine release that occurs after the anti-IgE-IgE interaction, but before the actual (second-stage) release process, and that is comparable to the phenomenon of desensitization.
F Kern, L M Lichtenstein
The effect of specific immunotherapy on several in vitro responses to ragweed antigen E has been evaluated in 17 atopic patients with ragweed hayfever. The methods employed were leukocyte histamine release, measurement of specific IgE anti-ragweed antibody and specific IgG anti-ragweed antibody, lymphocyte proliferation, and the production of two lymphocyte mediators (migration inhibitory factor and mitogenic factor). The duration of treatment and symptom improvement were also recorded for comparison. Immunotherapy was associated with a decrease in leukocyte sensitivity for histamine release to ragweed antigen E in a majority of the patients. In addition, there was a significant decrease in IgE anti-ragweed antibody and a significant increase in IgG anti-ragweed antibody. Immunotherapy also resulted in a significant decrease in lymphocyte responsiveness to ragweed antigen E as measured by proliferation and the production of mediators. Symptomatic improvement was best correlated with the presence of IgG anti-ragweed antibody responses. The production of this antibody was also associated with a decrease in lymphocyte responsiveness. The results of this study indicate that specific immunotherapy in ragweed-sensitive patients induces alterations in immunologic reactivity to ragweed antigen in vitro. This response is antigen specific, includes elements of both humoral and cellular immunity, and may account for the clinical improvement that is often observed in patients who undergo this form of therapy.
R Evans, H Pence, H Kaplan, R E Rocklin
Four patients with common, variable agammaglobulinemia were preveiously reported to have normal numbers of circulating B lymphocytes which synthesized normal amounts of IgG in tissue culture but failed to secrete the newly synthesized IgG. The B lymphocytes of these patients fail to incorporate [3H]mannose and/or [3H]glucosamine into newly synthesized IgG, whereas such incorporation appears to occur just before IgG secretion in cultures of normal B lymphocytes.
F Ciccimarra, F S Rosen, E Schneeberger, E Merler