Analysis of 24-h urinary steroid excretion was performed by capillary gas chromatography in six patients (five men, one woman) with adrenocortical insufficiency. Ten healthy subjects (five men, five women) served as controls. A complete absence of all 21-hydroxylated steroid metabolites was seen in patients with adrenocortical insufficiency, whereas the excretion of several steroids lacking hydroxylation in the 21-position (pregnenolone, pregnenetriol, and 11-ketoandrosterone) was markedly increased. In addition, the presence of 11 beta-hydroxyandrosterone was confirmed by mass-spectrometry in the urine of three patients. This pattern of steroid excretion was unchanged in patients with adrenocortical insufficiency, both after stimulation by 1-24 adrenocorticotropin (ACTH) and after short-term (3-d) suppression with dexamethasone. We conclude that patients with adrenocortical insufficiency present a pattern of steroid excretion characterized by the absence of 21-hydroxylated metabolites. In the absence of functional adrenocortical tissue, long-term pathologically elevated concentrations of ACTH apparently stimulate early steps of steroid synthesis, most likely in the gonads. In addition, the presence of 11-hydroxylated steroid metabolites (11-ketoandrosterone, 11 beta-hydroxyandrosterone) in the urine of patients with adrenocortical insufficiency demonstrates that chronic ACTH excess in this disorder may induce some activity of 11 beta-hydroxylase, an enzyme not found in the gonads under physiological conditions.
H Vierhapper, P Nowotny, W Waldhäusl
Plasma cholesterol metabolism was investigated in normotriglyceridemic patients with end-stage renal disease treated by hemo- or continuous ambulatory peritoneal dialysis (CAPD), and compared with that in a control group with normal renal function. A reversed net transport of free cholesterol from plasma to cultured fibroblasts, as well as greatly reduced levels of plasma cholesterol esterification and cholesterol ester transfer rates to low and very low density lipoproteins (LDL and VLDL), was found in the hemodialysis group compared to the controls. The LDL and VLDL contained increased amounts of free cholesterol and inhibited cholesterol ester transfer when recombined with control plasma. The LDL triglyceride content was doubled in the hemodialysis group, whereas cholesterol esters were decreased. Patients treated by CAPD, in marked contrast, had cholesterol metabolic rates that were within the normal range, as well as normal lipoprotein composition.
H Dieplinger, P Y Schoenfeld, C J Fielding
The cerebral capillary endothelium is unique and functions as an effective blood-brain barrier (BBB) owing to its intercellular tight junctions and rare pinocytotic vesicles. To assess how bacterial meningitis alters the BBB, rats were inoculated intracisternally with three encapsulated meningeal pathogens (Escherichia coli K1+, Streptococcus pneumoniae type III, Haemophilus influenzae type b) and an unencapsulated mutant strain (H. influenzae Rd). After defined infection durations, the morphologic alterations of the cerebral capillary endothelium were quantitatively assessed by transmission electron microscopy. Results revealed a significant increase in pinocytotic vesicle formation (P less than 0.001) early after meningitis induction (4 h) that was sustained with longer infection durations (10 h, 18 h) for all encapsulated strains tested. In addition, there was a progressive increase in completely separated intercellular junctions with increasing infection duration, (P less than 0.05). 4 h after induction of meningitis with H. influenzae Rd, cerebrospinal fluid (CSF) bacterial concentrations, cerebral capillary morphologic changes, and functional BBB permeability to circulating 125I-albumin were similar to those observed with H. influenzae type b. However, prolonging the H. influenzae Rd infection to 18 h allowed for CSF clearance of the organism, thereby precluding the significant increase in separated junctions or progression of functional BBB permeability seen with the encapsulated H. influenzae type b. These data suggest a uniform morphologic explanation for altered BBB permeability in meningitis with a reproducible temporal sequence. Encapsulation does not appear essential for BBB injury, but may facilitate its progression by allowing the organism to evade host clearance.
V J Quagliarello, W J Long, W M Scheld
In passive Heymann nephritis (PHN) in rats, antibody (anti-Fx1A) reacts in situ with a glomerular epithelial antigen and induces complement (C)-mediated cell-independent proteinuria. To assess the role of the membrane attack complex (MAC), we determined the need for C8 in the pathogenesis of proteinuria in an autologous-phase model of PHN. Isolated rat kidneys, containing nonnephritogenic, non-C-fixing gamma 2 sheep anti-Fx1A (planted antigen), when perfused in vitro with C-fixing guinea pig anti-sheep IgG and a source of C (fresh human plasma 50% vol/vol in buffer containing bovine serum albumin), developed marked proteinuria after 20 min (0.58 +/- 0.08 mg/min X g, n = 8) that increased further to 3.20 +/- 0.93 mg/min X g after 80 min. In contrast, identical kidneys perfused with antibody and heat-inactivated or C8-deficient human plasma and normal kidneys perfused with antibody and fresh plasma excreted only 0.27 +/- 0.03 (n = 6), 0.27 +/- 0.04 (n = 5), and 0.40 +/- 0.05 mg/min X g (n = 6) after 20 min, and 0.13 +/- 0.02, 0.22 +/- 0.03, and 0.32 +/- 0.05 mg/min X g after 80 min, respectively. When C8-deficient plasma was reconstituted with sources of C8 (n = 3), proteinuria was restored to the level observed with fresh normal plasma. Differences in protein excretion could not be explained by quantitative differences in glomerular antigen or antibody content. Extensive ultrastructural damage to glomerular visceral epithelial cells was exclusively seen in antigen-containing kidneys perfused with antibody and C8-replete plasma. Thus, glomerular injury in this model results from an antigen-specific, antibody-directed, C8-dependent reaction involving assembly of the MAC. The ultrastructural findings argue in favor of MAC-induced cytotoxicity of the glomerular visceral epithelial cells.
A V Cybulsky, H G Rennke, I D Feintzeig, D J Salant
Aortic atheromatous plaques regress slowly in cholesterol-fed rabbits that have been returned to normal laboratory diet. To delineate metabolic factors potentially responsible for persistence of atherosclerosis under these conditions, the physical, chemical, and metabolic characteristics were determined for lipoproteins of d less than 1.006 g/ml; such lipoproteins are thought to be the major determinant of progression of atherosclerotic lesions in cholesterol-fed rabbits. At the time of return to a normal laboratory diet regimen after 3 mo of feeding with cholesterol-enriched laboratory diet, plasma cholesterol concentrations were 2,275 +/- 252 mg/dl, mostly attributable to cholesteryl ester-rich very low density lipoproteins (VLDL). On the hypercholesterolemic diet, fractional catabolic rates of plasma clearance of 125I-labeled VLDL were reduced (0.011 +/- 0.002 vs. 0.151 +/- 0.015 h-1), but the total VLDL catabolic rate was increased considerably (17.1 +/- 2.2 vs. less than 1.2 +/- 0.4 mg of protein/kg X d), because of the expansion of the endogenous pool of cholesteryl ester-rich VLDL. The total catabolic rate of VLDL was maintained above estimated control values (5.8 +/- 0.7 mg protein/kg X d) even 10 wk after return of the rabbits to a normal chow regimen, an effect attributable to continued high rates of cholesteryl ester-rich VLDL synthesis in liver. Accumulation of cholesteryl ester-rich VLDL into aortic tissue persisted at a high rate. Thus the persistence of aortic atheromatous lesions after cessation of cholesterol feeding was attributable in part to continued high rates of hepatic production of cholesteryl ester-rich VLDL and its persistent delivery into the aortic wall.
A Daugherty, G Schonfeld, B E Sobel, L G Lange
Antibody reactivity against cultured allogeneic melanoma Y-Mel 81:180 was studied in 43 patients who participated in an adjuvant trial for stage I and II melanoma. Serum samples were obtained at trial entry within 2 mo of definitive surgery. At the time of serum acquisition, all patients were free of disease by physical examination and routine radiologic and laboratory parameters. Serum antibody reactivity was tested for by protein A hemadsorption before and after acid dissociation and ultrafiltration of serum. We have previously shown that this technique for immune complex dissociation augments autologous antibody reactivity. Results of serum antibody reactivity were scored by an investigator blinded to the patient's clinical status. Of the 43 patients studied, 15 relapsed and 28 remained disease-free. At study entry, there were 25 stage I patients and 18 stage II patients. Breslow depth was 3.25 +/- 2.5 mm in relapse patients and 1.67 +/- 1.1 mm in disease-free patients. The presence and titer of antibody directed against melanoma in either native serum or serum dissociated from immune complexes was found to be associated with eventual relapse (P = 0.0001). When results were subgrouped by stage of disease, Breslow depth, and hypopigmentation, antibody reactivity was still correlated with eventual relapse. The incidence and titer of antibody reactivity against melanoma appears to be a new prognostic factor in predicting eventual disease recurrence.
D R Vlock, R DerSimonian, J M Kirkwood
The plasma metabolic clearance of biologically active luteinizing hormone (bioactive LH) was studied using the rat interstitial cell testosterone (RICT) bioassay in six hypogonadotropic men after single bolus injection of highly purified human LH and during continuous steady-state infusions of three graded doses of LH. The LH bolus disappearance curves provided estimates of metabolic clearance rates (MCR) of 24.1 +/- 4.7 (+/- SD) ml/min for bioactive LH vs. 56.2 +/- 12 ml/min for immunoactive LH in the same men (P = 0.03). A lower MCR of bioactive LH compared with immunoactive LH was also observed during continuous infusions of physiological doses of LH; for example, the mean steady-state MCRs for bioactive and immunoactive LH were, respectively, 26.1 +/- 3.1 and 34.2 +/- 3.2 ml/min (P = 0.02). Moreover, the stepped-dose infusion regimens permitted us to demonstrate that increasing doses of pure human LH resulted in progressive and parallel decreases in the apparent MCRs of both bioactive and immunoactive LH. Based on the respective steady-state MCRs calculated at physiological plasma concentrations of immunoactive and bioactive LH, we estimate a mean endogenous production rate for bioactive hormone of 1,937 IU/24 h, and for immunoactive LH of 589 IU/24 h in normal men. These results indicate that previous estimates of LH production rates from immunoassay data alone markedly underestimate the quantity of biologically active hormone secreted in man.
J D Veldhuis, F Fraioli, A D Rogol, M L Dufau
The metabolism of pentose-phosphate was investigated in Plasmodium falciparum-infected normal and glucose-6-phosphate dehydrogenase (G6PD)-deficient human red blood cells in vitro. 5'-Phosphoribosyl-1-pyrophosphate (PRPP) content of infected normal red blood cells was increased 50-60-fold at the parasite trophozoite growth stage over that of uninfected cells. The PRPP increment in infected G6PD-deficient cells at comparable stage and parasitemia was only 40% of the value in normal infected cells. Red blood cell PRPP synthetase activity did not change during the growth cycle of the parasite and was similar in both normal and G6PD-deficient cells. Reduced glutathione (GSH) content of G6PD-deficient cells under conditions of culture fell to low or undetectable levels. These low levels of GSH were shown to inhibit the function of red blood cell PRPP synthetase, which requires GSH for full activity. Measurements of the incorporation of 1-14C or 6-14C selectively labeled glucose into parasite nucleic acids revealed that in normal infected red cells, approximately 20% of the pentose was produced via the oxidation of glucose-6-phosphate, whereas in infected G6PD-deficient cells (Mediterranean type), none of the pentose was produced via the oxidative pathway. It is concluded that the low level of reduced GSH found in G6PD deficiency and the resultant partial inhibition of PRPP synthetase together with the missing oxidative pathway for ribose phosphate production can account fully for the reduced parasite growth rate in G6PD-deficient red blood cells described previously. Of these two mechanisms, the predominant one is the impaired PRPP synthetase activity due to low GSH levels in enzyme-deficient red blood cells. The contribution to the ribose-phosphate pool by the hexose monophosphate shunt is relatively minor. A co-existing oxidative stress (which is often hypothesized to mediate the destruction of parasitized red blood cells) is not required to explain growth inhibition in this scheme and does not represent the most straight-forward explanation of the data described in this report.
E F Roth Jr, R M Ruprecht, S Schulman, J Vanderberg, J A Olson
We examined the effects of physiologic infusions of arginine vasopressin (AVP) on cardiovascular hemodynamics and on reflex responses initiated by decreasing cardiopulmonary baroreceptor stimulation (with lower body negative pressure) in 10 healthy, captopril-pretreated young men (19-27 yr). Their responses were compared with those of four volunteers given isosmotic infusion. Heart rate, stroke volume, blood pressure, and forearm blood flow were measured by electrocardiography, impedance cardiography, radial artery cannulation, and strain gauge plethysmography. Two 55-min infusions of AVP at rates of 0.15 and 0.40 ng/kg per min increased average plasma concentrations from control levels of 5 pg/ml to 18 and 36 pg/ml, respectively. These infusions resulted in progressive reductions of heart rate and cardiac output and increases of forearm and total peripheral resistance. Blood pressure increases were significant only during the larger AVP infusion rate. Lower body negative pressure provoked reflex increases of total peripheral resistance. These increases were enhanced 60% during AVP infusion compared with increases during control (pre-AVP). Baseline measurements and reflex responses were unchanged by isosmotic infusions. These results demonstrate that AVP has profound effects on cardiovascular function and augments cardiopulmonary baroreflex-mediated increases of peripheral resistance in man.
T J Ebert, A W Cowley Jr, M Skelton
Using a monoclonal anti-tubular basement membrane antibody (alpha TBM-Ab) affinity column, we isolated from collagenase-solubilized human renal tissue (HSRTA) a predominantly 48,000-mol-wt moiety (H3M-1) which is selectively recognized by antisera from two patients with alpha TBM-Ab-associated interstitial nephritis (alpha TBM disease). Whereas both antisera had alpha TBM-Ab titers of 1:64-1:128 by immunofluorescence on tissue sections, their reactivity with H3M-1 in a solid-phase radioimmunoassay was demonstrable at dilutions up to 1:10,000. While these sera displayed some reactivity with pre-column HSRTA, this was markedly less than with H3M-1. HSRTA depleted of H3M-1 by passage over the alpha TBM-Ab affinity column was almost completely depleted of reactivity. Neither pooled normal human sera nor sera from patients with a variety of renal lesions not associated with alpha TBM-Ab (including interstitial nephritis and antiglomerular basement membrane disease) were reactive with H3M-1. Both patient antisera containing alpha TBM-Ab were also highly reactive with R3M-1, the 48,000-mol-wt rabbit glycoprotein antigen of experimental alpha TBM disease. Furthermore, a competitive inhibition radioimmunoassay revealed that alpha TBM-Ab from rodents with experimental alpha TBM disease could inhibit 45-98% of the R3M-1 binding reactivity of patient antisera and 85% of the H3M-1 binding reactivity of patient antisera, thus suggesting paratypic cross-reactivity. We conclude, therefore, that tubular basement membrane target epitopes and their paratypic recognition are highly conserved among mammals.
M D Clayman, L Michaud, J Brentjens, G A Andres, N A Kefalides, E G Neilson