Specificity and stability of guinea pig anti-progesterone antibodies.

Antibodies against progesterone were induced in guinea pigs of both sexes by injection of progesterone-y beta-hemisuccinate conjugated to bovine serum albumin (BSA) in a ratio of 16 moles of steroid per mole of protein. The concentration of antibody binding sites for progesterone of the animals studied ranged from 5 to 20 microM. The expected heterogeneity of binding affinity for progesterone was observed with two major populations apparently predominating. On bound progesterone with an average affinity greater than 2 X 10(9) M-1 and the other showed an average affinity less than or equal to 6 X 10(6) M-1. The antibodies were fond to be stable to extremes of pH and temperature in serum as well as in solutions of ammonium sulfate precipitates. The antibodies were not stable, however, in a more highly purified form. Attempts to obtain active preparations in high yield by purification beyond the ammonium sulfate step were unsuccessful. Competition studies and direct analysis with radiolabeled steroids showed the high-affinity population to be relatively specific for progesterone binding, whereas other steroids were bound according to the polarity rule indicating that the binding forces are predominantly hydrophobic.

Comparative metabolic effects of acetate and dichloroacetate infusion in the anesthetized dog.

The comparative effects of acetate (10 mmol/h/kg) and dichloroacetate (DCA) (1 mmol/h/kg and 10 mmol/h/kg) on acid-base and intermediary metabolism were assessed using the fasted anesthetized dog, undergoing controlled ventilation, as a metabolic model. Infusion of acetate resulted in a marked metabolic alkalemia and a decline in PaO2, while DCA had minimal effects on acid-base state and oxygen consumption. Serum glucose decreased with both DCA and acetate infusion, although only significantly with the latter. At infusion rates of 10 mmol/h/kg, acetate caused marked decreases, while DCA caused marked increases, in serum potassium and phosphorus. Acetate and DCA also had opposing effects on lactate and citrate levels, the former caused increases and the latter decreases in both metabolites. Pyruvate levels decreased similarly in response to both infusates. Acetoacetate and beta-hydroxybutyrate levels increased significantly with both acetate and DCA infusions; however, the increases were much greater with acetate than with DCA infusion. Blood alanine levels decreased significantly during the infusion of both acetate and DCA, whereas, free fatty acids tended to increase with acetate infusion, remained unchanged with low dose DCA and fell significantly with high dose DCA. Plasma insulin levels were sustained during acetate infusion, but fell abruptly with termination of infusion. In contrast, insulin levels fell markedly with DCA infusion and remained depressed throughout the infusion and recovery periods. Blood levels of acetate and DCA rose markedly during infusion; however, while acetate levels decreased nearly to control values during the recovery period, DCA levels remained elevated.(ABSTRACT TRUNCATED AT 250 WORDS)

Endotoxin and bacterial contamination of dialysis center water and dialysate; a cross sectional survey.

The bacterial and endotoxin levels of purified water and effluent dialysate were examined in a cross section of dialysis centers in the central United States. All samples were collected within a four-hour drive of the University of Louisville and were collected, processed and analyzed by our personnel, to eliminate variability in sample handling. A medium capable of higher bacteria recovery from aqueous environments than those ordinarily employed in clinical assays was used. Endotoxins were determined by a quantitative colorimetric assay. By the more sensitive bacterial assay 53% of the centers had bacterial counts above the AAMI standard of 200 colony-forming units per ml (CFU/ml) for water and 35% of the centers had bacterial counts above the 2000 CFU/ml standard for dialysate in at least one sampling period. The samples showed 35% and 19% of water and dialysate above the standards, respectively. While there are no standards for endotoxin concentrations in water used to prepare dialysate, 2% of the centers had endotoxin levels in their water above five endotoxin units per ml (5 EU/ml = 1 ng/ml in our assay kit), the limit set by the AAMI standards for reprocessor water. Both bacterial and endotoxin levels tended to be elevated in dialysate, with the highest levels of endotoxin in dialysates posing an obvious potential risk when high-flux dialyzers are used.

Pharmacokinetics and pharmacodynamics of atrial natriuretic peptide after bolus and infusion administration in the isolated perfused rat kidney.

We used the isolated perfused rat kidney to test the hypothesis that the renal pharmacokinetics and pharmacodynamics of atrial natriuretic peptide (ANP) are saturable and dependent on the method of administration. Wistar rat kidneys were perfused for 90 min after a bolus dose or continuous infusion of 45, 180, 450 ng of ANP. ANP clearance ranged from 3.27 to 2.28 ml/min after bolus administration. ANP clearance fell after infusion, resulting in a disproportionate increase in the ANP concentration with increasing infusion rate. The ANP half-life was unaffected by dose in the bolus group averaging 18 min. Increasing the ANP dose also increased the amount of Na excreted into the urine, but there were no differences between experimental groups. However, the area under the curve responsible for the natriuresis was 36 to 41% lower after infusion. Exogenous creatinine clearance, renal perfusion pressure and flow and renal vascular resistance were not affected. We conclude that the renal pharmacokinetics of ANP are saturable and are altered by the method of administration due to a down regulation of the ANP receptor. Furthermore, infusion of ANP should result in a greater net natriuresis due to resulting greater ANP concentrations at steady state and an apparent increased sensitivity of the kidney to ANP after infusion.

Flow microfluorometric analysis of murine spermatozoa fails to detect H-2 antigens.

The presence of class 1 and class 2 histocompatibility antigens on murine sperm was investigated by flow microfluorometry. Monoclonal anti-H-2Kk (class 1), anti-Iak (specificity 2, class 2) and allo-anti-Iak (class 2) antisera were used in direct or indirect fluorescence labelling experiments to probe the expression of class 1 and class 2 antigens on epididymal mouse spermatozoa. Sperm-specific antibodies were generated by intraperitoneal immunization of both male and female C3H/HeN mice with syngeneic spermatozoa. Sperm-specific antigens were detected in 68-85% of syngeneic mouse sperm and 65-90% of allogeneic mouse sperm examined. Conversely, these antibodies did not stain syngeneic or allogeneic lymphocytes above the background of the negative control. Mouse sperm samples failed to exhibit specific fluorescence above the background of negative control values with antibodies against either class 1 or class 2 MHC antigens. We have established the sensitive, objective and economical assay of sperm membrane antigens with fluorochrome-labelled antibodies by flow microfluorometry. By use of this sensitive and objective technique we have not detected MHC antigens on murine sperm. We conclude that these MHC antigens are not expressed on sperm at a level to be practically detectable.

Stimulus intensity effects in electrodermal habituation.

Experiment 1 presented human subjects with 25 shocks of the same (.5, 1.5, or 2.5 mA:between-subjects design) or different (.5, 1.0, 1.5, 2.0, and 2.5 mA:within-subjects design) intensities to test predictions of the dual-process, cortical-model, and adaptation-level theories concerning terminal electrodermal response (EDR) magnitudes in an habituation paradigm. Dual-process theory correctly predicted terminal EDR magnitudes and relative EDR habituation rates. Dual-process theory was further supported in Experiment 3 when EDR magnitude to a standard intensity shock (1.25 mA) decreased with the intensity of a second comparison shock (0, .5, 1.25, or 2.5 mA) only up to the 1.25-mA level, as the common-elements construct of the dual-process theory predicts. Adaptation level incorrectly predicted that standard stimulus EDR magnitude would decrease as comparison intensity, hence adaptation level, increased. Forewarning subjects of each shock intensity increased EDR magnitude in Experiments 2 and 3 contrary to the cortical-model theory's prediction based on subjective stimulus uncertainty.

Bacteriology of hemodialysis fluids: are current methodologies meaningful?

Reports of increasing endotoxic reactions in dialysis centers using high-flux dialyzers and high contamination in liquid bicarbonate concentrates have resulted in concern for the microbial contamination of dialysate. The influence of salt-supplemented media on the recovery of bacterial contaminants from the fluids used in hemodialysis has been examined. This study found a negative influence of a 2% NaCl supplementation of growth media for both purified water and dialysate. Salt-supplemented pour plate cultures of bicarbonate concentrate samples were not statistically different from nonsupplemented cultures (p = 0.2). The influence of the bicarbonate salt on recovery in the pour plates was not addressed. The different media recommended for monitoring microbial contamination of dialysis fluids were compared. As previously reported, both water and dialysate collected from a relatively large geographic area showed higher recoveries on Reasoner's R2A agar than on media recommended by the Association for Advancement of Medical Instrumentation (AAMI) standards (p < 0.0001). Standard methods agar (SMA) and trypticase soy agar (TSA) produced the next highest recovery for water and dialysate, respectively. The higher recoveries generally observed on R2A or SMA suggest that to provide better patient safety these media should be selected for monitoring bacterial contamination of water, and R2A, SMA, or TSA for dialysate. The variability in the species identified across the three fluids and variability in counts observed in the different fluids suggest that significant dialysate contamination may occur from sources other than the water and bicarbonate concentrates.

Calcium carbonate precipitation in bicarbonate hemodialysis.

Calcium carbonate has been observed to precipitate in the fluid pathway of dialysate delivery systems dispensing bicarbonate-containing dialysates. Such precipitation can occlude the fluid pathway, leading to system malfunction and increased maintenance requirements. We show that commercial supplies of sodium bicarbonate are contaminated by trace amounts of sodium carbonate. This contamination may result in immediate precipitation of calcium carbonate on formulation of the dialysate, since bicarbonate-containing dialysates, as formulated, are metastable with respect to calcium carbonate. Sparging of the bicarbonate-containing concentrate with carbon dioxide converts any carbonate to bicarbonate, thus avoiding the formation of precipitates on addition of calcium ions.

Culture of dialysis fluids on nutrient-rich media for short periods at elevated temperatures underestimate microbial contamination.

Recommended culture methods for monitoring bacterial contamination of H2O, dialysate and bicarbonate concentrate in dialysis centers in the USA involves culturing these fluids for 48 h at 37 degrees C. A variety of media and commercial culture methods are accepted for monitoring these fluids. Over a 3-month a comparison was made between an acceptable culture method, tryptic soy agar (TSA) employing the pour plate (PP) technique at 37 degrees C for 48 h, and PP cultures on standard methods agar (SMA) and R2A agar, incubated at ambient temperature (23 degrees C) for 48, 72, 168 h. Increases in the colony counts over time occurred for all three fluids. However, counts wee greater on SMA and R2A than on TSA. The increases over the standard 48-hour TSA cultures ranged as high as 10(4) times for 23 degrees C cultures at 7 days of incubation. Endotoxin levels even in the most contaminated samples were found to be below the acceptable 5 EU/ml recommended for reprocessor water. Bacterial colonies that appeared at 48, 72 and 168 h were isolated and identified. Pseudomonas, Moraxella, Acinetobacter and CDC group VI C-2 were among some of the common bacteria isolated. This study indicates that the media utilized, the time and temperature of incubation may result in a significant underestimation of the bacterial population of water and dialysis fluids, thus potentially placing the patient at a higher risk.

Response of complement and neutrophils to hydrophilized synthetic membranes.

Membranes prepared from hydrophobic synthetic polymers adsorb proteins. Alloying these polymers with hydrophilic polymers increases water wetability and reduces hydrophobic interactions. The authors tested the hypothesis that alloying could affect the biocompatibility of the resultant membrane. Polymer films were cast from solutions containing 12% polysulfone and 4, 8, or 12% polyvinylpyrrolidone in dimethylacetamide. The effect of the films on the complement system and neutrophil function was assessed by incubating them in normal plasma and whole blood at 37 degrees C. Complement activation was followed by measuring plasma concentrations of C3a des Arg and C5a des Arg. Flow cytometric measurements of phagocytic capacity, H2O2 production, and expression of the C3bi receptor were used to assess neutrophil function. The degree of complement activation was found to depend on the composition of the polymer film. C3a des Arg and C5a des Arg generation decreased 75% and 86%, respectively, as the polyvinylpyrolidone content of the casting solution was increased from 25 to 50%. This was accompanied by a decrease in stimulation of neutrophil H2O2 production and C3bi receptor expression. From these data, the authors conclude that alloying with polyvinylpyrrolidone can affect the biocompatibility of polysulfone.

Bacterial contamination of hemodialysis center water and dialysate: are current assays adequate?

Many dialysis centers depend on clinical laboratories or a commercially available dip culture to determine the contamination levels in water and dialysate. To determine whether these standard clinical culture procedures adequately quantitate bacterial contamination in hemodialysis center water and dialysate, test results of two routine clinical media was compared, trypticase soy agar (TSA) and plate count agar (PCA), with those of nutrient-poor R2A medium. Dialysate samples demonstrated significant differences in media, the temperature of incubation, and plating techniques (pour plate versus spread plates). Purified water for dialysis demonstrated significant differences only for media; however, temperature was an important variable. Selective growth on R2A agar of some water- and dialysate-contaminating species was studied by velvet disk and loop transfer of colonies. A strong selectivity for water-borne bacteria was demonstrated by R2A agar; the bacteria that did not grow on TSA and PCA have been identified.

Mechanism of steroid binding to serum proteins.

Association and dissociation rate constants of steroid complexes with progesterone-binding globulin (PBG) and with corticosteroid-binding globulin have been determined, utilizing the fluorescence quenching phenomenon observed on steroid binding to protein. Stopped-flow techniques were used in most cases. The dissociation rates of the complexes with steroid-binding proteins of serum are much greater than those of steroid-receptor complexes, in accordance with the biological functions of these two types of proteins. Association of steroids with PBG is accompanied by conformational changes in both components of the complexes. Chemical modification of tryptophan, lysine, and tyrosine in PBG results in inactivation of the binding site; complex formation with progesterone protects against this inactivation. A comparison of the affinity constants of PBG complexes with steroids of different structures leads to a conceptual image of the binding site and to localization of the various forces of interaction over the binding site area.

Kinetic and equilibrium studies on steroid interaction with human corticosteroid-binding globulin.

Kinetic and equilibrium studies on the interaction of steroids with human corticosteroid-binding globulin (CBG, transcortin) were performed with pH, temperature, and steroid structure as variables. Dissociation rate constants were determined fluorometrically; the values for cortisol, corticosterone, deoxycorticosterone, and progesterone are 0.031, 0.047, 0.10, and 0.16 s-1, respectively, at 20 degrees C, pH 7.4. The pH dependence of the dissociation rate constant for the corticosterone complex below pH 10.5 at 20 degrees C is given by koff = 0.043 (1 + [H+]/10(-6.50)) s-1; above pH 11, koff = 0.030 (1 + 10(-12.15/[H+] s-1. A temperature-dependence study of koff for the cortisol and progesterone complexes gave values of 0.0028 s-1 and 0.012 s-1 at 4 degrees C, respectively, and 0.88 s-1 and 4.5 s-1 at 37 degrees C, with progesterone dissociating about four to five times faster over the entire temperature range. The affinity constants, determined by equilibrium dialysis, for the binding of cortisol, corticosterone, and progesterone at 4 degrees C were 7.9, 7.2, and 7.0 X 10(8) M-1; values of 0.40 and 0.26 X 10(8) M-1 were determined at 37 degrees C for cortisol and progesterone. The close similarity of the affinity constants of the three steroids combined with differing dissociation rates implies that the association rate changes with steroid structure, in contrast to our earlier findings with progesterone-binding globulin.

Steroid-protein interactions. Human corticosteroid-binding globulin: characterization of dimer and electrophoretic variants.

Human corticosteroid-binding globulin (CBG) forms a dimer that was isolated by gel filtration, has full binding affinity and capacity, and can be dissociated to the monomer. Monomeric CBG consists of two distinct molecular variants, which were detected by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The two monomeric CBG species were separated by preparative gel electrophoresis and were found to bind cortisol, as well as progesterone, with equal affinity. They have one steroid binding site per CBG molecule. Amino acid and carbohydrate analyses are essentially the same for both of the CBG variants. Removal of sialic acid or 90% of the carbohydrate did not affect the existence of the two molecular forms. The two CBG species were isolated from each of the sera from five individual donors, indicating that the observed heterogeneity does not result from pooling genetic variants. The two species are immunologically identical. A possible explanation for the existence of the two electrophoretic variants is a difference in amidation.

Microbial and endotoxin contamination in water and dialysate in the central United States.

The purified water supplies and randomly selected dialysates of 51 chronic and acute dialysis centers in the central United States were surveyed to assess the relative risks to dialysis patients from microbial and endotoxin contamination. A culture medium more sensitive than those generally employed in routine quality assurance assays was used for recovery of bacteria from water. With this medium, 35.3% of the water samples and 19% of the dialysate samples were out of compliance with the Association for the Advancement of Medical Instrumentation (AAMI) standards: 200 and 2,000 colony forming units (CFU)/ml, respectively. There was no correlation observed between the type of water purification system or the frequency of disinfection of the system and the bacterial and endotoxin contamination levels. There was also no correlation found between the bacterial and fungal CFU per ml and the endotoxin concentration per ml (EU/ml). It is recommended that more sensitive culturing methods be used to provide adequate bacterial monitoring of dialysate center water supplies. Dialysis centers should monitor endotoxin in dialysate on a regular schedule and immediately after any endotoxemic-like patient reactions. Yeast and fungi were observed in 10% and 64% of the water systems, respectively. Dialysate was contaminated by yeast and fungi in 30% and 70% of the centers, respectively. The concentrations of these microbes in both fluids were much lower than bacteria. However, they were observed often enough to warrant further investigation of their impact on the well-being of dialysis patients.