Hydraulically-induced convective solute transport across the rabbit peritoneum.

Transport of solutes during osmotically-induced transperitoneal ultrafiltration is less than would be predicted based upon rates of transperitoneal solute diffusion. Previous workers have hypothesized that osmotically-induced convective solute transport occurs only in small pores at the arteriolar end of peritoneal capillaries, whereas solute diffusion occurs only through large venular pores. We tested this heteroporosity hypothesis in the eviscerated New Zealand White rabbit by determining sieving coefficients (S) for creatinine, p-aminohippurate (PAH) and neutral dextran during hydraulically-induced transperitoneal ultrafiltration (N = 13). A hydraulically-induced driving force directs convective solute transport through the same capillary pores employed for diffusion; therefore S for all solutes should approach unity if the heteroporosity hypothesis is valid. S for creatinine and PAH were respectively 0.72 +/- 0.03 and 0.67 +/- 0.05, values lower than unity and not different from those previously determined during osmotically induced ultrafiltration. Mean S for dextran were relatively independent of molecular size, ranging from 0.50 at 13 A to 0.40 at 50 A. Thus, dextran S were higher than those previously determined during osmotically induced ultrafiltration yet still less than unity. Control experiments (N = 6) suggested that only surface area and not transport characteristics were altered by evisceration. These observations demonstrate that the heteroporosity hypothesis fails to completely describe both diffusive and convective transport properties of the peritoneum.

Dialysate to blood transport of macromolecules during peritoneal dialysis.

Asymmetrical transport of macromolecules between plasma and the peritoneal cavity results primarily from unidirectional lymphatic removal from the peritoneal cavity. Recent work suggests, however, that macromolecular transport across the peritoneal-plasma barrier via the capillary wall (i.e., the peritoneal membrane) may also be asymmetrical. We determined the diffusive and convective transport properties for creatinine, p-aminohippurate, and neutral dextran (13-40 A) across the peritoneal membrane in the dialysate to blood direction during peritoneal dialysis using isotonic and hypotonic solutions in awake New Zealand White rabbits. Values of the diffusive permeability-area product that were calculated during the isotonic exchange were similar to, yet somewhat smaller than, those previously determined in the blood to dialysate direction for all test solutes. Solute reflection coefficients that were calculated during the hypotonic exchange were variable, yet the resulting mean solute reflection coefficient values for all the test solutes were similar to those previously determined in the blood to dialysate direction. We conclude that asymmetrical peritoneal transport of macromolecules with radii less than 40 A is not due to asymmetrical transport across the peritoneal membrane.

Dialysate volume measurements required for determining peritoneal solute transport.

Solute transport parameters for the peritoneal membrane have been previously determined using dialysate volumes measured by the indicator dilution method. Recent work has shown that the indicator dilution volume (IDV) exceeds true dialysate volume (TV) because the indicator or index solute is lost from the peritoneal cavity. A peritoneal transport model that includes significant solute loss from the peritoneal cavity is here described. Theory suggests that simultaneous measurements of both IDV and TV are required to calculate solute transport parameters for the peritoneal membrane when solutes are lost from the peritoneal cavity. The magnitude of systematic errors incurred by the use of either IDV or TV alone was determined in the calculated diffusive permeability-area product (PA) for creatinine during a two hour exchange in a rabbit model of peritoneal dialysis. IDV was measured using dextran (2 X 10(6) daltons) and TV by the dilution of multiple injections of Evans blue-albumin complex. Best estimates of PA using either or both volume measurements were determined in the blood to dialysate direction with isotonic (N = 9) and hypertonic (N = 7) solutions and in the dialysate to blood direction with isotonic (N = 4) and hypotonic (N = 4) solutions. Systematic errors in PA using either IDV alone or TV alone were small with either isotonic or hypertonic solutions but were increased with hypotonic solutions. Moreover, systematic errors were larger when using TV alone thaN IDV alone. When solute transport parameters for the peritoneal membrane are approximately determined employing only a single volume measurement, the use of IDV leads to less systematic error than TV.

Temperature dependence of macromolecular sieving across plasma fractionating membranes.

The temperature dependence of macromolecular sieving coefficients (S) across a plasma fractionating membrane due to changes in the intrinsic process of membrane filtration was determined using dextrans as test macromolecules. Sieving coefficients were determined in an in vitro test circuit as a function of time, temperature, flow conditions, and plasma protein exposure. Sieving coefficients for albumin, gamma G immunoglobulin (IgG), and dextrans using normal human plasma as the test solution were comparable for dextrans and proteins of equivalent molecular size, independent of time after 15 min, lower at 8 degrees than at 37 degrees C, and higher at lower filtration flow rates. At 8 degrees C and high filtration flow rates, S for albumin and IgG also increased with increasing perfusate flow rate. Dextran S using a protein-free test solution before exposure to plasma were higher than those determined from plasma, and were independent of temperature. Dextran S from a protein-free test solution after exposure to plasma were similar to those determined from plasma at 37 degrees C, but higher than those determined from plasma at 8 degrees C. We conclude that sieving coefficients across plasma fractionating membranes can be altered by temperature due to changes in the process of membrane filtration. The present observations suggest that at low temperature a polarized layer or gel becomes a discriminating solute transport barrier.

Peritoneal dialysate volume determined by indicator dilution measurements.

Dialysate volume was simultaneously determined by two different indicator dilution technique as a function of dwell time in a rabbit model of peritoneal dialysis using isotonic, hypertonic and hypotonic solutions. After a single injection of a large molecular weight index solute (SIIS) to the dialysis solution at a known concentration, the first indicator dilution technique determined dialysate volume by the change in the index solute concentration. In the second technique, dialysate volume was determined after multiple injections of a different index solute (MIIS) by measuring the change in concentration of the index solute two minutes after its injection into the dialysis solution. The volumes determined by SIIS were similar during isotonic, but larger during both hypertonic and hypotonic exchanges, than those determined by MIIS. Drained volume was dependent upon the peritoneal catheter used, was not different from that determined by MIIS, but was significantly smaller than that determined by SIIS. The present results suggest that systematic errors in volume measurements when using indicator dilution result from the loss of the index solute from the peritoneal cavity and are greater for SIIS than for MIIS. A model for fluid transfer during peritoneal dialysis showed that dialysate volumes determined by SIIS were useful, however, when estimating the rate of fluid movement across the peritoneal membrane.

Molecular size dependence of peritoneal transport.

The nature of the barrier between blood and peritoneal dialysate was studied by determining peritoneal solute transport rates as a function of molecular size using neutral dextrans, polymers with different molecular weights but uniform chemical composition. Transport rates for creatinine, p-aminohippurate (PAH), and neutral dextran (3,000 to 50,000 daltons) were measured during peritoneal dialysis in the awake rabbit using sequential isotonic and hypertonic dialysis solutions. The permeability-area product (PA) for the peritoneum was determined from the dependence of the dialysate concentration on time during the isotonic exchange. The dependence of PA on molecular size showed no additional hindrance by the peritoneum as molecules of larger size were considered. By accounting for convective solute transport during the hypertonic exchange, the solute reflection coefficient (sigma) and PA were simultaneously determined. The values of PA for creatinine and PAH were similar to those determined during the isotonic exchange, and sigma values for creatinine and PAH were 0.18 +/- 0.20 and 0.14 +/- 0.14, respectively. Dextran sigma values (3,000 to 22,000 daltons) were near unity (0.9 to 1.0) and relatively independent of molecular size, suggesting substantial hindrance to convective transport for this size range. This work demonstrates that the paradoxical transport properties of an "open" diffusive yet "tight" convective peritoneal barrier are primarily reflective of the unique structural properties of this biologic tissue and are not related to test solute heterogeneity.

A novel bioassay to predict the clinical response to cryofiltration treatment. A preliminary report.

Phagocytosis by normal human mononuclear monocytes grown in vitro was examined after incubation with known concentrations of aggregated human gamma-globulin (AHG) and in plasma obtained from patients suffering from immunologic diseases. When the phagocytes were pretreated with AHG, an inverse relationship between amounts of AHG and phagocytosis of radiolabeled Candida albicans was found. Inhibition of phagocytosis by patient plasma was independent of other laboratory determinations including antinuclear antibody, C3, C4, CH50, DNA-binding and circulating immune complexes. Plasma from 5 patients subjected to cryofiltration treatment (CFT) demonstrated less inhibition after than before CFT. This lessened inhibition of phagocytosis in vitro correlated with the clinical response due to CFT. The response to CFT could not be predicted by the conventional laboratory parameters investigated. Improvement between pre- and post-CFT plasma estimated by using the present bioassay may provide a quantitative description of which patient may benefit from plasmapheresis treatment procedures.

Dopamine-beta-hydroxylase: structural comparisons of membrane-bound versus soluble forms from adrenal medulla and pheochromocytoma.

Dopamine-beta-hydroxylase (DBH) in membrane-bound (mDBH) and water-soluble (sDBH) forms was isolated from chromaffin granules of bovine adrenal medullae and a human pheochromocytoma tumor. sDBH was purified by concanavalin A-agarose column chromatography followed by DEAE-Sepharose column chromatography. The final bovine preparation had a specific activity of 16.27 IU/mg; the human preparation had a specific activity of 9.16 IU/mg. mDBH was isolated in enzymatically inactive form by preparative polyacrylamide gel electrophoresis. The proteins were subjected to amino acid analysis, as well as digestion with trypsin, followed by separation of the resulting peptides by two-dimensional TLC/electrophoresis. No intraspecies differences between sDBH and mDBH were found from comparisons of amino acid composition or peptide maps. Thus the basis of the difference between sDBH and mDBH cannot easily be explained by differences in primary structure, within the resolution of these techniques.

Chromogranin A, the major catecholamine storage vesicle soluble protein. Multiple size forms, subcellular storage, and regional distribution in chromaffin and nervous tissue elucidated by radioimmunoassay.

Chromogranin A (CgA), the major catecholamine storage vesicle (CSV) soluble protein, may index exocytotic sympathoadrenal secretion. To explore CgA in adrenergic tissues, we developed a radioimmunoassay for bovine CgA. Within adrenal medulla CSV, several minor chromogranins had similar amino acid compositions and peptide maps to that of CgA and also showed parallel, partial cross-reactivity in the CgA radioimmunoassay. CgA immunoreactivity represented 7 +/- 1% of total adrenal medulla cell protein and was localized to adrenal CSV, representing 46 +/- 2% of CSV soluble protein. In brain, there was 1000-fold less CgA than in adrenal medulla, with a widespread regional distribution (maximal in neocortex) and an unusual subcellular distribution (maximal in cytosol), both of which differ from reported catecholamine distribution. Brain chromogranin immunoreactivity also had a lower Stokes radius than adrenal CgA. Sympathetic nerve and serum had 6,000-fold and 30,000-fold less CgA than that in adrenal medulla. The results suggest a "family" of adrenal medulla chromogranins, similar structurally and immunoligically. Adrenal medulla and brain chromogranin differ in concentration, subcellular localization, and molecular size. Finally, CgA in serum may provide a useful tool for sympathoadrenal studies in intact organisms.

Human chromogranin A. Purification and characterization from catecholamine storage vesicles of human pheochromocytoma.

Chromogranin A is the quantitatively major soluble protein in catecholamine storage vesicles of the adrenal medulla and sympathetic nerve, and has been a useful index of exocytosis during sympathoadrenal neurosecretion. To probe human catecholamine storage and release, we isolated chromogranin A from chromaffin tissue in human pheochromocytoma, and compared it to chromogranin A isolated from chromaffin tissue in bovine adrenal medulla. The preparation included catecholamine storage vesicle isolation by sucrose gradient centrifugation, removal of dopamine-beta-hydroxylase by affinity chromatography on Concanavalin A-Sepharose, and preparative polyacrylamide gel electrophoresis. Human and bovine chromogranin A displayed considerable interspecies homology. Human chromogranin A is a 68,000 dalton monomeric protein with an unusual amino acid composition (31.53 weight % glutamic acid); an acidic, microheterogeneous isoelectric point (4.57-4.68); a characteristic tryptic digest peptide map; and marked dissimilarity to dopamine-beta-hydroxylase in all properties studied. A new probe of human sympathoadrenal function is available in chromogranin A.

Homologous radio-immunoassay of human plasma dopamine-beta-hydroxylase: analysis of homospecific activity, circulating plasma pool and intergroup differences based on race, blood pressure and cardiac function.

We used a homologous human dopamine-beta-hydroxylase (DBH) radio-immunoassay (RIA) to explore reported differences in plasma DBH enzymatic activity among patient groups stratified for race, blood pressure and cardiac function, as well as to determine plasma immunoreactive DBH protein pool and the relative activity of the enzyme in plasma versus human chromaffin tissue storage vesicles. Plasma DBH activity was lower in patients with congestive heart failure than in control subjects (19.9 +/- 4.0 versus 34.4 +/- 5.9 iu/l, P less than 0.05), paralleled by lower immunoreactive plasma DBH protein concentration (3.50 +/- 0.73 versus 6.34 +/- 1.05 micrograms/ml, P less than 0.05). All subject groups had similar plasma DBH homospecific activity (plasma DBH enzymatic activity/immunoreactive plasma DBH protein), ranging from 5.03 +/- 0.28 to 5.84 +/- 0.44 iu/mg. For the entire subject group, there was a significant relationship between plasma DBH activity and plasma DBH immunoreactive protein (r = 0.89, n = 78, P less than 0.01) from which no subgroup deviated systematically. Black hypertensives had lower plasma DBH activity than white hypertensives (23.0 +/- 5.2 versus 42.9 +/- 4.8 iu/l, P less than 0.01), though their plasma DBH homospecific activities and activity/immunoreactive protein plots were indistinguishable. Total circulating plasma DBH pools were large (from 13.1 +/- 3.7 to 27.5 +/- 4.8 mg).(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoreactive calcitonin in catecholamine storage vesicles of human pheochromocytoma.

The subcellular storage site for catecholamines in normal adrenal gland and pheochromocytoma tissue is the secretory granule or chromaffin granule (vesicle). Since some pheochromocytomas also contain calcitonin, we examined the occurrence and subcellular storage of calcitonin in 15 tumors from 12 patients with pheochromocytoma using a sensitive human calcitonin RIA. Only 1 of these patients had medullary thyroid carcinoma. The tumors of 9 patients contained calcitonin, from 1,705-3,602,457 pg/tumor. Highly purified chromaffin granules were prepared by sucrose density gradient centrifugation. The granules from 9 tumors in 8 patients contained calcitonin in amounts varying from 1,156-201,426 pg/tumor, accounting for 24 +/- 8% of the total calcitonin. Dilutions of chromaffin granule calcitonin immunoreactivity were parallel to the standard calcitonin RIA curve, which indicated antigenic similarity between pheochromocytoma calcitonin and synthetic human calcitonin; on gel filtration, chromaffin granule calcitonin coeluted with [125I]human calcitonin monomer, indicating size similarity. Thus, calcitonin seems to be present in the chromaffin secretory granules of most pheochromocytomas.

Human catecholamine storage vesicle proteins.

Catecholamines in the adrenal medulla and sympathetic nerve are stored in vesicles, along with numerous proteins and peptides; both catecholamines and proteins are released by and are markers of exocytosis during sympathoadrenal neurosecretion. The proteins include the enzyme dopamine-beta-hydroxylase and the chromogranins, a group of molecules of largely undetermined function. We have isolated catecholamine storage vesicles (chromaffin granules) from bovine adrenal medulla and human pheochromocytoma. The numerous soluble proteins in the granules: (a) have a spectrum of sizes; (b) have a spectrum of charges; and (c) display considerable interspecies qualitative homology. Dopamine-beta-hydroxylase (DBH) was isolated from human and bovine vesicles by Concanaval in A affinity chromotography. DBH is a tetrameric glycoprotein consisting of 2 non-covalently joined dimeric subunits, each of which is 2 disulfide linked monomers; interspecies molecular weight differences were noted. Ongoing studies concern chromogranin A, the quantitatively major vesicle protein, in bovine and human vesicles.