Transport of charged dipeptides by the intestinal H+/peptide symporter PepT1 expressed in Xenopus laevis oocytes.

The cloned intestinal peptide transporter is capable of electrogenic H+-coupled cotransport of neutral di- and tripeptides and selected peptide mimetics. Since the mechanism by which PepT1 transports substrates that carry a net negative or positive charge at neutral pH is poorly understood, we determined in Xenopus oocytes expressing PepT1 the characteristics of transport of differently charged glycylpeptides. Transport function of PepT1 was assessed by flux studies employing a radiolabeled dipeptide and by the two-electrode voltage-clamp-technique. Our studies show, that the transporter is capable of translocating all substrates by an electrogenic process that follows Michaelis Menten kinetics. Whereas the apparent K0.5 value of a zwitterionic substrate is only moderately affected by alterations in pH or membrane potential, K0.5 values of charged substrates are strongly dependent on both, pH and membrane potential. Whereas the affinity of the anionic dipeptide increased dramatically by lowering the pH, a cationic substrate shows only a weak affinity for PepT1 at all pH values (5.5-8.0). The driving force for uptake is provided mainly by the inside negative transmembrane electrical potential. In addition, affinity for proton interaction with PepT1 was found to depend on membrane potential and proton binding subsequently affects the substrate affinity. Furthermore, our studies suggest, that uptake of the zwitterionic form of a charged substrate contributes to overall transport and that consequently the stoichiometry of the flux-coupling ratios for peptide: H+/H3O+ cotransport may vary depending on pH.

Functional analysis of a chimeric mammalian peptide transporter derived from the intestinal and renal isoforms.

l. Recently two genes have been identified by expression cloning that encode mammalian epithelial peptide transporters capable of translocating di- and tripeptides and selected peptidomimetics by stereoselective and rheogenic substrate-H+ cotransport. PepT1 from rabbit or human small intestine induces a transport activity with high transport capacity but rather low substrate affinity when expressed in Xenopus oocytes. In contrast, the renal carrier PepT2 is a high affinity-type transporter with a lower maximal transport capacity. In addition, both transporters show differences in pH dependence and substrate specificity. 2. As a first approach to identify structural components of the transport proteins that determine their phenotypical characteristics, we constructed a recombinant chimeric peptide transporter (CH1Pep) in which the aminoterminal region (residues 1-401) is derived from PepT2 whereas the carboxyterminal region (residues 402-707) starting at the end of transmembrane domain 9 is derived from PepT1. Expression of PepT1, PepT2 and CH1Pep in Xenopus oocytes allowed the characteristics of the transporters to be determined by flux studies employing a radiolabelled dipeptide and by the two-electrode voltage clamp technique. 3. Our studies indicate that CH1Pep conserves the characteristics of PepT2 including the high affinity for dipeptides and peptidomimetics, the substrate specificity, the pH dependence of transport activation and the electrophysiological parameters. We conclude that the phenotypical characteristics of the renal peptide transporter are determined by its amino-terminal region.

Evaluation of serum estradiol concentrations in alopecic ferrets with adrenal gland tumors.

Seventeen ferrets were examined because of progressive bilaterally symmetric alopecia that was nonpruritic. Dermatologic and endocrinologic testing were used to determine the cause of the alopecia. Resting cortisol, testosterone, and thyroxin concentrations and results of ACTH stimulation tests were found to be within reference range limits established for this species. High serum estradiol concentrations were found to be a reliable indicator of adrenal cortical neoplasia in these ferrets.

[Continuous ambulatory peritoneal dialysis. 5-year clinical experience]. / Kontinuierliche ambulante Peritonealdialyse. Fünfjährige klinische Erfahrung.

Continuous ambulatory peritoneal dialysis (CAPD) is, next to conventional home dialysis, a world-wide method of treating chronic renal failure. This is the report of clinical experience of 34 CAPD patients who, from October 1978 to the end of 1983, had undergone 642 treatment months. The incidence of peritonitis after introduction of a new connector system in 1983 was reduced to 1 per 46 CAPD treatment months. In only 7 patients was it necessary to discontinue CAPD for reasons connected with the type of dialysis. No patient requested discontinuance of CAPD. Suitable bag and tube materials, adequate space, and well trained personnel are decisive for a successful CAPD programme, in addition to careful selection of patients. The latter aims at optimal patient motivation for the treatment method which, in principle, is more cost effective than home dialysis. In so far as patients fulfil the requirements for home dialysis they can be excellently rehabilitated.

[Biochemical changes with continuous ambulant peritoneal dialysis (CAPD)]. / Biochemische Veränderungen unter kontinuierlicher ambulanter Peritonealdialyse (CAPD).

During the past few years continuous ambulatory peritoneal dialysis (CAPD) has become well established in the home treatment of uremia. CAPD, however, may induce certain biochemical abnormalities. Using glucose as an osmotic agent of the dialysate the peritoneal glucose load may vary between 75 and 200 g per day, depending upon how often high osmotic dialysate is needed. If the latter is restricted to one bag per day a long-term disturbance of the glucoregulatory hormones insulin, GIP an glucagon seem to be inprobable. Investigations into glucose tolerance after 23 til 34 months of CAPD treatment in 4 patients did not indicate any exhaustion of the pancreatic beta-cells. Long-term evaluation into the metabolism of lipoproteins, total plasma proteins, aminoacids and trace elements did not show significant abnormalities induced by CAPD itself. Biochemical alterations observed are more or less related to the uremic state of the patients.

[First observations with continuous ambulatory peritoneal dialysis (author's transl)]. / Erste Beobachtungen mit kontinuierlicher ambulanter Peritonealdialyse.

Continuous ambulatory peritoneal diaglysis (CAPD) represents a new method for treatment of chronic renal failure whereby patients carry two litres of dialysate with them permanently and are freely mobile. Dialysis is only interrupted by exchange of dialysate in approximately 6-hourly intervals. Due to the long presence of the dialysate in the peritoneal cavity clearance values are superior to intermittent peritoneal dialysis for small-molecular substances and reach elimination values of haemofiltration for medium-molecular substances. The main technical requirements consist of a permanent peritoneal dialysis catheter linked to a plastic dialysate bag by way of a connecting tube. In order to avoid frequent change of bag and thus increased risk of peritonitis bags are folded and carried on the body of the patient during the interval. Thereafter the bag is unfolded, filled with used dialysate and exchanged for a new bag. Six patients were thus treated for over 34 patient-months so far. Changes of chemical pathology were readily acceptable. Even though CAPD has several advantages over conventional dialysis as they are mainly founded on the high degree of rehabilitation of patients, risk of peritonitis is still a considerable factor of uncertainty.

Capabilities of the Redy cartridge for regeneration of hemofiltrate.

Capabilities of the Redy cartridge for hemofiltrate regeneration were tested. Electrolytes, creatinine, BUN, uric acid, acid-base, glucose, heparin, fluoride and amino acids were measured in the cartridge inflow and outflow (V = 70 ml/min) over a four-hour period. There was complete adsorption of potassium, calcium, magnesium, creatinine, BUN, uric acid, phosphate and heparin. Sodium ions, hydrogen ions, fluoride ions and possibly other trace elements are released by the cartridge. Amino acid absorption is almost complete if the amino acids are aromatic, have two or more N-atoms or if they have S-atoms. When using hemofiltrate after sorbent regeneration for reinfusion to the patient, one has to consider electrolyte substitution (i.e., K, Ca, Mg), sodium balance, trace element metabolism, acid-base problems, amino-acid profiles and ammonia overload.

[Calcium and phosphate metabolism in hemofiltration (author's transl)]. / Calcium-phosphat-stoffwechsel und Hämofiltration.

In 10 patients undergoing hemofiltration treatment acute changes of parameters in the calcium-phosphate metaboism were investigated. Balance studies were also performed in all patients. Control studies were conducted after a 3-month interval in 7 patients. Whereas ionized calcium and 25-HCC remained constant, there was a significant decrease in phosphate, magnesium, fluoride and parathyroid hormone. Corresponding to these results, negative balances could be seen during the course of a hemofiltration treatment: for phosphate a mean value of -593 mg, for magnesium -8.4 mEq and for fluoride -458 microgram. When a calcium content of 3.75 mEq/l was used in the substitution solution, an only slightly positive calcium balance of +1.51 mEq/l (mean value) was found. A significant correlation between calcium and fluid balance was demonstrated by means of 197 filtration treatments of one patient: the calcium balance became negative whenever the fluid loss was greater than 3.86 liters. After a 3-month period no significant changes in the above parameters were found, which indicates, that disturbances in the calcium-phosphate-parathyroid hormone metabolism do not only lie in a reduced renal elimination. Even though our results do not indicate that hemofiltration treatment induces or increases the chances of renal osteodystrophy, the calcium concentration of the substitution solution should be increased to 4.0 mEq/l, in order to guarentee a positive calcium balance even by forced filtration.

[Biological availability of fluoride after combined administration with vitamin D3 in man (author's transl)]. / Bioverfügbarkeit von Fluorid bei kombinierter Gabe mit Vitamin D3 beim Menschen.

Six adults ingested 1 mg fluoride, first as NaF in a watery solution and, secondly, in tablet form (D-Fluoretten 1000). Fluoride concentration was almost identical in the two series. After initial values within the normal range concentrations rapidly rose to a maximum (six times normal) reached on average after 30 minutes, followed by an, at first steep then more gradual, fall in concentration over eight hours. The area under the curve was similar in the two series. These results indicate that the biological availability of fluoride is not influenced by vitamin D3 in the adult and, presumably, in the small child as well.

Effect of dialysate calcium concentration on plasma parathyroid hormone during hemodialysis.

The effect of changes in the dialysate calcium concentration on calcium fractions and parathyroid hormone species in plasma during hemodialysis has been examined. C-terminal immunoreactive parathyroid hormone was suppressed when plasma calcium increased by greater than 25% whereas N-terminal fragments demonstrated an increase. The significance of this is discussed in light of present knowledge of parathyroid hormone metabolism.

Parathyroid hormone, calcium and phosphate balance in hemofiltration.

The acute changes in calcium, phosphate and parathyroid hormone have been examined in chronic renal failure patients under-going hemofiltration therapy and the results compared to a similar group treated by hemodialysis. In both groups there was a significant increase in Catot (0.32 mEq/1 for hemodialysis; 0.56 m Eq/1 for hemofiltration) with Ca++ remaining constant. Plasma phosphate and parathyroid hormone decreased during hemofiltration. Calcium balances were slightly positive and phosphate balances distinctly negative in all cases. To date there is no indication of induced osteodystrophy during hemofiltration therapy, although long-term studies are needed. However, the present results indicate, that hemofiltration more closely approaches the physiological situation than conventional hemodialysis.

Comparative calcium ion determinations in plasma and whole blood with a new calcium ion analyzer.

A new Ca2+ analyzer has been used for the determination of Ca2+ in plasma and whole blood from normal subjects and patients with disorders of calcium metabolism. The results were nearly identical: the equation of the regression line was y=1.02x-0.037 and the coefficient of correlation r=0.998. The possibility of using whole blood for measurement with concomitant simplification of the anaerobic sample preparation is an obvious advantage.

Fluoride determination in plasma by ion selective electrodes: a simplified method for the clinical laboratory.

A potentiometric method for the determination of fluoride (F-) in serum and plasma is proposed; it is based on a combination of the single-known-addition method and the electrode slope-by-dilution method. This procedure provides reliable results in extremely low measuring ranges down to 2.5 mug/l, where the electrode slope deviates markedly from Nernstian behaviour. In this method no electrode calibration is required and only one standard is necessary. 1 ml of plasma is sufficient for one analysis. Apart from a 5% enrichment of all samples with a concentrated total ionic strength adjustment buffer, no further preparation of the sample is required. The simplicity of the various pipetting and analytical steps, and also of the evaluation of the readings, may render this method highly suitable for the clinical laboratory. Investigations into the accuracy and precision of the method produced satisfactory results. The recovery in plasma amounted to 99.7%, even in the low measuring ranges. The discrimination capacity of the method amounts to 0.1 mug/l. With the apparatus and experimental procedures described, 18 plasma analyses per day can be performed even at low F- concentrations with which longer electrode stabilization periods are required. Storage of the plasma samples frozen at --20 degrees C for up to 8 weeks exerts no effect on the F- concentration. Problems of sample contamination and other disturbances are discussed. Determinations of normal values in 20 test subjects resulted in a mean value of 10.4 plus or minus 4.01 mug/l (Mean plus or minus S.D.). The modal value amounted to 9.5 mug/l, and the range was between 5.9 and 18.8 mug/l. The F- content of the drinking water supplied to this group of persons amounted to 180 mug/l. The importance of the method has been illustrated using a clinico-nephrological study as an example.