Avoiding the use of long-term parenteral support in patients without intestinal failure: A position paper from the European Society of Clinical Nutrition & Metabolism, the European Society of Neurogastroenterology and Motility and the Rome Foundation for Disorders of Gut-Brain Interaction.

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Avoiding the use of long-term parenteral support in patients without intestinal failure: A position paper from the European Society of Clinical Nutrition & Metabolism, the European Society of Neurogastroenterology and Motility and the Rome Foundation for Disorders of Gut-Brain Interaction.

The role of long-term parenteral support in patients with underlying benign conditions who do not have intestinal failure (IF) is contentious, not least since there are clear benefits in utilising the oral or enteral route for nutritional support. Furthermore, the risks of long-term home parenteral nutrition (HPN) are significant, with significant impacts on morbidity and mortality. There has, however, been a recent upsurge of the use of HPN in patients with conditions such as gastro-intestinal neuromuscular disorders, opioid bowel dysfunction, disorders of gut-brain interaction and possibly eating disorders, who do not have IF. As a result, the European Society of Clinical Nutrition and Metabolism (ESPEN), the European Society of Neuro-gastroenterology and Motility (ESNM) and the Rome Foundation for Disorders of Gut Brain Interaction felt that a position statement is required to clarify - and hopefully reduce the potential for harm associated with - the use of long-term parenteral support in patients without IF. Consensus opinion is that HPN should not be prescribed for patients without IF, where the oral and/or enteral route can be utilised. On the rare occasions that PN commencement is required to treat life-threatening malnutrition in conditions such as those listed above, it should only be prescribed for a time-limited period to achieve nutritional safety, while the wider multi-disciplinary team focus on more appropriate biopsychosocial holistic and rehabilitative approaches to manage the patient's primary underlying condition.

The PDZ-interaction of the intestinal anion exchanger downregulated in adenoma (DRA; SLC26A3) facilitates its movement into Rab11a-positive recycling endosomes.

Electroneutral NaCl absorption in the ileum and colon is mediated by downregulated in adenoma (DRA) (Cl⁻/HCO3⁻ exchanger; SLC26A3) and Na⁺/H⁺ exchanger 3 (NHE3, SLC9A3). Surface expression of transport proteins undergoes basal and regulated recycling by endo- and exocytosis. Expression and activity of DRA in the plasma membrane depend on intact lipid rafts, phosphatidylinositol 3-kinase (PI3-kinase), and the PDZ interaction of DRA. However, it is unknown how the PDZ interaction of DRA affects its trafficking to the cell surface. Therefore, the (re)cycling pathway of DRA was investigated in HEK cells stably expressing enhanced green fluorescent protein (EGFP)-DRA or EGFP-DRA-ETKFminus (a mutant lacking the PDZ interaction motif). Early, late, and recycling endosomes were immunoisolated by precipitating stably transfected mCherry-hemagglutinin (HA)-Rab5a, -7a, or -11a. EGFP-DRA and EGFP-DRA-ETKFminus were equally present in early endosomes. In recycling endosomes, wild-type DRA was preferentially present, whereas, in late endosomes, DRA-ETKF-minus dominated. Correspondingly, EGFP-DRA colocalized with mCherry-HA-Rab11a in recycling endosomes, whereas EGFP-DRA-ETKFminus colocalized with mCherry-HA-Rab7a in late endosomes. Functionally, this different distribution was reflected by a shorter half-life of the mutant DRA. Transient expression of dominant-negative Rab11a(S25N) inhibited the activity (-17%, P < 0.05) and the cell surface expression of DRA (-30%, P < 0.05). Transient transfection of Rab4a or its dominant-negative mutant Rab4a(S22N) was without effect and thus excluded participation of the rapid recycling pathway. Taken together, the PDZ interaction of DRA facilitates its movement into Rab11a-positive recycling endosomes, from where it is inserted in the plasma membrane. A scenario emerges where specific PDZ adaptor proteins are present along several compartments of the endocytosis-recycling pathway.

[Current aspects of sepsis caused by bacterial translocation]. / Neue Erkenntnisse zur Sepsis durch bakterielle Translokation.

Bacterial translocation has been put forward as a concept to explain sepsis without an infectious focus, but it has been difficult to prove in humans. Dysfunction of the intestinal barrier, which is composed of physical, biochemical and immunological factors, is the pathophysiological prerequisite for bacterial translocation. Recent findings indicate that not only viable bacteria but also pathogen associated molecular patterns may translocate and cause sepsis. Molecular detection methods for bacteria or their components have been developed to address these new concepts, but they have not yet become widely available. Specific therapeutic interventions within the sepsis cascades and signaling pathways of the innate and specific immune system so far have not been successful. Selective oral decontamination (SOD) und selective digestive tract decontamination (SDD) are efficacious prophylactic measures against nosocomial septic complications. An increased incidence of resistant pathogens has not been encountered. The use of probiotics as prophylaxis against septic complications is controversial and has led in some studies to a worse prognosis.

A multi-national survey of experience and attitudes towards commencing home parenteral nutrition for patients with advanced cancer.

INTRODUCTION: Advanced cancer (AC) is increasingly an indication for home parenteral nutrition (HPN) but an area with possible variation in practice between geographical locations. The aims of this study are to explore the views and experiences of international multi-disciplinary teams to determine opinions and practices. METHODS: An online questionnaire was developed with members of the Home Artificial Nutrition and Chronic Intestinal Failure interest group of the European Society for Clinical Nutrition and Metabolism (ESPEN) and distributed to colleagues involved in managing patients with AC on HPN. RESULTS: A total of 220 responses were included from 5 continents including 36 countries, with 90% of all responses from Europe. Predicted survival was a key factor influencing the decision to commence HPN for most respondents 152/220 (75%), with the majority of participants reporting that patients should have a predicted survival of ≥3 months if considered for HPN (≥3 months: n = 124, 56% vs. <3 months: n = 47, 21%, p < 0.001). However, most respondents were not confident about predicting overall survival in more than 50% of cases (confident n = 40, 23% vs not confident n = 135, 77%, p < 0.001). Barriers to utilising HPN in AC included colleagues' objections (n = 91, 46%), lack of local expertise (n = 55, 28%) and funding restrictions (n = 34, 17%). CONCLUSIONS: Significant consensus was observed regarding AC as indication for HPN, while areas of variation exist. Survival prognostication is often used as an indication for commencing HPN in people with AC, although the majority of respondents were not confident in prognosticating, suggesting better clinical prognostication tools will be of assistance. Further studies are also required to better understand the obstacles faced by clinical teams to commencing HPN that may explain variations in clinical practice between countries, as well as adressing variation in funding.

Characterization of refractory port-related blood stream infections in intestinal failure patients on parenteral nutrition.

BACKGROUND AND AIMS: Parenteral nutrition is life-saving for patients with severe intestinal failure. Line-related blood stream infection is the most frequent complication and strategies have been developed to sterilize central lines. Nevertheless, failures of attempted sterilization are not well understood. METHODS: 19 ports were explanted from 19 patients receiving parenteral nutrition because of port-related blood stream infection and failed sterilization, defined as a) recurrence of the same organism after a recent sterilization attempt <90 days), b) recovery of the causative organism after 10 days of proper antibiotic therapy or c) insufficient clinical improvement. Port chambers were opened and swabs were examined by culture. RESULTS: Pathogens resembled those typically found in successfully treated line-related blood stream infection. Despite proper therapy for a median of 6.5 days the same pathogen was recovered from 18/19 chambers. In 9/19 chambers visible debris were found, from which the pathogen could be cultured. CONCLUSIONS: Infected debris or infected biofilms in the chamber are the reason for failure to sterilize a port. Lock techniques, single lumen tunneled catheters or in certain settings the exchange of only the port chamber may be approaches to prevent, circumvent or treat failures of attempted sterilization of an infected port system.

Activity and PI3-kinase dependent trafficking of the intestinal anion exchanger downregulated in adenoma depend on its PDZ interaction and on lipid rafts.

The Cl/HCO(3) exchanger downregulated in adenoma (DRA) mediates electroneutral NaCl absorption in the intestine together with the apical Na/H exchanger NHE3. Lipid rafts (LR) modulate transport activity and are involved in phosphatidylinositol 3-kinase (PI3-kinase)-dependent trafficking of NHE3. Although DRA and NHE3 interact via PDZ adaptor proteins of the NHERF family, the role of LR and PDZ proteins in the regulation of DRA is unknown. We examined the association of DRA with LR using the nonionic detergent Triton X-100. DRA cofractionated with LR independently of its PDZ binding motif. Furthermore, DRA interacts with PDZK1, E3KARP, and IKEPP in LR, although their localization within lipid rafts is independent of DRA. Disruption of LR integrity resulted in the disappearance of DRA from LR, in a decrease of its surface expression and in a reduction of its activity. In HEK cells the inhibition of DRA by LR disruption was entirely dependent on the presence of the PDZ interaction motif. In addition, in Caco-2/BBE cells the inhibition by LR disruption was more pronounced in wild-type DRA than in mutated DRA (DRA-ETKFminus; lacking the PDZ binding motif)-expressing cells. Inhibition of PI3-kinase decreased the activity and the cell surface expression of wild-type DRA but not of DRA-ETKFminus; the partitioning into LR was unaffected. Furthermore, simultaneous inhibition of PI3-kinase and disruption of LR did not further decrease DRA activity and cell surface expression compared with LR disruption only. These results suggest that the activity of DRA depends on its LR association, on its PDZ interaction, and on PI3-kinase activity.

[Cystic fibrosis in adults]. / Mukoviszidose im Erwachsenenalter.

Cystic fibrosis (CF) is a common autosomal-recessive inherited disease, which often results in premature death. Due to treatment advances, life expectancy has however continuously improved in recent years. Currently about half of all patients are adults. There are also "atypical" variants of CF with symptoms occurring in late adulthood. CF is caused by a mutation in the gene coding for a chloride ion channel, known as the cystic fibrosis transmembrane conductance regulator (CFTR). This mutation results in abnormally viscous mucosal secretions, leading to multi-organ disease with particular emphasis in the respiratory and digestive tracts. Impaired mucociliary clearance results in bacterial colonization of the airways (e. g. Pseudomonas aeruginosa) and consequently in chronic pulmonary inflammation, inevitably leading to progressive bronchiectasis and combined ventilatory disorders. Typical acute complications are infective exacerbations - the most frequent cause of death in cystic fibrosis - along with allergic bronchopulmonary aspergillosis, haemoptyses and pneumothoraces. Involvement of the gastrointestinal tract generally manifests as exo- and later endocrine pancreatic insufficiency with diabetes mellitus, malabsorption and sometimes biliary liver cirrhosis. Typical acute complications are pancreatitis and ileus. The article describes epidemiology and pathophysiology of CF and focuses on the signs and symptoms, as well as the diagnostic and multi-modal therapeutic strategies used in adult patients.

After Intestinal Transplantation Kidney Function Is Impaired by Downregulation of Epithelial Ion Transporters in the Ileum.

BACKGROUND: Intestinal transplantation is a treatment option for intestinal failure. Although nephrotoxic medication after transplantation is a major cause for posttransplant renal insufficiency, it remains unclear why kidney dysfunction is particularly frequent after intestinal transplantation. METHODS: This study analyzed messenger RNA expression of NHE3, DRA, and CFTR in 404 biopsies obtained between day 2 and 1508 from the terminal ileum of 10 adult intestinal transplant recipients. RESULTS: The time courses of immunosuppression and glomerular filtration rate were correlated. In the first posttransplant year, expression of NHE3 and DRA, which mediate NaCl absorption, was diminished to a greater degree than that of CFTR, which mediates chloride secretion. Reduced NHE3 and DRA expression was associated with high tacrolimus trough levels. Titration of tacrolimus to low levels by year 2 was paralleled by partially restored NHE3 and DRA expression. In cell culture experiments, similar effects of tacrolimus on transporter expression were detected. In patients, both reduced tacrolimus levels and recovery of NHE3 and DRA expression were associated with stabilization of renal function. CONCLUSIONS: Our data strongly suggest that tacrolimus impairs absorption of NaCl and water from the transplanted ileum, leading to volume depletion and impaired renal function. This may be reversible by reduction of tacrolimus to lower levels without increased rates of rejection or chronic graft failure.

Expression and function of Na+HCO3- cotransporters in the gastrointestinal tract.

The stomach, duodenum, colon, and pancreas secrete HCO3- ions into the lumen. Although the importance of HCO3- secretion for the maintenance of mucosal integrity, a normal digestion, and the reabsorption of Cl- has been well established, the molecular nature of the apical and basolateral HCO3- transporting proteins has remained largely unknown. Functional studies have suggested that a Na+HCO3- cotransport system, similar but not identical to the well-characterized Na+HCO3- cotransporter in the basolateral membrane of the kidney proximal tubule, is present in duodenal and colonic enterocytes, pancreatic ducts cells, and gastric cells and involved in HCO3- uptake from the interstitium. This report describes our work towards understanding the molecular nature, cellular origin, and functional relevance of the Na+HCO3- cotransporter(s) in the stomach and intestine and reviews work by others on the function and localization of Na+HCO3- cotransport processes in the gastrointestinal tract.

Short-term regulation of NHE3 by EGF and protein kinase C but not protein kinase A involves vesicle trafficking in epithelial cells and fibroblasts.

NHE3 is an intestinal epithelial isoform Na+/H+ exchanger that is present in the brush border of small intestinal, colonic, and gallbladder Na(+)-absorbing epithelial cells. NHE3 is acutely up- and downregulated in response to some G protein-linked receptors, tyrosine kinase receptors, and protein kinases when studied in intact ileum, when stably expressed in PS120 fibroblasts, and in the few studies reported in the human colon cancer cell line Caco-2. In most cases this is due to changes in Vmax of NHE3, although in response to cAMP and squalamine there are also changes in the K'(H+)i of the exchanger. The mechanism of the Vmax regulation as shown by cell surface biotinylation and confocal microscopy in Caco-2 cells and biotinylation in PS120 cells involves changes in the amount of NHE3 on the plasma membrane. In addition, in some cases there are also changes in turnover number of the exchanger. In some cases, the change in amount of NHE3 in the plasma membrane is associated with a change in the amount of plasma membrane. A combination of biochemical studies and transport/inhibitor studies in intact ileum and Caco-2 cells demonstrated that the increase in brush border Na+/H+ exchange caused by acute exposure to EGF was mediated by PI 3-kinase. PI 3-kinase was also involved in FGF stimulation of NHE3 expressed in fibroblasts. Thus, NHE3 is another example of a transport protein that is acutely regulated in part by changing the amount of the transporter on the plasma membrane by a process that appears to involve vesicle trafficking and also to involve changes in turnover number.

Genotoxic effects of crude juices from Brassica vegetables and juices and extracts from phytopharmaceutical preparations and spices of cruciferous plants origin in bacterial and mammalian cells.

Crude juices of eight Brassica vegetables as well as juices and extracts of spices and phytopharmaceutical preparations from cruciferous vegetables were tested for induction of point mutations in Salmonella TA98 and TA100, repairable DNA damage in E.coli K-12 cells and clastogenic effects in mammalian cells. In bacterial assays, all juices caused genotoxic effects in the absence of metabolic activation, the ranking order being: Brussels sprouts > white cabbage > cauliflower > green cabbage > kohlrabi > broccoli > turnip > black radish. In experiments with mammalian cells, six juices induced structural chromosome aberrations. Brussels sprouts, white and green cabbage caused the strongest effects (800 microliters of juice induced a 5-fold increase over the background). In sister chromatid exchange assays, positive results were measured as well, but the effects were less pronounced. With all juices the genotoxic effects seen in mammalian cells were paralleled by a pronounced decrease in cell viability. Column fractionation experiments showed that 70-80% of the total genotoxic activity of the juices is found in the fraction which contains isothiocyanates and other breakdown products of glucosinolates, whereas phenolics and flavonoids contributed to a lesser extent to the overall effects. On the basis of these findings, and considering the negative results obtained with non-cruciferous vegetables (tomato, carrot and green pepper), it seems likely that the genotoxic effects of the juices are due to specific constituents of cruciferous plants such as glucosinolates and/or their breakdown products, in particular, isothiocyanates, which we found previously to be potent genotoxins in bacterial and mammalian cells. Finally, spices (mustards and horse radish paste) and phytopharmaceutical preparations were tested in bacterial assays. Mustards and horse radish caused very weak effects while most of the pharmaceutical preparations gave negative results, except cabbage tablets, which caused a strong and dose dependent induction of his revertants in Salmonella TA100. The present findings clearly indicate that cruciferous vegetables contain DNA damaging constituents. These observations are in contrast to earlier findings, which emphasized the antimutagenic effects of vegetable juices and also raise the question whether greatly increased consumption of Brassica vegetables or their concentrated constituents as a means for cancer prevention is indeed recommendable.

Detection of mutagenic activity in textiles with Salmonella typhimurium.

A hundred and ninety-six textile samples were tested in a modified version of the Salmonella/microsome assay for release of mutagenic contaminants. As heat sterilization of the samples can result in reduction of mutagenic activity, tests were performed with streptomycin resistant derivatives of Salmonella tester strains TA98 and TA100. Textile samples were preincubated in buffered saline (PBS), DMSO or ethanol. Subsequently, the fabrics were placed on streptomycin supplemented selective agar plates. In total, 18 samples (9.2%) exerted mutagenic activity. DMSO was the most effective solvent (15 positives) followed by ethanol (9 positive samples) and PBS (7 positives). Most fabrics (16) caused mutagenic effects only upon metabolic activation with liver S9 mix. Chemical analysis indicates that the positive results obtained with PBS are not due to release of histidine or formaldehyde. Three directly active samples gave negative results in strain TA98NR which is devoid of classical nitroreductase. With one exception all other textiles were negative in strain TA98/1,8-DNP6 (which lacks O-acetyltransferase). These findings indicate that nitroaromatics and amines might be responsible for the mutagenic effects of the textiles.

A computer program for the simulation of colony-like proliferation of fibroblasts and their cell cycle behavior.

Several models have been constructed which describe the population dynamics of fibroblast cultures by means of their collective behavior. In contrast, we present here the first example of a program which also allows a topographical simulation of colony-like growth. Starting with one cell, each clonal population consists of single 'intelligent' cells running through a fictive cell cycle. Within the cycle several parameters (position of daughter cells, duration of cycle, etc.) are randomly chosen within given intervals, thus leading to an extremely large number of different clones which can be simulated. Therefore, an experimental-like procedure is possible. Although not being a true reflection of the cell cycle, the model is in good accordance with the clonal growth of fibroblasts. Our model has several aims, for example the application to prenatal diagnosis and the appearance of aberrant clones (in vivo versus in vitro occurrence of chromosome abnormalities in amniotic fluid cell clones). However, it seems to be well suited to check theories related to cellular senescence in vitro, and in particular the commitment theory.

[Short bowel syndrome in Germany. Estimated prevalence and standard of care]. / Das Kurzdarmsyndrom in Deutschland. Geschätzte Prävalenz und Versorgungssituation.

OBJECTIVE: As data about prevalence and standard of care in short bowel syndrome (SBS) are not available for Germany, this study estimated the prevalence and assessed the medical infrastructure to potentially improve care of SBS patients. METHODS: In a validated approach for prevalence estimation in rare diseases, a randomized census of 478 size-stratified hospitals with surgical, internal medicine and pediatric departments was conducted to estimate SBS prevalence. The number of SBS patients, specialized outpatient clinics and caregiver expertise were assessed. RESULTS: The response rate was 85 % of randomized hospitals (405/478). Strata-derived estimation yielded a total of 2,808 SBS patients in Germany for 2011/2012 (95 % CI: 1750.3865), translating into a prevalence estimation for 34/million inhabitants (95 % CI: 21.47). Overall expertise in SBS treatment was only rated "satisfactory" by most caregivers. While 86 specialized outpatient clinics were identified, there was no central registry to access these resources. CONCLUSION: Short bowel syndrome, with a newly estimated prevalence of 34/million inhabitants is not a very rare medical condition in Germany. The interdisciplinary approach needed for optimal care for SBS patients would be greatly facilitated by a central registry.

Enantioselective analysis of R- and S-propafenone in plasma by HPLC applying column switching and liquid-liquid extraction.

Two HPLC methods are described for the enantioselective analysis of R- and S-propafenone in plasma. In a column switching approach, centrifuged plasma was injected onto a silica-based strong acid cation-exchanger and the fraction containing propafenone was switched on-line onto an enantioselective Chiralcel column for separation of the enantiomers. In another approach, propafenone was extracted from plasma by liquid-liquid extraction at pH 11.4. The extracted components were transferred into aqueous medium and separated on a Chiralcel ODR. Both methods were validated and showed comparable performance. Within-day and between-day precision was better than 5% for both methods. Linear calibration functions were obtained (r(2)>0.999), and the limit of detection for each enantiomer was 0.2 microg/mL for column switching and 0.55 microg/mL for liquid-liquid extraction. The analysis methods were applied for the determination of the effect of physical exercise on the enantiomeric ratio of R- and S-propafenone in plasma of healthy volunteers. During exercise, the concentration of both enantiomers reached a maximum, followed by a significant decrease during recovery.

The emerging role of PDZ adapter proteins for regulation of intestinal ion transport.

In the gastrointestinal tract, CFTR, in conjunction with one or several members of the SLC26 anion exchanger family, mediates electrogenic Cl- and HCO3- secretion. Na+/H+ exchanger isoform NHE3, on the other hand, coupled to one or several of the SLC26 isoforms, mediates electroneutral NaCl absorption. The agonist-induced activation of anion secretion and inhibition of salt absorption causes secretory diarrhea. Current dogma sees the formation of a multiprotein complex of transport proteins, postsynaptic density-95/discs large/zonula occludens-1 (PDZ) adapter proteins, anchoring proteins, the cytoskeleton, and the involved protein kinases as one crucial step in the regulation of these transport processes. Data obtained in heterologous expression studies suggest an important role of these PDZ adapter proteins in trafficking, endocytic recycling, and membrane retention of the respective transmembrane proteins. This article reviews recent advances in our understanding of the role of the PDZ adapter proteins NHERF, E3KARP, PDZK1, IKEPP (NHERF-1 to NHERF-4), CAL, and Shank-2 that bind to CFTR, NHE3, and the intestinal SLC26 members in the regulation of intestinal fluid transport. Current concepts are mostly derived from heterologous expression studies and studies on their role in organ physiology are still in infancy. Recently, however, PDZ adapter protein-deficient mice and organ-specific cell lines have become available, and the first results suggest a more cell-type and possibly signal-specific role of these adapter proteins. This opens the potential for drug development targeted to PDZ domain interactions, which is, in theory, one of the most efficient antidiarrheal strategies.

Rapid method for the isolation of hydroxylysylpyridinoline and lysylpyridinoline from concentrated bone hydrolysates by liquid chromatographic techniques.

A rapid method for the isolation of hydroxylysylpyridinoline and lysylyridinoline from bone by liquid chromatographic methods is described. Decalcified bone is hydrolysed in 7 M hydrochloric acid. After evaporation of the acid, the high molecular mass and dark coloured degradation products are removed by adsorption on non-polar adsorbents. The pyridinolines are separated from the majority of the amino acids by adsorption on cellulose. Separation of HP and LP is performed either by cation-exchange chromatography or by reversed-phase ion-pair chromatography. The pyridinoline containing fractions are desalted by size-exclusion chromatography. The progress of the hydrolytic cleavage of collagen and the optimal parameters for purification and separation were examined. As a result the existing method allows the isolation of high amounts of pyridinolines with low amounts of adsorbents and chemicals within a short time.

Intracellular pH-regulating ion transport mechanisms in parietal cell basolateral membrane vesicles.

Na(+)-H+ and Cl(-)-base exchangers on the parietal cell have been demonstrated by several authors. Controversy exists concerning a basolateral Na(+)-HCO3- cotransporter in the parietal cell. To clarify this issue, we prepared highly enriched basolateral membrane (BLM) and apical-tubulovesicular membrane (to serve as negative controls) vesicles from rabbit fundic mucosa. Na(+)-H+ exchange was demonstrated by measuring pH gradient-driven amiloride-sensitive 22Na+ uptake and Na+ gradient-driven proton uptake into voltage-clamped BLM but not into apical-tubulovesicular vesicles. Anion exchange was demonstrated by measuring 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-inhibitable influx of 36Cl- into Cl(-)- or HCO3(-)-loaded voltage-clamped BLM vesicles. Na(+)-HCO3- cotransport was assessed by comparing HCO3(-)-driven 22Na uptake with uptake driven by an identical pH gradient. No significant difference was found between 22Na uptake in the presence and absence of HCO3-; 1 mM amiloride inhibited 22Na uptake > 90% in both conditions, whereas 2 mM DIDS had no effect. In BLM vesicles prepared from rabbit renal cortex, however, a HCO3- gradient stimulated 22Na uptake much more than an equivalent pH gradient, and DIDS inhibited this HCO3- gradient-driven 22Na uptake. This indicates that our experimental setup was suitable to detect a Na(+)-HCO3- cotransporter if present. Our data suggest that the parietal cell BLM contains Na(+)-H+ exchangers and Cl(-)-HCO3- exchangers but no Na(+)-HCO3- cotransporter.

Assay of neopterin in serum by means of two-dimensional high-performance liquid chromatography with automated column switching using three retention mechanisms.

An automated two-dimensional HPLC method for the determination of neopterin in serum is described. Neopterin is separated from proteins on a short octadecylsilica column by size exclusion and from the majority of the other serum components by adsorption. The fraction containing neopterin is transferred by column switching to a solvent-generated cation-exchange column using dodecylsulfonic acid as surface activator. Parameters influencing the separation performance and sensitivity of the fluorescence detection are discussed. The efficiency of the cleaning of the first column was optimized. The method was validated. It achieves a precision of 1% (R.S.D.) and a detection limit of about 0.3 nmol/l. The accuracy is nearly 100%. The method allows a high sample throughput, requiring 15 min per sample.