Can a nutrition intervention improve albumin levels among hemodialysis patients? A pilot study.

OBJECTIVE: To determine the effect of a tailored intervention on albumin levels among hemodialysis patients. DESIGN: Randomized controlled trial. SETTING: Eight freestanding chronic hemodialysis units in northeast Ohio. SUBJECTS: Eighty-three randomly selected adult patients who had been on dialysis for at least 6 months and had a mean albumin <3.7 g/dL (bromcresol green method) or <3.4 g/dL (bromcresol purple method) for the last 3 months. To better elucidate the feasibility and outcomes of the intervention, we selected more intervention than control patients. INTERVENTION: Dietitians of the 52 intervention patients determined whether any of the following potential barriers to adequate protein nutrition were present for each patient: (1) poor knowledge of protein-containing foods, (2) poor appetite, (3) needing help shopping or cooking, (4) low fluid intake, and (5) inadequate dialysis. Depending on the specific barriers present, the dietitians (1) educated patients on protein-containing foods, (2) recommended snacks for which patients had preserved appetite, (3) helped set up social supports, (4) provided recommendations on fluid intake, and/or (5) arranged for improved dialysis. Dietitians of the 31 control patients continued to provide usual care. MAIN OUTCOME MEASURES: Change in albumin after 6 months, stratified as minimal change (less than.25 g/dL increase or decrease), moderate improvement (.25 to.49 g/dL increase), and large improvement (increase of .50 g/dL or more). To examine the role of inflammatory states, we also determined serum C-reactive protein levels at the beginning and end of the trial. RESULTS: Among intervention patients, 29% had a minimal change in albumin, 44% had a moderate improvement, and 27% had a large improvement. Among control patients, 74% had a minimal change in albumin, 19% had a moderate improvement, and 6% had a large improvement (P <.001 for comparison of intervention and control subjects). About 60% of subjects had high baseline C-reactive protein levels (> 10 mg/L). However, there was little relationship between change in albumin and either baseline C-reactive protein levels or changes in C-reactive protein levels (P = .83). CONCLUSION: A nutrition intervention tailored to patient-specific barriers resulted in improved albumin levels even among patients with high C-reactive protein levels. Further work is needed to refine and test this intervention on a larger sample.

Production of stable isotope enriched antimicrobial peptides in Escherichia coli: an application to the production of a 15N-enriched fragment of lactoferrin.

A method is described for the production of recombinant isotopically enriched peptides in E. coli. Peptides are produced in high yield as fusion proteins with ketosteroid isomerase which form insoluble inclusion bodies. This insoluble form allows easy purification, stabilizes the peptide against degradation and prevents bactericidal activity of the peptide. Cyanogen bromide cleavage released peptide which was conjugated with alkylamines to form lipopeptide. An important advantage of this system is that it allows production of peptides that are toxic to bacteria, which we have demonstrated on a dodecapeptide based on residues 21-31 of human bactericidal protein lactoferrin.

Bacterial expression and refolding of different fragments of human CD14.

We have investigated bacterial expression of several fragments of CD14, a human cellular receptor for lipopolysaccharides (LPS). Despite binding of CD14 to the LPS, a vital constituent of bacterial outer membrane, we have succeeded in producing full length recombinant hCD14 in E. coli. High level of production of CD14 resulted in deposits of aggregated CD14 in bacteria in form of inclusion bodies, which made production of this protein possible. We have also produced N-terminal fragments consisting of 134 and 152 residues, which comprise N-terminal domain with 2 and 3 leucine rich repeats, respectively and a fragment that contains only leucine rich repeats. Production of the N-terminal domain consisting of 69 residues could not be detected, probably due to the degradation of the produced protein within the bacteria. Full length CD14 and a fragment of 152 residues from inclusion bodies were refolded, while the 134 residues fragment and the one with 10 leucine-rich repeats could not be refolded. Those results confirm that the minimal folding unit of CD14 must include N-terminal domain and at least 3 leucine rich repeats.

Expression and refolding of functional fragments of the human lipopolysaccharide receptor CD14 in Escherichia coli and Pichia pastoris.

CD14 is a high-affinity cellular receptor specific for bacterial lipopolysaccharides (LPS), present in the bacterial cell wall. Binding of LPS to CD14 initiates the innate component of immune response and triggers a response that can lead to septic shock. In order to provide recombinant protein for the study of LPS-CD14 molecular interactions we have expressed human CD14 in Escherichia coli and Pichia pastoris. In bacteria, the protein was produced in high yield in the form of inclusion bodies. We have optimized the procedure for its refolding and generated correctly folded protein. A procedure to monitor the refolding efficiency by using conformation-specific anti-human CD14 monoclonal antibody has been established. A fragment of 152 amino acids of CD14 which retains the ability to bind LPS has been produced in a methylotrophic yeast, P. pastoris, expression system. The recombinant protein from yeast is glycosylated and secreted into the medium. The CD14 fragment was purified to homogeneity by immunoaffinity chromatography. Recombinant CD14 from both bacteria and yeast bind to LPS.