Human hemofiltrate as a source of circulating bioactive peptides: Determination of amino acids, peptides and proteins

Abstract

Human hemofiltrate (HF) was evaluated regarding its content of free amino acids, proteins, and regulatory peptides. Human HF was obtained from patients with end stage renal disease (ESRD). In contrast to plasma it mainly contains low and middle weight molecules < or = 45 kDa. The content of free amino acids, peptides, and proteins in pooled filtrate was determined by amino acid analysis using ortho-phthaldialdehyde/fluorenyl methyl chloroformate (OPA/FMOC) precolumn derivatization. The total amount of peptides and proteins in human HF is 49.4 mg/L (n = 8). The levels of all free amino acids (230 mg/L) and the concentration of some regulatory peptides like insulin, endothelin, gastrin, vasopressin and angiotensin II were similar compared with blood plasma. The amount of peptides and proteins detected in the filtrate was around 0.07% of total plasma proteins, and consisted mainly of smaller proteins and peptides as shown by size exclusion chromatography (SEC). The presence of large proteins in plasma is reduced by a factor of 1500 after filtration. We conclude that human hemofiltrate is a valuable source for the large-scale extraction of regulatory peptides.

hBD‐1: a novel β‐defensin from human plasma

“…About 3 mg of hBD-1 were isolated from 4800 litres of HF, so hBD-1 is present in nanomolar concentration in HF. Concentrations of small peptides in HF with molecular masses below 10 kDa are the same as in plasma, since they are readily filtered during hemofiltration [17]. Therefore it is likely that plasma concentrations of hBD-1 in patients with renal failure, from whom HF is obtained, are in nanomolar range.…”

“…SSDI 0014-5793(95)00687-7 station (Millipore, Eschborn, Germany) with 6 M HC1 for 1 h at 160°C. Amino acids were analyzed on a 1090L AminoQuant Analyzer (Hewlett Packard, Waldbronn, Germany) using precolumn derivatisation with OPA and Fmoc [17]. Norvaline and sarcosine were used as internal standards at final concentrations of 50 pM.…”

“…HF is obtained from patients with end stage renal disease (ESRD) undergoing hemofiltration for the elimination of uremic toxins [16]. HF quantitatively contains all small peptides present in plasma and can be considered to be a prepurified source of peptides from blood plasma [17]. We started a systematic investigation of peptides from HF [18].…”

Advances in Urinary Proteome Analysis and Biomarker Discovery

“…Spent dialysate and hemofiltrate fluid is an excellent source for proteomic analysis because it contains little albumin and other interfering large proteins (90). In 1994, Forssmann and colleagues (90,91) used an advanced LC-MS approach to identify proteins from hemofiltrate fluid using a "peptide bank" with up to 300 different chromatographic fractions that were prepared from 10,000 L of human hemofiltration fluid.…”

“…Spent dialysate and hemofiltrate fluid is an excellent source for proteomic analysis because it contains little albumin and other interfering large proteins (90). In 1994, Forssmann and colleagues (90,91) used an advanced LC-MS approach to identify proteins from hemofiltrate fluid using a "peptide bank" with up to 300 different chromatographic fractions that were prepared from 10,000 L of human hemofiltration fluid. Several additional peptides with various biochemical functions were isolated (e.g., human peptide hormone guanylin, endostatin and resistin as angiogenesis inhibitors, a proopiomelanocortin-derived peptide with lipolytic activity) (92,93).…”