Separation and quantification of the "middle molecules" in uremia

A modification of a two-stage chromatographic procedure (molecular sieve followed by ion-exchange) to separate potentially toxic uremic "middle molecules" from body fluids has been established. The procedure has an analysis time less than half that previously reported with improved resolution. Elution volumes have been found to be reproducible to within 2% and concentrations (as measured by peak height) to within 10%. Careful attention, however, must be paid to artifacts that may arise from sample preparation, drug therapy, and dialysis conditions. Up to ten identifiable subpeaks were observed in sera, urine, and red cell hemolysate samples following ion-exchange separation of a molecular sieve peak in the middle molecule range (300 to 2000 daltons). Red cell concentrations of five of these moieties were significantly higher than those in serum. Four peaks were not detected at all in red cells, and one peak was not detected in sera or urine samples. In addition, although serum concentrations are elevated in uremia, red cell levels in patients with uremia are comparable with those obtained in normal subjects. If any of these species are subsequently shown to be uremic toxins, then their two-compartment distribution within the body has important ramifications in the choice of an appropriate mathematic model to program optimal dialysis therapy.