Separation, identification of uremic middle molecules, and preliminary study on their toxicity

Compounds accumulating in uremic serum with molecular mass from 300 to 5000 Da are called uremic middle molecules (UMMs). In our previous work, two UMM fractions A and B were obtained from uremic sera, urine, and normal urine by gel permeation chromatography (GPC), and six UMMs from subfraction A3 of uremic plasma and normal urine were purified and characterized.

Urine and serum samples from uremic patients and healthy subjects were isolated by GPC, ion exchange chromatography (IEC), and reversed-phase high-performance liquid chromatography (RP-HPLC). Moreover, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) were used to characterize the compounds. The effects of subfraction A3 on renal function were studied in rabbit models with chronic renal failure (CRF).

A compound with molecular weight 1007.94 in subfraction A3 was determined to be an octapeptide by mass spectrometry, with an amino acid sequence of Val-Val-Arg-Gly-Cys-Thr-Trp-Trp. Two CRF rabbits injected with A3 died in 5 days, while the other two CRF rabbits (no injection) survived a few days. By multistep chromatography and MALDI-TOF MS, another 11 endogenous compounds were found not only in the subfraction B9 of uremic sera but also in that of normal urine.

Seventeen endogenous middle molecular compounds were found in fractions A and B of uremic plasma and normal urine, among them an octapeptide with M(W) 1007.94 in subfraction A3. Preliminary experimental results on rabbits indicate that subfraction A3 could accelerate the death of rabbits with CRF.