[Determination of "middle molecules" presenting vitamin B12 molecular size in normal and uremic body fluids (author's...

Uremic solutes with the molecular size of vitamin B12 are assumed to be toxic. An analytical method is proposed to detect and separate these solutes in body fluids using two combined techniques: gel filtration on Sephadex G-15 and ion-exchange chromatography on DEAE-Sephadex A-25. The vitamin B12 molecular size has been localized by ultrafiltration through membranes with a defined cut-off. Normal and uremic body fluids (urine, plasma, hemodialysis fluid) have been separated into 9 ultraviolet-absorbing peaks (a to i) by high-speed gel filtration. Peaks b and c present the molecular size of vitamin B12, 10--15 A molecular diameter in pH7 aqueous solution. Peak b, which correlates with uremic neuropathy, is separated into 6 sub-peaks (b1 to b6) by ion-exchange chromatography, sub-peak b4.2 is the only one to correlate with uremic neuropathy. The coefficient of variation in the integrated area of a single peak is 16%. This method gives the chromatographic prolife of the vitamin B12 molecular size from 500 microliter of uremic plasma or 100 microliter of normal urine within one hour.