We have isolated from pathological sera a bilirubin fraction (delta) that is very tightly, if not covalently, bound to protein, most likely albumin. This delta fraction absorbed at a lambda max of 433 nm in the visible spectrum, between the lambda max of unconjugated (alpha) and that of conjugated (Bc) bilirubin when measured in solutions containing albumin. However, unlike the other bilirubin species, this fraction could not be separated from the proteins in serum by exhaustive ultrafiltration in the presence of caffeine/benzoate solution. In the Jendrassik-Grof diazo procedure for bilirubin analysis, the delta fraction gave a large direct reaction (76-89% of the total reaction). Yet, when relatively hydrophobic azo dyes were formed by reaction of the delta fraction with the diazonium salt of dichloroaniline, only 50% of the dyes were extractable from aqueous solution. On chromatography the rest remained associated with protein. Of the extractable dye, more than 70% was accounted for by two liquid-chromatographic peaks with retentions identical with those of azo dyes formed from unconjugated bilirubin. This delta fraction was not appreciably separated from protein by treatment with strong acid or base, or by prolonged digestion with various enzymes. Finally, in a highly denaturing solvent (urea/mercaptoethanol), this fraction was not dialyzable through a membrane with a 12 000-dalton cutoff.
We describe a "high-performance" liquid chromatographic (HPLC) method for accurately determining creatinine in serum. After prechromatographic precipitation of protein, we performed isocratic ion-exchange chromatography with ultraviolet detection (234 nm). Analytical results showed linearity up to 1770 mumol/L, a detection limit of 22 mumol/L, an average analytical recovery of 101%, and a CV ranging from 3% to 11%. We used certified human serum (National Institute of Standards and Technology), and additional lyophilized serum pools also assayed by definitive isotope-dilution mass spectrometry, to validate the accuracy of the HPLC method. In addition, the isocratic HPLC results showed close agreement with those obtained with a step-gradient HPLC method. We also compared the isocratic HPLC method with alkaline picrate and enzymatic methods. Our findings with samples from nonuremic, uremic, and diabetic ketoacidotic patients confirmed the positive bias previously reported with the alkaline picrate method. Interlaboratory transferability of the method was demonstrated with various commercial instruments and analytical columns. We evaluated column stability and possible interference from endogenous or exogenous compounds. On the basis of our analytical findings, we recommend the isocratic HPLC method as a candidate Reference Method for determining creatinine in serum.
Earlier we described a "high-performance" liquid-chromatographic procedure for separating the four distinct fractions of bilirubin (unconjugated, monoconjugated, diconjugated, and protein-bound) in pathological human serum (J. Chromatogr. 226: 391-402, 1981). We have modified the prechromatography precipitation of the serum globulins required in that method and have measured the bilirubin content of the precipitate spectrophotometrically. On average, the precipitate contained less than 10% of the total bilirubin in the serum samples. Adding the value obtained for the precipitate to that obtained by chromatography for the individual bilirubin fractions gave an estimate of the concentration of the total bilirubin in the sample. For 357 samples from 132 patients, this total value correlated well with that obtained by the Jendrassik-Gróf diazo procedure (slope = 1.00; r = 0.995, linear least-squares fit). The CV for the total and fractional bilirubin measurements was, on average, less than or equal to 5% for pathological sera. Serum sampled at different times from the same patient showed significant changes in the distribution of bilirubin among the four fractions.
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