Specific determination of PAH and its N-acetyl metabolite by HPLC increases the accuracy and precision of PAH clearance measurements

Abstract

PAH (N-(4-aminobenzoyl)-glycin) clearance measurements have been used for 50 years in clinical research for the determination of renal plasma flow. The quantitation of PAH in plasma or urine is generally performed by colorimetric method after diazotation reaction. Although straightforward, the measurements must be corrected for the nonspecific residual response observed in blank plasma. We have therefore developed an HPLC method for the specific determination of PAH and its metabolite NAc-PAH using a gradient elution ion-pair reverse-phase chromatography with UV detection. The Nacetyltransferase (NAT-1 or NAT-2 dependent) activity does not seem clinically relevant nor does it affect notably PAH clearances, although NAc-PAH represents 10.2 +/- 2.7% of the PAH excreted unchanged in 12 healthy subjects. The performance of the HPLC technique has been compared with the colorimetric method using urine and plasma samples collected from 12 healthy volunteers following a priming dose of PAH followed by a constant rate infusion. Good correlations (r = 0.94 and 0.97, for plasma and urine respectively) are found between the results obtained with both techniques. However, the colorimetric method gives higher concentrations of PAH in urine while the concentrations in plasma are lower than those determined by HPLC. Hence, both renal (CLR = U x V/P) and systemic (CLS = Rinf/Css) clearances are systematically higher (35.1%, resp. 17.8%) with the colorimetric method. The fraction of PAH excreted by the kidney CLR/CLS calculated from HPLC data (n = 143) is, as expected, always < 1 (mean = 0.73 +/- 0.11), whereas the colorimetric method gives a mean extraction ratio of 0.87 +/- 0.13 implying unphysio-logical values (> 1) in some cases. In conclusion HPLC not only enables the simultaneous quantitation of PAH and NAc-PAH, but may also provide more accurate and precise PAH clearance measurements.