SPECIFIC DETERMINATION OF PAH AND ITS N-ACETYL METABOLITE BY HPLC INCREASES THE ACCURACY AND PRECISION OF PAH-CLEARANCE MEASUREMENTS

PAH (N-(4-aminobenzoyl)-glycin) clearance measurements have been used for 50 years in clinical research for the determination of renal plasm a flow. The quantitation of PAH in plasma or urine is generally perfor med by colorimetric method after diazotation reaction. Although straig htforward, the measurements must be corrected for the nonspecific resi dual 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 if affect notably PAH clearances, although NAc-PAH represents 10.2 +/- 2.7% of the PAH e xcreted unchanged in 12 healthy subjects The performance of the HPLC t echnique has been compared with the colorimetric method using urine an d plasma samples collected fr om 12 healthy volunteers following a pri ming dose of PAH followed by a constant rate infusion. Good correlatio ns (r = 0.94 and 0.97 for plasma and urine respectively) are found bet ween the results obtained with both techniques; However, the colorimet ric method gives higher concentrations of PAH in urine while the conce ntrations in plasma are lower than those determined by HPLC. Hence, bo th renal (CLR = UxV/P) and systemic (CLS = R-inf/C-ss) clearances are systematically higher (35.1% resp. 17.8%) with the colorimetric method . The fraction of FAH excreted by the kidney CLR/CLS calculated front HPLC data (n = 143) is, as expected, always < 1 (mean = 0.73 +/- 0.11) , whereas the colorimetric method gives a mean extraction ratio of 0.8 7 +/- 0.13 implying unphysio-logical values (> 1) in some cases. In co nclusion HPLC not only enables the simultaneous quantitation ofPAH and NAc-PAH, but may also provide more accurate and precise PAH clearance measurements.