La. Decosterd et al., SPECIFIC DETERMINATION OF PAH AND ITS N-ACETYL METABOLITE BY HPLC INCREASES THE ACCURACY AND PRECISION OF PAH-CLEARANCE MEASUREMENTS, Renal failure, 20(2), 1998, pp. 311-317
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.