Alternative high-performance liquid chromatographic assay for p-aminohippuric acid (PAH): effect of aging on PAH excretion in the isolated perfused rat kidney

Para-aminohippuric acid (PAH), an indicator of renal plasma flow, is a comm only used marker of organic anion transport by the kidney. An analytical me thod for PAH using HPLC was developed. The method is simple, fast and requi res a minimum amount of organic solvent. Sample preparation involved protei n precipitation with zinc sulfate. Para-amino benzoic acid was utilized as an internal standard (IS). Chromatography was performed using a reversed-ph ase phenyl column with UV detection at a wavelength of 254 nm. Mobile phase consisted of 0.1 M acetic acid and acetonitrile (99:1) at a flow rate of 1 ml/min. The assay was validated over a standard concentration range from I to 25 mug/ml. Accuracy, precision, reproducibility and specificity of the method was established with coefficients of variation < 10%. The method was sensitive and showed linear response in peak height ratio (analyte:IS) ove r the concentration range studied (r(2) > 0.99). The assay was used to stud y the effect of aging on PAH excretion in the isolated perfused rat kidney model. Experiments were conducted in kidneys from young (2-3 months., n = 6 ), adult (6-9 months, n = 5) and aged (12-16 months, n = 3) male Sprague-Da wley rats at an initial drug concentration of 20 mug/ml. Significant differ ences in kidney function (e.g. glomerular filtration rate and glucose reabs orption) were observed in aged kidneys. Despite a 5-fold reduction in glome rular filtration rate, PAH renal clearance (kidney weight-corrected) decrea sed by only 2-fold in aged (2.2 +/- 0.42 ml/min per gram) compared to young (4.6 +/- 0.70 ml/min per gram, P < 0.05) rats. Furthermore. renal excretio n ratio was significantly higher in aged rats (27 +/- 8.0 vs. 15 +/- 5.0, P < 0.05). These preliminary findings challenge the 'Whole Nephron Hypothesi s' that assumes parallel reductions in renal filtration and secretory capac ity secondary to disease or aging. (C) 2001 Elsevier Science B.V. All right s reserved.