title: Alternative high-performance liquid chromatographic assay for p-aminohippuric acid (PAH): effect of aging on PAH excretion in the isolated perfused rat kidney.
abstract: Para-aminohippuric acid (PAH), an indicator of renal plasma flow, is a commonly used marker of organic anion transport by the kidney. An analytical method for PAH using HPLC was developed. The method is simple, fast and requires a minimum amount of organic solvent. Sample preparation involved protein precipitation with zinc sulfate. Para-amino benzoic acid was utilized as an internal standard (IS). Chromatography was performed using a reversed-phase 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 1 to 25 microg/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) over the concentration range studied (r(2)>0.99). The assay was used to study 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-Dawley rats at an initial drug concentration of 20 microg/ml. Significant differences in kidney function (e.g. glomerular filtration rate and glucose reabsorption) were observed in aged kidneys. Despite a 5-fold reduction in glomerular filtration rate, PAH renal clearance (kidney weight-corrected) decreased 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 excretion ratio was significantly higher in aged rats (27+/-8.0 vs. 15+/-5.0, P<0.05). These preliminary findings challenge the 'Whole Nephron Hypothesis' that assumes parallel reductions in renal filtration and secretory capacity secondary to disease or aging.
authors.affiliation: Division of Pharmaceutics and Industrial Pharmacy, Long Island University, 1 University Plaza, Brooklyn, New York, NY 11201, USA.
authors.forename: I A
authors.initials: IA
authors.lastname: Savant
authors.forename: M
authors.initials: M
authors.lastname: Kalis
authors.forename: H
authors.initials: H
authors.lastname: Almoazen
authors.forename: S R
authors.initials: SR
authors.lastname: Ortiz
authors.forename: M
authors.initials: M
authors.lastname: AbuTarif
authors.forename: D R
authors.initials: DR
authors.lastname: Taft
mesh_terms: D000375:Aging / Q000378:metabolism*; D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D066298:In Vitro Techniques; D007668:Kidney / Q000254:growth & development* / Q000502:physiology; D007677:Kidney Function Tests; D008297:Male; D010477:Perfusion; D051381:Rats; D017207:Rats, Sprague-Dawley; D012015:Reference Standards; D015203:Reproducibility of Results; D012996:Solutions; D010130:p-Aminohippuric Acid / Q000652:urine*
chemical_list: D012996:Solutions; D010130:p-Aminohippuric Acid