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

• Published in: Journal of pharmaceutical and biomedical analysis 2001-12, Vol.26 (5), p.687-699
• Main Authors: Savant, Ishani A, Kalis, Michelle, Almoazen, Hassan, Ortiz, Stephan R, AbuTarif, Malaz, Taft, David R
• Format: Article
• Language: English
• Subjects: Aging; Aging - metabolism; Analysis; Animals; Biological and medical sciences; Calibration; Chromatography, High Pressure Liquid; General pharmacology; In Vitro Techniques; Kidney - growth & development; Kidney - physiology; Kidney Function Tests; Male; Medical sciences; p-Aminohippuric Acid - urine; Para-aminohippurate; Perfused rat kidney; Perfusion; Pharmacology. Drug treatments; Rats; Rats, Sprague-Dawley; Reference Standards; Reproducibility of Results; Reversed-phase chromatography; Solutions
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/S0731-7085(01)00440-X

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 μg/ml. Accuracy, precision, reproducibility and specificity of the method was established with coefficients of variation 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 μg/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