Alternative high-performance liquid chromatographic assay for p-aminohippuric acid (PAH)

Effect of aging on PAH excretion in the isolated perfused rat kidney

Ishani A. Savant, Michelle Kalis, Hassan Almoazen, Stephan R. Ortiz, Malaz AbuTarif, David R. Taft

Research output: Contribution to journalArticle

6 Citations (Scopus)

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 μg/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 (r2 > 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<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.

Original languageEnglish (US)
Pages (from-to)687-699
Number of pages13
JournalJournal of Pharmaceutical and Biomedical Analysis
Volume26
Issue number5-6
DOIs
StatePublished - Oct 25 2001

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p-Aminohippuric Acid
Rats
Assays
Aging of materials
Kidney
Liquids
Zinc Sulfate
Plasma flow
Benzoic Acid
Chromatography
Glomerular Filtration Rate
Acetic Acid
Organic solvents
Anions
Flow rate
Renal Plasma Flow
Glucose
Wavelength
Nephrons
Pharmaceutical Preparations

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Pharmaceutical Science
  • Drug Discovery
  • Spectroscopy
  • Clinical Biochemistry

Cite this

Alternative high-performance liquid chromatographic assay for p-aminohippuric acid (PAH) : Effect of aging on PAH excretion in the isolated perfused rat kidney. / Savant, Ishani A.; Kalis, Michelle; Almoazen, Hassan; Ortiz, Stephan R.; AbuTarif, Malaz; Taft, David R.

In: Journal of Pharmaceutical and Biomedical Analysis, Vol. 26, No. 5-6, 25.10.2001, p. 687-699.

Research output: Contribution to journalArticle

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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 μg/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 (r2 > 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<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.",
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