Kidney function before, during and after compensatory hypertrophy.

PAH (N-(4-aminobenzoyl)-glycin) clearance measurements have been used for 50 years in clinical research for the determination of renal plasma flow. The quantitation of PAH in plasma or urine is generally performed by colorimetric method after diazotation reaction. Although straightforward, the measurements must be corrected for the nonspecific residual 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 it affect notably PAH clearances, although NAc-PAH represents 10.2 +/- 2.7% of the PAH excreted unchanged in 12 healthy subjects. The performance of the HPLC technique has been compared with the colorimetric method using urine and plasma samples collected from 12 healthy volunteers following a priming dose of PAH followed by a constant rate infusion. Good correlations (r = 0.94 and 0.97, for plasma and urine respectively) are found between the results obtained with both techniques. However, the colorimetric method gives higher concentrations of PAH in urine while the concentrations in plasma are lower than those determined by HPLC. Hence, both renal (CLR = U x V/P) and systemic (CLS = Rinf/Css) clearances are systematically higher (35.1%, resp. 17.8%) with the colorimetric method. The fraction of PAH excreted by the kidney CLR/CLS calculated from HPLC data (n = 143) is, as expected, always < 1 (mean = 0.73 +/- 0.11), whereas the colorimetric method gives a mean extraction ratio of 0.87 +/- 0.13 implying unphysio-logical values (> 1) in some cases. In conclusion HPLC not only enables the simultaneous quantitation of PAH and NAc-PAH, but may also provide more accurate and precise PAH clearance measurements.

Aminohippuric Acids↗

PubMed1997

The cytotoxic effect of paraquat to isolated renal proximal tubular segments from rabbits.

Paraquat (PQ) induces lung, liver and kidney damage. Since PQ mainly is eliminated by the kidney, the kidney damage is of particular importance to the outcome of PQ poisoning. The exact toxic mechanism of PQ is still unclear but it is assumed to involve redox cycling and formation of reactive oxygen species. In this study, further investigations on the toxic mechanism and metabolic effects of PQ were performed using isolated renal proximal tubules from rabbits. Proximal tubules were isolated using a combined iron perfusion and collagenase method. Suspended tubules were incubated for varying periods and concentrations of PQ at 25 or 37 degrees C in Krebs-Ringer phosphate buffer or HCO3-/CO2 buffer. The cytotoxic effect of PQ was evaluated by (1) markers of oxidative stress: status of glutathione (GSH/GSSG) and formation of malondialdehyde (MDA); and (2) markers of tubular metabolism: oxygen consumption (QO2), transport of 14C-p-aminohippuric acid (PAH) and 14C-tetraethylammonium (TEA). Using 0.5 and 5 mM PQ, the GSH/GSSG ratio decreased whereas formation of MDA increased indicating oxidative stress. PQ reduced the accumulation of PAH and TEA, the basal QO2 and the ouabain sensitive QO2 indicating inhibition of the Na/K-ATPase. Nystatin-stimulated QO2 was reduced by PQ, excluding inhibition of Na+ entry as a possible cytotoxic mechanism and suggesting mitochondrial injury. This was confirmed by measuring FCCP-uncoupled QO2. Thus high concentrations of PQ appear to disrupt mitochondrial electron chain transfer resulting in reduction of metabolic functions.

Aminohippuric Acids↗

PubMed1996

Assessment of the accuracy of quantitative urine collection in mink (Mustela vison) using osmotic pumps for continuous release of p-amino-hippuric acid and inulin.

A method is described to assess the accuracy of quantitative collection of urine in small experimental animals using implanted Alzet osmotic pumps for continuous release of specific urinary markers. The nominal pumping rate (10.00 +/- 0.15 microliters/h; mean +/- SEM) of 10 osmotic pumps was verified (9.96 +/- 0.12 microliters/h) in a 10-day in vitro assay in isotonic saline at 39.0 degrees C. Ten adult female mink (1100 +/- 34 g) had a 2-ml osmotic pump implanted intraperitoneally for 7 days while maintained in metabolic cages on a conventional mink diet. In 5 mink the pumps contained [3H]-labelled p-aminohippuric acid (PAH) only. The remaining 5 animals received a pump containing [3H]-PAH and [14C]-labelled inulin. The experiment was well tolerated by all animals. In fed animals, the amount of urine collected per day was not influenced by the osmotic pumps, whereas 24 h of fasting (water allowed) caused a dramatic fall in urinary volume. In 4 consecutive 24-h collections of urine (n = 10 animals) the recovery of [3H]-PAH was 70.8 +/- 3.6% (range: 52.0-87.2%), and urinary plus faecal water (= total) recovery of [3H]-PAH averaged 77.0 +/- 3.7% (range: 60.3%-94.3%). For [14C]-inulin (n = 5 animals) the urinary and total recoveries were 68.4 +/- 2.2% and 77.2 +/- 2.4%, respectively. In urine the 14C to 3H counts-ratio was almost identical to that of the infusion solution, indicating that metabolic decomposition of the markers was negligible. The results indicate that the daily recovery of suitable urinary markers, released by implanted osmotic pumps, provides a reproducible and valid measure of the accuracy achieved in quantitative collection of urine in mink and probably also in other animal species. Hence, this technique may be useful in future studies on animal nutrition and/or drug disposition.

Aminohippuric Acids↗