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PubMed1998

Chromatographic estimation of iothalamate and p-aminohippuric acid to measure glomerular filtration rate and effective renal plasma flow in humans.

Iothalamate (IOT) clearance and p-aminohippuric acid (PAH) clearance are used for estimation of glomerular filtration rate (GFR) and effective renal plasma flow (ERPF). A simple and rapid method is described for simultaneous determination of IOT and PAH in the same chromatogram in the serum and urine of humans. The mobile phase consisted of methanol-50 mM sodium monobasic phosphate with 0.5 mM tetrabutyl ammonium hydrogen sulfate (18:82, v/v), pumped at a rate of 0.8 ml/min on a C18 reversed-phase column. Samples of serum and urine were deproteinized with two volumes of acetonitrile containing the internal standard, p-aminobenzoic acid (PABA). The UV detector was set at 254 nm and peak height ratios of PAH or IOT to PABA were calculated with an integrator. Precision and accuracy were within 15% for both PAH and IOT. The recovery of PAH in urine and serum were 94% and 91%, respectively. For IOT the corresponding recoveries were 93% and 92%, respectively. This method clearly distinguishes acetyl-PAH from PAH and has been validated in healthy volunteers.

Adult↗
PubMed1999

Simultaneous determination of inulin and p-aminohippuric acid (PAH) in human plasma and urine by high-performance liquid chromatography.

Inulin and p-aminohippuric acid (PAH) clearances are used for the estimation of glomerular filtration rate (GFR) and effective renal plasma flow (ERPF). A simple and rapid high-performance liquid chromatography (HPLC) method with UV detection is described for the simultaneous determination of inulin and PAH in the same chromatogram in the plasma and urine of humans. Plasma and urine samples were hydrolyzed with perchloric acid (0.7%) in boiling water. The mobile phase consisted of 0.01 M potassium dihydrogenphosphate with 0.02 M tetramethylammonium chloride and o-phosphoric acid (pH 3)-acetonitrile (94:6, v/v), pumped at a rate of 1.2 ml min-1 on a C8 reversed-phase column. Tannic acid was used as the internal standard and UV detection at 285 nm was employed. The calibration curves were linear over the concentration range of 12.5-100 mg l-1 for inulin and 6.25-50 mg l-1 for PAH with determination coefficients greater than 0.997. The method is accurate (bias < 13%) and reproducible (intra- and inter-day relative standard deviation less than 11%), with a limit of quantitation of 12.5 mg l-1 and 6.25 mg l-1 for inulin and PAH, respectively. Analytical recoveries from urine and plasma were ranged from 81 to 108% for both compounds. This fully validated method, which allows the simultaneous determination of inulin and PAH clearances, is simple, rapid (total run time < 10 min) and requires only a 200 microliters plasma or urine sample.

Chromatography, High Pressure Liquid↗
PubMed1989

[Polyfructosan and para-aminohippuric acid clearance, comparison of 2 methods for determining glomerular filtration and plasma renal flow].

Glomerular Filtration Rate (GFR) and Renal Plasma Flow (RPF) were measured respectively, Polyfructosan and Para-aminohippuric acid clearances, in 13 patients with different degrees of renal insufficiency. Two different methods were used, the standard method and one without urine collection. The mean GFR (66.4 +/- 27.8 vs. 72.2 +/- 19.0 ml/m, r = 0.74) and RPF (431 +/- 204 vs 490 +/- 188 ml/m, r = 0.76) as well as Filtration Fraction (17.7 +/- 8.3 vs. 16.6 +/- 6.6%) were well correlated. These data show that within the normal range of GRF and RPF the method determining only plasma levels is as reliable as the standard method, giving the advantage to be less troublesome for patients and staff, requiring no bladder catheterization.

Adult↗
PubMed1983

[Dose- and age-dependence of the renal tubular transport of p-aminohippuric acid (PAH) in rats after injection of single doses].

The renal excretory capacity for tubularly eliminated foreign substances can be determined with certainty by measuring the excretion of p-aminohippuric acid (PAH). To make superfluous the laborious estimation of the maximum tubular transport capacity in the framework of screening programs, the authors established those doses of PAH the single application of which permits to assess the renal tubular transport capacity of rats of differing ages. Because of its selective renal excretion, it is possible to calculate for PAH a half-time value in urine (t1/2, urine) such as commonly indicated for the serum. Except for extremely high dosages (500 mg PAH/100 g body mass: intraperitoneally), the t1/2, urine value for PAH in adult rats is independent of the dose applied. The t1/2, urine value increases in rats 5 and 10 d of age with increasing PAH doses. In rats of all age-groups, the glomerularly filtered PAH proportion increases with increasing dosage (kinetics in decreasing blood level). In rats 5 and 10 d of age, the proportion of glomerularly filtered PAH in the total amount of excreted PAH is greater than in older rats.

Aging↗
PubMed1987

Comparative study of colorimetric method using diazotization reaction and high-performance liquid chromatographic method in determination of para-aminohippuric acid.

In this study, colorimetric method and high-performance liquid chromatographic (HPLC) method were improved and established, respectively, in order to minimize analytical errors in determination of para-aminohippuric acid (PAH) in rat urine and plasma. In terms of the colorimetric method, an operative step following addition of Tsuda reagent was modified as follows: after the addition of Tsuda reagent, reaction mixture was kept at 40 degrees C for 70 min before spectrophotometry. Linearities were observed both in the higher range of 0 and 2.5 to 12.5 micrograms and in the lower range of 0 and 100 to 1,000 ng per test tube, and its practical detection limit was 100 ng per test tube. In terms of HPLC method, using a reversed-phase column (Nucleosil 5 C18), PAH was separated by a mobile phase of acetonitrile/50 mM KH2PO4 (pH 2.8) = 9/95. Linearities were observed in the higher range of 0 and 10 ng to 2 micrograms and in the lower range of 0 and 1 to 10 ng per injection, and its practical detection limit was 1 ng per injection. These results denote that the above two methods are applicable to routine PAH determination. In addition, our HPLC method is considered to be applicable to microassay of PAH, because its sensitivity is more sensitive and minimization of volume system is more easily achieved as compared with the colorimetric method.

Aminohippuric Acids↗
PubMedDate not supplied

[Measure of splanchnic blood flow by marker dilution: comparison of 4 methods of para-aminohippuric acid determination].

The objective of this study was to determine the influence of the analytical method of para aminohippuric acid (PAH) on splanchnic blood flows, as measured by a dye dilution method in ovines. Four different analytical methods of PAH were compared. They differed in the pretreatment of the samples and in the presence or absence of a deacetylation step to account for the partial acetylation of PAH in the liver (13%). The optimum conditions of deacetylation were defined as 60 min of incubation at 90 degrees C in presence of HCl (5N). The four analytical methods were checked to be linear (from 0 to 35 mg/L), repeatable (CV = 0.70%) and reproducible (CV = 2.1%). It appeared necessary to prepare the standards in the same matrix as that of the samples. The choice of the analytical method was responsible for differences in PAH concentrations and in the absolute values for portal and hepatic blood flows, which could reach 21%. The presence of a deacetylation step with HCl significantly reduced the contribution of the hepatic artery to the total hepatic blood flow (from 24 to 10%). Consequently, this study showed that the nature of the analytical method chosen can highly influence the measurements of splanchnic nutrient fluxes. The recommended analytical method is the one which includes a deacetylation step.

Acetylation↗
PubMed1975

[Stimulation of renal excretion of p-aminohippuric acid by repeated administration of drugs in young and adult rats].

The velocity of renal excretion of p-aminohippuric acid (PAH) could be raised by repeated administration of phenol red, probenecid and penicillin, which are actively transported by the acid carrier into the urine like PAH. These drugs produced an effect in fairly lower doses than PAH. The renal excretion of PAH can be accelerated by repeated pretreatment with the lipid-soluble drugs sulfaclomide, sulfamethoxypyridazine and cyclopenthiazide. It could be demonstrated that the stimulation of renal excretion of PAH by repeated administration of the investigated compounds is less pronounced in young rats than in adult animals.

Aminohippuric Acids↗
PubMed1981

[Stimulation of the renal excretion of p-aminohippuric acid by probenecid homologues (author's transl)].

Probenecid and five of its homologues showed increased lipophilicity with increasing chain length of the substituents. Parallel to this, the toxicity increased about 30 times. All the probenecide homologues under study stimulated the excretion of p-aminohippuric acid (PAH) when applied repeatedly. If a threshold dose is exceeded, an increase of the pretreatment dose will not result in a further increase in PAH excretion. As compared to non-pretreated control animals, the highest possible increase in PAH excretion lies between 40 and 80% independently of the structure of the respective probenecide homologue. Due to their more favourable therapeutic range (LD50 divided by D40-50), the probenecide homologues with shorter chains are better suited to stimulate the excretion of PAH, though the extent of stimulation is the same with all the probenicide homologues under study.

Aminohippuric Acids↗
PubMed1987

Lack of effect of alpha-human atrial natriuretic polypeptide on volume reabsorption and p-aminohippuric acid secretion in the rabbit proximal straight tubule.

To assess a possible direct tubular action of alpha-human atrial natriuretic peptide (alpha-hANP), we studied the effects of alpha-hANP on volume reabsorption (JV), transepithelial voltage (VT), and p-aminohippuric acid (PAH) secretion in the rabbit proximal straight tubules (PST) by in vitro microperfusion technique. In superficial PST (SFPST), addition of alpha-hANP at various concentrations (10(-7), 10(-9), 10(-11) M) to the bath solution did not alter JV significantly. Bath alpha-hANP (10(-7) M) did not change VT in SFPST, either. In juxtamedullary PST (JMPST), addition of alpha-hANP (10(-7) M) to the bath solution changed neither VT nor JV significantly. The alpha-hANP (10(-7) M) in the luminal fluid caused no changes in either VT or JV in SFPST. Furthermore, alpha-hANP (10(-7) M) in the bath did not change the rate of PAH secretion in SFPST. Thus, we could not obtain any evidence for direct tubular action of alpha-hANP in rabbit PST. Accordingly, alpha-hANP may increase solute excretion without directly affecting solutes transport in PST of the rabbit kidney.

Aminohippuric Acids↗
PubMed1984

Technetium-99m-labeled p-aminohippuric acid analog: a new renal agent: concise communication.

A renal agent labeled with Tc-99m and quantitatively secreted by the tubules has been sought for many years. To meet this need, a PAH analog (PAHIDA), has been synthesized by the coupling of p-aminohippuric acid with nitrilotriacetic acid anhydride, using Sn(II) reduction. This yields a stable complex with Tc-99m at a pH of 5.8. The purity and stability of the Tc-99m complex have been established by ITLC and high performance liquid chromatography (HPLC). Urinary excretions of the Tc-99m PAHIDA and I-131 hippurate, determined in mice and rats at different time intervals, are similar. The new compound is clearly excreted but its total clearance is lower than that of hippurate as a result of high protein binding. Rat urine analysis by ITLC and HPLC suggests that the agent excreted is similar to the complex administered to the animals. The Tc-99m-labeled agent is rapidly excreted in urine with no significant extrarenal pathway, thus providing excellent renal scintigrams in a rabbit model. The Tc-99m PAHIDA contains the R-CO-NH-CH2-COOH grouping, analogous to that in hippurate, and consequently may provide the substrate specificity for renal excretion of this new class of agents labeled with technetium-99m.

Aminohippuric Acids↗
PubMed2000

High-performance liquid chromatographic determination of p-aminohippuric acid and iothalamate in human serum and urine: comparison of two sample preparation methods.

A high-performance liquid chromatography method applied to determine p-aminohippuric acid (PAH) and iothalamate (IOT) in serum and urine samples of patients was evaluated according to recovery, reproducibility and linearity utilizing narrow-bore columns. The mobile phase consisted of 0.15 M sodium dihydrogenphosphate with 1.2 mM tetrabutylammonium sulphate, the pH was adjusted to pH 4.6, acetonitrile was added to a final ratio of 95:5 (v/v), the flow-rate was set at 0.3 ml/min. The separation was achieved on a ODS Hypersil column (200 x 2.1 mm I.D.). The UV detector was set at 254 nm. PAH and IOT are used for evaluation of kidney function [effective renal plasma flow (ERPF) and glomerular filtration rate (GFR)]). Under the described chromatographic conditions two sample preparation techniques, ultrafiltration and acetonitrile precipitation were compared. The results demonstrate the accuracy of both methods in evaluation of ERPF and GFR. Due to its cost-effectiveness we recommend the acetonitrile precipitation method in clinical routine.

Chromatography, High Pressure Liquid↗
PubMed2002

Sex differences in p-aminohippuric acid transport in rat kidney: role of membrane fluidity and expression of OAT1.

Sex differences in the kinetic parameters of p-aminohippuric acid (PAH) transport in brush border (BBMV) and basolateral (BLMV) membrane vesicles from kidney cortex have been observed. Membrane fluidity of BBMV was higher in females as compared with male rats as indicated by anisotropy values (0.1897 +/- 0.0010 vs. 0.2003 +/- 0.0014, p < 0.05, for females and males respectively). Membrane fluidity of BLMV were similar in both sexes. Western blot studies revealed that OAT1 protein in female BLMV was present at only 40% of level found in BLMV from male rats. The lower expression of OAT1 in BLMV in association with the higher BBMV fluidity (which may affect the affinity of PAH transporter in this membrane domain) observed in females may be responsible, at least in part, for the gender difference described in renal PAH secretion.

Alkaline Phosphatase↗
PubMed2001

Secretory transport of p-aminohippuric acid across intestinal epithelial cells in Caco-2 cells and isolated intestinal tissue.

The intestinal transport of an organic anion, p-aminohippuric acid (PAH), was studied in Caco-2 cell monolayers and rat intestinal tissue mounted in Ussing chambers. In both experimental methods, PAH exhibited vectorial transport with significantly greater permeability in the secretory direction than the absorptive direction, indicating net secretion. This secretory transport required metabolic energy, but protons or hydroxyl ions were not involved as the driving force. In Caco-2 monolayers, secretory transport of [3H]PAH was decreased, and the intracellular accumulation of PAH was increased with increasing concentration of unlabelled PAH at the basolateral side. Addition of probenecid and genistein at the basolateral side decreased the secretory transport of [3H]PAH; the accumulation was not changed by probenecid, but was increased by genistein. In addition, the initial uptake rate of [3H]PAH from the basolateral side was decreased by both PAH and probenecid, but not by genistein. Therefore, it is suggested that the transport of PAH in Caco-2 cells is regulated by several transporters: a genistein-sensitive transporter on the apical membrane and probenecid-sensitive transporters on both the basolateral and apical membranes. In rat intestinal tissues, the transport rate of PAH showed regional variation (ileum > jejunum > duodenum), suggesting that secretory transporters with high activity exist predominantly in the lower region of the small intestine. The results suggest that PAH transport in both Caco-2 cells and rat intestinal tissues is regulated by multiple transporters on the apical and basolateral membranes, and these transporters have different characteristics.

Animals↗
PubMed1997

Selective brain to blood efflux transport of para-aminohippuric acid across the blood-brain barrier: in vivo evidence by use of the brain efflux index method.

Efflux transport of para-aminohippuric acid (PAH) across the blood-brain barrier (BBB) has been demonstrated by use of the brain efflux index (BEI) method. PAH was eliminated from the ipsilateral cerebrum extensively with an apparent efflux rate constant of 0.0587 (min-1) after microinjection into a cerebral cortex region termed Par2. This efflux transport showed a saturation with the Michaelis constant of approximately 400 microM. No more than 3% dose of PAH and carboxyl-inulin, used as a reference compound showing limited permeability at the BBB, were found in the contralateral cerebrum, cerebellum or cerebrospinal fluid up to 20 min after administration. Under saturated conditions for carrier-mediated efflux of PAH via the blood-cerebrospinal fluid barrier, the BEI value of PAH did not change significantly, which suggested that blood-cerebrospinal fluid barrier was not responsible for the elimination of PAH from the brain after microinjection. No significant metabolism of PAH was demonstrated in the brain for at least 20 min after microinjection, and most of the radioactivity in the ipsilateral and contralateral carotid veins was as the intact form. With the distribution volume of PAH, 0.800 ml/g brain, obtained from the brain slice uptake experiment, the apparent efflux clearance was calculated as 46.9 microl/min/g brain. In addition, the influx clearance of PAH across the BBB determined by the in vivo brain uptake index method was much smaller than the efflux clearance, which demonstrates that BBB transports PAH selectively from the brain to the circulating blood.

Animals↗
PubMed1975

[Accumulation of p-aminohippuric acid and cyclopenthiazide in renal cortex slices from rats of various ages and their dependence on the energy supply].

The accumulation of p-aminohippuric acid (PAH) and cyclopenthiazide, two drugs of acidic character and extremely different physico-chemical properties, was determined in renal cortical slices of rats aged 5, 15, 33, 55, 105, and 240 days. PAH is accumulated in the cortical slices of 5- and 15-day-old animals to a lesser extent than in those of adult rats. Cyclopenthiazide is accumulated in much higher amounts than PAH in all age groups. The age dependence of cyclopenthiazide accumulation is not so pronounced as with PAH accumulation. There is no accumulation of PAH in the cortical slices of all age groups through active tubular transport, when the energy supply is inhibited by means of 2,4-dinitrophenol (DNP) or nitrogen 2 times bubbling (i.e. anaerobic incubation). By subsequent addition of DNP to the incubation medium or subsequent N2 atmosphere bubbling, an already existing PAH accumulation may be completely nullified. Contrary to PAH accumulation, cyclopenthiazide accumulation can be neither prevented nor abolished by inhibiting the energy supply.

Age Factors↗
PubMed1991

[Reproducible determination of the glomerular filtration rate (GFR) and the effective renal plasma flow (ERPF) in conscious rats using inulin and p-aminohippuric acid].

Estimation of the glomerular filtration rate (GFR) and of the effective renal plasma flow (ERPF) by means of the test substances inulin and p-aminohippuric acid according to the steady state clearance measurement during the drop of the plasma level after the initially injection of an depot of the indicators was examined methodologically. The procedure should be repeatable in the same animal under conscious conditions. The results demonstrate that the described method is adequate with regard to accuracy, reproducibility and sensitivity for the estimation of the GFR, whereas limitations apply to calculation of the ERPF.

Animals↗