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Simultaneous determination of p-aminohippuric acid, acetyl-p-aminohippuric acid and iothalamate in human plasma and urine by high-performance liquid chromatography.

A sensitive and specific high-performance liquid chromatographic assay was developed for the simultaneous determination of p-aminohippuric acid (PAH), acetyl-p-aminohippuric acid (aPAH), and iothalamate in human plasma and urine. Plasma samples were prepared by protein precipitation with acetonitrile followed by evaporation, reconstitution in mobile phase, and injection onto a C18 reversed-phase column. Urine samples were diluted with 3 volumes of mobile phase prior to injection. Column effluent was monitored by UV detection at 254 nm. The lower limits of quantification in plasma were 0.5 mg/l for PAH and aPAH, and 1.0 mg/l for iothalamate. The within-day and between-day coefficients of variation in plasma and urine were < or =7.8% for all analytes. This method is well suited for renal function studies using iothalamate and PAH, whether administered as a bolus dose or by continuous infusion, to measure glomerular filtration rate and effective renal plasma flow, respectively.

Acetonitriles↗

Paired ion reversed-phase HPLC assay for the determination of iothalamic acid and para aminohippuric acid in urine.

A paired ion reversed-phase high performance liquid chromatographic method for simultaneous determination of iothalamic acid (Io) and para aminohippuric acid (PAH) in urine is described. The method uses a single internal standard for both drugs. The only sample preparation required is dilution of urine (1:100 or 1:500) with deionized water. The internal standard is added to a small aliquot of the diluted specimen and injected. For HPLC, a C8 column and a mobile phase consisting of potassium phosphate buffer with dodecyl triethylammonium phosphate IP reagent, 25% organic modifier with UV detection at 254 nm was used. Within day and between day variation for the assay were in the range of 1.48-9.46% for iothalamic acid and 1.84-10.36% for para aminohippuric acid for four levels of concentration. Limits of quantitation were 50.0 micrograms ml-1 for iothalamic acid and 75.0 micrograms ml-1 for para aminohippuric acid. Mean recovery was 98.55% for Io and 97.79% for PAH. This isocratic HPLC assay is simple, rapid and relatively inexpensive.

Chromatography, High Pressure Liquid↗

Simultaneous determination of p-aminobenzoic acid, acetyl-p-aminobenzoic acid and p-aminohippuric acid in serum and urine by capillary gas chromatography with use of a nitrogen-phosphorus detector.

In various studies during recent years, the use of p-aminobenzoic acid has been described in screening tests for exocrine pancreatic function. A synthetic three-unit compound N-benzoyl-L-tyrosyl-p-aminobenzoic acid has been administered orally and hydrolysed in the small intestine in the presence of chymotrypsin to N-benzoyl-L-tyrosine and p-aminobenzoic acid. This study describes a convenient procedure in which, after a selective extraction and derivatization with diazomethane, capillary gas chromatography is used combined with nitrogen-sensitive detection. With the proposed procedure, p-aminobenzoic acid and its major metabolites, acetyl-p-aminobenzoic acid and p-aminohippuric acid, can be monitored in serum and in urine samples.

4-Aminobenzoic Acid↗

System identification of the low-dose kinetics of p-aminohippuric acid.

The renal clearance of p-aminohippuric acid, due to tubular secretion in addition to glomerular filtration, can only be determined by kinetic experiments. Maximal information can be gained from observed temporal marker concentration profiles by fitting dynamic mathematical models of the processes involved, such as absorption, distribution, and elimination, to the kinetic data. Thereby the values of the system constants, such as fractional elimination or fractional distribution rates, and their accuracy measures are determined by methods which are based firstly on measured time-dependent data elicited in an individual test object by perturbing inputs and secondly, on mathematical formulations of prior knowledge of the underlying physiological system. Such methods of model adaptation are called system identification. In this context a computer-based method of system identification and error estimation for the system constants of two-compartment models matched a dynamic concentration profiles of p-aminohippuric acid is presented. The method is used of single-injection experiments to demonstrate that such a technique is able to correctly estimate the clearance of p-aminohippuric acid if sufficiently long experimental protocols are chosen, and to ascertain the sufficient length of a protocol for an individual subject. The renal clearance of p-aminohippuric acid is known to exhibit concentration-dependence generally, but to achieve its maximal value when low doses are applied. The present study deals with the low-dose kinetics of p-aminohippuric acid.

Adult↗

Probenecid infusion in mares: effect on para-aminohippuric acid clearance.

Para-aminohippuric acid (PAHA, 0.1 mg/min/kg of body weight) was infused IV into 2 mares, followed by concurrent IV infusion of PAHA and probenecid (0.075, 0.15, 0.25, or 0.35 mg of probenecid/min/kg). Probenecid infusion reduced the clearance of PAHA at serum probenecid concentrations greater than 55 micrograms/ml. At 12-hour intervals, probenecid (in 5 repeated doses - 50, 75, 100, or 200 mg/kg) was administered by gavage to 2 mares. Mean serum probenecid concentration was greater than 55 micrograms/ml for all dosages. At dosages less than 200 mg/kg, accumulation of probenecid in the serum was minimal from the 1st to the 5th dose. At a dosage of 200 mg/kg, probenecid accumulated in the serum from the 1st to the 5th dose. Intragastric administration of 5 doses of probenecid (75 mg/kg) at 12-hour intervals to 6 mares reduced the clearance of PAHA by 50%. Bioavailability of probenecid was 117 and 102% for 2 mares after a single intragastric dose, compared with a single IV dose.

Administration, Oral↗

The intrarenal distribution of tritiated para-aminohippuric acid determined by a modified technique of section freeze-dry radioautography.

Section freeze-dry radioautography has been used to examine the intrarenal distribution of a water-soluble organic acid (para-aminohippuric acid (PAH-3H)) under constant-infusion, steady-state conditions in mouse and rat kidney in vivo. The technique described here has the following advantages: (a) Sectioning and freeze-drying are accomplished in a closed cryostat at temperatures below -40 degrees C; (b) Handling of the section is facilitated by mounting of the section-to-be on adhesive-coated Saran Wrap prior to cutting; (c) Unembedded freeze-dried sections are attached to photographic film at ambient temperature in the dark room; (d) Fixation follows completion of radioautographic exposure and precedes photographic development; (e) Permanent close contact is maintained between tissue and film. Morphologic preservation compared favorably with that obtained by optimal fixation techniques, which, however, permit diffusion. Cellular accumulation of PAH-3H during secretion was demonstrated in the proximal tubule under steady-state conditions in vivo. The cellular concentration of PAH-3H was uniform throughout the length of the proximal tubule in mouse and rat kidney.

Aminohippuric Acids↗

p-Aminohippuric acid transport into brush border vesicles isolated from flounder kidney.

p-Aminohippuric acid (PAH) transport was investigated in brush border vesicles isolated from renal proximal tubules of the winter flounder. Three characteristics of carrier-mediated transport were demonstrated: 1) unlabeled PAH inhibited the uptake of [3H]PAH; 2)[3H]PAH efflux from the vesicles was stimulated in the presence of unlabeled PAH in the extravesicular medium; and 3) PAH influx was inhibited by 2,4-dinitrophenol (DNP) and 4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene (SITS). D-Glucose plus a sodium gradient stimulated PAH uptake, as did a K2SO4 gradient plus valinomycin, suggesting that PAH is transported as an anion. In contrast, PAH uptake into a membrane fraction containing mainly basal-lateral plasma membranes exhibited a larger inhibition by probenecid but a smaller inhibition by unlabeled PAH and SITS. Thus, carrier-mediated transfer of PAH driven by the electrochemical potential difference for PAH is demonstrated in the brush border membrane of the flounder kidney.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

[Extent and duration of drug-induced stimulation of renal excretion of p-aminohippuric acid].

Repeated pretreatment with p-aminohippuric acid (PAH), probenecide, cyclopenthiazide, and phenobarbital stimulates the renal excretion of PAH. For an interval of at least 6 hrs following i.p. application pretreated rats excrete more PAH excretion than the controls. All the drugs studied were found to stimulate renal PAH excretion within a period of 3 hrs by 50-60% of the control value. The required duration of pretreatment varies with the substance used. With cyclopenthiazide, the excretion of PAH is demonstrable for 2-3 weeks. Phenobarbital has a brief stimulating action. Correlations of a binding of drugs to structures of their tubular cell and the length of the stimulating action are discussed.

Aminohippuric Acids↗

Rapid determination of p-aminohippuric acid in serum and urine by high-performance liquid chromatography.

We report a simple and rapid HPLC procedure for measuring p-aminohippuric acid in serum and urine. After deproteinization with acetonitrile and addition of p-aminobenzoic acid as an internal standard, the chromatographic run is performed on a C18 column with the absorbance detector set at 275 nm. The separation is carried out in 10 min at a flow-rate of 1.0 ml/min with a mobile phase composed of 7 mmol/l 1-decanesulfonic acid, pH 3.70, and acetonitrile (82:18, v/v). The relationship between p-aminohippuric acid concentration and the p-aminohippuric acid/internal standard peak area is linear up to 100 microg/ml. Within-run precision measured at three different p-aminohippuric acid concentrations ranges from 1.73 to 1.98% in serum and from 0.72 to 1.32% in urine. Between-run precision varies from 1.80 to 4.06% in serum and from 1.05 to 2.66% in urine. Analytical recovery is between 98.26 and 99.44% in serum and from 99.57 to 100.45% in urine. The method is very simple, sensitive, and requires small volumes of sample for the assay (100 microl). Therefore, it could be a useful tool for the analysis of p-aminohippuric acid in the evaluation of renal plasma flow.

Adult↗

Simple and micro high-performance liquid chromatographic method for simultaneous determination of p-aminohippuric acid and iothalamate in biological fluids.

A simple, rapid and micro high-performance liquid chromatographic method was developed for separate or simultaneous determination of p-aminohippuric acid and iothalamate in plasma and urine using p-aminobenzoic acid as an internal standard. The method involved deproteinizing samples with two volumes of acetonitrile followed by injection of 5 microliters of deproteinized supernatant onto a C18 reversed-phase column. The mobile phase contained 3.5% acetonitrile in 0.04% phosphoric acid and flowed at a rate of 1.5 ml/min. The column effluent was monitored by an ultraviolet detector at 254 nm. Retention times for p-aminohippuric acid, iothalamate and p-aminobenzoic acid were approximately 4.5, 6 and 8 min, respectively. This method requires as little as 5 microliters of sample and can be used to measure accurately down to 1 microgram/ml p-aminohippuric acid and 0.5 microgram/ml iothalamate in plasma samples. The coefficients of variation of the assay with or without the use of internal standard were generally low (below 7%). No interferences from endogenous substances or any drugs tested were found.

4-Aminobenzoic Acid↗

Conventional measurement of renal function utilizing serum creatinine, creatinine clearance, inulin and para-aminohippuric acid clearance.

Inulin and para-aminohippuric acid clearances, determined by the conventional method of continuous intravenous infusion with blood and urine sample collections, are the gold standards for estimating glomerular filtration rate and renal plasma flow, respectively. Creatinine clearance provides a reasonably good estimate of glomerular filtration rate but is still subject to errors in accuracy and precision. However, novel methods employing cimetidine to block renal tubular creatinine secretion hold promise for improving the accuracy of estimates. More importantly, a large (and growing) number of studies have consistently demonstrated that estimating glomerular filtration rate by creatinine clearance calculated from the Cockcroft-Gault formula is better than measuring creatinine clearance with a 24-h urine collection. Until newer, more simple methods are developed, calculating creatinine clearance using fasting serum creatinine level, body weight, age and sex provides a reasonable and clinically useful bedside measure of glomerular filtration rate for the practising clinician.

Creatinine↗

Transport of p-aminohippuric acid, uric acid and glucose in highly purified rabbit renal brush border membranes.

A procedure for preparing highly purified brush border membranes from rabbit kidney cortex using differential and density gradient centrifugation is described. Brush border membranes prepared by this procedure were substantially free of basal-lateral membranes, mitochondria, endoplasmic reticulum and nuclear material as evidenced by an enrichment factor of less than 0.3 for (Na+ + K+)-ATPase, succinate dehydrogenase, NADPH-cytochrome c reductase and DNA. Alkaline phosphatase was enriched ten fold indicating that the membranes were enriched at least 30 fold with respect to other cellular organelles. The yield of brush border membranes was 20%. Transport of D-glucose by the membranes was identical to that previously reported except that the Arrhenius plot for temperature dependence of transport was curvilinear (EA = 11.3--37.6 kcal/mol) rather than biphasic. Transport of p-aminohippuric acid and uric acid were increased by the presence of NaCl, either gradient or preequilibrated. However, no overshoot was obtained in the presence of a NaCl gradient, and KCl and LiCl also produced equivalent stimulation of transport suggesting a nonspecific ionic strength effect. Uptakes of p-aminohippuric acid and uric acid were not saturable, and were increased markedly by reducing the pH from 7.5 to 5.6. Probenecid (1 mM) reduced p-aminohippuric acid and uric acid (50 muM) uptake by 49% and 21%, respectively. We conclude that the uptake of uric acid and p-aminohippuric acid by renal brush border membranes of the rabbit occurs primarily by a simple solubility-diffusion mechanism.

Aminohippuric Acids↗

The change of p-aminohippuric acid disposition kinetics accompanied by growth in mice.

The change of disposition kinetics for p-aminohippuric acid (PAH) was studied following intravenous administration of p-[glycyl-1-14C]aminohippuric acid to 1-day-old, 1-week-old, 3-week-old and 8-week-old mice. The expiratory excretion of 14CO2 in 24 h following the administration was almost negligible in 1-day-old and 1-week-old mice in contrast to 3-week-old (6.8 +/- 1.8%) and 8-week-old (8.8 +/- 1.9%) mice. The ability to metabolize PAH may not be developed in these infant mice. The elimination of blood radioactivity following the administration was considerably delayed in 1-day-old and 1-week-old mice, especially in 1-day-old mice, suggesting that the renal tubular secretory function for PAH might not have been developed in the infant mice. Whole-body autoradiographic data showed that the transfer of PAH from blood to muscle was enhanced in 1-day-old and 1-week-old mice, especially in 1-day-old mice, compared to 3-week-old and 8-week-old mice. The enhanced muscular cell membrane permeability to PAH in 1-day-old and 1-week-old mice was considered to be the most plausible explanation for this result.

Age Factors↗

Technetium-99m labeled p-aminohippuric acid analogues: renal function agents.

A number of p-aminohippuric acid analogues were synthesized in order to develop clinically useful 99mTc-labeled radiopharmaceuticals for evaluation of renal function measurements. Stable 99mTc-labeled complexes were formed at pH 5.7 using a Sn(II) reduction method with all derivatives. The newly synthesized complexes were screened utilizing biodistribution studies in small animals. All complexes were excreted via the GU tract within 60 min post iv administration, with no significant activity in GI tract and liver. The [99mTc]methyl-PAHIDA complex showed optimal biodistribution among these analogues. Further investigation is needed to determine if these derivatives may be used to replace [131I]o-iodohippuric acid for the evaluation of renal function.

Aminohippuric Acids↗

Effect of splanchnicotomy on the renal excretion of para-aminohippuric acid in the anaesthetized dog.

Renal excretion of para-aminohippuric acid (PAH) was studied during PAH loading on unilaterally splanchnicotomized ("denervated") anaesthetized dogs. Urine flow, sodium excretion of denervated kidneys were significantly increased. Below a plasma concentration of 20 mg% there were no differences between intact and denervated kidneys in urinary excretion and in calculated tubular transport of PAH. However, maximum secretion rate on the splanchnicotomized side was significantly decreased (innervated: 34.8, denervated: 25.2 mg/100 ml GFR, respectively). Although both Na reabsorption and PAH secretion are impaired by denervation, the exact mechanism of action of renal sympathectomy is not elucidated as yet.

Aminohippuric Acids↗