Transport of p-aminohippuric acid by plasma membrane vesicles isolated from rat kidney cortex

Summary Basal-lateral plasma membrane vesicles and brush border membrane vesicles were isolated from rat kidney cortex and the uptake of p-amino-hippuric acid (PAH) into these vesicles was studied by Millipore filtration techniques.Both membrane preparations take up PAH into an osmotically reactive intravesicular space. The transport across the brush border membrane seems to involve only simple diffusion whereas in the basal-lateral plasma membrane in addition a specific transport system exists which is inhibited competitively by probenecid. The apparent affinity of this transport system for PAH is 5.4×10^–4 M and for probenecid 5.4×10^–5 M.PAH uptake into basal-lateral plasma membrane vésicles is influenced by alteration of the membrane potential. Maneuvers which render the intravesicular space more positive-as for example replacement of chloride by sulfate in the presence of a sodium gradient directed into the vesicles and addition of valinomycin in the presence of a potassium gradient directed into the vesicles-stimulate the uptake of PAH. Replacement of a sodium chloride gradient by a sodium thiocyanate gradient reduces the uptake probably by reducing the inside positive membrane potential.In the absence of salt gradients anion replacement and replacement of sodium by potassium does not affect PAH transport by basal-lateral plasma membranes.These results suggest that in isolated basal-lateral membranes transfer of PAH across the membrane is accompanied by a transfer of negative charge. They furthermore provide no evidence for the existence of a sodium-PAH cotransport system in this membrane preparation.Part of these data have been presented on the Spring Meeting of the German Physiological Society in Bochum, 1975 [Pflügers Arch., Suppl.355, 99 (1975)].     

Simultaneous determination of inulin and p-aminohippuric acid in plasma and urine by reversed-phase high-performance liquid chromatography

A simple, accurate and sensitive high-performance liquid chromatographic method with UV detection was carried out to measure simultaneously plasma and urine concentrations of both p-aminohippuric acid and inulin. Following a simplified acid hydrolysis of the sample, the separation was carried out in 4 min using a C18 reversed-phase column with a flow-rate of 1 ml/min, and monitoring the absorbance at 280 nm. Within the investigated concentration ranges of inulin (0.1-3.2 mg/ml) and p-aminohippuric acid (0.0097-0.3 mg/ml), good linearity (r>0.99) was obtained. Within-run RSD ranged from 2.9 to 6.1% and between-run RSD ranged from 6.4 to 10%. Analytical recoveries were 101-112%, with little differences between plasma and urine samples. The detection limit was 1 microg/ml for all the analytes studied. This method might be ideal for renal function studies where a rapid and reproducible assessment of both renal glomerular filtration rate and blood flow-rate is required.     

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.     

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.     

An amino acid transporter involved in gastric acid secretion

Gastric acid secretion is regulated by a variety of stimuli, in particular histamine and acetyl choline. In addition, dietary factors such as the acute intake of a protein-rich diet and the subsequent increase in serum amino acids can stimulate gastric acid secretion only through partially characterized pathways. Recently, we described in mouse stomach parietal cells the expression of the system L heteromeric amino acid transporter comprised of the LAT2-4F2hc dimer. Here we address the potential role of the system L amino acid transporter in gastric acid secretion by parietal cells in freshly isolated rat gastric glands. RT-PCR, western blotting and immunohistochemistry confirmed the expression of 4F2-LAT2 amino acid transporters in rat parietal cells. In addition, mRNA was detected for the B⁰AT1, ASCT2, and ATB(0+) amino acid transporters. Intracellular pH measurements in parietal cells showed histamine-induced and omeprazole-sensitive H⁺-extrusion which was enhanced by about 50% in the presence of glutamine or cysteine (1 mM), two substrates of system L amino acid transporters. BCH, a non-metabolizable substrate and a competitive inhibitor of system L amino acid transport, abolished the stimulation of acid secretion by glutamine or cysteine suggesting that this stimulation required the uptake of amino acids by system L. In the absence of histamine glutamine also stimulated H⁺-extrusion, whereas glutamate did not. Also, phenylalanine was effective in stimulating H⁺/K⁺-ATPase activity. Glutamine did not increase intracellular Ca²⁺ levels indicating that it did not act via the recently described amino acid modulated Ca²⁺-sensing receptor. These data suggest a novel role for heterodimeric amino acid transporters and may elucidate a pathway by which protein-rich diets stimulate gastric acid secretion.P. Kirchhoff and M.H. Dave contributed equally to this study and therefore share first authorship     

Improved method for simultaneous determination of ascorbic acid and dehydroascorbic acid, isoascorbic acid and dehydroisoascorbic acid in food and biological samples.

The total vitamin C amount in different food and plasma samples was determined by a dual detection system, after HPLC separation, with direct detection of ascorbic acid and indirect fluorimetric detection of dehydroascorbic acid after a post-column O-phenyldiamine derivatisation. The two active forms of vitamin C and their D-isomers were separated within 10 min. The repeatability was determined by measurement of several fruits and vegetables and ranged from 0.3 to 1.9% (relative standard deviation) for vitamin C. The reproducibility, based on double determinations, ranged from 1.9 to 3.6% for vitamin C, depending on the matrix. The reproducibility, based on several determinations of reference materials, ranged from 2.4 to 3.7% for ascorbic acid and from 4.3 to 5.8% for dehydroascorbic acid, again depending on the matrix.     

Antagonism of sulfadoxine and pyrimethamine antimalarial activity in vitro by p-aminobenzoic acid, p-aminobenzoylglutamic acid and folic acid

The activity of pyrimethamine and sulfadoxine against two strains of Plasmodium falciparum has been studied in vitro by a radioisotopic technique. Low level antagonism of pyrimethamine resulted from the inclusion of p-aminobenzoic acid, p-aminobenzoylglutamic acid or folic acid in the test medium. Sulfadoxine activity was antagonised slightly by p-aminobenzoic but not by p-aminobenzoylglutamic acid, and antagonised markedly by folic acid at concentrations above 4 X 10(-8) M. At 10(-7) M folic acid, a concentration lower than that of normal RPMI medium 1640, sulfadoxine activity was reduced 7000 to 9000-fold in comparison with controls. These results are of importance in terms of the utilisation of folates by P. falciparum, the susceptibility of the parasite to antifolate drugs and the in vitro determination of parasite susceptibility.     

Heats of mixing of polyriboadenylic acid and polyribouridylic acid in aqueous NaCl solution

The heats of mixing of polyriboadenylic acid (Poly A) and polyribouridylic acid (Poly U) in various proportions by changing the mole ratio n = [Poly U]/[Poly A] in 0,001, 0,1, and 0,5 molar NaCl solutions containing 0,1 molar tris-HCl buffer solution were measured. Constant values for the heats of mixing of the (Poly A+n Poly U) system are found for n ≤ 1 and n ≥ 2. From the results of UV and CD spectra, it may be considered that the mixing of Poly A and n (Poly U) leads to the formation of Poly(A+U) for n ≤ 1, and to that of Poly(A+2U) for n ≥ 2, even at [NaCl] = 0,001 mol/l. The enthalpies of formation of Poly(A+U) and that of Poly(A+2U) depend on the concentration of NaCl. This dependence may be attributed to the differences in conformation of Poly(A+U) in NaCl solution.     

Microstructure of styrene-methacrylic acid and styrene-acrylic acid copolymers, 4. 1H NMR study of phenyl rings in methylated styrenemethacrylic acid and styrene-acrylic acid copolymers

The sequence distribution in methacrylic acid-styrene and acrylic acid-styrene copolymers of various composition prepared in benzene and emusion were studied by using the aromatic proton region in the ¹H NMR spectra of methylated copolymers. A digital computer was used to resolve overlapping peaks, and one Lorentzian and two Gaussian curves were fitted for their approximation. The study of random copolymers shows that styrene sequences as small as two and three units are contained. The run numbers and number-average sequence lengths of styrene calculated for emulsion methacrylic acid-styrene and acrylic acid-styrene copolymers show that these copolymers do not have long sequences of styrene units.     

Cyclopolycondensations. III. Solution polymerization of aromatic diaminodicarboxylic acid in polyphosphoric acid

Polyamide amino acids of high molecular weight were prepared by the self-polycondensation of aromatic diaminodicarboxylic acids in polyphosphoric acid. Polyamide amino acids thus obtained were converted to the fully aromatic polymer containing benzoxazinone rings by the cyclodehydration in polyphosphoric acid. The resulting aromatic polymer shows excellent thermal stability up to 450°C in nitrogen. The fully aromatic heterocyclic polymers were characterized by good thermal stability and the molecular weight attained was high enough to exhibit film-forming properties for the products.