Urinary fluorescent metabolite O-aminohippuric acid is a useful biomarker for lung cancer detection.

INTRODUCTION: Urine contains diagnostically important metabolites that can act as natural fluorophores. However, whether these fluorescent metabolites can be used in lung cancer diagnosis is unknown. OBJECTIVES: This study was conducted to determine whether fluorescent urinary metabolites could be useful biomarkers for lung cancer detection. METHODS: A total of 46 lung cancer patients and 185 volunteers without cancer were evaluated between November 2013 and November 2014. Samples of the first urine of the day were collected from lung cancer patients and diagnosed at the Hamamatsu University School of Medicine and the Hamamatsu Medical Center prior to cancer treatment, and from volunteers without cancer at the Hamamatsu Medical Imaging Center. Fluorescent urinary metabolites were screened by high-performance liquid chromatography and select effective fluorescent substances for distinguishing cancer from non-cancer status. RESULTS: The fraction of patients at each stage of cancer severity were: 41.3% stage I, 8.7% stage II, 19.6% stage III, and 30.4% stage IV. A robust predictive biomarker for lung cancer was selected by the multivariate logistic analysis of fluorescent metabolites and identified to be O-aminohippuric acid (OAH). The area under the curve (AUC) data for OAH was 0.837 (95% CI 0.769-0.898, P < 0.001). CONCLUSION: We identified a fluorescent urinary metabolite that can predict lung cancer. OAH exceeds the AUC (0.817) of lung cancer detection by AminoIndex® cancer screening, can be analyzed non-invasively without additional sample processing, and may be a valuable addition to existing lung cancer prediction models.

Simultaneous determination of p-aminobenzoic acid and its metabolites in the urine of volunteers, treated with p-aminobenzoic acid sunscreen formulation.

p-Aminobenzoic acid (PABA) and its metabolites (p-aminohippuric acid, p-acetamidobenzoic acid, and p-acetamidohippuric acid) were detected using high-performance liquid chromatography with an electrochemical (carbon paste) detector (HPLC-ECD). For direct current (dc) mode, with the current at a constant potential, and measurements with suitable experimental parameters, a linear concentration from 0.125 to 1.80 microg/ml was found. The detection limit was approximately 2.0 ng/ml. A carbon paste coulometric detector was used to demonstrate that PABA and its metabolites are electrochemically oxidized in acidic media, and to determine, by analyzing human urine, the percutaneous absorption of PABA and its metabolites. Findings using HPLC-ECD and HPLC with an ultraviolet detector (HPLC-UV) were comparable.

Simple HPLC-UV method for determination of iohexol, iothalamate, p-aminohippuric acid and n-acetyl-p-aminohippuric acid in human plasma and urine with ERPF, GFR and ERPF/GFR ratio determination using colorimetric analysis.

A simple high-performance liquid chromatographic (HPLC) method was developed for the simultaneous determination of iohexol, iothalamate, p-aminohippuric acid (PAH) and n-acetyl-p-aminohippuric acid (n-acetyl-PAH) in human plasma and urine. A C(18) column at a flow rate of 1 ml/min with an aqueous mobile phase of trifluoroacetic acid (0.1% TFA in deionized water (pH 2.2), v/v) and methanol gradient was used for component separation. The plasma and urine assay demonstrated linearity from 10 to 50 microg/ml for iohexol and iothalamate, 5 to 40 microg/ml for PAH and 2.5 to 40 microg/ml for n-acetyl-PAH. The HPLC plasma and urine results obtained for PAH were used to calculate the subject kidney effective renal plasma flow (ERPF) and the iohexol results were used to calculate the subject kidney glomerular filtration rate (GFR). The HPLC results for PAH were then compared to an alternative colorimetric method for analyzing PAH to determine if subject metabolism (acetylation) of PAH affected the ERPF results obtained using the colorimetric method, the subsequent ERPF/GFR ratio and clinical impression of subject patient kidney function. The method was utilized in several different clinical studies evaluating the effect of kidney function from medications (phase IV evaluations) marketed for patients with cardiovascular disease.

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.

Characteristics of renal p-aminohippurate and urate excretion in rabbits.

Urate and p-aminohippurate (PAH) excretion was studied in New Zealand white rabbits to determine the extent to which the renal tubular handling of these organic anions by intact kidneys paralleled observations made in single isolated perfused tubules in vitro. At low plasma concentrations (less than 60 mumol/L) net reabsorption of urate was observed in the majority of animals; as plasma urate concentration was increased by infusing lithium urate, net urate secretion was demonstrated, the fractional excretion of urate (FE urate) reaching 270%, and net urate secretion (filtered urate minus urinary concentration of urate times volume flow rate) 1.8 mumol/min at mean plasma urate levels 200 and 310 mumol/L. A maximal urate secretion rate was not demonstrated. Probenecid (250 mumol/kg) reduced FE urate to 27%, thereby revealing concealed reabsorption despite high plasma urate levels. By contrast, net secretion of PAH at all plasma levels was achieved by infusing NaPAH. The highest secretory rate was 38.6 mumol/min at plasma PAH levels between 1000 and 1750 mumol/L. Probenecid reduced fractional excretion of PAH (FEPAH) from 796% to 265%, no evidence of PAH reabsorption being found after inhibition of secretion with probenecid. At comparable plasma levels (220 mumol/L), net PAH secretion exceeded net urate secretion (reflected by the decrement in urate and PAH excretion caused by probenecid) by a factor of 5.3:1. These experiments indicate that urate and PAH are filtered and secreted by the rabbit kidney; however, only urate is significantly reabsorbed over the range of plasma organic anion levels examined. The configuration of the relation between plasma levels and net secretion rates and the greater secretory rate for PAH for urate in the living rabbit are consonant with the characteristics of PAH and urate transport determined previously in studies of isolated perfused proximal tubules.

The correlation between certain tryptophan metabolites and the N-nitrosamine content in the urine of bilharzial bladder cancer patients.

The present work is an up-to-date approach to study the correlation between the excretion pattern of tryptophan metabolites along the kynurenine pathway (after loading with 2 gm. L-tryptophan), and the N-nitrosamine content in urine of bilharzial bladder cancer patients. The control group was composed of healthy subjects who had no reported history of S. haematobium infection and no current bacterial cystitis. The N-nitrosamine content was determined by the colorimetric method of Eisebrand and Preussmann (1970). It was demonstrated that 64 per cent of the patients metabolized the tryptophan load abnormally and the others metabolized it almost normally. Moreover, the N-nitrosamines were present in 43 per cent of controls and 93 per cent of patients have these derivatives in higher values. The presence of an inverse correlation between certain tryptophan metabolites, shown previously to be bladder carcinogens, and the N-nitrosamine content, especially after loading, was interpreted in view of the possible conversion of some tryptophan metabolites into N-nitrosamines either under endovesical conditions or during the execution of the colorimetric determination of these compounds. Therefore, thorough investigation is urgently needed to study the origin of these urinary N-nitrosamines. Moreover, improved method(s) for their colorimetric determination are also urgently needed.

PAH excretion in two species of cancroid crab Cancer irroratus and C. borealis.

To determine the characteristics of p-aminohippuric acid (PAH) excretion in Cancer irroratus and C. borealis, crabs were given a single injection of radiolabeled PAH and polyethylene glycol (PEG), and serum, urine, tissue, and seawater concentrations of each were measured over the next 6 days. In both species, PEG clearances averaged 5-10 ml serum/day; serum PAH concentrations fell exponentially after a 1-day equilibration period. In C. irroratus, urine PAH rose over the first 2 days and then fell; during days 2-6, mean urine PAH averaged two to four times mean serum PAH. In C. borealis, urine-to-serum ratios for PAH were 20-40 during days 2-6. PAH-to-PEG clearance ratios exceeded unity in both species, with ratios in C. borealis about three to six times those in C. irroratus. Tissue distribution data showed that C. irroratus midgut gland accumulated up to 50% of injected PAH, with gland-to-serum concentration ratios of 15-45; in this species, stomach fluid-to-serum PAH concentration ratios averaged 87, and nonurinary routes accounted for more than half of excreted PAH. In C. borealis, midgut gland-to-serum ratios never exceeded 2, and the urinary routes accounted for all excreted PAH. Thus, in C. irroratus, injected PAH accumulated in the midgut gland, with slow excretion through renal and nonrenal routes; in C. borealis, PAH remained in hemolymph, and only the renal route of excretion was utilized.

Effects of indomethacin and naproxen on renal p-amino-hippurate (PAH) excretion in rats during postnatal development.

Effects of the cyclooxygenase inhibitors indomethacin and naproxen on renal PAH excretion were studied in volume-expanded and sodium-loaded, conscious rats of different ages. Indomethacin and naproxen reduce renal PAH excretion in 5- and 10-day-old rats but not in rats of older ages. Findings can be explained by a decrease in glomerular filtration rate. In addition, a competitive inhibition of PAH transport by the organic acids indomethacin and naproxen must be mentioned. Until now it is not known whether or not there is a direct or indirect influence of prostaglandins on processes involved in tubular PAH transport.

[Inhibition of the renal excretion of PAH by probenecid homologs]. / Hemmung der renalen PAH-Ausscheidung durch Probenecid-Homologe.

The authors studied the effects of probenecid and five of its homologues on the renal excretion of p-amino-hippuric acid (PAH). All the compounds under study inhibited the excretion of PAH. Probenecid and its homologues were injected 15 min before the administration of PAH. With all the substances tested, the inhibition of the excretion of PAH was most marked during the first 30 min of the diuresis experiment. The extent of efficiency increases within the homologous series up to the diethyl compound; after that, the inhibitory effect decreases with the increase in chain length. In the dosage range under study, the probenecid homologues show linear dose-response relationships. With due regard to toxicity and efficiency, the authors conclude from the results obtained that the diethyl compound is the most potent substance; probenecid itself is less efficient, being twice as toxic.

[Behavior of some functional parameters of acute incomplete drainage-impeded kidney in animal experiments]. / Das Verhalten einiger Funktionsparameter der akut inkomplett abflussbehinderten Niere im Tierexperiment.

The 131-hippuran clearance was determined preoperatively in 24 female Alsatian dogs. At three times (days 1, 7 and 28) after antireflux grafting operation, PAH, inulin, and 131I-hippuran clearances of the two kidneys were determined separately on both sides with a radioisotope nephrogram. There was a postoperative functional restriction of the operated or contralateral kidney at no time and for none of the clearance substances. However, there was a significant increase of the tubulosecretory partial function of both kidneys on the 1st postoperative day. The single findings of the radioisotope nephrograms of interest are discussed.

[Tryptophan metabolism in patients with vitiligo]. / Studi sul metabolismo del triptofano in pazienti con vitiligine.

Our previous research showed that tryptophan is an important precursor in the biogenesis of melanins. Therefore, with the purpose of observing whether a relationship exists between tryptophan metabolism and diseases characterized by an altered process of skin pigmentation in man, we studied the metabolism of this aminoacid along the kynurenine pathway in 29 vitiliginous patients (11 males and 18 females) and in 21 control subjects (11 males and 10 females) by determining 10 urinary metabolites after an oral loading of 50 mg/kg body weight L-tryptophan. The mean total excretion of the metabolites in patients resulted similar to that of the controls. However, considering the individual metabolites one can observe a decreased excretion of 3-hydroxykynurenine, o-aminohippuric acid and 3-hydroxyanthranilic acid and an increased excretion of xanthurenic acid and of its 8-methyl ether in the group of vitiliginous patients in respect to the controls. These results seem to indicate a decreased formation of nicotinic acid from tryptophan. Moreover, in relation to the fact that 3-hydroxykynurenine could be the metabolite through which tryptophan is involved in melanin biosynthesis, this study supports the hypothesis of a connection of tryptophan metabolism with the lack of pigmentation in vitiligo.

[Stimulation of the renal excretion of p-aminohippuric acid by probenecid homologues (author's transl)]. / Stimulation der renalen PAH-Ausscheidung durch Probenecid-Homologe.

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.

Urinary tryptophan metabolites and cancer of the bladder in humans.

Urinary excretion of six aromatic amine metabolites of tryptophan was compared between a group of 30 patients with bladder cancer and a group of 33 controls selected from the general population of the study area. Measurements were based on a single overnight urine specimen collected at home by each subject, without a "loading" does of L-tryptophan. For each metabolite, both the distribution and the mean amount excreted were nearly identical for cases and controls. The small cases-control differences in mean values were virtually unchanged by adjustment for age, sex, and smoking status. Generally, excretion levels were higher for females than for males. Excretion levels also tended to be higher for older than for younger subjects and for smokers compared to nonsmokers. However, most of these differences also were small.

Influence of protein biosynthesis inhibitors on stimulated renal p-aminohippurate excretion in rats.

The renal excretion of p-aminohippurate (PAH) can be stimulated by repeated administration of cyclopenthiazide (5 mg/100 g b.w., i.p. for 3 days, twice daily). The reason for this seems to be an increased renal tubular transport capacity for weak organic acids. Inhibitors of protein biosynthesis as azauracil, actinomycin D, neomycin and cycloheximide influence the cyclopenthiazide stimulated p-aminohippurate excretion. Azauracil (5 mg/100 g b.w.), an inhibitor of replication is effective in the same way as inhibitors acting on transcription and translation (actinomycin D 7.5-60 micrograms/100 g b.w.; neomycin 5-10 mg) 100 g b.w.; cycloheximide 0.02-0.05 mg/100 g b.w., i.p. for 4 days, once daily). Probably these substances inhibit the de novo synthesis of carrier proteins, increased by stimulation of renal tubular transport processes.

Stimulation of renal excretion of p-aminohippurate (PAH) after unilateral nephrectomy in adult and ageing rats.

Unilateral nephrectomy (UNX) is followed by a significant decrease of excreted amount of PAH in rats. In 105 and 240-day-old rats, we characterized the time course of restitution of the PAH transport process., Furthermore, we studied whether or not the regeneration of kidney function can be accelerated by repeated administration of cyclopenthiazide. After stimulation of tubular transport of PAH by repeated administration of cyclopenthiazide, the loss of one kidney after UNX can be compensated more rapidly as in nephrectomized rats without pretreatment. In 105-day-old rats the regeneration and the extent of stimulation are more marked than in 240-day-old rats.

[Is the inulin (and PAH) clearance test obsolete in renal functional diagnosis?]. / Ist die Inulin (und PAH-) Clearance in der renalen Funktionsdiagnostik überholt?

1. It could be confirmed that the endogenic creatinine clearance overestimates the GFR. This overestimation increases with the restriction of the glomerular filtration. 2. By the biphasic pump-regulated permanent infusion of inulin and PAH a constant equilibrium was reached and the clearance was calculated according to the classical formula (output method) as well as from the addition of the test substance (input method) and compared. 3. The comparison of the two clearance techniques results in significantly higher value for inulin as well as for PAH in the input technique. 4. By clearance equations the relation of input and output clearance can be made. The correlation coefficient for the different investigation techniques for the estimation of the inulin clearance was 0.83, for the PAH-clearance 0.88.