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.

A fluorescence-based protocol for quantifying angiotensin-converting enzyme activity.

The determination of angiotensin-converting enzyme (ACE) activity represents a useful tool in the study of different health pathologies, such as hypertension. This protocol describes a fluorescent assay for measuring ACE activity in vitro with high precision and sensitivity. The method relies on the ability of ACE to hydrolyse the internally quenched fluorescent substrate o-aminobenzoylglycyl-p-nitro-L-phenylalanyl-L-proline. The generation of the fluorescent product o-aminobenzoylglycine can be continuously monitored, preferably using a microtiter-plate fluorometer, though the use of a conventional cuvette fluorometer would also be possible. The method has important advantages with respect to other assays, because it involves only a one-step reagent, is easy to carry out and allows the analysis of an elevated number of samples in shorter times. It can be completed in one and a half hours. In addition, the fact that all reagents are commercially available allows the rapid introduction of the assay into the laboratory.

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.

Mutagenicity and PAH content of airborn particulates and of fallen dusts from two Hungarian towns and emission samples from aluminum reduction and power plants.

Urban air particulates (suspended particles and settling dust), furthermore dust emitted by a Soderberg aluminum reduction plant and a coal burning power plant from an industrial town, Ajka (30,000 inhabitants) were analysed for PAH content (liquid chromatography) and mutagenicity (Salmonella microsome test). Air particulates from Papa--a town of similar size without considerable heavy industry--and corresponding plant emission from Inota, a third town in the study, served as controls. The dust content and the PAH concentration, as well as the mutagenic potency of the air in Ajka were higher than in Papa. Mutagenicity of the airborne particulates showed a clear seasonality with a winter maximum and a summer minimum in both towns. The mutagenic potency of air correlated well with the air BaP and total PAH content in Ajka, but not in Papa. The amounts of extractable organic material and mutagenic potency as calculated for unit quantity of airborne particulate matter was higher in the Papa samples. Similar differences between the two towns were observed in the case of fallen dust, too. On the basis of examination of emitted dust, it can be stated, that in the mutagenicity of urban air, the aluminum plant emission plays a considerably higher role than the power plant emission, which is the main component of air dust pollution in Ajka.

Chemical assay of p-aminohippuric acid simplified by use of dimethylaminocinnamaldehyde in ethanol.

We have developed a new and simple method of p-aminohippuric acid determination by use of dimethylaminocinnamaldehyde (DACA). It differs from previous methods using DACA in that the reaction is carried out in ethanol rather than in dilute acid. This results in deeper and more stable color development. We have used this method successfully to determine effective renal plasma flow in a clinical study of this variable in pregnant women.

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.

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.

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.

[Size distribution of PAH and of lead in atmospheric particles]. / Distribution de hap et du plomb en fonction de la dimension des particules atmosphériques.

An analysis of an atmospheric urban aerosol with regard to the nature and particle size distribution for different locations has been undertaken. Results indicate that 67% of total suspended matter, 87% of lead, and 70--80 % of polycyclic aromatic hydrocarbons (PAH) penetrate the respiratory tracts.

A study of the Bratton and Marshall hydrolysis procedure utilizing high performance liquid chromatography.

Most analytical methods for conjugated aromatic amines require hydrolysis to free the amine group. The rate of conversion of N-acetyl-p-aminohippuric acid to its deacetylated and deglycinated derivatives is directly related to the hydrolysis reaction time and the concentration of hydrochloric acid used in the reaction. Variations in methodology involving either of these hydrolysis parameters produced artifacts which were not detectable by the standard colorimetric procedures. High performance liquid chromatography offers an improved tool for detecting and quantitating the changes produced during hydrolysis.

Permeability of chorion laeve to succinylcholine in vitro.

The simultaneous diffusion of 14C-succinylcholine and p-amino hippurate across human placental tissue in vitro was measured. Column chromatographic purification of the radioactivity followed by paper chromatographic analysis showed little alteration of the succinylcholine during the course of these experiments. Similar chloroform-buffer partition coefficients for the two solutes were measured. Thus in these experiments, the nearly identical transfer rates of these two similar sized, lipid-insoluble compounds indicated that succinylcholine crosses human placental tissue in quantitites compatible with its molecular size and lipophobic nature, and that the diffusion rate is not significantly influenced by its increased molecular charge.

[Renal production and excretion of AMPc after intravenous infusion of salmon calcitonin in man]. / Etude de la production et de l'excrétion rénale d'AMPc après perfusion intraveineuse de calcitonine de Saumon chez l'Homme?29H

Intravenous infusion of salmon calcitonin in man produced an increase in the plasma levels and urinary excretion of cyclic AMP. This study demonstrates a net extraction of cyclic AMP from plasma by the kidneys but salmon calcitonin does not act only on the kidney and stimulates the production of cyclic AMP in extra renal tissues. The excess of cyclic AMP formed is catabolized by the kidneys.

Comparison of hilar and capsular renal lymph.

The sites of origin of renal lymph were studied by analysis of simultaneously collected samples of lymph and plasma. The samples included renal hilar (HL) and capsular lymph (CL), thoracic duct lymph (TD), renal venous (RVP) and arterial plasma (AP), and were analyzed for Na+, Cl-, K+, urea, glucose, inulin, and PAH concentrations. The glucose concentrations of HL (76 mg/100 ml, SE +/- 1.9), and CL (92 mg/100 ml, SE +/- 2.5) were significantly different (P less than 0.01) from that of RVP (86 mg/100 ml, SE +/- 2.2). Concentrations of both inulin and PAH in CL and HL fell between the AP and RVP values. The concentration of inulin in CL (CL/RVP = 1.27) exceeded that in HL (HL/RVP = 1.10), but no such difference was detected for PAH. The Na+ (152 meq/liter) and Cl- (129 meq/liter) concentrations in HL were significantly (P less than 0.01) higher than those in CL (Na+ = 148 meq/liter; Cl- = 120 meq/liter), in TD (Na+ = 146 meq/liter; Cl- = 121 meq/liter), and in RVP (Na+ = 144 meq/liter; Cl- = 114 meq/liter. These differences remained highly significant when the electrolyte concentrations were converted to milliequivalents per kilogram H2O. In contrast, no significant differences were detected between the concentrations of urea in the various fluids sampled. It is concluded that CL and HL do not drain a perfectly homogeneous intrarenal pool, and that they do not equilibrate with RVP or AP within the kidney. The results also indicate that CL and HL derive a small component from tubular reabsorbate; that for CL stemming from the cortex, and that for HL from the deeper cortex and outer medulla.

High K+ effects on PAH transport and permeabilities in isolated snake renal tubules.

Effects of high potassium concentrations on para-aminohippurate (PAH) transport by isolated, perfused snake (Thamnophis spp.) distal-proximal renal tubules were studied. Increasing the potassium concentration in bath from 3 mM TO 10 mM OR 40 MM led to about 50% decrease in net PAH transport from bath to lumen in less than 10 min, but transport still occurred against concentration gradient. Cell water PAH concentration was not significantly depressed in 10 mMpotassium and was nearly double control level in 40 mM potassium. Apparent luminal membrane permeability to PAH, calculated from perfusion studies, averaged about 3.5 x 10(-5) cm sec(-1) in 3 mM potassium, 1.15 x 10(-5) cm sec(-1) in 10 mM potassium, and 0.48 x 10(-5) cm sec(-1) in 40 mM potassium. Apparent peritubular membrane permeability, determined from PAH efflux from tubules with oil-filled lumens averaged about 0.5 x 10(-5) cm sec(-1) in 3 mM potassium, 0.29 x 10(-5) cm sec(-1) in 10 mM potassium, and 0.17 x 10(-5) cm sec(-1) in 40 mM potassium. These data suggest that high potassium concentrations depress transepithelial PAH transport primarily by reducing luminal and peritubular membrane permeabilities. Effect of high potassium on PAH transport was reversed within 20 min after restoration of control potassium concentration.

Renal transport of organic acids and bases in genetically obese mice.

Accumulation of p-aminohippurate (PAH) and N-methylnicotinamide (NMN) by renal cortical slices was used to estimate transport capacity for organic anions and cations, respectively. In a previous study, renal organic anion transport appeared to be selectively depressed in animals rendered obese by high fat feeding. However, the effects of obesity could not be discretely separated from the effects of the 30% fat diet used to produce the obesity. Genetically obese hyperglycemic mice provided a model to determine the effect of obesity on renal transport systems without the complication due to diet. Accumulation of both PAH and NMN was depressed in renal cortical slices from genetically obese mice. Addition of plasma from thin or obese animals increased PAH accumulation by slices from thin animals. Accumulation by slices from obese animals was unaffected by addition of plasma. Oxygen consumption with acetate in the medium was less in kidneys from obese mice than kidneys from thin mice. Thus, in addition to inhibition of transport capacity, renal cortex of genetically obese mice has a biochemical defect that prevents response to stimulators. It is concluded that several renal functions are depressed in the genetically obese hyperglycemic mouse. Whether the depressed function results from the obesity or is concomitant with the gene for obesity is as yet undertermined.