Influence of para-aminohippuric acid analysis on net portal-drained viscera flux of nutrients in pigs.

In nutrition studies, para-aminohippuric acid (PAH) is a marker frequently used to measure blood flow in pigs, which is essential for estimating portal-drained viscera (PDV) flux of nutrients. The aim of this study was to evaluate the PAH analytical method by means of qualimetric statistical procedures to estimate the matrix effect and the accuracy and limits of quantitation of the method. Net PDV flux of nutrients was determined in five multi-catheterized pigs using water, plasma or commercial serum as standard matrix. A proportional systematic error due to matrix effect was found for plasma and serum. Mean recovery was 99.4%, and intra- and inter-day precision of the method was 2.4% and 3.8% relative standard deviation, respectively. The limit of quantification was 0.22 mg PAH/l. Use of water for the PAH standard curves underestimated portal blood flow compared with PAH standards prepared with plasma or commercial serum (706, 954 and 927 ml/min; P<0.05, respectively). Consequently, PDV O2 consumption, glucose and amino acids fluxes were underestimated by 33% (P<0.001). In conclusion, our results stress the importance of using plasma from pigs not infused with PAH or alternatively commercial pig serum to prepare PAH standards to determine blood flow in pigs to avoid underestimation of blood flow.

Comparación de la concentración urinaria de orto-cresol y de ácido hipúrico como biomarcadores de exposición laboral a tolueno / Comparison of urinary concentration of ortocresol and hippuric acid as biomarkers of occupational exposure to toluene

La exposición a tolueno en ambientes laborales puede ser biomonitoreada a través de la concentración de ácido hipúrico en orina. Sin embargo, se sabe que este metabolito puede provenir de fuentes distintas al tolueno, pudiendo causar errores en la estimación de la exposición a tolueno. En este estudio, se compararon los niveles de ácido hipúrico urinario con los de otro metabolito del tolueno, el orto-cresol, con el fin de establecer si éste último es mejor biomarcador de exposición. Se cuantificaron ambos biomarcadores por la técnica de HPLC en muestras de trabajadores de empresas que utilizan tolueno y de personas no expuestas. La validación de la metodología analítica demostró la inestabilidad del ácido hipúrico y que su ensayo de cuantificación no es robusto. La concentración de ácido hipúrico urinaria tuvo una gran variabilidad en las muestras tanto de trabajadores expuestos como del grupo control así como una falta de relación con la concentración ambiental de tolueno, lo que no le permite cumplir con los requerimientos de un biomarcador. Por otro lado, orto-cresol demostró especificidad y menor variabilidad interindividual. Se propone que orto-cresol es una mejor alternativa para el biomonitoreo de exposición laboral a tolueno evitando los falsos positivos que se puedan generar en el biomonitoreo por ácido hipúrico (AU)

Comparison of urinary concentration of ortocresol and hippuric acid as biomarkers of occupational exposure to toluene Occupational exposure to toluene may be evaluated by the quantification of hippuric acid in urine. However, this metabolite is not exclusive to toluene which may lead to bias in exposure assessment. In this study, urinary hippuric acid was compared to urinary ortho-cresol, another toluene metabolite to assess if the latter is a better biomarker of exposure to the solvent. Urine samples from workers exposed to toluene and from a control population were collected and both metabolites were quantified by HPLC. Validation of analytical methods showed that hippuric acid is unstable and that the analytical assay is not robust. Hippuric acid had highly variable concentrations in both workers and control population and was not related to environmental concentrations of the solvent, showing its limited validity as a biomarker. Ortho-cresol, on the other hand, showed specificity and less variability among the individuals tested. We propose that ortho-cresol is a better predictor of occupational exposure to toluene and that it may be used to avoid false positive determinations when hippuric acid is used as biomarker (AU)

Influence of the para-aminohippuric acid analysis method on the net hepatic flux of nutrients in lactating cows.

Para-aminohippuric acid (pAH) is a marker frequently used to measure plasma or blood flow. In sheep studies, it is recognized that its determination must include a deacetylation step to counteract the hepatic acetylation of pAH. Such a procedure is not of common usage in cattle studies although a recent suggestion of hepatic pAH acetylation in dairy cows may have important consequences for hepatic nutrient fluxes. The aims of this study were to evaluate pAH analytical methods according to international procedures of metrology and confirm hepatic acetylation of pAH in dairy cows. The effect of the matrix used to prepare the standard curve was tested, and the influence of the pAH analytical method on blood flows and subsequent net hepatic fluxes of nutrients was determined. For the first objective, accuracy profiles were established and bias, indicators of precision, and limits of quantification were reported for 2 analytical methods (without and with a pAH deacetylation step) using 2 different standard matrices (water and plasma). Second, the net hepatic flux of different nutrients was determined including or not the deacetylation step and preparing the standard curves in plasma using samples collected from 5 multicatheterized lactating Jersey cows. The choice of the matrix had a significant impact on plasma pAH concentrations as illustrated by accuracy profiles. Water matrix decreased (P < 0.01) the slope, y-intercept, and the absorbance at concentration 0 mg pAH/L of the standard curve in both methods (without and with the deacetylation), supporting that standards prepared in water should not be used to analyze plasma samples. Samples collected on cows confirmed hepatic acetylation of pAH across the liver. Deacetylation, performed using plasma as the standard matrix, increased (P < 0.05) plasma pAH concentrations from 18.4, 26, and 23.5 to 21.4, 28.9, and 27.3 mg/L in the artery, portal vein, and hepatic vein, respectively. Deacetylation decreased the hepatic venous and arterial plasma and blood flows (P < 0.05) by 9 and 55%, respectively, modifying the net hepatic flux of acetate, total amino acid, and oxygen by more than 19% (P < 0.05). In conclusion, our results highlight the importance of including a deacetylation step in the pAH analysis method in cattle studies and of using plasma as standard matrix.

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.

Determination of uric acid and p-aminohippuric acid in human saliva and urine using capillary electrophoresis with electrochemical detection: potential application in fast diagnosis of renal disease.

The monitoring of uric acid (UA) and p-aminohippuric acid (PAH) levels in biological samples is routinely carried out in clinical laboratories as an indication of renal disease. With the aim of investigation of the correlation between the trace amounts of UA and PAH in human saliva or urine and renal diseases, we carried out the determination of UA and PAH in human saliva and urine by using capillary electrophoresis with electrochemical detection (CE-ED) in this work. Under the optimum conditions, UA, PAH and three coexisting analytes could be well separated within 21 min at the separation voltage of 14 kV in 80 mmol/L borax running buffer (pH 9.2). Good linear relationship was established between peak current and concentration of analytes over two orders of magnitude with detection limits (S/N = 3) ranged from 5.01 x 10(-7) to 2.00 x 10(-6) mol/L for all analytes. The result shows that this proposed method could be successfully applied for the study on the correlation between the levels of UA and PAH in human saliva and urine and renal diseases, and provide an alternative and convenient method for the fast diagnosis of renal disease.

Segmentary effects on the renal proximal tubule due to hexachloro-1,3-butadiene in rats: biomarkers related to gender.

Renal tissue biomarkers (glutamine synthetase and p-aminohippuric acid uptake) were studied in male and female rats after treatment with hexachloro-1,3-butadiene. Reduced glutathione content also was also determined in liver and kidney. Histopathological examination (light microscopy) was then performed. The aim was to define sex differences in nephrotoxic effects caused by the solvent injected i.p. at 50, 100 and 200 mg kg(-1) dose. The rats were sacrificed 24 and 48 h after treatment; after 24 h a significant (P < 0.05) dose-dependent depletion of liver reduced glutathione was observed in male rats only; after 48 h male and female rats showed a significant (P < 0.05) increase at 50 and 100 mg kg(-1) doses. Reduced glutathione in the kidney was increased in male but not in female rats 24 and 48 h after treatment. Glutamine synthetase activity in renal tissue showed a significant (P < 0.05) dose-dependent decrease 24 and 48 h after treatment in both sexes, but is was significantly (P < 0.05) greater in female rats after 48 h. p-Aminohippuric acid uptake in renal cortical slices appeared significantly (P < 0.05) decreased in both sexes at the higher dose 24 h after treatment but this was significantly (P < 0.05) greater in female rats. A further significant (P < 0.05) impairment was observed after 48 h in males treated with a 200 mg kg(-1) dose. In addition, a slight but significant (P < 0.05) loss of p-aminohippuric acid uptake was observed 48 h after treatment with a 100 mg kg(-1) dose in both sexes. Light microscopy showed that the pars recta of the proximal tubule was mainly affected and tubular damage increased according to dose and time, involving the inner medulla and cortex. In conclusion, female rats show a significantly earlier and higher susceptibility of the kidney to toxic effects of hexachloro-1,3-butadiene.

Tubular segment-specific biomarkers of nephrotoxicity in the rat.

Segment-specific localization of p-aminohippuric acid accumulation and glutamine synthetase activity along the proximal tubule was investigated in kidneys of rats treated with segment-specific nephrotoxicants such as potassium dichromate (pars convoluta) and hexachloro-1:3-butadiene (pars recta). Potassium dichromate and the highest dose (200 mg/kg b.w.) of hexachloro-1:3-butadiene caused a significant, dose-dependent decrease of p-aminohippuric acid uptake in the renal cortical slices 24 and 48 h after the treatment. In contrast, hexachloro-1:3-butadiene and only the highest dose (40 mg/kg b.w.) of potassium dichromate, caused a significant dose-dependent decrease of glutamine synthetase activity in the kidney beginning 24 h after treatment. Finally, potassium dichromate and the highest dose (200 mg/kg b.w.) of hexachloro-1:3-butadiene (48 h after the treatment) caused a significant dose-dependent loss of kidney protein content. The results suggest that p-aminohippuric acid accumulation is localized in the pars convoluta and confirm that glutamine synthetase is in the pars recta of the rat proximal tubule. p-Aminohippuric acid uptake impairment and glutamine synthetase activity loss caused by the highest doses of hexachloro-1:3-butadiene and potassium dichromate, respectively, suggests that high doses of segment-specific chemicals may involve other portions of the proximal tubule; in addition, the decrease of glutamine synthetase activity caused by potassium dichromate may be related to the protein content loss.

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.

Analysis of aromatic DNA adducts in laryngeal biopsies.

Epidemiological studies have confirmed the correlation between tobacco smoking, environmental pollution and the incidence of cancers of the respiratory tract. The occurrence of laryngeal cancer in Poland is relatively high compared to other European countries. Since 1969 the mortality related to larynx cancer appears to be increasing. Tobacco smoke contains an abundance of such carcinogenic compounds as polycyclic aromatic hydrocarbons (PAH), aromatic amines and N-nitrosoamines, which can react with DNA and form adducts. We analyzed aromatic DNA adducts in laryngeal tissues from patients with primary laryngeal, which was confirmed histopathologically to be squamous cell carcinoma. The group consisted of 33 patients (5 women and 28 men). Total laryngectomy was performed in patients. A detergentphenol method was used for DNA isolation. Aromatic DNA adducts were analyzed by a 32P-postlabelling technique with butanol extraction and high performance liquid chromatography. The presence of aromatic DNA adducts was demonstrated in all tissues. Large interindividual differences of DNA adduct levels were seen in each tissue studied. There was a higher mean level of DNA adducts in interarytenoid area non-tumors (51.96/10(8) +/- 91.71 NN) than in non-tumor tissue elsewhere (46.91/10(8) +/- 46.36 NN) and tumor tissue (43.52/10(8) +/- 45.88 NN). Adduct levels were correlated with age, sex, cigarette smoking and TNM stage.

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.

Regional aspects and statistical characterisation of the load with semivolatile organic compounds at remote Austrian forest sites.

Spruce needles and humus layer of 25 remote forest sites spread all over Austria were investigated for their concentrations of PCDD/F, PCB, HCH, HCB, PCP, DDX and PAH. Influences of the sites' location on the detected concentrations have been identified: The north of Austria was characterised by a comparably higher overall pollutant load. In addition, altitudinal aspects were addressed. Between several compounds significant positive correlations have been identified, which were more pronounced for compounds with a stronger causal relationship. Pattern analyses allowed to identify--even for the remote sites--differences between the relative PCDD/F, HCH, DDX and PAH patterns of the sites. Partly, these different patterns were related to significantly higher or lower corresponding absolute concentrations of the sites.

An assessment of indoor air contaminants in buildings with recreational activity.

Indoor air quality measurements were carried out during three concerts and one ice hockey game in three different halls. Gas phase components consisted of CO2, CO, and NO whereas for particulate indicators, measurements of particle mass distributions (0.05-9 microm), particle number distributions (0.75-10 microm), and particle bound polycyclic aromatic hydrocarbons (pPAH) were carried out. The calculated ventilation rates did not meet the ventilation requirements for rooms with occupants who smoke to be perceived as acceptable by 80% of the occupants. Average PM9 (mass of particulate matter with an aerodynamic diameter < 9 microm) concentrations throughout the events ranged from 318 to 2000 microg m(-3). Particle concentrations in the size range < 0.4 microm measured 203-696 microg m(-3), the majority of it being attributed to environmental tobacco smoke (ETS). For particle numbers > 0.75 microm concentrations ranged from 2 x 10(4) to 1.9 x 10(5) particles per l while for pPAH, concentrations from 336 to 990 ng m(-3) were observed. The average event concentrations for the gaseous component CO2 ranged from 1110 to 1700 ppm, for CO 2-3.1 ppm and for NOx 237 ppb. The event to baseline concentration ratios for gaseous components ranged from 1.1 to 4.3 while for particulate indicators generally much greater ratios between 0.7 and 140 were found. Possible health effects inflicted by an exposure based on the measured concentrations of the various parameters are discussed.

Tidal creek and salt marsh sediments in South Carolina coastal estuaries: II. Distribution of organic contaminants.

Twenty-eight tidal creeks along the South Carolina coast were sampled during the summer of 1995 to determine the levels of sediment contamination including organic chemicals (i.e., polycyclic aromatic hydrocarbons [PAHs], polychlorinated biphenyls [PCBs], and DDT and its metabolites) associated with different types and varying levels of watershed development (i.e., industrial/urban, suburban, forested, and salt marsh). Organic analysis utilized high-performance liquid chromatography (HPLC) with fluorescence detection and capillary gas chromatography-ion trap mass spectrometry (GC-ITMS) for PAHs, and gas chromatography with electron capture detection (GC-ECD) for pesticides and PCBs. Results indicated that creeks with industrial/urban watersheds had significantly higher concentrations of PAHs, PCBs, and DDT compared with creeks with suburban and forested (reference) watersheds. The suburban watershed class of creeks had concentrations of half the PAH analytes and the total PCBs which exceeded the concentrations found in the forested watershed class of creeks. The spatial distribution of organic contaminants was evaluated in four of these tidal creek-salt marsh systems representing urban/industrial, suburban, and forested watersheds, from the creek channel to the adjacent uplands. The distribution of organic contaminants within each representative creek was not concordant with the total organic carbon or the clay content of the sediment. The representative industrial/urban creek-marsh system, Diesel Creek, had the highest concentration of PAHs in the creek channel and the highest concentration of PCBs and DDT on the marsh surface, primarily in the upper portion of the system. The representative suburban creek-marsh system, Shem Creek, had elevated levels of both PAHs and PCBs throughout the entire system. This system also had one site with a total PAH concentration of 324,000 ppb and a total DDT concentration that was 20-100 times higher than the other sites. One of the representative forested creek-marsh systems, Rathall Creek, had low levels of the three organic contaminants except for one sampling site that had PAH concentrations a factor of 10 higher than the other sites. The other representative forested creek-marsh system, Long Creek, had low levels of PAHs and PCBs, but elevated levels of DDT were observed, particularly in the upper portion on the marsh surface. The results of this study suggest that (1) anthropogenic alteration of the land cover in the watersheds of tidal creek-salt marsh systems may increase the organic contaminant loadings in the sediment, and (2) tidal creek-salt marsh sediments, particularly in the creek channel, are repositories and potentially conduits of organic contaminants from the upland environment to the deeper estuarine areas.http://link.springer-ny. com/link/service/journals/00244/bibs/37n4p458.html

Effects of inhibitors and substitutes for chloride in lumen on p-aminohippurate transport by isolated perfused rabbit renal proximal tubules.

The transport step for p-aminohippurate (PAH) from cell to lumen across the luminal membrane of rabbit proximal tubules has not been adequately defined. To examine this process more closely, we determined the effects of possible transport inhibitors and substitutes for chloride on PAH secretion in isolated perfused S2 segments of rabbit proximal tubules. The addition of 4-acetamido-4'-isothiocyano-2,2' disulfonic stilbene (10(-4) M) to the perfusate irreversibly inhibited PAH secretion, whereas the addition of probenecid (10(-4) M) to the perfusate reversibly inhibited PAH secretion. PAH secretion was unaffected by thiocyanate replacement of chloride in the luminal perfusate, reversibly inhibited by 15 to 20% by methyl sulfate replacement, and irreversibly inhibited by isethionate replacement. Because the luminal membrane is at least as permeable to thiocyanate as to chloride, less permeable to methyl sulfate, and much less permeable to isethionate, these data suggest that the PAH transport step from cells to lumen does not require chloride in the lumen but does require a highly permeant anion. During inhibition of PAH transport from cells to lumen, PAH uptake across the basolateral membrane was also reduced, suggesting some type of feedback inhibition. The data are compatible with PAH transport across the luminal membrane by an anion exchanger, a potential-driven uniporter, both carriers, or a carrier that can function in both modes.

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.

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.

Pitfalls in measuring inulin and para-amino-hippuric acid clearances.

Various carbohydrates and a variety of widely used medicines interfere with the generally used laboratory methods for determining inulin and para-aminohippuric acid (PAH). When these pitfalls are not recognized, false measurements of inulin and PAH clearances, which represent glomerular filtration rate and renal plasma flow respectively, are obtained. When performing these tests a careful history of dietary habits and oral drug therapy must be taken.

[Measure of splanchnic blood flow by marker dilution: comparison of 4 methods of para-aminohippuric acid determination]. / Mesure des débits splanchniques par dilution de marqueur: comparaison de quatre méthodes de dosage de l'acide para-amino-hippurique.

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.

Acute pulmonary toxicity of urban particulate matter and ozone.

We have investigated the acute lung toxicity of urban particulate matter in interaction with ozone. Rats were exposed for 4 hours to clean air, ozone (0.8 ppm), the urban dust EHC-93 (5 mg/m3 or 50 mg/m3), or ozone in combination with urban dust. The animals were returned to clean air for 32 hours and then injected (intraperitoneally) with [3H]thymidine to label proliferating cells and killed after 90 minutes. The lungs were fixed by inflation, embedded in glycol methacrylate, and processed for light microscopy autoradiography. Cell labeling was low in bronchioles (0.14 +/- 0.04%) and parenchyma (0.13 +/- 0.02%) of air control animals. Inhalation of EHC-93 alone did not induce cell labeling. Ozone alone increased (P < 0.05) cell labeling (bronchioles, 0.42 +/- 0.16%; parenchyma, 0.57 +/- 0.21%), in line with an acute reparative cell proliferation. The effects of ozone were clearly potentiated by co-exposure with either the low (3.31 +/- 0.31%; 0.99 +/- 0.18%) or the high (4.45 +/- 0.51%; 1.47 +/- 0.18%) concentrations of urban dust (ozone X EHC-93, P < 0.05). Cellular changes were most notable in the epithelia of terminal bronchioles and alveolar ducts and did not distribute to the distal parenchyma. Enhanced DNA synthesis indicates that particulate matter from ambient air can exacerbate epithelial lesions in the lungs. This may extend beyond air pollutant interactions, such as to effects of inhaled particles in the lungs of compromised individuals.

High-performance liquid chromatography of the renal blood flow marker p-aminohippuric acid (PAH) and its metabolite N-acetyl PAH improves PAH clearance measurements.

PAH (N-(4-aminobenzoyl)glycin) clearance measurements have been used for 50 years in clinical research for the determination of renal plasma flow. The quantitation of PAH in plasma or urine is generally performed by colorimetric method after diazotation reaction but the measurements must be corrected for the unspecific residual response observed in blank plasma. We have developed a HPLC method to specifically determine PAH and its metabolite NAc-PAH using a gradient elution ion-pair reversed-phase chromatography with UV detection at 273 and 265 nm, respectively. The separations were performed at room temperature on a ChromCart (125 mmx4 mm I.D.) Nucleosil 100-5 microm C18AB cartridge column, using a gradient elution of MeOH-buffer pH 3.9 1:99-->15:85 over 15 min. The pH 3.9 buffered aqueous solution consisted in a mixture of 375 ml sodium citrate-citric acid solution (21.01 g citric acid and 8.0 g NaOH per liter), added up with 2.7 ml H3PO4 85%, 1.0 g of sodium heptanesulfonate and completed ad 1000 ml with ultrapure water. The N-acetyltransferase activity does not seem to notably affect PAH clearances, although NAc-PAH represents 10.2+/-2.7% of PAH excreted unchanged in 12 healthy subjects. The performance of the HPLC and the colorimetric method have been compared using urine and plasma samples collected from healthy volunteers. Good correlations (r=0.94 and 0.97, for plasma and urine, respectively) are found between the results obtained with both techniques. However, the colorimetric method gives higher concentrations of PAH in urine and lower concentrations in plasma than those determined by HPLC. Hence, both renal (ClR) and systemic (Cls) clearances are systematically higher (35.1 and 17.8%, respectively) with the colorimetric method. The fraction of PAH excreted by the kidney ClR/ClS calculated from HPLC data (n=143) is, as expected, always <1 (mean=0.73+/-0.11), whereas the colorimetric method gives a mean extraction ratio of 0.87+/-0.13 implying some unphysiological values (>1). In conclusion, HPLC not only enables the simultaneous quantitation of PAH and NAc-PAH, but may also provide more accurate and precise PAH clearance measurements.