Determination of oxalic acid in urine by high-performance liquid chromatography

A new method for determination of oxalic acid in urine is described. The method encloses sample purification prior to the treatment by High Performance Liquid Chromatography (HPLC). The purification step consists in the passage of acidified urine through Sep-pak C18 cartridge (Waters), followed by the precipitation of the oxalic acid eluted with CaCl2, new dilution of the calcium oxalate precipitate, oxalic acid extraction with diethyl ether and total dryness of the sample. The losses of oxalic acid during this process are evaluated by the addition of oxalic acid (U-14C) before the precipitation step. The dried samples are redissolved in mobile phase (o-H3PO4, 0.05 M) and injected into a HPLC chromatograph, with reversed phase column (Lichrosorb RP-8, Merck). Oxalate peak is detected spectrophotometrically at 220 nm, with a retention time of 3.20 minutes. The method shows a mean recovery value of 98.25%, with an intra-run and between-run values of 5.13 and 8.06 respectively. The oxalic acid measured in urine by this method is 35.52 +/- 9.42 mg/24 h in normal subjects.     

Simultaneous detection of 35S- and 32P-labeled proteins on electrophoretic gels

A new method for the determination of oxalic acid in urine, which does not require isolation of oxalic acid, was developed by derivatizing oxalic acid and separating and quantitating the product by automated liquid chromatography. Oxalic acid in urine was reacted with o-phenylenediamine to form the strongly uv-absorbing compound 2,3-dihydroxyquinoxaline. Isolation and quantitation of this derivative were accomplished using a reverse-phase C₈ column, 5% methanol in 0.1 m ammonium acetate buffer (pH 6.6) as eluant, and absorption at 314 nm. The method was linear from 1 to 151 μg oxalic acid/ml of sample and the conversion of oxalic acid to the dihydroxyquinoxaline over this concentration range was 94.9%. The precision of duplicates averaged ±1.1%. Analyses of urine before and after treatment with oxalate decarboxylase were employed to differentiate actual urinary oxalic acid from oxalogenic compounds. Under the conditions employed, no urine was found to contain inhibitors of oxalate decarboxylase. No significant contribution to the method was found in a study of 19 potentially interfering urinary constituents. Levels of oxalic acid found in 27 urine samples from patients by this method averaged 71% of levels found using an earlier colorimetric method.     

Determination of plasma oxalic acid by high-pressure liquid chromatography

A new specific and sensitive method for determination of oxalic acid in plasma by High Performance Liquid Chromatography (HPLC) is described. The plasma sample is deproteinized by ultrafiltration. The oxalic acid in the ultrafiltrate is purified by precipitation with CaCl2, new dilution of calcium oxalate precipitate, oxalic acid extraction with diethyl-ether and total dryness of the sample. The losses of oxalic acid during this process are evaluated by the addition of oxalic acid (U-14C) before the precipitation step. The dried samples are redissolved in mobile phase (o-H3PO4, 0.05 M) and injected into a HPLC chromatograph, with reversed phase column (Lichrosorb RP-8, Merck). Oxalate peak is detected spectrophotometrically at 220 nm with a retention time of 3.20 minutes. The method shows a mean recovery value of 82.11, with an intra-run and between-run CV values of 2.54 and 6.95 respectively. The oxalic acid measured in plasma by this method is 291 +/- 89 micrograms/100 ml plasma ultrafiltrate, in 16 normal subjects.     

Excretion of trimethylselenonium ion in human urine

A method to permit isolation and measurement of trimethylselenonium ion [TMSe, (CH3)3Se+] from 1 liter of human urine was developed. The method was based on precipitation of TMSe with ammonium reineckate, preseparation with anion-exchange resin, and final thermal decomposition and collection of the product in HNO3. It was tested for recovery and separation from other selenium moieties present in urine using both in vivo-labeled rat urine and human urine spiked with unlabeled TMSe. Recoveries from the former were in the range 76.8-87.0% (mean +/- SD: 81.8 +/- 3.7%, n = 5), while for the latter they were in the range 72.0-93.0% (mean +/- SD for three occasions (%): 80.9 +/- 5.5, 81.4 +/- 7.8, and 78.9 +/- 1.0). The reliability of the method was tested against an HPLC procedure using in vivo-labeled rat's urine. The mean (+/- SD) percentage of urine radioactivity appearing as TMSe was 36.0 +/- 5.7% for the present and 36.2 +/- 6.6% for the HPLC method. The mean of deviations, as percentage of the HPLC method, was -0.03 +/- 8.8%. The linear regression equation for the two methods was y = -0.805 + 1.029x (r2 = 0.81). Excretion of TMSe was measured in urine samples from several persons (range: 0.18-0.37 micrograms Se/liter; mean +/- SD: 0.26 +/- 0.07, n = 9). One subject consumed three separate doses of unlabeled selenite on alternate days (Day 1, 197 micrograms Se; Day 3, 395; and Day 5, 592). For the first 24 h of each period, TMSe excretions (micrograms Se/24 h) were 0.24, 0.53, and 0.97, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversed-phase liquid chromatography method to determine COL-3, a matrix metalloproteinase inhibitor, in biological samples

A reversed-phase high-performance liquid chromatographic (HPLC) method with ultraviolet (UV) detection was developed and validated for the quantification of 6-deoxy-6-demethyl-4-dedimethylamino-tetracycline (COL-3), a matrix metalloproteinase (MMPs) inhibitor, in rat serum. This simple, sensitive, rapid and reproducible assay involved a preliminary serum deproteinization by adding a mixture of acetonitrile-methanol-0.5 M oxalic acid (70:20:10 (v/v)), as the combined precipitant and metal blocking agent, into serum samples (2:1 (v/v)). An aliquot (20 microl) of the supernatant was injected into the HPLC system linked to a Waters XTerra RP(18) column (150 mm x 4.6 mm i.d., particle size 5 microm). The compound was eluted by a mixture of acetonitrile-methanol-0.01 M oxalic acid (40:10:50 (v/v), pH 2.00), as the mobile phase, and detected at the wavelength of 350 nm. The total running time was 10 min. The low and high concentration calibration curves were linear in the range of 50-1200 ng/ml and 1200-12,000 ng/ml, respectively. The intra- and inter-day coefficients of variation at three quality control concentrations of 100, 1200, and 12,000 ng/ml were all less than 6%, while the percent error ranged from -2.5 to 6.6%. The limit of quantitation (LOQ) for COL-3 in serum was 50 ng/ml. This assay was successfully employed to study the serum concentration-time profiles of COL-3 after its intravenous and oral administration in rats. The method with some minor modifications in sample pretreatment was also applicable to the determination of the concentrations of COL-3 in rat bile, urine and feces.     

Rapid and convenient determination of oxalic acid employing a novel oxalate biosensor based on oxalate oxidase and SIRE technology

A new method for rapid determination of oxalic acid was developed using oxalate oxidase and a biosensor based on SIRE (sensors based on injection of the recognition element) technology. The method was selective, simple, fast, and cheap compared with other present detection systems for oxalate. The total analysis time for each assay was 2-9 min. A linear range was observed between 0 and 5 mM when the reaction conditions were 30 degrees C and 60 s. The linear range and upper limit for concentration determination could be increased to 25 mM by shortening the reaction time. The lower limit of detection in standard solutions, 20 microM, could be achieved by means of modification of the reaction conditions, namely increasing the temperature and the reaction time. The biosensor method was compared with a conventional commercially available colorimetric method with respect to the determination of oxalic acid in urine samples. The urine oxalic acid concentrations determined with the biosensor method correlated well (R=0.952) with the colorimetric method.     

Determination of evolved 14CO2 in decarboxylase reactions with application to measurement of [14C]oxalic acid.

An assay is described for the determination of the radioactive purity of [14C]oxalic acid preparations and the quantity of [14C]oxalic acid in biological samples. In this method oxalate decarboxylase is used to convert oxalate to formate and CO2. The entire procedure is carried out in a scintillation vial. The 14CO2 released in the enzymic reaction is allowed to diffuse off in a fume hood following acidification. Scintillation fluid is added to reacted and unreacted vials and the radioactivity measured. The loss of radioactivity from the reacted versus the unreacted vials provides the quantity of evolved 14CO2. This value is equal to 50% of the [14C]-oxalate (dpm) present. The radioactive purity of four preparations of [U-14C]oxalic acid was 99.0% while a fifth batch had a purity of 88%. A single batch of [U-14C]oxalic acid had a radioactive purity of 99.0% following storage of an aqueous solution, at -20 degrees C for 7 years. Recovery of [14C]oxalic acid from rat fecal extracts was 101.3%. Eight replicate analyses of a [U-14C]oxalic acid preparation gave a coefficient of variation of 0.3%. Following subcutaneous infusion of [U-14C]oxalic acid to rats, 100.2 +/- 2.9%, mean +/- SD, of the 14C in fecal extracts was present as [14C]oxalic acid (n = 10). The procedure provides a rapid, sensitive, and specific method to determine [14C]oxalic acid. It avoids the time consuming and inconvenient procedure for trapping and counting the evolved 14CO2. The approach used to determine the evolved 14CO2 may find application in other radiochemical methods that require its measurement.     

Measurement of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine by high-performance liquid chromatography with direct ultraviolet detection

A new high-performance liquid chromatographic (HPLC) method is described for the determination of urinary N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC), the final product of the conjugation reaction between a metabolic intermediate of N,N-dimethylformamide (DMF) and glutathione. Urine samples were purified by C(18) solid-phase extraction and then directly analysed by HPLC with an Aminex Ion Exclusion HPX-87H column maintained at 25 degrees C and a UV detector set at 196 nm. Under isocratic conditions (2.4 mM sulphuric acid, flow-rate=0.6 ml/min) AMCC eluted at 20.2 min. The reproducibility (C.V.%) was 1.3-2.7% (intra- and inter-assay, N = 5); the accuracy was 98.0+/-1.7% at 10 mg/l and 101.9+/-1.5% at 800 mg/l (mean+/-SD, N = 3). AMCC was measured in urine from 22 exposed subjects. A strong correlation was found between AMCC and environmental DMF [AMCC (mg/g creatinine)=3.40xDMF (mg/m(3)) + 3.07; r=0.95], while in the urine of 20 unexposed subjects the concentration of AMCC was constantly below the detection limit of the method (0.9 mg/l in urine). The method described appears to be useful for the biological monitoring of DMF exposure.     

13-cis-retinoic acid is an endogenous compound in human serum

The occurrence of 13-cis-retinoic acid as an endogenous component in human serum has been confirmed by cochromatography with standards in both normal-phase and reverse-phase high-performance liquid chromatographic (HPLC) system, by the lambda max of its UV spectrum recorded simultaneously with the HPLC run, and by chromatography of its methyl derivative. The method using solid-phase extraction followed by a gradient reverse-phase HPLC procedure with an internal standard and sensitive UV detector, provides an efficient and sensitive technique for the separation and quantification of serum 13-cis- and all-trans-retinoic acid. Serum levels of 13-cis- and all-trans-retinoic acid in 26 fasting volunteers ranged from 1.0 to 2.2 ng/ml (mean +/- SEM = 1.4 +/- 0.3 ng/ml) and from 1.1 to 1.9 ng/ml (mean +/- SEM = 1.4 +/- 0.2 ng/ml), respectively. The levels determined by a liquid-liquid double-phase extraction method were 90% higher in both 13-cis- and all- trans-retinoic acid than those from a solid-phase extraction. Human small intestine can isomerize all-trans-retinoic acid. 13-cis-Retinoic acid is the predominant cis isomer after incubation of intestinal mucosa homogenates with all-trans-retinoic acid. Moreover, the concentration of retinoic acid in serum is related to diet in that the level of total retinoic acid was 36% higher (n = 10) 2 h after a nonstandard breakfast than in fasting subjects.     

A sensitive high performance liquid chromatographic method for determining small amounts of glycosylproteins

We developed a simple isocratic high performance liquid chromatography (HPLC) method for the quantitative determination of 5-hydroxymethyl-2-furfuraldehyde (5-HMF) liberated by mild hydrolysis of small amounts of glycosyl proteins. The absorbance of hydrolysate components after HPLC separation was recorded at 280 nm. To detect substances possibly interfering with the 5-HMF peak we always recorded the ratio of the peak heights A280 nm/A254 nm which was a constant value of 4.4. For each sample the blank was obtained by reduction with NaBH4 before hydrolysis with oxalic acid 1 mol/l. The best NaBH4/protein ratio was found to be 4 mg/mg. With this method we measured the nonenzymatic glycosylation (glycation) as 5-HMF in samples with a protein concentration as low as 0.8 mg/ml. 5-HMF produced per milligram of protein was independent from protein concentration for a wide range (0.8-10 mg/ml). The mean coefficient of variation for within assay and between precision was 6.8 and 11.6%, respectively. The 5-HMF measured on plasma proteins from normal subjects (n = 7) was 0.16 +/- 0.04 nmol/mg. Protein from insulin-dependent diabetic patients was 0.31 +/- 0.07 nmol/mg. With this method we succeeded in detecting an excessive glycation of platelet membrane proteins in 13 type-I diabetic patients.     

Picric acid methods greatly overestimate serum creatinine in mice: more accurate results with high-performance liquid chromatography

The creatinine levels of blood and urine from humans, rats, and mice were measured by high-performance liquid chromatography. These were compared to the alkaline picrate analysis of creatinine performed by standard colorimetric, kinetic, and AutoAnalyzer techniques. For human serum and urine the values obtained using the HPLC technique gave good agreement with four out of five alkaline picrate techniques. For black or white mice, the serum creatinine concentration was 8.7 +/- 0.4 microM by HPLC but 44.9 +/- 1.9 microM by the lowest alkaline picrate method. Mouse urine creatinine concentrations were 3.24 +/- 0.19 mM by HPLC and 4.59 +/- 0.39 mM by the nearest alkaline picrate method. Rat serum creatinine concentrations analyzed by HPLC were about half the values obtained by AutoAnalyzer. Mouse and rat samples seemed to have substances which gave nonspecific color and thus interfered with the analysis of creatinine by the alkaline picrate methods. While the alkaline picrate analysis of creatinine was adequate for human samples, it was necessary to use HPLC to accurately measure rodent creatinine. The fractional excretion of creatinine was determined by measuring creatinine in mouse urine and plasma by both the kinetic and HPLC methods and comparing these values to urine and plasma inulin. Using the kinetic method, creatinine was cleared at 43 +/- 3% of the rate of inulin. Using the HPLC method, creatinine was cleared at 170 +/- 11% of the rate of inulin.     

Use of a homologous internal standard for the quantification of the major metabolite of prostaglandin F2 alpha in human urine by multiple ion analysis

A gas chromatographic-mass spectrometric method for quantitative determination of 9 alpha, 11 alpha-dihydroxy-15-oxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid, the major urinary metabolite of prostaglandin F2 alpha (PGF-M), was developed. The metabolite was analyzed as the dimethyl ester-O-methyloxime-bis-trimethylsilyl ether derivative. The internal standard consisted of a mixture of diethyl ester + monoethyl ester-delta-lactone of PGF-M. Those two species were converted to the 1-methyl-20-ethyl ester derivative during the analytical process. Linear standard curves were developed in the range 0 to 100 ng of injected prostaglandin. The method comprised extraction with Amberlite XAD-2, methylation, chromatography over octadecasilyl-silica, delactonization, remethylation, and chromatography over silicic acid and Lipidex-5000, followed by methoximation, trimethylsilylation, and instrumental analysis. Interassay coefficient of variation, for the analysis of four identical urine specimens, was 7% and intraassay coefficient of variation, when each sample was injected four times, ranged from 3.2 to 6.0%. Specificity, accuracy, and precision of the method were verified by recovery of the metabolite from two different urine pools. The recovery of authentic, underivatized PGF-M added to urine was 99.1 +/- 2.4% (mean +/- SE, N = 6). The plot of recovered versus added metabolite followed the equation y = 0.936 x + 25.8, with r = 0.9918.     

Metabolism of carnitine in phenylacetic acid-treated rats and in patients with phenylketonuria

The effect of metabolites accumulating in phenylketonuria (PKU) was investigated on carnitine metabolism in rats and in patients with PKU. Of phenylacetic acid (PEAA), phenylpyruvic acid and homogentisic acid the PEAA was found to be the most effective in inhibiting carnitine biosynthesis in rats. Following 60 min, a single intraperitoneal dose of PEAA the relative conversion rate, i. e. the hydroxylation, of tracer [Me-(3)H]butyrobetaine to [Me-(3)H]carnitine decreased from 62.2+/-6.00% to 39.4+/-5.11% (means+/-S.E.M., P<0.01) in the liver, in the only organ doing this conversion in rats. The conversion of loading amount of unlabeled butyrobetaine to carnitine was also markedly reduced. The impaired hydroxylation of butyrobetaine was reflected by a reduced free and total carnitine levels in the liver and a reduced total carnitine concentration in the plasma. PEAA decreased the hepatic level of glutamic acid and alpha-ketoglutaric acid (alpha-KG), suggesting a mechanism for the reduced flux through the butyrobetaine hydroxylase enzyme, because alpha-KG is an obligatory co-enzyme. In the plasma and urine of PKU patients on unrestricted diet, markedly decreased total carnitine levels were detected. In the liver of PEAA-treated rats and urine of PKU patients, a novel carnitine derivative, phenacetyl-carnitine was verified by HPLC and gas chromatography-mass spectrometry.     

An Improved Fluorometric High-Performance Liquid Chromatography Method for Sialic Acid Determination: An Internal Standard Method and Its Application to Sialic Acid Analysis of Human Apolipoprotein E

An improved fluorometric HPLC method for sialic acid determination was developed by employing synthetic N-propionylneuraminic acid (NPNA) as an internal standard. A fixed amount of NPNA was added to a sialoglycoconjugate sample. After hydrolyzing sialioglycoconjugates with diluted sulfuric acid, the released sialic acids and NPNA were derivatized with a fluorogenic compound, 1,2-diamino-4,5-(methylenedioxy)benzene (DMB), followed by fluorometric HPLC. The fluorescent derivative of NPNA was separated from those of N-acetylneuraminic acid, N-glycolylneuraminic acid, 2-keto-3-deoxy-D-glycero-D-galacto-nonoic acid, and 2-keto-3-deoxyoctanoate on HPLC. The separation of NPNA derivative on HPLC was not interfered by components of biological samples such as human sera. Using this internal standard method, low amounts of NANA (0.15-1.0 ng) were quantified with the coefficient of variation values below 4%. Using this method, the sialic acid content of human apolipoprotein E was successfully determined. The present method is useful for sensitive and accurate quantification of sialic acids of different molecular species in biological samples.     

Simultaneous determination of oxalic and citric acids in urine by high-performance liquid chromatography

A simple method for the simultaneous determination of oxalic and citric acids has been developed using reversed-phase HPLC. An aqueous solution containing potassium dihydrogenphosphate (0.25%) and tetrabutylammonium hydrogensulphate (2.5 mmol) at pH 2.0 was used as mobile phase. Under these conditions both the components were well resolved and detected at 210 nm. The recovery for oxalic and citric acids was 97% and 102%, respectively. The method presented here was applied to urine specimens of a large number of urolithic patients and control subjects. Because of the simplicity of the method its application provides better means of monitoring the concentration of oxalic and citric acids in the formation of renal calculi.     

Improvement and validation of a liquid chromatographic method for the determination of levosulpiride in human serum and urine

A rapid, selective and highly sensitive reversed-phase high-performance liquid chromatography (HPLC) method was developed for the determination of levosulpiride, 5-(aminosulfonyl)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-methoxy benzamide, in human serum and urine. The method involved the extraction with a dichloromethane followed by back-extraction into 0.025 M sulfuric acid. HPLC analysis was carried out using reversed-phase isocratic elution with a Luna C(18)(2) 5 microm column, a mobile phase of acetonitrile-0.01 M potassium hydrogen phosphate (30:70, v/v, adjusted to pH 8.5 with triethylamine), and a fluorescence detector with excitation at 300 nm and emission at 365 nm. The chromatograms showed good resolution and sensitivity and no interference of human serum and urine. The calibration curves were linear over the concentration range 0.25-200 ng/ml for serum and 0.2-20 microg/ml for urine with correlation coefficients greater than 0.997. Intra- and inter-day assay precision and accuracy fulfilled the international requirements. The mean absolute recovery for human serum was 89.8+/-3.7%. The lower limits of quantitation in human serum and urine were 0.25 ng/ml and 0.2 microg/ml, respectively, which were sensitive enough for pharmacokinetic studies. Stability studies showed that levosulpiride in human serum and urine was stable during storage, or during the assay procedure. This method was successfully applied to the study of pharmacokinetics of levosulpiride in human volunteers following a single oral administration of levosulpiride (25 mg) tablet.     

Liquid chromatography-electrospray ionization ion trap mass spectrometry for analysis of mesocarb and its metabolites in human urine

A method is described for the determination of metabolites of mesocarb in human urine by combining gradient liquid chromatography and electrospray ionization (ESI)-ion trap mass spectrometry. Seven metabolites (two isomers of hydroxymesocarb, p-hydroxymesocarb, two isomers of dihydroxymesocarb and two isomers of trihydroxymesocarb) and parent drug were detected in human urine after the administration of a single oral dose 10 mg of mesocarb (Sydnocarb, two tablets of 5 mg). Various extraction techniques (free fraction, enzyme hydrolyses and acid hydrolyses) and their comparison were carried out for investigation of the metabolism of mesocarb. After extraction procedure the residue was dissolved in methanol and injected into the column HPLC (Zorbax SB-C18 (Narrow-Bore 2.1 x 150 mm i.d., 5 microm particles)) with mobile phase (0.2 ml/min) of methanol/0.2 mM ammonium acetate. Conformation of the results and identification of all metabolites are performed by LC-MS and LC-MS/MS. The major metabolites of mesocarb in urine of the human were p-hydroxylated derivative of the phenylcarbamoyl group of the parent drug (p-hydrohymesocarb) and dihydroxylated derivative of mesocarb (two isomers of dihydroxymesocarb). This analytical method for dihydrohymesocarb was very sensitive for discriminating the ingestion of mesocarb longer than the parent drug or other metabolites in human urine. The dihydroxymesocarb was detected in urine until 168-192 h after administration of the drug.     

A competitive enzyme-linked immunosorbent assay-like method for the measurement of urinary hyaluronan.

A highly sensitive method for measurement of urinary hyaluronan with a minimum molecular mass of 2,000 Da was developed without using HPLC or radioisotopes. This competitive enzyme-linked immunosorbent assay-like method, used competitive binding of free hyaluronan in the sample and biotin-labeled standard hyaluronan to hyaluronan binding protein in solid phase. A total of 150 healthy individuals from both sexes at ages from 0 to 100 years was examined by the established method. Hyaluronan of 384 +/- 80 ng/mg creatinine (mean +/- SD) was constantly excreted into urine of 24-40-year-old healthy adults. The urinary hyaluronan levels were significantly higher before age 1 (p < 0.001) and rather high after 90 years compared to the other groups. The average molecular weight of urinary hyaluronan (5,500 Da) was constant through all generations. Sex difference of urinary hyaluronan was not observed both quantitatively or qualitatively.     

Identification of 2-guanidinoethanol in human urine

Guanidino compounds in normal human urine were analyzed by high-performance liquid chromatography; an unknown peak was observed in the chromatogram that was identical to the peak of synthetic 2-guanidinoethanol. In another experiment, the substance was purified from human urine by successive use of strongly acidic ion-exchanger, thin-layer chromatography and then weakly acidic ion-exchanger. After this it was reacted with acetylacetone to form dimethylpyrimidyl derivative. After further reaction of this derivative with trifluoroacetic anhydrate, it was analyzed by gas chromatography/mass spectrometry. The mass chromatogram and mass spectrum were identical to those of the trifluoroacetylated dimethylpyrimidyl derivative of synthetic 2-guanidinoethanol. This is the first report on the identification of 2-guanidinoethanol in human urine. The concentration of 2-guanidinoethanol in the urine of healthy humans was 5.7 +/- 1.8 (mean +/- SD) mumol/g creatinine.     

Urinary high performance reverse phase chromatography cortisol and cortisone analyses before and at the end of a race in elite cyclists

A functional and basic method for the quantitative analysis of urine cortisol (F) and cortisone (E) using a Solid-Phase Extraction column and HPLC with ultraviolet detection is here described and validated to analyse urine samples. Urine specimens were analysed to study F and E relation and ratio in athletes and healthy sedentary subjects. The F and E concentrations in random urine specimens were significantly higher in the post exercise versus pre exercise condition in cyclists (F: 136+/-93 nmol/l versus 67+/-50 nmol/l (p<0.001); E: 797+/-400 nmol/l versus 408+/-252 nmol/l (p<0.001)). The F/E ratio was 0.18+/-0.11 versus 0.16+/-0.07, respectively, and a significant difference was only demonstrated comparing sedentary (0.11+/-0.07) and cyclist individuals at rest (p<0.05).