Allantoin In Plasma Research Articles

Abstract P167: Altered Gut Microbiome Contributes to Metabolite Biomarkers in Patients With Pulmonary Hypertension

Objectives: Pulmonary arterial hypertension (PAH) is a cardiopulmonary disease without effective cure. The objective of this study is to test the hypothesis that PAH patients have a distinct gut microbiome composition acting as a driving force for the increased PAH metabolic biomarkers, acyl carnitines and urate. Methods: Fecal samples from 18 type 1 PAH patients (mean pulmonary arterial pressure [mPAP] 57.4±16.7 mmHg) and 13 reference subjects in Brazil were collected for comparison of microbiome using shotgun metagenomics. Plasma samples from 33 type I PAH patients (mPAP 51.7± 13.5 mmHg) and 19 reference subjects in the USA were used for untargeted metabolomic analysis. Results: More than 80 bacterial taxa were differentially enriched in the microbiomes of the PAH cohort. Random forest machine-learning algorithm predicted PAH patients with 82.4% accuracy from their gut microbiome composition. The most increased taxa in the PAH microbiome were trimethylamine (TMA) producing bacteria like Collinsella (P=0.0037). The PAH metabolome also showed increases of gut microbiome-derived short chain acyl carnitines like isubutyrylcarnitine (P=0.010) and valerylcarnitine (P=0.030), which are the precursors of TMA. In addition, increased urate (P=0.0004) and allantoin (P=0.027) in PAH plasma correlated with significantly increased enzymes producing urate in the PAH microbiome (purine metabolism (P=0.017) and xanthine oxidase (P<0.05)). Conclusion: These findings suggest that the random forest modeling of the gut microbiome composition can be used for prediction of PAH and that the unique gut microbiome of PAH influences acyl carnitines and urate metabolism to contribute to pathophysiology.

A monitoring of allantoin, uric acid, and malondialdehyde levels in plasma and erythrocytes after ten minutes of running activity.

Uric acid is the final product of human purine metabolism. It was pointed out that this compound acts as an antioxidant and is able to react with reactive oxygen species forming allantoin. Therefore, the measurement of allantoin levels may be used for the determination of oxidative stress in humans. The aim of the study was to clarify the antioxidant effect of uric acid during intense exercise. Whole blood samples were obtained from a group of healthy subjects. Allantoin, uric acid, and malondialdehyde levels in plasma and erythrocytes were measured using a HPLC with UV/Vis detection. Statistical significant differences in allantoin and uric acid levels during short-term intense exercise were found. Immediately after intense exercise, the plasma allantoin levels increased on the average of 200 % in comparison to baseline. Plasma uric acid levels increased slowly, at an average of 20 %. On the other hand, there were no significant changes in plasma malondialdehyde. The results suggest that uric acid, important antioxidant, is probably oxidized by reactive oxygen species to allantoin. Therefore allantoin may be suitable candidate for a marker of acute oxidative stress.

Myeloperoxidase and oxidative stress in rheumatoid arthritis

To determine whether MPO contributes to oxidative stress and disease activity in RA and whether it produces hypochlorous acid in SF. Plasma and where possible SF were collected from 77 RA patients while 120 healthy controls supplied plasma only. MPO and protein carbonyls were measured by ELISAs. 3-Chlorotyrosine in proteins and allantoin in plasma were measured by mass spectrometry. Plasma MPO concentrations were significantly higher in patients with RA compared with healthy controls [10.8 ng/ml, inter-quartile range (IQR): 7.2-14.2; P<0.05], but there was no significant difference in plasma MPO protein concentrations between RA patients with high disease activity (HDA; DAS-28 >3.2) and those with low disease activity (LDA; DAS-28 ≤ 3.2) (HDA 27.9 ng/ml, 20.2-34.1 vs LDA 22.1 ng/ml, 16.9-34.9; P>0.05). There was a significant relationship between plasma MPO and DAS-28 (r=0.35; P=0.005). Plasma protein carbonyls and allantoin were significantly higher in patients with RA compared with the healthy controls. MPO protein was significantly higher in SF compared with plasma (median 624.0 ng/ml, IQR 258.4-2433.0 vs 30.2 ng/ml, IQR 25.1-50.9; P<0.0001). The MPO present in SF was mostly active. 3-Chlorotyrosine, a specific biomarker of hypochlorous acid, was present in proteins from SF and related to the concentration of MPO (r=0.69; P=0.001). Protein carbonyls in SF were associated with MPO protein concentration (r=0.40; P=0.019) and 3-chlorotyrosine (r=0.66; P=0.003). MPO is elevated in patients with RA and promotes oxidative stress through the production of hypochlorous acid.

Detection of allantoin in clinical samples using hydrophilic liquid chromatography with stable isotope dilution negative ion tandem mass spectrometry

Allantoin is the major oxidation product of urate in humans and is a potential biomarker of oxidative stress. Several methods are used to measure allantoin in biological samples but they have inherent issues that can include lack of specificity and sensitivity, difficulty in sample preparation, or artefactual generation of allantoin. We have developed a method for measuring allantoin using hydrophilic liquid chromatography with stable isotope dilution tandem mass spectrometry (HILIC–MS/MS). It was validated for measuring allantoin in plasma, synovial fluid and urine from human subjects. The limit of quantification was determined to be 10fmol and the assay displayed excellent linearity for the wide range of concentrations found in clinical samples. Relative standard deviations were <5% for between-day and <7% for within-day variation. Accuracy was between 100% and 104%. Concentrations of allantoin in plasma of healthy controls (2.0μM; interquartile range 1.4–3.6μM, n=35) was significantly lower (p<0.001) than that in plasma from patients with rheumatoid arthritis (3.7μM; IQR 3.0–5.6μM, n=43) and in synovial fluid of patients with gout (3.3μM; IQR 2.8–5.8μM, n=10). This newer HILIC–MS/MS method is a simple and highly sensitive assay for detection of allantoin. It can be used to assess the level of oxidative stress in human pathologies.

Field-amplified sample injection combined with pressure-assisted capillary electrophoresis UV detection for the simultaneous analysis of allantoin, uric acid, and malondialdehyde in human plasma

The allantoin/uric acid (All/UA) ratio and malondialdehyde (MDA) plasma levels have been proposed as important markers for monitoring oxidation triggered by the action of free radicals (FR). Here, we describe an easy field amplified sample injection capillary electrophoresis method with UV detection for the separation and quantification of All, UA, and free MDA in human plasma. The plasma samples were simply filtered through centrifugation membrane tubes for protein elimination and directly injected on a capillary without complex cleanup and/or sample derivatization procedures. The use of a run buffer composed of 300 mmol/L sodium borate at pH 10 with 50 mmol/L of N-methyl-D: -glucamine and an overimposed pressure/voltage of 0.1 psi during the electrophoretic run allows basline resolution of the analytes within 17 min. The electrokinetic injection allows a detection limit of 15 nmol/L for All, 20 nmol/L for UA and 10 nmol/L for MDA in a plasma sample, thus significantly improving the LOD of previous described methods based on capillary electrophoresis. Precision tests indicate a good repeatability of our method both for migration times (CV = 1.85%) and areas (CV = 2.87%). Moreover, a good reproducibility of intra- and inter-assay tests was obtained (CV = 4.63% and CV = 6.59% respectively). The suitability of the method was tested by measuring analyte levels in 40 healthy volunteers.

Allantoin in Human Plasma, Serum, and Nasal-Lining Fluids as a Biomarker of Oxidative Stress: Avoiding Artifacts and Establishing Realin vivoConcentrations

Urate is the terminal product of purine metabolism in primates, including humans. Urate is also an efficient scavenger of oxidizing species and is thought to be an important antioxidant in human body fluids. Allantoin, the major oxidation product of urate, has been suggested as a candidate biomarker of oxidative stress because it is not produced metabolically. Although urate is converted to allantoin under strongly alkaline pH, such conditions have been used in the past to facilitate extraction of allantoin. We evolved a method for the determination of allantoin concentrations in human plasma and serum by gas chromatography-mass spectrometry without such artifact. With this method, we show that alkaline conditions do indeed cause breakdown of urate, leading to significant overestimation of allantoin concentration in human samples. By using our alternative method, serum samples from 98 volunteers were analyzed, and allantoin levels were found to be significantly lower than was previously reported. The in vivo utility and sensitivity of our method was further evaluated in human nasal-lining fluids. We were able to demonstrate an ozone-induced increase in allantoin, in the absence of increases in either ascorbate or glutathione oxidation products.

Allantoin as a marker of oxidative stress in human erythrocytes

Uric acid is the final product of purine metabolism in humans. It was determined that this compound has important antioxidative properties and it may be oxidized to allantoin by various reactive oxygen species. Therefore, the measurement of allantoin may be useful for the determination of oxidative stress in humans. We measured allantoin and uric acid in human plasma and erythrocytes obtained from patients with chronic renal failure before hemodialysis (n=30) and blood donors (n=30). We used a method based on selective isolation of allantoin from deproteinized plasma and erythrocyte lysate samples on AG 1-X8 resin and its derivatization to glyoxylate-2, 4-dinitrophenylhydrazone. Separation of glyoxylate-2, 4-dinitrophenylhydrazone from interfering substances was achieved on reversed phase HPLC with gradient elution and UV/VIS detection at 360 nm. Uric acid was determined by reversed phase HPLC with UV/VIS detection at 292 nm. We found significant differences in allantoin and uric acid concentration between the patients with chronic renal failure and the control group both in plasma (20.5+/-6.5 micromol/L and 323.9+/-62.9 micromol/L vs. 2.1+/-1.1 micromol/L and 270.1+/-62.3 micromol/L, p<0.05) and erythrocytes [82.8+/-39.1 nmol/g hemoglobin (Hb) and 110.7+/-28.8 nmol/g Hb vs. 20.1+/-6.1 nmol/g Hb and 82.1+/-23.7 nmol/g Hb, p<0.05]. Significant higher levels of allantoin in both plasma and erythrocytes of patients with chronic renal failure indicate that allantoin may be used as a good marker of oxidative stress.

Monitoring of antioxidant properties of uric acid in humans for a consideration measuring of levels of allantoin in plasma by liquid chromatography

Uric acid is the end product of purine metabolism in humans. It has been pointed out that uric acid acts as an antioxidant and is capable to react with biologically relevant oxidants to form allantoin. Therefore, measurement of allantoin in humans was proposed as a marker of oxidative stress. We estimated allantoin in human plasma obtained from the patients with chronic renal failure before hemodialysis ( n = 30), patients with non-insulin dependent diabetes mellitus ( n = 30) and blood donors ( n = 30) using a method based on selective isolation of allantoin from deproteinized plasma samples on AG 1-X8 resin and its derivatization to glyoxylate-2,4-dinitrophenylhydrazone. The method is free from urate and glyoxylate interferences. Separation of glyoxylate-2,4-dinitrophenylhydrazone from other hydrazones was achieved on reversed phase HPLC with gradient elution and UV/VIS detection at 360 nm. The analytical performance of this method is satisfactory with intra-assay CV 5.7%, inter-assay CV 8.3% and recovery 94.1%. We have determined other parameters of oxidative stress (malondialdehyde, total antioxidant status, superoxide dismutase and glutathione peroxidase) too. The preliminary reference range of allantoin in a group of blood donors is 4.76 ± 2.99 μmol/L. In the patients with chronic renal failure and the patients with non-insulin dependent diabetes mellitus we found allantoin levels in plasma (27.1 ± 13.8) μmol/L and (11.08 ± 5.90) μmol/L, respectively. It seems that allantoin is a possible indicator of free radical damage in vivo.

Allantoin, the oxidation product of uric acid is present in chicken and turkey plasma

Urate oxidase is not present in birds yet allantoin, a product of this enzyme, has been measured in birds. Studies were designed to compare the concentrations of plasma purine derivatives in chickens and turkeys fed inosine-supplemented diets. The first study consisted of 12 male chicks that were fed diets supplemented with 0.6 mol inosine or hypoxanthine per kilogram diet from 3- to 6-week-old. Study 2 consisted of 12 turkey poults (toms) fed inosine-supplemented diets (0.7 mol/kg) from 6- to 8-week-old. Plasma allantoin and oxypurines concentrations were measured weekly using high performance liquid chromatography. Plasma uric acid (PUA) in chickens fed inosine-supplemented diets increased from 0.31 to 1.34 mM ( P<0.05) at the end of week 2. In turkeys, those fed control diet had 0.17 mM PUA concentration compared to 0.3 mM in those fed the inosine diet at week 2 ( P<0.05). Allantoin concentration increased in chickens from week 1 to 2 while a decrease was observed in turkeys ( P<0.005) for both treatments. These data show that allantoin is present in turkey and chicken plasma. The presence of allantoin in avian plasma is consistent with uric acid acting as an antioxidant in these species.

Estimation of allantoin flux using continuous infusion of [14C]allantoin: sensitivity to plasma loading with unlabelled allantoin.

Allantoin net flux through the plasma allantoin compartment was determined in sheep given a roughage diet by means of a continuous infusion of [4,5-14C]allantoin for 17 h. Unlabelled allantoin was infused intravenously during the last 7 h of the tracer infusion to increase the allantoin flux by approximately 75 %. When unlabelled allantoin was infused, the specific radioactivity of allantoin in plasma and urine declined exponentially to approach a lower plateau some 2-3 h later. The estimate of net flux during the infusion of unlabelled allantoin, estimated from blood plasma and urine, was on average 79 and 90 % of expected values. Expected values of allantoin net flux during infusion of unlabelled allantoin were calculated as the sum of allantoin net flux pre-loading plus the known rate of infusion of the allantoin load. It is probable that endogenous allantoin synthesis was decreased by the infusion of allantoin: allosteric inhibition of uricase appears a plausible explanation for this observation, and for lower estimates of net flux. Appearance of labelled allantoin-C in ruminal or blood bicarbonate was negligible. Our results indicate that net flux of allantoin through blood plasma is a good predictor of the entry rate of allantoin into the primary compartment and should be a better predictor of rumen microbial outflow than urinary allantoin excretion. However, measurements of allantoin-specific radioactivity, during continuous infusion, should be taken after a period of 24 h, at which time the true plateau specific radioactivity value of allantoin in plasma would be attained.

Kinetics of allantoin metabolism in sheep

The kinetics of allantoin metabolism were studied in rumen-cannulated sheep by means of a single intravenous injection of [4,5-(14)C]allantoin. The decline in the specific radioactivity of allantoin in plasma following the injection of tracer was best described by a double exponential function, indicating that allantoin moves in and out of two or more kinetically distinct compartments. Sequestering of tracer in secondary or tertiary compartments in the body water appears likely to have resulted in overestimation of net flux of allantoin through the blood in the present study. In future studies, sampling of blood for several days after administration of tracer should alleviate this problem. About 80 % of the [(14)C]allantoin injected was recovered as [(14)C]allantoin in urine during the 12 h after tracer injection, increasing to 94 % after 4 d. Allantoin-C also passed through the blood bicarbonate pool, suggesting that allantoin is degraded in the gastrointestinal tract. A small amount of allantoin-C (4 % of the net flux of allantoin through the blood pool) was apparently degraded to form bicarbonate-C in the rumen and postruminally, and subsequently appeared in blood bicarbonate-C. Transfer of allantoin-C into the rumen via saliva was insignificant. In view of these findings, the net flux of allantoin through the blood should be a better predictor of rumen microbial outflow than urinary allantoin excretion, because urinary excretion of purine derivatives must be adjusted for conversion of allantoin-C to blood bicarbonate when used to predict the flow of microbial protein from the rumen.

Does a relationship exist between the urate pool in the body and lipid peroxidation during exercise?

In this study, we investigated whether a relationship exists between the levels of urate in vivo and lipid peroxidation during exercise. Seven healthy male subjects performed exhaustive cycling exercise under the following three conditions. The levels of urate, thiobarbituric acid reactive substances (TBARS) and allantoin in plasma and urine were examined before exercise and during a 3 h recovery period. (1) Benzbromarone administration experiment: benzbromarone (an uricosuric agent) was administered orally the day before exercise. (2) IMP administration experiment: inosine 5′-monophosphate disodium salt (a precursor of urate) was administered orally the day before exercise. (3) Control experiment: no test substance was administered. The main results obtained were as follows. Plasma urate levels and total peroxyl radical-trapping antioxidant parameter (TRAP) for deproteinized plasma in the resting period significantly decreased depending on the treatment, in the order of IMP > control > benzbromarone. A significant positive correlation was evident between plasma urate levels and TRAP values for deproteinized plasma. The increase in plasma levels of allantoin was observed only in the case of IMP treatment. A significant negative correlation between plasma levels of urate in the resting period and the amounts of urinary TBARS excreted during the recovery period was recognized. These results suggest that the urate level in vivo before exercise is a factor influencing lipid peroxidation during exhaustive exercise. Furthermore, these findings support the view that urate may serve as an important free-radical scavenger in vivo.

Simultaneous measurement of allantoin, uric acid, xanthine and hypoxanthine in blood by high-performance liquid chromatography

A high-performance liquid chromatographic method for determining catabolism products of nucleic acids and purines, such as oxypurines (i.e. uric acid, xanthine and hypoxanthine) and allantoin in the blood plasma of ruminants was developed. The plasma was deproteinized with 10% trichloroacetic acid. The method enabled determination of oxypurines without derivatization. Allantoin was determined after conversion with 2,4-dinitrophenylhydrazine to a hydrazone (GLX-DNPH). Separation of converted allantoin, uric acid, xanthine and hypoxanthine derivatives was carried out using two reversed-phase C 18 columns. The combination of pre-column derivatization and gradient elution with monitoring of the effluent at 205, 254 and 360 nm provides a simple and selective analytical tool for studying oxypurines and allantoin in plasma. The total run time of the HPLC analysis was 60 min. The recovery of the purine derivatives (i.e. oxypurines and allantoin) added to the plasma was between 95 and 106%. Purine derivatives were stable when the processed samples were stored for 7 days at −10°C. The low values of the intra-assay coefficient of variations (2.5–4.6%) and the low values of the detection limits (0.187–0.004 nmol) point to the satisfactory precision and sensitivity of the method.

Accumulation of allantoin and uric acid in plasma of exercising trotters

Summary Plasma concentrations of hypoxanthine, uric acid, and allantoin, which are breakdown products of adenine nucleotides, were measured in Standardbred and Finnhorse trotters during and after an exercise test on a high-speed treadmill, after an incremental exercise test performed on a racetrack, and after a racing competition. Fiber-type composition of the middle gluteal muscle and the muscle concentrations of adenine nucleotides and inosine monophosphate were measured after the racetrack test. Changes in the concentration of hypoxanthine were not observed in any of the tests. Peak concentration of uric acid was measured between 5 and 30 minutes after exercise, and it was three- to tenfold higher than the value at rest. The variability can be explained by intensity of the exercise test and variation among horses. The concentration of allantoin after exercise was 2 to 3 times as high as that at rest, depending on the intensity of the exercise, although the absolute increase was about 10 times as high as the increase in the concentration of uric acid. Peak values of allantoin for the treadmill and the racetrack tests were obtained 4 to 6 minutes after exercise and &lt; 30 minutes after the races. Peak concentration of allantoin correlated positively with the percentage of type-II (IIA + IIB) fibers in the middle gluteal muscle. Significant correlations were not observed between plasma concentration of uric acid or allantoin and muscle concentrations of adenosine triphosphate (atp) or inosine monophosphate. It can be concluded that in horses, breakdown of atp during and after exercise continues until allantoin is produced. The peak concentration of allantoin increases with the intensity of exercise, is reached rapidly after exercise, and the variation in the time to the peak value is small among horses. It is suggested that the main source of allantoin is the fast-twitch, type-II fibers and that the mixed muscle concentrations of adenine nucleotides are of limited value when estimating the effects of exercise on atp content of the muscle tissue.

Measurement of allantoin in urine and plasma by high-performance liquid chromatography with pre-column derivatization

A method is reported for determination of allantoin in urine and plasma based on high-performance liquid chromatography (HPLC) and pre-column derivatization. In the derivatization procedure, allantoin is converted to glyoxylic acid which forms a hydrazone with 2,4-dinitrophenylhydrazine. The hydrazone appears as syn and anti isomers at a constant ratio. These derivatives are separated by HPLC using a reversed-phase C 18 column from hydrazones of other keto acids possibly present in urine and plasma and then monitored at 360 nm. All components were completely resolved in 15 min. Both the reagents and derivatization products are stable. Recovery of allantoin added to urine and plasma was 95 ± 3.7% ( n = 45) and 100 ± 7.5% ( n = 64), respectively. The lowest allantoin concentration that gave a reproducible integration was 5 μmol/l. The between-assay and within-day coefficients of variation were 2.8 and 0.6%, respectively.

Improved HPLC method for the simultaneous measurement of allantoin and uric acid in plasma.

Uric acid is generated by the oxidation of purines, but no enzyme is present to oxidize it further. It is present in plasma at a concentration approaching 500 J.lIIlollL and has been proposed as an important antioxidant. 1 In agreement with this proposal experiments in vitro have shown the protective effect of uric acid against oxidative damage. The first and major product of uric acid oxidation is allantoin, and it has been suggested that allantoin may be useful as a 'marker' of free radical reactions in vivo.2,3 However, methods currently used for the analysis of allantoin in body fluids are tedious and complicated, and since allantoin is very polar' it is difficult to obtain resolution from other serum constituents that have a similar polarity on a reverse-phase HPLC column. In the method of Grootveld and Halliwelluric acid is first analysed by HPLC, and allantoin is then measured in a separate procedure which involves initial separation and fraction collection by HPLC, hydrolysis and derivatization of allantoin, and finally separation of the derivatives by HPLC. We have developed a unique chromatographic assay to determine uric acid and allantoin simultaneously in plasma ultrafiltrate which is a significant improvement on previous HPLC methods.

Simultaneous determination of allantoin and oxypurines in biological fluids by high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the separation and quantification of allantoin and oxypurines in plasma and urine samples. Urine was analyzed directly and plasma after acid deproteinisation with perchloric acid. Separation and quantification of purine derivatives was achieved using two Spherisorb ODS-5 column (250 mm × 4.6 mm I.D.) connected in series together with a NH 4H 2PO 4—NH 4H 2PO 4acetonitrile (80:20) gradient and monitoring the effluent at 205 nm. The average recoveries of standard compounds added to urine and plasma samples were 96 and 97%, respectively, using allopurinol as internal standard. The within-day variability was less than 7% and the day-to-day coefficient of variation less than 11% indicating a good precision of the method.

Purin- und Pyrimidinmetaboliten zur Beurteilung des Pansenstoffwechsels: HPLC-Analysen in Milch und Blutplasma

In milk and blood plasma samples of 6 German Simmental and 12 German Black and White heifers it was investigated, whether purine and pyrimidine compounds are suitable indicators of the microbial protein synthesis in the rumen. Therefore the secreted quantities in milk and the concentration in blood plasma are correlated with energy intake. The results indicated significant correlation coefficients for both the secretion quantity of allantoin in milk (r = 0.942) and the concentration of allantoin in blood plasma (r = 0.694). Other investigated compounds appeared more suitable for evaluating the mammary gland metabolism (uridine-lactose synthesis, pseudouridine-protein synthesis). In an experiment with 7 male castrated pigmy goats subjected to a four-day fasting period the decrease of plasma allantoin, which was already apparent after 12 hours of fasting, was closely correlated with the increase of plasma free fatty acids.

Kinetic analysis of anaerobic metabolism in rats during acute cyanide poisoning

In order to investigate the changes in energy metabolism during acute anoxia, blood levels of various metabolites were analysed in cyanide-poisoned rats. After intraperitoneal injection of a sublethal dose of potassium cyanide (5 mg/kg), blood samples were obtained by cervical dislocation at intervals of 5 min until 30 min. Lacatate and lactate/pyruvate ratio (L/P) in plasma concomitantly changed with cyanide; increased rapidly at 5 min, remained fairly constant until 20 min and then began to decrease at 25 min. In contrast, the products of ATP degradation, oxypurines and inorganic phosphate (Pi), increased gradually until 25 min and then began to decrease at 30 min. Allantoin in plasma scarcely increased throughout the experiments. The results indicate that the rapid activation of anaerobic ATP formation by glycolysis was followed by the increase in ATP degradation in cyanide-poisoned rats. Thus, increase in plasma oxypurines could be regarded as an indicator for severe anoxic states in tissues with massive ATP degradation.

High-performance liquid chromatographic method for the determination of plasma allantoin

A new method has been devised for the determination of nanomole levels of allantoin in human plasma. Allantoin was converted into xanthylallantoin, which was chromatographed on a reversed-phase silica gel using a mixture of acetonitrile—water (27:73) as mobile phase. The eluted compound was measured using an ultraviolet detector. The detection limit of the assay for plasma was about 100 ng/ml. This method was applied successfully to the determination of allantoin in human plasma after oral administration of 100 mg of aldioxa.