International Archives of
Olcational (k
Int Arch Occup Environ Hlth 37,205-217 (1976)
aml EnvilonlenItal Health t by Springer-Verlag 1976
The Absorption, Metabolism, and Excretion of Xylenes in Man V EDIVEC and J FLEK Institute of Hygiene and Epidemiology (Director: Prof F Janca, M D , Dr Sc ), Center of Industrial Hygiene and Occupational Diseases (Head: Prof B Svestka, M.D , Member of the Czechoslovak Academy of Sciences), Vinohrady, Srobarova 48, v Prague 10, CSSR
Summary Fifteen exposure experiments were carried out, in which four persons each, were simultaneously exposed to a defined concentration of o-, m-, and p-xylene vapors and also to their mixture at a ratio of 1:1:1 The concentrations were in all cases around O 2 mg/l (Czechoslovakian MAC) or around double the amount The period of exposure amounted to exactly 8 h. It was ascertained that the pulmonary retention is practically identical in all persons and in all isomers and amounts to 63 6 ± 4 2% Its extent does not depend upon inoculation level, nor upon the duration of the exposure. During the period of desaturation the lungs excrete approximately 5% of the total amount retained in the organism Xylene excretion via urine is quite negligible and amounts to a thousandth of 1%. The chief metabolites are toluic acids which are excreted in conjugated form with glycine as so-called toluric (=methylhippuric) acids Free toluic acids, toluylglucuronic acids, and hydroxytoluic acids do not occur in the urine of persons exposed to a reasonable concentration of xylene vapors. The excreted amounts of toluric acids reach a maximum in the portion of urine collected at the end of exposure; then they decrease rapidly, but trace amounts can be still observed even after 4-5 days The excretion takes a similar course in different persons and at different intensity of exposure. During the last 2 h of exposure a mean of 23 6% is excreted and in 8 h of exposure 71 7% of the amount excreted-within 24 h. The side metabolites of xylenes are compounds hydroxylated on the aromatic nucleus After exposure to o-xylene the presence of 2 3 and 3 4-xylenol was observed in urine; after exposure to m-xylene the presence of 2 4 xylenol, and finally after exposure to p-xylene the presence of 2 5-xylenol The excretion of xylenols reaches a maximum as a rule just after termination of exposure. It was proved by balance calculation that of the total amount of xylene retained in the organism during exposure, more than 95% is excreted in the form of toluric acid (o-97 1; m-99 2; p-95 1%) and only a small part in form of xylenol (o-0 86 ; m-1 98 ; p-0 05%). Key words:
Xylenes
Absorption
Metabolism
Excretion.
205
INTRODUCTION The metabolism of xylenes was studied in experimental animals after peroral administration of a relatively high dose l2,3,5l. It was proved that all isomers are oxidized in the organism to the corresponding toluic (=methylbenzoic) acids, which are then excreted in urine in different forms: m and p-toluic acid predominantly conjugated with glycine as so-called toluric (=methylhippuric) acid, whereas o-toluic acid occurs predominantly in free form or bound to glucuronic acid In the urine of intoxicated animals the presence of metabolites hydroxylated on the aromatic nucleus was also proved; some of them were later isolated and identified 'i, 5, Considerably less is known about the metabolic' changes of xylenes in the human organism It has been ascertained in experiments in volunteers exposed to adequate vapor concentrations that about 72% of the retained m-xylene is excreted within 26 h as m-toluric acid, the determination of which in urine may serve as an exposure test l8l The form in which the remaining share of m-xylene is excreted is not known, data on the excretion of p-toluric acid are lacking, and the metabolism of o-xylene has not been studied in man yet There are also no data available on the retention of isomeric xylenes in the respiratory system and on the quantity of unchanged hydrocarbons eliminated by exhaling. Studies of the metabolism of xylenes and also studies of the kinetics of excretion of metabolites were accompanied by a number of difficulties The classical analytical methods did not allow reliable determination of the metabolites in the presence of the related normal components of urine (e g , hippuric acid or phenol or p-cresol); this is the reason why the majority of findings was only of qualitative or semiquantitative character. The application of gas chromatography made it possible to derive the quantitative relations.
EXPERIMENTAL PART Methods Preparation of the Atmosphere with Defined Contents of Xylene Vapors
The
exposure experiments were carried out in a laboratory of 5 4 x 3.4 x 3 5 m in size The concentration of xylene vapors in the atmosphere was automatically analyzed by gas chromatography at 5-min intervals and automatically adapted to the required value by special apparatus l12 l. Subjects, Time and Intensity of Exposure
Six healthy men aged 28-50
years, body weight 72-90 g, were at our disposal for the exposure experiments Four of them were simultaneously exposed 206
to a known concentration of isomers (about 0 2 mg/1 and 0 4 mg/l) and in later experiments also to a mixture of known ratio of components The time of exposure was exactly 8 h (without interruption). Analysis of the Air Exhaled during Exposure
The experimental subject
breathed through a mouthpiece connected to a two-way valve The exhaled air was conducted into a glass vessel (7 1 volume), partitioned by a metal sieve (homogenization of the sample), leaving the vessel through a one-way valve In order to prevent condensation of the water vapors, the vessel was externally heated by an electric bandage to 400 C The mean sample was drawn from the vessel by perforated metal tubing and continuously conducted through the pipette of the gas sampling and introduction device of the chromatograph by means of a membrane pump The contents of the gas pipette were introduced at suitable time intervals (after 10-15 min of breathing) into the column and the chromatogram was recorded Each analysis was repeated twice and the concentration of xylene vapors in the inhaled air was determined simultaneously (Fig 1). Analysis of the Air Exhaled after Exposure
The person exhaled the lung
contents into one arm of a T-shaped metal tube Wright's respirometer was connected to the other arm During exhaling, i e , while the dial pointer of the respirometer was revolving, the sample of exhaled air was gradually sucked from the third arm into the connected large (200 ml) glass syringe, immediately introduced into the gas sampling and introduction device, and the chromatogram was recorded Although this procedure did not guarantee as perfect a homogenization of the exhaled air as the previous method, it was possible, however, to study the rapid changes of the xylene concentration (especially during the first phases after termination of exposure), which would be distorted if an apparatus with large volume capacity would have been used. Determination of the Breathing Volume
The experimental subject was
breathing through a mouthpiece into which a Wright's respirometer was placed The volume of exhaled air was read from the dial exactly after 10 min and measurement was repeated at least four times during exposure; the mean was calculated from the measured values. Collection of Urine Samples The urine was sampled at 2-h intervals
during and after termination of exposure and later collected at 8-h intervals. Determination of Free Xylene in Urine
A measured volume of urine
(ob-
tained in an environment free of xylene vapors) was saturated with ammonium sulphate and thoroughly shaken with 1/5 of carbon
207
disulphide volume After centrifugation in a sealed test tube, 3 l of the extract were drawn into an injection microsyringe and the chromatogram was recorded using flame ionization detection The quantitative relations were derived by the method of direct calibration. Determination of the Total Toluic Acids l6l An equal volume of 50 % wt/vol sodium hydroxide was added to 2 ml urine and the mixture was heated for 60 min to 130 0 C in a silicon oil bath After cooling, acidification with dilute sulphuric acid, and saturation with ammonium sulphate, the freed toluic acid was extracted by ethyl acetate and converted into methylester by diazomethane The methylester was chromatographed on a column packed with 10% polyethyleneglycol 1500 at 130 0 C Acetophenone was used as internal standard The total toluic acid (either o-, or m-, or p-) can be determined irrespective whether it is present in the urine free, bound to glucuronic acid, or conjugated with glycine. Determination of Free Toluic Acids
The procedure was the same as in
the previous case, but the alkaline hydrolysis of urine was omitted The extract of urine contained besides toluic acid also toluric and hippuric acid, the methylesters of which have very long retention times under the given conditions The working temperature was increased after recording the peak of methylester of toluic acid, in order to accelerate the elution of the higher boiling esters. Determination of Toluric Acids l10l
Slightly acidified urine
(5 ml)
was saturated by ammonium sulphate and extracted with ethyl acetate The acids which passed into the extract were converted by diazomethane into methylesters, which were chromatographed on a column packed with 3% neopentylglycolsuccinate at 190 0 C using flame ionization detection Phenacetin (p-acetophenetide) served as internal standard The method afforded determination of m and p-toluric acid The methylester of o-toluric acid eluted simultaneously with the methylester of hippuric acid and the chromatographic peaks overlapped l10 l The procedure was therefore modified: the hippuric acid was decomposed by alkaline hydrolysis under mild conditions (20 min of heating urine with the same volume of 50% wt/vol sodium hydroxide to 100 0 C); the resistent o-toluric acid was only decomposed to a small part. The share not decomposed (about 85%) was extracted from the reaction mixture, converted into methylester and chromatographed. The calibration curve was constructed in the same way (i e , including alkaline hydrolysis under mild conditions).
208
Determination of Xylenols One ml of diluted (1:1) sulphuric acid was added to 5 ml of urine and the mixture was heated in a closed vessel in a boiling water bath for 20 min After cooling the liquid was saturated with ammonium sulphate and thoroughly shaken with 2 ml carbon disulphide Three microliters of the extract were analyzed on columns packed with 5% polyethyleneglycol 20 M, 10% didecylphthalate, or 3% neopentylglycolsuccinate at a temperature of about 150 0 C The quantitative relations were derived by the method of direct calibration.
RESULTS AND DISCUSSION Saturation of Organism The first question was, what amount of xylenes is retained in the lungs when inhaling contaminated atmosphere The lung retention (in %) can be calculated from the concentration of xylene vapors (C) in the inhaled and exhaled air according to the equation: lung retention
Cexhalation atmosphere lung retention (%) atmosphere
100
Because the concentration of vapors is directly proportional to the height of the corresponding chromatographic peak, it was possible to substitute in the formula the values (in mm) read on the chromatographic record (Fig 1) In order to achieve reproducible results it appeared essential to homogenize the exhaled air perfectly prior to analysis (in the first part of exhaling there is always a higher concentration than in the last part, and during desaturation the opposite is the case) and to determine the lung retention at quite normal breathing Both these conditions were well fulfilled, if the exhaled air was collected by the previously described apparatus. The lung retention was measured always at two different vapor concentrations, whereby the following mean values were ascertained: o-xylene 62 4 %, m-xylene 64 2%, and p-xylene 64 3 % The results disclosed that retention is almost the same in all isomers and that it is independent of the level of exposure within the studied limits Its extent did not change even with the time of exposure; the values determined between the 1st and 3rd h and between the 5th and 7th h were identical From the theoretical aspect the retention should be somewhat higher in the first phases and should decrease to the mentioned value after a certain time, when equilibrium has been established between the concentration in the inhaled air and in the blood This first stage is obviously short, because several measurements performed after 15 min of exposure yielded practically the same results as
209
Fig 1 Determination of lung retention. Chromatogram obtained in analysis of inhaled and exhaled air during exposure to m-xylene (mean concentration in atmosphere 0 397 mg/l) Volume of sample 3 ml, stationary phase PEG 20 M, working temperature 1100 C, sensitivity 1/16 at input resistance of 1010 ohm
mentioned above In experiments repeated after a few days or even weeks the same values of retention in the same person were In different persons, however, the retention always ascertained differed somewhat; the greatest difference (67 4 and 58 6 %) was observed in persons with very different lung ventilation (7 9 and 11 7 1/min) It seems therefore that the value of retention decreases somewhat as ventilation increases The mean value 63.6 ± 4 2% (standard deviation) was calculated from all the results (48 measurements) and it was used in all further considerations. The total amount of xylene, which was retained in the organism during exposure was calculated according to the equation: X = C
V
R/100
t
where X is the amount of xylene (in mg) retained in organism during exposure, C is the concentration of xylene vapors in the atmosphere (mg/l), V is the mean lung ventilation of the examined person (1/min), R is the mean lung retention (= 63 6 %), and t is the period of exposure (in min).
Elimination of Xylenes by the Lungs After termination of exposure, the exhaled air was analyzed in short (10 min) intervals, which were later prolonged (30 min); on the 2nd day the time interval between the individual analyses was 4 or even 8 h The studies were continued as long as the If the logpresence of xylene could be observed in exhaled air arithm of the xylene concentration in exhaled air is plotted on a diagram against the time that passed since termination of ex-
210
12
16
20
24 hours
Fig 2 Concentration of m-xylene in exhaled air after exposure Subject was exposed for 8 h to mean m-xylene concentration of 0 373 mg/1 Dependence of concentration in exhaled air upon time of desaturation demonstrated on scale loglin (lower curve) and scale log-log (upper curve)
posure
(Fig 2),
the usual desaturation curve is obtained,
its
trend being interpreted as the sum of the three exponential curves corresponding to the desaturation of the different compartments (probably the respiratory ways, blood, tissue) It is evident that during the first phases after termination of the exposure, the concentration in the exhaled air decreases very steeply and as time proceeds the decrease slows down significantly The elimination takes a relatively long time; a trace amount of xylene can be still observed in the exhaled air even after 48 h. It is possible to calculate approximately (from the concentrations in the exhaled air, from the lung ventilation, and the time of elimination), the total amount of xylene, which is exhaled after exposure If this amount is expressed in percent of xylene retained in organism, the value for the o-xylene is 5 3%, for m-xylene 5 8%, and for p-xylene 3 5% The lower value with the last-mentioned isomer is probably due to the more rapid metabolization of p-xylene At exposure to a mixture of isomers in ratio 1:1:1 a value of 4 9% was found, this being in good agreement with the mean of the individual isomers The amount of xylene eliminated by the lungs is considerably smaller when compared with toluene, of which an average of 16 3% is exhaled l 13 l1
211
Excretion of Unchanged Xylene via Urine Trace amounts of xylene appear already in the first portion of urine collected after 2 h of exposure In the further samples (i.e , after 4, 6, and 8 h of exposure) its concentration increases only slightly The excretion terminates shortly after termination of exposure, and the presence of xylene cannot be observed any more in the further portions of urine If the total amount of xylene excreted via urine is related to the amount retained in the organism during exposure, it appears that this way of detoxication is of small importance The excreted share amounts to thousands of percent (values ascertained in the individual isomers and persons: o 0 0046 and 0.0036%, m 0 0047 and 0 0024%, p 0 0026 and O 0012%), thus being considerably smaller than in the nearest lower homologuetoluene (0 06 %) 13 l.
Identification of the Excretion Forms of Toluic Acids A further question was in what form toluic acids are excreted from the human organism, whether free, bound to glucuronic acid, or conjugated with glycine. The analysis of fresh excreted urine of exposed subjects did not disclose the presence of free toluic acids After storing the urine samples for several days at laboratory temperature, however, clearly visible peaks started to appear on the chromatograms, the retention times of which were identical with the retention times of toluic acids Microbial or enzymatic splitting of toluric acids into toluic acids occurred and could be prevented by slight acidification of the samples (0 4 ml 40% sulphuric acid to 100 ml urine) or by storage at low temperature It was clear that subjects exposed to vapors of o-, m-, and p-xylene do not excrete free toluic acids. The search for acids bound to glucuronic acid was somewhat more difficult No direct method is known so far and for this reason two expediently combined analyses were applied In one sample of urine, the amount of total toluic acid (after alkaline hydrolysis) was determined by the first analysis and the amount of toluric acid by the second analysis Because the amount of total toluic acid was in all cases (i e , after exposure to m-, p-, and also o-xylene) exactly equivalent to the amount of toluric acid, it is obvious that all excreted toluic acids are bound to glycine and that no other forms (i e , free acids or toluylglucuronic acids) are present in the urine of exposed subjects. The fact that man excretes after exposure to xylene only toluric acids contradicts the results of experiments in rab-
212
bits
23
The difference could be attributed to the different It seems, however, that the size of the
course of metabolism
Whereas experimental subjects administered dose is decisive retained a maximum of 0 019 g xylene/kg weight during 8 h of exposure, a dose of up to 0 6 g/kg (i e , about x 30 higher) was The formation of derivatives of administered once to rabbits glucuronic acid is some sort of "emergency" mechanism 2,9l which only sets in at a high load, when the organism is not Such a conlonger able to conjugate all acids with glycine dition is not achieved by far at exposure to adequate concenIt cannot be ex0 4 mg/l) trations of xylene vapors (0 2 cluded, of course, that in special cases toluylglucuronic acids It is therefore expedient for practical could appear in urine purposes to determine the total toluic acids, because the result comprises not only toluric acids but also toluylglucuronic acids and free toluic acids.
The Course of Excretion of Toluric Acids The excretion of toluric acids takes a comparatively rapid A significant amount of metabolites is already present course It increases in the first portion of urine collected after 2 h 2-h sample of in the its maximum and reaches as time proceeds that moment From of exposure on termination urine collected amount but a small onward the excretion decreases again rapidly can still traces remains in the urine for a considerable time; Figure 3 demonstrates the course of 5 days be proved after 4 excretion of m-toluric acid (expressed as the total m-toluic and p-toluric acid takes a quite acid); the excretion of o on the curve are the mean values asThe points similar course in 4 experimental persons, the extreme values are Although the experimental subjects were marked by the abscissa in homogeneous atmosphere, and for the exposed simultaneously, same time, the excreted amount differs nevertheless somewhat in This is caused by the fact that the persons each individual and retain unequal amounts of xyventilation differ in lung certained
lenes during exposure. The course of the excretion curves is very similar in all This can be dempersons and at different levels of exposure amount of total mexample: If the onstrated by the following toluic acid excreted in 24 h (8 h of exposure + further 16 h) is considered to equal 100%, then the amount excreted during 8-h exposure is 72 1 % and the amount excreted in the last 2 h The deviations between various individof exposure is 22 7% The total o-toluic acid (68 5 and 23 0%) uals are very small and the total p-toluic acid (74 6 and 25 0%) are excreted with The mean values 71 7 and 23 6% can be calsimilar regularity culated from the mentioned data and may be of certain import-
213
Fig 3 Excretion of total m-toluic acid in urine Curve is mean of values ascertained in 4 subjects simultaneously exposed to m-xylene of concentration 0.397 mg/l; extreme values marked by abscissa
ance for practical
purposes
If for example the amount of metab-
olite excreted during the last 2 h of exposure is known, it is possible to calculate what amount would be excreted during 8 h of exposure or during 24 h from the start of the 8-h exposure. Recalculation yields reliable results if the concentration of xylene vapors is constant during the entire period of exposure. If the concentration changes significantly or if the exposure is interrupted, the conditions of excretion of metabolites are considerably complicated and the recalculation is less accurate or even fails.
Excretion of Toluric Acids after Exposure to a Mixture of Xylene Isomers Exposure to pure xylene isomers
is to be considered only rarely
under industrial conditions; the employees are much more frequently exposed to vapors of so-called technical xylene, i e , to a mixture of isomers in which usually m-xylene predominates. It was necessary to verify, whether the above-mentioned findings Experimental also apply at exposure to a mixture of isomers to a mixture of isomers at ratio 1:1:1 and subjects were exposed it was ascertained that toluic acids are excreted only in conjugated form with glycine (i e , as toluric acids) and that no free toluic acids or toluylglucuronic acids are present in the urine If the sum of total toluic acids was plotted in the diagram against the time of excretion, a similar excretion curve
214
was obtained as at exposure to pure isomers A rather interesting fact came to light, however, if the mutual ratio of excreted isomeric acids was compared Because the persons were exposed to isomeric xylenes at ratio 1:1:1, it could be excepted, that the ratio of excreted acids would be the same It appeared, however, that there is somewhat more total p-toluic acid (41 5%) in the first 2-h portion of urine and somewhat less o-toluic acid (23 8%); the total m-toluic acid was represented by an amount of 34 7%, which approaches the theoretical value In the further samples of urine the percentual amount of isomeric acids approached mutually, until it was the same (i e , about 33%) at the end of exposure, later total o-toluic acid predominated slightly The preferential excretion of the total p-toluic acid is probably caused by the fact that p-xylene is more easily oxidized in the organism than m or o-xylene Less probable is the possibility that the differences would be caused by different rapidity of excretion via the kidney.
Identification of Hydroxylated Metabolites and the Kinetics of Their Excretion The chromatograms of the extracts of hydrolyzed urine demonstrated several peaks, the retention times of which were longer than the retention times of the normal phenolic components of urine (i e , phenol and p-cresol) L11 l Because these peaks did not appear on the records of urine collected prior to exposure, it was obvious, that they belong to the substances formed in the metabolism of xylenes The identification was performed by comparison with the retention times of individual pure xylenols. In order to exclude the possibility of confusion, data obtained in two stationary phases with different polarity were compared. It is theoretically possible to derive from o-xylene two hydroxylated derivatives; 2 3-xylenol and 3 4-xylenol Both these compounds were ascertained in the urine of persons exposed to o-xylene It is remarkable that the mutual relation of both isomers was considerably variable between different subjects; in some individuals 2 3-xylenol predominated significantly over 3 4-xylenol (ratio about 3:1), in others the opposite was the case. There are three xylenols with the methyl groups in the meta position: 2 4-xylenol, 2 6-xylenol, and 3 5-xylenol But only the first of them was found in the urine of subjects exposed to m-xylene vapors The other two xylenols are not formed in the biotransformation of m-xylene. Only a single hydroxylated derivative (2 5-xylenol) can be derived from p-xylene It was proved according to expectations in the urine of subjects exposed to p-xylene. The amount of xylenols formed by the metabolic transformation of the individual xylenes differed considerably The greatest 215
amount of hydroxylated derivative (2 4-xylenol) was present in urine after exposure to m-xylene, the smallest amount (2 5-xylenol) was found after exposure to the same concentration of pxylene The excretion curves had a similar but not identical course as the excretion curves of the total toluic acids
The
chief difference was that the excreted amounts did not reach maximum at the end of exposure, but in the period immediately following exposure (2 h) For the sake of completeness, it must be added that xylenols formed by biotransformation had in all cases the same position of the methyl groups as the initial xylenes Migration of the methyl group was not observed l4,7l. the assumed metabolites of xylenes in Hydroxytoluic acids were not proved in the urine of exexperimental animals l2l posed persons.
Balance Calculations In order to make an estimation possible on the amount of xylene excreted in the form of toluric acid and in the form of xylenols, urine was collected in several experiments for the entire period during which the presence of the mentioned metabolites could be 5 proved With toluric acids the excretion took usually 4 days, with xylenols about 1 2 days The total amount of excreted metabolites was recalculated to xylene (1 mg of total toluic acid = 0 7798 mg xylene, 1 mg xylenol = 0 8690 mg xylene) and compared with the amount of xylene retained in organism during exposure The results disclosed that toluric acids are formed with almost quantitative yield (o-97 1%, m-99 2%, p-95 1%); the amount of xylenols is only small (o-0 86 %, m-1 98%, p-0 05 %). If the share of xylene eliminated in unchanged form by exhalation is added up with the shares excreted in urine in form of toluric acids and in form of xylenols (the excretion of unchanged xylene in urine is negligibly small), the value for the o-isomer comes to 103 3%, for the m-isomer 107 O%, and for the p-isomer 98 7% In all cases, the result approaches 100 % and this proves that no important way of detoxication and no important metabolite has been overlooked in the studies.
REFERENCES 1 2 3
216
Bakke, O M , Scheline, R R : Hydroxylation of aromatic hydrocarbons in the rat Toxicol appl Pharmacol 16, 691 (1970) Bray, H G , Humpris, B G , Thorpe, W V : Metabolism of derivatives of toluene 3 o-, m and p-xylenes Biochem J 45, 241 (1949) Bray, H G , Humpris, B G , Thorpe, W V : Metabolism of derivatives of toluene 5 The fate of the xylenols in the rabbit, with further obser47, 395 (1950) vations on the metabolism of the xylenes Biochem J
4
Daly, J W , Jerina, D M , Witkop, B : Arene oxides and the NIH shift: The metabolism, toxicity and carcinogenity of aromatic compounds Experientia 28, 1129 (1972)
5
Fabre, R , Truhaut, R , Laham, S : Recherches sur le metabolisme compare des xylenes ou dimethylbenzenes
Arch Mal prof
21, 314 (1960)
9
Flek, J , edivec, V : Determination of toxic substances and their metabolites in biological fluids by gas chromatography VII Toluric or toluic acids in urine Coll Czechoslov Chem Commun 38, 1754 (1973) Kaubisch, N , Daly, J W , Jerina, D M : Arene oxides as intermediates Isomerization of in the oxidative metabolism of aromatic compounds methyl-substituted arene oxides Biochemistry 11, 3080 (1972) Ogata, M , Tomokuni, K , Takatsuka, Y : Urinary excretion of hippuric acid and m or p-methylhippuric acid in the urine of persons exposed to vapours of toluene and m or p-xylene as a test of exposure Brit J. industr Med 27, 43 (1970) Pagnotto, L D , Lieberman, L M : Urinary hippuric acid excretion as an
10
28, 129 (1967) index of toluene exposure Amer industr Hyg Ass J Sedivec, V , Flek, J : Bestimmung toxischer Substanzen und ihrer Meta-
11
boliten in biologischen Flussigkeiten mittels der Gaschromatographie. V Hippursaure in Urin Coll Czechoslov Chem Commun 35, 3265 (1970) Sedivec, V , Flek, J : Bestimmung toxischer Substanzen und ihrer Meta-
6
7
8
boliten in biologischen Flissigkeiten mittels der Gaschromatographie.
12
II Phenol und p-Kresol in Urin Coll Czechoslov Chem Commun 35, 242 (1970) Sedivec, V , Flek, J , Mr&z, M : Preparation of an atmosphere with
13
defined contents of studied substances (in Czech , English summary). Pracovni Lkarstvf 26, 48 (1974) Srbov&, J , Teisinger, J : Absorption and elimination of toluene in man (in Czech , English summary)
Pracovn• Lekarstvi 4, 41
(1952)
Received April 22, 1976 / Accepted May 3, 1976
217
Olcational (k
Int Arch Occup Environ Hlth 37,205-217 (1976)
aml EnvilonlenItal Health t by Springer-Verlag 1976
The Absorption, Metabolism, and Excretion of Xylenes in Man V EDIVEC and J FLEK Institute of Hygiene and Epidemiology (Director: Prof F Janca, M D , Dr Sc ), Center of Industrial Hygiene and Occupational Diseases (Head: Prof B Svestka, M.D , Member of the Czechoslovak Academy of Sciences), Vinohrady, Srobarova 48, v Prague 10, CSSR
Summary Fifteen exposure experiments were carried out, in which four persons each, were simultaneously exposed to a defined concentration of o-, m-, and p-xylene vapors and also to their mixture at a ratio of 1:1:1 The concentrations were in all cases around O 2 mg/l (Czechoslovakian MAC) or around double the amount The period of exposure amounted to exactly 8 h. It was ascertained that the pulmonary retention is practically identical in all persons and in all isomers and amounts to 63 6 ± 4 2% Its extent does not depend upon inoculation level, nor upon the duration of the exposure. During the period of desaturation the lungs excrete approximately 5% of the total amount retained in the organism Xylene excretion via urine is quite negligible and amounts to a thousandth of 1%. The chief metabolites are toluic acids which are excreted in conjugated form with glycine as so-called toluric (=methylhippuric) acids Free toluic acids, toluylglucuronic acids, and hydroxytoluic acids do not occur in the urine of persons exposed to a reasonable concentration of xylene vapors. The excreted amounts of toluric acids reach a maximum in the portion of urine collected at the end of exposure; then they decrease rapidly, but trace amounts can be still observed even after 4-5 days The excretion takes a similar course in different persons and at different intensity of exposure. During the last 2 h of exposure a mean of 23 6% is excreted and in 8 h of exposure 71 7% of the amount excreted-within 24 h. The side metabolites of xylenes are compounds hydroxylated on the aromatic nucleus After exposure to o-xylene the presence of 2 3 and 3 4-xylenol was observed in urine; after exposure to m-xylene the presence of 2 4 xylenol, and finally after exposure to p-xylene the presence of 2 5-xylenol The excretion of xylenols reaches a maximum as a rule just after termination of exposure. It was proved by balance calculation that of the total amount of xylene retained in the organism during exposure, more than 95% is excreted in the form of toluric acid (o-97 1; m-99 2; p-95 1%) and only a small part in form of xylenol (o-0 86 ; m-1 98 ; p-0 05%). Key words:
Xylenes
Absorption
Metabolism
Excretion.
205
INTRODUCTION The metabolism of xylenes was studied in experimental animals after peroral administration of a relatively high dose l2,3,5l. It was proved that all isomers are oxidized in the organism to the corresponding toluic (=methylbenzoic) acids, which are then excreted in urine in different forms: m and p-toluic acid predominantly conjugated with glycine as so-called toluric (=methylhippuric) acid, whereas o-toluic acid occurs predominantly in free form or bound to glucuronic acid In the urine of intoxicated animals the presence of metabolites hydroxylated on the aromatic nucleus was also proved; some of them were later isolated and identified 'i, 5, Considerably less is known about the metabolic' changes of xylenes in the human organism It has been ascertained in experiments in volunteers exposed to adequate vapor concentrations that about 72% of the retained m-xylene is excreted within 26 h as m-toluric acid, the determination of which in urine may serve as an exposure test l8l The form in which the remaining share of m-xylene is excreted is not known, data on the excretion of p-toluric acid are lacking, and the metabolism of o-xylene has not been studied in man yet There are also no data available on the retention of isomeric xylenes in the respiratory system and on the quantity of unchanged hydrocarbons eliminated by exhaling. Studies of the metabolism of xylenes and also studies of the kinetics of excretion of metabolites were accompanied by a number of difficulties The classical analytical methods did not allow reliable determination of the metabolites in the presence of the related normal components of urine (e g , hippuric acid or phenol or p-cresol); this is the reason why the majority of findings was only of qualitative or semiquantitative character. The application of gas chromatography made it possible to derive the quantitative relations.
EXPERIMENTAL PART Methods Preparation of the Atmosphere with Defined Contents of Xylene Vapors
The
exposure experiments were carried out in a laboratory of 5 4 x 3.4 x 3 5 m in size The concentration of xylene vapors in the atmosphere was automatically analyzed by gas chromatography at 5-min intervals and automatically adapted to the required value by special apparatus l12 l. Subjects, Time and Intensity of Exposure
Six healthy men aged 28-50
years, body weight 72-90 g, were at our disposal for the exposure experiments Four of them were simultaneously exposed 206
to a known concentration of isomers (about 0 2 mg/1 and 0 4 mg/l) and in later experiments also to a mixture of known ratio of components The time of exposure was exactly 8 h (without interruption). Analysis of the Air Exhaled during Exposure
The experimental subject
breathed through a mouthpiece connected to a two-way valve The exhaled air was conducted into a glass vessel (7 1 volume), partitioned by a metal sieve (homogenization of the sample), leaving the vessel through a one-way valve In order to prevent condensation of the water vapors, the vessel was externally heated by an electric bandage to 400 C The mean sample was drawn from the vessel by perforated metal tubing and continuously conducted through the pipette of the gas sampling and introduction device of the chromatograph by means of a membrane pump The contents of the gas pipette were introduced at suitable time intervals (after 10-15 min of breathing) into the column and the chromatogram was recorded Each analysis was repeated twice and the concentration of xylene vapors in the inhaled air was determined simultaneously (Fig 1). Analysis of the Air Exhaled after Exposure
The person exhaled the lung
contents into one arm of a T-shaped metal tube Wright's respirometer was connected to the other arm During exhaling, i e , while the dial pointer of the respirometer was revolving, the sample of exhaled air was gradually sucked from the third arm into the connected large (200 ml) glass syringe, immediately introduced into the gas sampling and introduction device, and the chromatogram was recorded Although this procedure did not guarantee as perfect a homogenization of the exhaled air as the previous method, it was possible, however, to study the rapid changes of the xylene concentration (especially during the first phases after termination of exposure), which would be distorted if an apparatus with large volume capacity would have been used. Determination of the Breathing Volume
The experimental subject was
breathing through a mouthpiece into which a Wright's respirometer was placed The volume of exhaled air was read from the dial exactly after 10 min and measurement was repeated at least four times during exposure; the mean was calculated from the measured values. Collection of Urine Samples The urine was sampled at 2-h intervals
during and after termination of exposure and later collected at 8-h intervals. Determination of Free Xylene in Urine
A measured volume of urine
(ob-
tained in an environment free of xylene vapors) was saturated with ammonium sulphate and thoroughly shaken with 1/5 of carbon
207
disulphide volume After centrifugation in a sealed test tube, 3 l of the extract were drawn into an injection microsyringe and the chromatogram was recorded using flame ionization detection The quantitative relations were derived by the method of direct calibration. Determination of the Total Toluic Acids l6l An equal volume of 50 % wt/vol sodium hydroxide was added to 2 ml urine and the mixture was heated for 60 min to 130 0 C in a silicon oil bath After cooling, acidification with dilute sulphuric acid, and saturation with ammonium sulphate, the freed toluic acid was extracted by ethyl acetate and converted into methylester by diazomethane The methylester was chromatographed on a column packed with 10% polyethyleneglycol 1500 at 130 0 C Acetophenone was used as internal standard The total toluic acid (either o-, or m-, or p-) can be determined irrespective whether it is present in the urine free, bound to glucuronic acid, or conjugated with glycine. Determination of Free Toluic Acids
The procedure was the same as in
the previous case, but the alkaline hydrolysis of urine was omitted The extract of urine contained besides toluic acid also toluric and hippuric acid, the methylesters of which have very long retention times under the given conditions The working temperature was increased after recording the peak of methylester of toluic acid, in order to accelerate the elution of the higher boiling esters. Determination of Toluric Acids l10l
Slightly acidified urine
(5 ml)
was saturated by ammonium sulphate and extracted with ethyl acetate The acids which passed into the extract were converted by diazomethane into methylesters, which were chromatographed on a column packed with 3% neopentylglycolsuccinate at 190 0 C using flame ionization detection Phenacetin (p-acetophenetide) served as internal standard The method afforded determination of m and p-toluric acid The methylester of o-toluric acid eluted simultaneously with the methylester of hippuric acid and the chromatographic peaks overlapped l10 l The procedure was therefore modified: the hippuric acid was decomposed by alkaline hydrolysis under mild conditions (20 min of heating urine with the same volume of 50% wt/vol sodium hydroxide to 100 0 C); the resistent o-toluric acid was only decomposed to a small part. The share not decomposed (about 85%) was extracted from the reaction mixture, converted into methylester and chromatographed. The calibration curve was constructed in the same way (i e , including alkaline hydrolysis under mild conditions).
208
Determination of Xylenols One ml of diluted (1:1) sulphuric acid was added to 5 ml of urine and the mixture was heated in a closed vessel in a boiling water bath for 20 min After cooling the liquid was saturated with ammonium sulphate and thoroughly shaken with 2 ml carbon disulphide Three microliters of the extract were analyzed on columns packed with 5% polyethyleneglycol 20 M, 10% didecylphthalate, or 3% neopentylglycolsuccinate at a temperature of about 150 0 C The quantitative relations were derived by the method of direct calibration.
RESULTS AND DISCUSSION Saturation of Organism The first question was, what amount of xylenes is retained in the lungs when inhaling contaminated atmosphere The lung retention (in %) can be calculated from the concentration of xylene vapors (C) in the inhaled and exhaled air according to the equation: lung retention
Cexhalation atmosphere lung retention (%) atmosphere
100
Because the concentration of vapors is directly proportional to the height of the corresponding chromatographic peak, it was possible to substitute in the formula the values (in mm) read on the chromatographic record (Fig 1) In order to achieve reproducible results it appeared essential to homogenize the exhaled air perfectly prior to analysis (in the first part of exhaling there is always a higher concentration than in the last part, and during desaturation the opposite is the case) and to determine the lung retention at quite normal breathing Both these conditions were well fulfilled, if the exhaled air was collected by the previously described apparatus. The lung retention was measured always at two different vapor concentrations, whereby the following mean values were ascertained: o-xylene 62 4 %, m-xylene 64 2%, and p-xylene 64 3 % The results disclosed that retention is almost the same in all isomers and that it is independent of the level of exposure within the studied limits Its extent did not change even with the time of exposure; the values determined between the 1st and 3rd h and between the 5th and 7th h were identical From the theoretical aspect the retention should be somewhat higher in the first phases and should decrease to the mentioned value after a certain time, when equilibrium has been established between the concentration in the inhaled air and in the blood This first stage is obviously short, because several measurements performed after 15 min of exposure yielded practically the same results as
209
Fig 1 Determination of lung retention. Chromatogram obtained in analysis of inhaled and exhaled air during exposure to m-xylene (mean concentration in atmosphere 0 397 mg/l) Volume of sample 3 ml, stationary phase PEG 20 M, working temperature 1100 C, sensitivity 1/16 at input resistance of 1010 ohm
mentioned above In experiments repeated after a few days or even weeks the same values of retention in the same person were In different persons, however, the retention always ascertained differed somewhat; the greatest difference (67 4 and 58 6 %) was observed in persons with very different lung ventilation (7 9 and 11 7 1/min) It seems therefore that the value of retention decreases somewhat as ventilation increases The mean value 63.6 ± 4 2% (standard deviation) was calculated from all the results (48 measurements) and it was used in all further considerations. The total amount of xylene, which was retained in the organism during exposure was calculated according to the equation: X = C
V
R/100
t
where X is the amount of xylene (in mg) retained in organism during exposure, C is the concentration of xylene vapors in the atmosphere (mg/l), V is the mean lung ventilation of the examined person (1/min), R is the mean lung retention (= 63 6 %), and t is the period of exposure (in min).
Elimination of Xylenes by the Lungs After termination of exposure, the exhaled air was analyzed in short (10 min) intervals, which were later prolonged (30 min); on the 2nd day the time interval between the individual analyses was 4 or even 8 h The studies were continued as long as the If the logpresence of xylene could be observed in exhaled air arithm of the xylene concentration in exhaled air is plotted on a diagram against the time that passed since termination of ex-
210
12
16
20
24 hours
Fig 2 Concentration of m-xylene in exhaled air after exposure Subject was exposed for 8 h to mean m-xylene concentration of 0 373 mg/1 Dependence of concentration in exhaled air upon time of desaturation demonstrated on scale loglin (lower curve) and scale log-log (upper curve)
posure
(Fig 2),
the usual desaturation curve is obtained,
its
trend being interpreted as the sum of the three exponential curves corresponding to the desaturation of the different compartments (probably the respiratory ways, blood, tissue) It is evident that during the first phases after termination of the exposure, the concentration in the exhaled air decreases very steeply and as time proceeds the decrease slows down significantly The elimination takes a relatively long time; a trace amount of xylene can be still observed in the exhaled air even after 48 h. It is possible to calculate approximately (from the concentrations in the exhaled air, from the lung ventilation, and the time of elimination), the total amount of xylene, which is exhaled after exposure If this amount is expressed in percent of xylene retained in organism, the value for the o-xylene is 5 3%, for m-xylene 5 8%, and for p-xylene 3 5% The lower value with the last-mentioned isomer is probably due to the more rapid metabolization of p-xylene At exposure to a mixture of isomers in ratio 1:1:1 a value of 4 9% was found, this being in good agreement with the mean of the individual isomers The amount of xylene eliminated by the lungs is considerably smaller when compared with toluene, of which an average of 16 3% is exhaled l 13 l1
211
Excretion of Unchanged Xylene via Urine Trace amounts of xylene appear already in the first portion of urine collected after 2 h of exposure In the further samples (i.e , after 4, 6, and 8 h of exposure) its concentration increases only slightly The excretion terminates shortly after termination of exposure, and the presence of xylene cannot be observed any more in the further portions of urine If the total amount of xylene excreted via urine is related to the amount retained in the organism during exposure, it appears that this way of detoxication is of small importance The excreted share amounts to thousands of percent (values ascertained in the individual isomers and persons: o 0 0046 and 0.0036%, m 0 0047 and 0 0024%, p 0 0026 and O 0012%), thus being considerably smaller than in the nearest lower homologuetoluene (0 06 %) 13 l.
Identification of the Excretion Forms of Toluic Acids A further question was in what form toluic acids are excreted from the human organism, whether free, bound to glucuronic acid, or conjugated with glycine. The analysis of fresh excreted urine of exposed subjects did not disclose the presence of free toluic acids After storing the urine samples for several days at laboratory temperature, however, clearly visible peaks started to appear on the chromatograms, the retention times of which were identical with the retention times of toluic acids Microbial or enzymatic splitting of toluric acids into toluic acids occurred and could be prevented by slight acidification of the samples (0 4 ml 40% sulphuric acid to 100 ml urine) or by storage at low temperature It was clear that subjects exposed to vapors of o-, m-, and p-xylene do not excrete free toluic acids. The search for acids bound to glucuronic acid was somewhat more difficult No direct method is known so far and for this reason two expediently combined analyses were applied In one sample of urine, the amount of total toluic acid (after alkaline hydrolysis) was determined by the first analysis and the amount of toluric acid by the second analysis Because the amount of total toluic acid was in all cases (i e , after exposure to m-, p-, and also o-xylene) exactly equivalent to the amount of toluric acid, it is obvious that all excreted toluic acids are bound to glycine and that no other forms (i e , free acids or toluylglucuronic acids) are present in the urine of exposed subjects. The fact that man excretes after exposure to xylene only toluric acids contradicts the results of experiments in rab-
212
bits
23
The difference could be attributed to the different It seems, however, that the size of the
course of metabolism
Whereas experimental subjects administered dose is decisive retained a maximum of 0 019 g xylene/kg weight during 8 h of exposure, a dose of up to 0 6 g/kg (i e , about x 30 higher) was The formation of derivatives of administered once to rabbits glucuronic acid is some sort of "emergency" mechanism 2,9l which only sets in at a high load, when the organism is not Such a conlonger able to conjugate all acids with glycine dition is not achieved by far at exposure to adequate concenIt cannot be ex0 4 mg/l) trations of xylene vapors (0 2 cluded, of course, that in special cases toluylglucuronic acids It is therefore expedient for practical could appear in urine purposes to determine the total toluic acids, because the result comprises not only toluric acids but also toluylglucuronic acids and free toluic acids.
The Course of Excretion of Toluric Acids The excretion of toluric acids takes a comparatively rapid A significant amount of metabolites is already present course It increases in the first portion of urine collected after 2 h 2-h sample of in the its maximum and reaches as time proceeds that moment From of exposure on termination urine collected amount but a small onward the excretion decreases again rapidly can still traces remains in the urine for a considerable time; Figure 3 demonstrates the course of 5 days be proved after 4 excretion of m-toluric acid (expressed as the total m-toluic and p-toluric acid takes a quite acid); the excretion of o on the curve are the mean values asThe points similar course in 4 experimental persons, the extreme values are Although the experimental subjects were marked by the abscissa in homogeneous atmosphere, and for the exposed simultaneously, same time, the excreted amount differs nevertheless somewhat in This is caused by the fact that the persons each individual and retain unequal amounts of xyventilation differ in lung certained
lenes during exposure. The course of the excretion curves is very similar in all This can be dempersons and at different levels of exposure amount of total mexample: If the onstrated by the following toluic acid excreted in 24 h (8 h of exposure + further 16 h) is considered to equal 100%, then the amount excreted during 8-h exposure is 72 1 % and the amount excreted in the last 2 h The deviations between various individof exposure is 22 7% The total o-toluic acid (68 5 and 23 0%) uals are very small and the total p-toluic acid (74 6 and 25 0%) are excreted with The mean values 71 7 and 23 6% can be calsimilar regularity culated from the mentioned data and may be of certain import-
213
Fig 3 Excretion of total m-toluic acid in urine Curve is mean of values ascertained in 4 subjects simultaneously exposed to m-xylene of concentration 0.397 mg/l; extreme values marked by abscissa
ance for practical
purposes
If for example the amount of metab-
olite excreted during the last 2 h of exposure is known, it is possible to calculate what amount would be excreted during 8 h of exposure or during 24 h from the start of the 8-h exposure. Recalculation yields reliable results if the concentration of xylene vapors is constant during the entire period of exposure. If the concentration changes significantly or if the exposure is interrupted, the conditions of excretion of metabolites are considerably complicated and the recalculation is less accurate or even fails.
Excretion of Toluric Acids after Exposure to a Mixture of Xylene Isomers Exposure to pure xylene isomers
is to be considered only rarely
under industrial conditions; the employees are much more frequently exposed to vapors of so-called technical xylene, i e , to a mixture of isomers in which usually m-xylene predominates. It was necessary to verify, whether the above-mentioned findings Experimental also apply at exposure to a mixture of isomers to a mixture of isomers at ratio 1:1:1 and subjects were exposed it was ascertained that toluic acids are excreted only in conjugated form with glycine (i e , as toluric acids) and that no free toluic acids or toluylglucuronic acids are present in the urine If the sum of total toluic acids was plotted in the diagram against the time of excretion, a similar excretion curve
214
was obtained as at exposure to pure isomers A rather interesting fact came to light, however, if the mutual ratio of excreted isomeric acids was compared Because the persons were exposed to isomeric xylenes at ratio 1:1:1, it could be excepted, that the ratio of excreted acids would be the same It appeared, however, that there is somewhat more total p-toluic acid (41 5%) in the first 2-h portion of urine and somewhat less o-toluic acid (23 8%); the total m-toluic acid was represented by an amount of 34 7%, which approaches the theoretical value In the further samples of urine the percentual amount of isomeric acids approached mutually, until it was the same (i e , about 33%) at the end of exposure, later total o-toluic acid predominated slightly The preferential excretion of the total p-toluic acid is probably caused by the fact that p-xylene is more easily oxidized in the organism than m or o-xylene Less probable is the possibility that the differences would be caused by different rapidity of excretion via the kidney.
Identification of Hydroxylated Metabolites and the Kinetics of Their Excretion The chromatograms of the extracts of hydrolyzed urine demonstrated several peaks, the retention times of which were longer than the retention times of the normal phenolic components of urine (i e , phenol and p-cresol) L11 l Because these peaks did not appear on the records of urine collected prior to exposure, it was obvious, that they belong to the substances formed in the metabolism of xylenes The identification was performed by comparison with the retention times of individual pure xylenols. In order to exclude the possibility of confusion, data obtained in two stationary phases with different polarity were compared. It is theoretically possible to derive from o-xylene two hydroxylated derivatives; 2 3-xylenol and 3 4-xylenol Both these compounds were ascertained in the urine of persons exposed to o-xylene It is remarkable that the mutual relation of both isomers was considerably variable between different subjects; in some individuals 2 3-xylenol predominated significantly over 3 4-xylenol (ratio about 3:1), in others the opposite was the case. There are three xylenols with the methyl groups in the meta position: 2 4-xylenol, 2 6-xylenol, and 3 5-xylenol But only the first of them was found in the urine of subjects exposed to m-xylene vapors The other two xylenols are not formed in the biotransformation of m-xylene. Only a single hydroxylated derivative (2 5-xylenol) can be derived from p-xylene It was proved according to expectations in the urine of subjects exposed to p-xylene. The amount of xylenols formed by the metabolic transformation of the individual xylenes differed considerably The greatest 215
amount of hydroxylated derivative (2 4-xylenol) was present in urine after exposure to m-xylene, the smallest amount (2 5-xylenol) was found after exposure to the same concentration of pxylene The excretion curves had a similar but not identical course as the excretion curves of the total toluic acids
The
chief difference was that the excreted amounts did not reach maximum at the end of exposure, but in the period immediately following exposure (2 h) For the sake of completeness, it must be added that xylenols formed by biotransformation had in all cases the same position of the methyl groups as the initial xylenes Migration of the methyl group was not observed l4,7l. the assumed metabolites of xylenes in Hydroxytoluic acids were not proved in the urine of exexperimental animals l2l posed persons.
Balance Calculations In order to make an estimation possible on the amount of xylene excreted in the form of toluric acid and in the form of xylenols, urine was collected in several experiments for the entire period during which the presence of the mentioned metabolites could be 5 proved With toluric acids the excretion took usually 4 days, with xylenols about 1 2 days The total amount of excreted metabolites was recalculated to xylene (1 mg of total toluic acid = 0 7798 mg xylene, 1 mg xylenol = 0 8690 mg xylene) and compared with the amount of xylene retained in organism during exposure The results disclosed that toluric acids are formed with almost quantitative yield (o-97 1%, m-99 2%, p-95 1%); the amount of xylenols is only small (o-0 86 %, m-1 98%, p-0 05 %). If the share of xylene eliminated in unchanged form by exhalation is added up with the shares excreted in urine in form of toluric acids and in form of xylenols (the excretion of unchanged xylene in urine is negligibly small), the value for the o-isomer comes to 103 3%, for the m-isomer 107 O%, and for the p-isomer 98 7% In all cases, the result approaches 100 % and this proves that no important way of detoxication and no important metabolite has been overlooked in the studies.
REFERENCES 1 2 3
216
Bakke, O M , Scheline, R R : Hydroxylation of aromatic hydrocarbons in the rat Toxicol appl Pharmacol 16, 691 (1970) Bray, H G , Humpris, B G , Thorpe, W V : Metabolism of derivatives of toluene 3 o-, m and p-xylenes Biochem J 45, 241 (1949) Bray, H G , Humpris, B G , Thorpe, W V : Metabolism of derivatives of toluene 5 The fate of the xylenols in the rabbit, with further obser47, 395 (1950) vations on the metabolism of the xylenes Biochem J
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Daly, J W , Jerina, D M , Witkop, B : Arene oxides and the NIH shift: The metabolism, toxicity and carcinogenity of aromatic compounds Experientia 28, 1129 (1972)
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9
Flek, J , edivec, V : Determination of toxic substances and their metabolites in biological fluids by gas chromatography VII Toluric or toluic acids in urine Coll Czechoslov Chem Commun 38, 1754 (1973) Kaubisch, N , Daly, J W , Jerina, D M : Arene oxides as intermediates Isomerization of in the oxidative metabolism of aromatic compounds methyl-substituted arene oxides Biochemistry 11, 3080 (1972) Ogata, M , Tomokuni, K , Takatsuka, Y : Urinary excretion of hippuric acid and m or p-methylhippuric acid in the urine of persons exposed to vapours of toluene and m or p-xylene as a test of exposure Brit J. industr Med 27, 43 (1970) Pagnotto, L D , Lieberman, L M : Urinary hippuric acid excretion as an
10
28, 129 (1967) index of toluene exposure Amer industr Hyg Ass J Sedivec, V , Flek, J : Bestimmung toxischer Substanzen und ihrer Meta-
11
boliten in biologischen Flussigkeiten mittels der Gaschromatographie. V Hippursaure in Urin Coll Czechoslov Chem Commun 35, 3265 (1970) Sedivec, V , Flek, J : Bestimmung toxischer Substanzen und ihrer Meta-
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12
II Phenol und p-Kresol in Urin Coll Czechoslov Chem Commun 35, 242 (1970) Sedivec, V , Flek, J , Mr&z, M : Preparation of an atmosphere with
13
defined contents of studied substances (in Czech , English summary). Pracovni Lkarstvf 26, 48 (1974) Srbov&, J , Teisinger, J : Absorption and elimination of toluene in man (in Czech , English summary)
Pracovn• Lekarstvi 4, 41
(1952)
Received April 22, 1976 / Accepted May 3, 1976
217
