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A simple method for simultaneous determination of benzoic and hippuric acids in biological fluids
CLINICA CHIMICA ACTA
A SIMPLE
METHOD
AND HIPPURIC
S. N. SINHA
Research
FOR
ACIDS
SIMULTANEOUS IN BIOLOGICAL
DETERMINATION
OF BENZOIC
FLUIDS*
AND E. R. GABRIEL1
Division
(Received
313
of Clinical Labovatovies,
E. J, Mevev Memorial
Hospital,
Buflalo,
X.Y.
(C.S.A.)
October 3oth, 1967)
SUMMARY
A simple and sensitive
gel filtration-spectrophotometric
technique
for simul-
taneous determination of benzoic acid and hippuric acid in urine is described. The standard deviations in a series of assays using standard solutions are less than 2.7% and the recovery of added known amounts of benzoic and hippuric acids from urine is satisfactory.
The efficiency of benzoic acid conjugation with glycine to form hippuric acid, and the estimation of the latter in urine was proposed first by Quick1>2 as a clinical test of hepatic function. Since then, this test has been repeatedly used in liver function studies but has not been adapted in clinical laboratories, as a routine test, chiefly due to the lack of a simple and sensitive assay procedure for small amounts of hippuric acid in urine. At present, the metabolic fate of benzoic acid is being reinvestigated in our laboratories. [%]benzoic acid is administered to rats and urinary hippuric acid excretion is studied in clinical subjects. During the course of these studies, the need for a simple and reliable method to determine small amounts of benzoic and hippuric acids in the same specimen became acutely evident. The various micro-n~etllods3-7 reported in recent years in this area were not found satisfactory in our studies. The results of our metabolic studies in rats and human will be reported elsewhere. In this paper a gel filtration-spectrophotometric technique for simultaneous determination of benzoic and hippuric acids in urine is described. MATERIALS
AXD METHODS
For the separation of benzoic and hippuric acids, a column of dextran gel (Sephadex G-IO, Pharmacia Fine Chemicals; 45 x I cm) was used. Before packing the column, the Sephadex powder was washed several times with deinonized water; the fine particles were decanted and then, the slurry was equilibrated with 0.1 M phosphate buffer of pH 7.0. The same buffer was used for elution. All reagents in this *
Supported
by Grants from NIH
GiV 12662-03
and United Health Foundation
Clin. Chim.
Acta,
G-66
XZZIH-5.
19 (1968) 313-317
314
SINHA
study were prepared from analytical grade chemicals. Absorbance measurements were made with a Model DU-z Beckman spectrophotometer. In order to determine the elution pattern, pure solutions of hippuric acid, benzoic acid, uric acid, creatinine, amino acids (&tine, L-asparagine, L-cystine), glucuronic acid, oxalic acid and phenol were passed through the column and eluted with the phosphate buffer. The eluate was collected in r-ml aliquots. Each aliquot was diluted with the buffer to 5 ml and the absorbance was measured at 232 mp against the buffer blank. It was found that glucuronic acid, oxalic acid and phenol were retained while others passed through the column and mere clearly separated. After calibration, I ml of I : 5 diluted urine was applied to tlte column and eluted as above. The eluates of urine were found to follow the same pattern of separation as the mixture of known solutions (Fig. I, Curve A and B). Known concentrations of benzoic acid and hippuric acid, in aqueous solution were passed through the column in order to indicate the precision of the method. The results are recorded in Table I.
Fig. I .t : Elution pattrrn of a mixture of known substances in aqueous solution (0.2 mg each) passed through Scphadescolumn 45 cm x 1 cm. l’eaks, I, amino acids (glycine, z-cystinc; L-n~;psraof I ml ginc); I I, crwtinine; TIT, hippuric acid; I\‘, bcnzoic acid; \‘. uric acid. B: Fractionation dilutctl (1 ,.j) normal urine.
315
BENZOIC AND HIPPURIC ACID
Various
amounts
of benzoic
and hippuric
study and the results are presented TABLE RECOVERY
acids were added to urine for the recovery
in Table
II.
II OF
ADDED
HIPPURIC
SFECTROPHOTOMETRIC
Hippuric acid
3ND
BENZOIC
0.4
0.3
0.2
0.391 0.408
FROM
URINE
BY
GEL
FILTRATIOX-
0.4
IOO.fJ
0.202
101.0
0.396 0.408 0.385 0.388 0.382
0.2
97.8 102.0 90.0 95.0
0.396 0.380 0.406 0.294 0.288 0.301 0.194 0.*92 0.205 0.200
Benzoic acid
ACIDS
METHOD
0.201
0.194 0.196 0.190 0.204
101.5
98.0 90.0
97.0 90.0 102.5 IocP.0
99.0 102.0 9t1.3 97.0 95.5 100.5
97.0 98.2 95.0 102.0
After the outlined preliminary investigations, the routine procedure for the estimation of benzoic and hippuric acids in urine was as follows: One ml of diluted urine was placed on the column and eluted with the phosphate buffer. The first 30 ml of eluate was rejected and then 35 ml of eluate in I-ml aliquots was collected. Each aliquot was diluted to 5 ml with the buffer and the absorbance was measured at 232 rnp against the buffer blank. A standard curve was established by means of standard solutions and used in the urine assay. RESULTS
The curve A in Fig. I represents a typical elution pattern of a mixture of pure solutions of amino acids (glycine, L-cystine, and L-asparagine), creatinine, hippuric acid, benzoic acid, uric acid, glucuronic acid, oxalic acid and phenol (0.2 mg/ml each) applied to the column. Amino acids, creatinine, hippuric acid, benzoic acid and uric acid passed through the column and the respective peaks represent their separation : while glucuronic acid, oxalic acid and phenol were retained by the column. Curve B indicates the fractionation of I ml of I : 5 diluted normal urine. It is evident from Curve B that the eluates of urine follow the same pattern of separation as the known mixture (A). In order to test the specificity of the method, spectral absorbance curves of all eluate fractions that contained hippuric acid or benzoic acid, were determined Clin. Chim.
Acta,
19 (196X) 313-317
316
SINHA
and compared with the absorbance curves of the pure compounds. (Wavelength of maximum absorbance; hippuric acid z&S rnp; benzoic acid 225 mp). In all eluate portions only one peak was observed which corresponded to the pure compounds indicating that the absorbance is entirely due to hippuric acid or benzoic acid only. The data in Table I demonstrate the reproducibility in serial determinations of known solutions of hippuric acid and benzoic acid. The standard deviations of each set of five experiments were found to be less than 2.70/. The recovery studies in when various amounts of benzoic and hippuric Table II show a range of 95-102.5”/0 acids n-cre added to urine.
Several methods are reported in the literature
for the determination
of benzoic
acid and hippuric acid in biological fluids but they were not found satisfactory in our studies. I’riedmanns determined urinary hippuric acid gravimetrically after crystallization, a good but definitely a macromethod. Later workersrlyr2 modified the isolation procedure, using sodium chloride or ammonium sulphate to enhance the crystallization of hippuric acid, which is followed by titration of the acid with sodium hydroxide. The titrimetric procedures of Folin and Flanders? and Kingsbury et ~1.‘~ require continuous extraction of the urine with ether and titration of the benzoic acid, liberated on hydrolysis of the hippuric acid. Quick1 in his classical report also used these tech niques, and determined the amino nitrogen of the liberated glycine by formal titration. All these methods are cumbersome and only suitable for large quantities of hippuric acid. Nichollsr3, in his attempt to develop a color reaction, oxidized benzoic acid by hydrogen peroxide to salicylic acid, which can be estimated calorimetrically; this oxidation proceeds, however, only to about ro”,b and is, therefore, unsatisfactory for quantitative purposes. Dickens and co-workers7 estimated calorimetrically microquantities of benzoic and hippuric acids in biological materials, by nitration of these compounds at room temperature. This procedure is time-consuming and complicated for routine use in a clinical laboratory. Gaffney and co-workers3 reported a paper chromatographic separation of urinary hippuric acid but this technique is too timeconsuming for routine use. Elliott4 developed a spectrophotometric method based on a partial separation of hippuric acid from other urinary components by ion exchange chromatography while Ellman et al.5 estimated this compound by a fluorometric method. Roth these methods are sensitive and relatively simple but cannot be used for the simultaneous determination of benzoic and hippuric acids. The method of Tubis and co-workers6 of solvent extraction is also long and laborious. It may be seen from the foregoing brief discussion that the existing methods are not practical and/or cannot be used for simultaneous assay of hippuric and benzoic acids. The gel filtration-spectrophotometric procedure described here is simple, sensitive and has been found satisfactory for this purpose. It is recognized that some of the urinary components such as amino acids, creatinine, uric acid, phenols, glucuronic acid, oxalic acid and others, absorb ultraviolet light at the wavelength of 232 rnp and would interfere in the determination of benzoic and hippuric acids. In our procedure these sources of error are eliminated as amino acids, creatinine, hippuric acid, benzoic acid and uric acid after fractionation are clearly separated, whereas
BENZOIC
AND
HIPPURIC
ACID
317
glucuronic acid, phenol and oxalic acid are retained by the column. Interference by other components in the urine appeared to be negligible, either due to their low level or, because they do not absorb light at this wavelength. The described assay method was also applied successfully to plasma samples containing [%]benzoic and [14C]hippuric acids and the elution pattern of these two labelled compounds was found to be the same as determined in urine. REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13
A. J. QUICK, J. Viol. Chew, 67 (1926) 177. A. J. QUICK, Am. J. Med. Sci., 185 (1933) 630. G. W. GAFFNEY, K. SCHREUR, N. FERR~NTE AND K. ALTMAX, J. Biol. Chem., 206 (1954) 695. H. C. ELLIOTT, Jr., Anal. Chem., 29 (1957) 1712. G. ELLMAN, A. BURKH~LTER AND J. La Du, J. Lab. Clin. Med., 57 (1961) 813. &I. TUBIS, W. H. BLAHD, J. S. ENDOW AND S. S. Raw~~ay. J. Nuclear Med., 5 (1964) 532. F. DICKENS AND J. PEARSON, B&hem. J., 48 (1951) 216. E. FRIEDMANN, Hop@-Seylers Z. Physiol. Chem., 35 (1911) 49. 0. FOLIN AND F. F. FLANDERS, J. Biol. Chew, II (1912) 257. F. B. KINGSBURY AND W. W. SWANSON, 1. Biol. Chem., 48 (1921) I.~. L. &EVE, E. HILL AND S. NESBITT, J. Ldb. Clin. Med., 3k (;950) 705. T. E. WEICHSELBAUM AND J. G. PROBSTEIN, J. Lab. Clin. Med.. 24 (1939) 636 J. R. NICHOLLS, Analyst, 53 (1928) 19. Clin. Chim.
Acta,
19 (~968)
313-3
17
A SIMPLE
METHOD
AND HIPPURIC
S. N. SINHA
Research
FOR
ACIDS
SIMULTANEOUS IN BIOLOGICAL
DETERMINATION
OF BENZOIC
FLUIDS*
AND E. R. GABRIEL1
Division
(Received
313
of Clinical Labovatovies,
E. J, Mevev Memorial
Hospital,
Buflalo,
X.Y.
(C.S.A.)
October 3oth, 1967)
SUMMARY
A simple and sensitive
gel filtration-spectrophotometric
technique
for simul-
taneous determination of benzoic acid and hippuric acid in urine is described. The standard deviations in a series of assays using standard solutions are less than 2.7% and the recovery of added known amounts of benzoic and hippuric acids from urine is satisfactory.
The efficiency of benzoic acid conjugation with glycine to form hippuric acid, and the estimation of the latter in urine was proposed first by Quick1>2 as a clinical test of hepatic function. Since then, this test has been repeatedly used in liver function studies but has not been adapted in clinical laboratories, as a routine test, chiefly due to the lack of a simple and sensitive assay procedure for small amounts of hippuric acid in urine. At present, the metabolic fate of benzoic acid is being reinvestigated in our laboratories. [%]benzoic acid is administered to rats and urinary hippuric acid excretion is studied in clinical subjects. During the course of these studies, the need for a simple and reliable method to determine small amounts of benzoic and hippuric acids in the same specimen became acutely evident. The various micro-n~etllods3-7 reported in recent years in this area were not found satisfactory in our studies. The results of our metabolic studies in rats and human will be reported elsewhere. In this paper a gel filtration-spectrophotometric technique for simultaneous determination of benzoic and hippuric acids in urine is described. MATERIALS
AXD METHODS
For the separation of benzoic and hippuric acids, a column of dextran gel (Sephadex G-IO, Pharmacia Fine Chemicals; 45 x I cm) was used. Before packing the column, the Sephadex powder was washed several times with deinonized water; the fine particles were decanted and then, the slurry was equilibrated with 0.1 M phosphate buffer of pH 7.0. The same buffer was used for elution. All reagents in this *
Supported
by Grants from NIH
GiV 12662-03
and United Health Foundation
Clin. Chim.
Acta,
G-66
XZZIH-5.
19 (1968) 313-317
314
SINHA
study were prepared from analytical grade chemicals. Absorbance measurements were made with a Model DU-z Beckman spectrophotometer. In order to determine the elution pattern, pure solutions of hippuric acid, benzoic acid, uric acid, creatinine, amino acids (&tine, L-asparagine, L-cystine), glucuronic acid, oxalic acid and phenol were passed through the column and eluted with the phosphate buffer. The eluate was collected in r-ml aliquots. Each aliquot was diluted with the buffer to 5 ml and the absorbance was measured at 232 mp against the buffer blank. It was found that glucuronic acid, oxalic acid and phenol were retained while others passed through the column and mere clearly separated. After calibration, I ml of I : 5 diluted urine was applied to tlte column and eluted as above. The eluates of urine were found to follow the same pattern of separation as the mixture of known solutions (Fig. I, Curve A and B). Known concentrations of benzoic acid and hippuric acid, in aqueous solution were passed through the column in order to indicate the precision of the method. The results are recorded in Table I.
Fig. I .t : Elution pattrrn of a mixture of known substances in aqueous solution (0.2 mg each) passed through Scphadescolumn 45 cm x 1 cm. l’eaks, I, amino acids (glycine, z-cystinc; L-n~;psraof I ml ginc); I I, crwtinine; TIT, hippuric acid; I\‘, bcnzoic acid; \‘. uric acid. B: Fractionation dilutctl (1 ,.j) normal urine.
315
BENZOIC AND HIPPURIC ACID
Various
amounts
of benzoic
and hippuric
study and the results are presented TABLE RECOVERY
acids were added to urine for the recovery
in Table
II.
II OF
ADDED
HIPPURIC
SFECTROPHOTOMETRIC
Hippuric acid
3ND
BENZOIC
0.4
0.3
0.2
0.391 0.408
FROM
URINE
BY
GEL
FILTRATIOX-
0.4
IOO.fJ
0.202
101.0
0.396 0.408 0.385 0.388 0.382
0.2
97.8 102.0 90.0 95.0
0.396 0.380 0.406 0.294 0.288 0.301 0.194 0.*92 0.205 0.200
Benzoic acid
ACIDS
METHOD
0.201
0.194 0.196 0.190 0.204
101.5
98.0 90.0
97.0 90.0 102.5 IocP.0
99.0 102.0 9t1.3 97.0 95.5 100.5
97.0 98.2 95.0 102.0
After the outlined preliminary investigations, the routine procedure for the estimation of benzoic and hippuric acids in urine was as follows: One ml of diluted urine was placed on the column and eluted with the phosphate buffer. The first 30 ml of eluate was rejected and then 35 ml of eluate in I-ml aliquots was collected. Each aliquot was diluted to 5 ml with the buffer and the absorbance was measured at 232 rnp against the buffer blank. A standard curve was established by means of standard solutions and used in the urine assay. RESULTS
The curve A in Fig. I represents a typical elution pattern of a mixture of pure solutions of amino acids (glycine, L-cystine, and L-asparagine), creatinine, hippuric acid, benzoic acid, uric acid, glucuronic acid, oxalic acid and phenol (0.2 mg/ml each) applied to the column. Amino acids, creatinine, hippuric acid, benzoic acid and uric acid passed through the column and the respective peaks represent their separation : while glucuronic acid, oxalic acid and phenol were retained by the column. Curve B indicates the fractionation of I ml of I : 5 diluted normal urine. It is evident from Curve B that the eluates of urine follow the same pattern of separation as the known mixture (A). In order to test the specificity of the method, spectral absorbance curves of all eluate fractions that contained hippuric acid or benzoic acid, were determined Clin. Chim.
Acta,
19 (196X) 313-317
316
SINHA
and compared with the absorbance curves of the pure compounds. (Wavelength of maximum absorbance; hippuric acid z&S rnp; benzoic acid 225 mp). In all eluate portions only one peak was observed which corresponded to the pure compounds indicating that the absorbance is entirely due to hippuric acid or benzoic acid only. The data in Table I demonstrate the reproducibility in serial determinations of known solutions of hippuric acid and benzoic acid. The standard deviations of each set of five experiments were found to be less than 2.70/. The recovery studies in when various amounts of benzoic and hippuric Table II show a range of 95-102.5”/0 acids n-cre added to urine.
Several methods are reported in the literature
for the determination
of benzoic
acid and hippuric acid in biological fluids but they were not found satisfactory in our studies. I’riedmanns determined urinary hippuric acid gravimetrically after crystallization, a good but definitely a macromethod. Later workersrlyr2 modified the isolation procedure, using sodium chloride or ammonium sulphate to enhance the crystallization of hippuric acid, which is followed by titration of the acid with sodium hydroxide. The titrimetric procedures of Folin and Flanders? and Kingsbury et ~1.‘~ require continuous extraction of the urine with ether and titration of the benzoic acid, liberated on hydrolysis of the hippuric acid. Quick1 in his classical report also used these tech niques, and determined the amino nitrogen of the liberated glycine by formal titration. All these methods are cumbersome and only suitable for large quantities of hippuric acid. Nichollsr3, in his attempt to develop a color reaction, oxidized benzoic acid by hydrogen peroxide to salicylic acid, which can be estimated calorimetrically; this oxidation proceeds, however, only to about ro”,b and is, therefore, unsatisfactory for quantitative purposes. Dickens and co-workers7 estimated calorimetrically microquantities of benzoic and hippuric acids in biological materials, by nitration of these compounds at room temperature. This procedure is time-consuming and complicated for routine use in a clinical laboratory. Gaffney and co-workers3 reported a paper chromatographic separation of urinary hippuric acid but this technique is too timeconsuming for routine use. Elliott4 developed a spectrophotometric method based on a partial separation of hippuric acid from other urinary components by ion exchange chromatography while Ellman et al.5 estimated this compound by a fluorometric method. Roth these methods are sensitive and relatively simple but cannot be used for the simultaneous determination of benzoic and hippuric acids. The method of Tubis and co-workers6 of solvent extraction is also long and laborious. It may be seen from the foregoing brief discussion that the existing methods are not practical and/or cannot be used for simultaneous assay of hippuric and benzoic acids. The gel filtration-spectrophotometric procedure described here is simple, sensitive and has been found satisfactory for this purpose. It is recognized that some of the urinary components such as amino acids, creatinine, uric acid, phenols, glucuronic acid, oxalic acid and others, absorb ultraviolet light at the wavelength of 232 rnp and would interfere in the determination of benzoic and hippuric acids. In our procedure these sources of error are eliminated as amino acids, creatinine, hippuric acid, benzoic acid and uric acid after fractionation are clearly separated, whereas
BENZOIC
AND
HIPPURIC
ACID
317
glucuronic acid, phenol and oxalic acid are retained by the column. Interference by other components in the urine appeared to be negligible, either due to their low level or, because they do not absorb light at this wavelength. The described assay method was also applied successfully to plasma samples containing [%]benzoic and [14C]hippuric acids and the elution pattern of these two labelled compounds was found to be the same as determined in urine. REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13
A. J. QUICK, J. Viol. Chew, 67 (1926) 177. A. J. QUICK, Am. J. Med. Sci., 185 (1933) 630. G. W. GAFFNEY, K. SCHREUR, N. FERR~NTE AND K. ALTMAX, J. Biol. Chem., 206 (1954) 695. H. C. ELLIOTT, Jr., Anal. Chem., 29 (1957) 1712. G. ELLMAN, A. BURKH~LTER AND J. La Du, J. Lab. Clin. Med., 57 (1961) 813. &I. TUBIS, W. H. BLAHD, J. S. ENDOW AND S. S. Raw~~ay. J. Nuclear Med., 5 (1964) 532. F. DICKENS AND J. PEARSON, B&hem. J., 48 (1951) 216. E. FRIEDMANN, Hop@-Seylers Z. Physiol. Chem., 35 (1911) 49. 0. FOLIN AND F. F. FLANDERS, J. Biol. Chew, II (1912) 257. F. B. KINGSBURY AND W. W. SWANSON, 1. Biol. Chem., 48 (1921) I.~. L. &EVE, E. HILL AND S. NESBITT, J. Ldb. Clin. Med., 3k (;950) 705. T. E. WEICHSELBAUM AND J. G. PROBSTEIN, J. Lab. Clin. Med.. 24 (1939) 636 J. R. NICHOLLS, Analyst, 53 (1928) 19. Clin. Chim.
Acta,
19 (~968)
313-3
17