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The dependence of vanillic acid excretion on urinary pH
413
CLINICA CHIMICA ACTA
THE DEPENDENCE
OF VANILLIC
ACID EXCRETION
ON URINARY
pH
J. PRICE
Nuftield Unit of Medical Gevzetics and Department of Psychiatry, (Received
Ulaiversity of Livev$vol (U.K.)
June 23. xg6g)
SUMMARY
The amount of excretion of vanillic acid, a metabolite of protocatechuic acid (and a normal urinary constituent), correlates positively with urinary PH. The excretion of vanilloylglycine is not significantly related to urinary pH. The implications of these findings are discussed.
INTRODUCTION
It has been known since the work of Milner that a change in urinary pH will have a profound effect on the excretion of certain weak acids and bases. Reports of this type of effect have been well summarized by Weiner and Mudgea; such reports have been mainly concerned with drug excretion rather than with the excretion of naturally occurring compounds. The occurrence of phenolic acids in the urine is well known, but quantitative studies have been limited. Daily excretion rates are reported to vary within wide limits for many of the compounds studieds. Urinary pH does not appear to have been taken much account of, and this may be related to the practice of collecting samples straight into mineral acid prior to estimation of their phenolic acid content ; this, for example, is recommended practice in the estimation of homovanillic acid’, and of vanillylmandelic acid (VMA) 6+. Phenolic acids will certainly be more stable in acid conditions but will not necessarily deteriorate quickly at normal urinary pH : in the present study more vanillic acid (VA) was found in alkaline than in acid urine. METHODS
Thirty subjects were studied, 24 male and 6 female. Their ages ranged between 18 and 33. All were in good physical health. Each subject was given a dose of protocatechuic acid (PCA) under the following standardized conditions. During the day before PCA administration all the drugs and the following items known to yield sizeable amounts of phenolic acids (and particularly vanillic acid) were excluded from the diet: spinach, carrots, custard, blancmange, ice cream, all fruit, coffee, cocoa, beer, coca cola, fruit drinks. This list is somewhat empirical, C&n.Cl&.
LfCtU,
26
(19%)
413-418
PRICE
414
but, as shown in Table II, interference by phenolics other than PCA metabolites was of a low order. At the end of a x0-h overnight fast, the subjects consumed a dose of PCA*. The amount consumed was proportional to body weight, a IO-stone individual receiving a dose of I g. The PCA was administered in the form of a solution adjusted with alkali to a pH of about 6 and containing glucose which acted both as a sweetener and as a reducing agent (PCA being liable to oxidation to the corresponding quinone). No fluid was taken for 14 h after PCA administration. Thereafter fluid was taken freely. Immediately prior to the consumption of PCA the bladder was emptied, this specimen beingdiscarded (except in 6 subjects who each provided a pre-test specimen). Urine was collected thereafter into a polythene container containing 0.2 ml 5% aqueous sodium azide (this amount of azide does not appreciably affect urinary pH). At the end of 3 h the subjects were instructed to empty their bladder completely and the collection was thus completed. Smoking was forbidden on the day of the test until urine collection had been completed. The pH of each specimen of urine was measured on a Pye pH meter and its volume was measured at room temperature in a measuring cylinder. Three ml concentrated HCl were then added to the urine and an aliquot of this acidified urine was stored at --zoo until analysis was carried out. TABLE
I
THE MEASUREMENT
OF VA AND
VAG
Substancc(s j being measured
Melhod
I. VA plus VAG 2. VA 3. VAG
Acid hydrolysis, then VA measurement No acid hydrolysis, VA measurement Result I minus result z
ilcid hydrolysis was carried out by adding 3 ml concentrated HCl to 15 ml of urine and autoclaving at rg-lb/sq. inch for 14 h (Vanillylglycine is entirely broken down to vanillic acid and glycine under these conditions).
0I \ 0
HO CH30 Voniiltc acid (VA)
\ /
Isovonillic
HO
HO’ Vanilloylglycine
(vAG)
COOH
acid (IVAt
COOH
I
Protocotechuic
acid (PCA)
Fig. I. Structural formulae of the compounds
cited.
Analytical methods These are described in more detail elsewhere8. To summarize, VA estimations were carried out before and after acid hydrolysis, values for VA and vanillylglycine being derived as shown in Table I. Fig. I gives the structural formulae of the compounds cited. * Obtained from Sigma Chemical Company and used without further purification. C&z. Clti~z. Acta, 26 (1969) 413-418
URINARY
VANILLIC
ACID AND
pH
41.5
The pH was adjusted to 1.5 (in both hydrolysed and unhydrolysed specimens), and the solution saturated with sodium chloride. It was then extracted with ethyl acetate and an aliquot of the latter extracted with 10% NaHCO,. An aliquot of the bicarbonate was then acidified and the resulting acid solution was extracted with a small volume of ethyl acetate, this small volume constituting the final ethyl acetate extract. After paper chromatography (ascending) in a system which separates VA from VAG and PCA (chloroform-glacial acetic acid-water, 2: I: I, lower organic phase)9, the area of the paper known (by staining of a duplicate) to contain the vanillic acid was cut out and eluted, and colour developed by means of the modification of the Emerson reaction used by Sumere 10.Standard aqueous solutions of vanillic acid* of varying concentration were carried through the entire procedure so as to enable a calculation to be made of the amount of vanillic acid originally in the urine. An error is introduced because VA and VAG as estimated here are contaminated by small amounts of other PCA metabolites, namely isovanillic acid and its glycine conjugate but the amounts of these metabolites will lead to an overestimate of VA and VAG by only about 4%, and they have been ignored in the present study. All estimations on samples and standard urine were done in duplicate and not accepted unless there was agreement between duplicates within 5%. Six samples of pre-test urine were also studied; a calculation of blank values was made by estimating how much VA and VAG would have been calculated to be present in an amount of pretest urine containing as much creatinine as the test specimen. It will be noted from Table II that these values are very small. TABLE THE
II
PERCENTAGE
Means f
EXCRETION
OF
VA
AND
VAG
IN THE
3-h
URINE
VAG as y. total dose
S.D.
24 male subjects 6 female subjects 6 pre-test urines (all from male subjects)
24.27 + 3.65 22.71 i_ 4.50 0.123 + 0.029
COLLECTION
VA as % total dose 3.31 2 1.486 3.16 + 1.868 0.041 f 0.007
Note: All percentages represent the amount of PCA from which VA and VAG were derived as a percentage of the administered dose.
RESULTS
The results are shown graphically in Figs. z and 3. In Fig. z, vanillylglycine as a percentage of the administered dose is plotted against urinary pH. In Fig. 3, vanillic acid as a percentage of the administered dose is plotted against urinary pH. The lines in these figures are the regression lines obtained for the 24 male subjects only. The slope of the regression line in Fig. 3, but not in Fig. 2, deviates significantly from zero (see Table III). It will be noted that the slope of the line in Fig. 3 is considerable. At pH 5, the value given by the line for VA x Ioo/totaldose is 1.74; at pH 7, 4.91 * Obtained from Koch-Light
Chemical Company. C&-Z. China. A&,
26 (1969) 413-418
416
PRICE
24.
. 14
I 6.0 Urinary pH
5.0
7.0
Fig. 2. The excretion of vanilloylglycine, plotted against urinary pH. Ordinate: vanillylglycine in a 3-h collection as o/0 of dose of PCA. a, female volunteers; 0, male volunteers.
I
I
5.0
6.0
7.0
pH of urine
Fig. 3. The excretion of vanillic acid, plotted against urinary pH. Ordinate: free vanillic acid in a 3-h urine collection as o/0 of dose of PCA. n , 6 female volunteers; 0, 24 male volunteers. TABLE SOME
III
STATISTICAL
Subjects
24 male
6 female all 30
pH
RESULTS
OBTAINED
IJ~YSUSVA
FROM
THE
DATA
as 0/0total dose
Y
t
0.608 0.873 0.657
3.596 3.583 4.612
pH
versus
VAG
as 0/0total dose
P
1
t
P
0.01 > p > O.OOI 0.05 > p > 0.02 O.OOI >p
-0.209
- I.002 0.795 -0.r94
N.S. N.S. N.S.
0.369
-0.037
It might be expected that the volume of a test solution should be related to its pH (since more dilute urine specimens will tend to be less strongly acidic). The relationship did not attain statistical significance in the present study, although the correlation was in the expected direction (Y = 0.293, t = 1.621, p > 0.1). There was, however, a significant association between VA excretion and urine volume for the total group of 30 subjects. For this reason, partial correlation coefficient+ were calculated: for VA excretion against pH the partial regression coefficient, after adjustClin. Chim.
Acta,
26 (1969)
413-418
URINARY
VANILLIC
ACID AND
pH
417
ment for the effect of volume, was r = 0.616 ($J < 0.001) and for VA excretion against volume, after adjustment for the effect of pH, was Y = 0.295 ($ > 0.1). This confirms that it is pH rather than volume that determines the effect that is here described. DISCUSSION
Weiner and Mudge2>‘2 indicate in their comprehensive studies of the subject under discussion here that an influence of urinary pH on the excretion of an organic acid is generally more likely to occur if this acid is lipid soluble. That VA is more lipid soluble than VAG may be reflected in the high RF (0.78) of the former in the chlorofornl-containing solvent system used compared with the much lower RF (0.10) of the latters. PCA, the excretion of which also appears to be unrelated to urinary pH, has an even lower RF in this solvent system than VAG has. Although VA and PCA have very similar pK, values (4.52 and 4.48 respectively)*3, it is only in the case of VA that excretion is related to urinary pH. These observations are consistent with principles stated by Weiner and Mudge 2~12,namely that both a suitable pK, and a sufficient degree of lipid solubility are necessary if an effect of urinary pH on the excretion of a compound is to occur. Future quantitative studies of phenolic acid excretion under normal and pathological conditions ought to take more account of urinary pH. This is not an impracticable suggestion. In a metabolic ward with accurate collection facilities each specimen of urine making up a 24-h collection could be split immediately into equal parts, half being kept in mineral acid for phenolic acid estimation (and amine analysis, if required), the other half being stored in a preservative, e.g. sodium azide, which will not invalidate the measurement of urinary pH. An example of a recently studied lipid-soluble carboxylic acid to which the above statements may have relevance is 3,4-dimethoxyphenylacetic acid (DMPAA) (refs. 14, IS). DMPAA excretion has been of interest in connection with the disputed excretion of the amine from which DMPAA can derive, 3,4-dimethoxyphenylethylamine (DMPE) in schizophrenic individuals. The judgement that an individual’s DMPAA excretion falls “within the normal range” may need to make some reference to urinary pH. ACKNOWLEDGEMENTS
The author is grateful to the Central Photographic Service, University of Liverpool, and to Mrs. T. Williams for help with the preparation of Figs. 2 and 3. Mr. M. C. K. Tweedie gave valuable statistical advice. This work was initiated while the author was a Bates Research Fellow of the Mental Health Research Fund. The Schizophrenia Research Fund has also provided financial assistance which the author is happy to acknowledge. REFERENCES I M. D. MILNE, B. H. SC~BNER AND M. A. CRAWFORD, Aster. J. Med., 24 (1958) 7og. z I. M. WEINER AND G. H. MUDGE, Amer. J. Med., 36 (1964) 743. 3 C. M. WILLIAMS AND C. C. SWEELEY, in H. A. SZYMANSKI (Ed.), BiocLemicaZApplicatioms Chromalograj$zy, Plenum Press, New York, 1964. C&z. Chim. Acta,
of&s
26 (1969) 413-418
PRICE
4x8 1 C, R. J. RUTHVEN AND M. SANDLER, Cr’ilz. Chiw. A&t, 14 (1~66) 511. H, VA&BY, PvacticaL i%nical ~~~c~~rn~s~~~, 4th ed,, W. Heinemann, London,
5 6 7 8
I. D. P. WOOTTON, Microanalysis ia MedicaE Biochemistry, E. C. BATE-SMITH,The Pharmacology ofPlant Phenolics, J, PRICE,Clin. Chim. Acta, 25 (1969) 31.
g A. N. Boarn, 0. H. EMERSON, F. T.
Churchill, Academic
1967, p. 692. London, ~$4. p. 189. Press, New York, 1959.
JONES AND F. de EDS, J. Biol. Chcm.,
229 (1957) 51. 6 (1961) 484. New York, 1952,p.284. 12 I.M. WEINER, K. C. BLANEH~RD AND G. H. MUDGE, Amer. J. Physiol., 207 (1964)953. 13 R. T. WILLMMS, in E. C. BATE-SMX~K(Ed.), The ~~arrna~~~ogy of P&z& PhsnoKcs, Academic Press, New Yark, r959., p. 13. IO C. I?. SUMERE, F. PARMENTIER AND H. TEUCHY, J. Chromatog., rI L. I-I. C.TIPPETT, The Methods of Stakistics, 4th ed., John Wiley,
r4 F.A. Ku~~~L,R.E.ORMOND AND W.J.A.VANDENHEUVEL,N~~~~,ZII xg A. J. FRIEDRQFFAND K. FURIYA,Natwe, z14(rg67)1127. Cdi%Ckint.b%
26 (1969)413-418
(1966) 606.
CLINICA CHIMICA ACTA
THE DEPENDENCE
OF VANILLIC
ACID EXCRETION
ON URINARY
pH
J. PRICE
Nuftield Unit of Medical Gevzetics and Department of Psychiatry, (Received
Ulaiversity of Livev$vol (U.K.)
June 23. xg6g)
SUMMARY
The amount of excretion of vanillic acid, a metabolite of protocatechuic acid (and a normal urinary constituent), correlates positively with urinary PH. The excretion of vanilloylglycine is not significantly related to urinary pH. The implications of these findings are discussed.
INTRODUCTION
It has been known since the work of Milner that a change in urinary pH will have a profound effect on the excretion of certain weak acids and bases. Reports of this type of effect have been well summarized by Weiner and Mudgea; such reports have been mainly concerned with drug excretion rather than with the excretion of naturally occurring compounds. The occurrence of phenolic acids in the urine is well known, but quantitative studies have been limited. Daily excretion rates are reported to vary within wide limits for many of the compounds studieds. Urinary pH does not appear to have been taken much account of, and this may be related to the practice of collecting samples straight into mineral acid prior to estimation of their phenolic acid content ; this, for example, is recommended practice in the estimation of homovanillic acid’, and of vanillylmandelic acid (VMA) 6+. Phenolic acids will certainly be more stable in acid conditions but will not necessarily deteriorate quickly at normal urinary pH : in the present study more vanillic acid (VA) was found in alkaline than in acid urine. METHODS
Thirty subjects were studied, 24 male and 6 female. Their ages ranged between 18 and 33. All were in good physical health. Each subject was given a dose of protocatechuic acid (PCA) under the following standardized conditions. During the day before PCA administration all the drugs and the following items known to yield sizeable amounts of phenolic acids (and particularly vanillic acid) were excluded from the diet: spinach, carrots, custard, blancmange, ice cream, all fruit, coffee, cocoa, beer, coca cola, fruit drinks. This list is somewhat empirical, C&n.Cl&.
LfCtU,
26
(19%)
413-418
PRICE
414
but, as shown in Table II, interference by phenolics other than PCA metabolites was of a low order. At the end of a x0-h overnight fast, the subjects consumed a dose of PCA*. The amount consumed was proportional to body weight, a IO-stone individual receiving a dose of I g. The PCA was administered in the form of a solution adjusted with alkali to a pH of about 6 and containing glucose which acted both as a sweetener and as a reducing agent (PCA being liable to oxidation to the corresponding quinone). No fluid was taken for 14 h after PCA administration. Thereafter fluid was taken freely. Immediately prior to the consumption of PCA the bladder was emptied, this specimen beingdiscarded (except in 6 subjects who each provided a pre-test specimen). Urine was collected thereafter into a polythene container containing 0.2 ml 5% aqueous sodium azide (this amount of azide does not appreciably affect urinary pH). At the end of 3 h the subjects were instructed to empty their bladder completely and the collection was thus completed. Smoking was forbidden on the day of the test until urine collection had been completed. The pH of each specimen of urine was measured on a Pye pH meter and its volume was measured at room temperature in a measuring cylinder. Three ml concentrated HCl were then added to the urine and an aliquot of this acidified urine was stored at --zoo until analysis was carried out. TABLE
I
THE MEASUREMENT
OF VA AND
VAG
Substancc(s j being measured
Melhod
I. VA plus VAG 2. VA 3. VAG
Acid hydrolysis, then VA measurement No acid hydrolysis, VA measurement Result I minus result z
ilcid hydrolysis was carried out by adding 3 ml concentrated HCl to 15 ml of urine and autoclaving at rg-lb/sq. inch for 14 h (Vanillylglycine is entirely broken down to vanillic acid and glycine under these conditions).
0I \ 0
HO CH30 Voniiltc acid (VA)
\ /
Isovonillic
HO
HO’ Vanilloylglycine
(vAG)
COOH
acid (IVAt
COOH
I
Protocotechuic
acid (PCA)
Fig. I. Structural formulae of the compounds
cited.
Analytical methods These are described in more detail elsewhere8. To summarize, VA estimations were carried out before and after acid hydrolysis, values for VA and vanillylglycine being derived as shown in Table I. Fig. I gives the structural formulae of the compounds cited. * Obtained from Sigma Chemical Company and used without further purification. C&z. Clti~z. Acta, 26 (1969) 413-418
URINARY
VANILLIC
ACID AND
pH
41.5
The pH was adjusted to 1.5 (in both hydrolysed and unhydrolysed specimens), and the solution saturated with sodium chloride. It was then extracted with ethyl acetate and an aliquot of the latter extracted with 10% NaHCO,. An aliquot of the bicarbonate was then acidified and the resulting acid solution was extracted with a small volume of ethyl acetate, this small volume constituting the final ethyl acetate extract. After paper chromatography (ascending) in a system which separates VA from VAG and PCA (chloroform-glacial acetic acid-water, 2: I: I, lower organic phase)9, the area of the paper known (by staining of a duplicate) to contain the vanillic acid was cut out and eluted, and colour developed by means of the modification of the Emerson reaction used by Sumere 10.Standard aqueous solutions of vanillic acid* of varying concentration were carried through the entire procedure so as to enable a calculation to be made of the amount of vanillic acid originally in the urine. An error is introduced because VA and VAG as estimated here are contaminated by small amounts of other PCA metabolites, namely isovanillic acid and its glycine conjugate but the amounts of these metabolites will lead to an overestimate of VA and VAG by only about 4%, and they have been ignored in the present study. All estimations on samples and standard urine were done in duplicate and not accepted unless there was agreement between duplicates within 5%. Six samples of pre-test urine were also studied; a calculation of blank values was made by estimating how much VA and VAG would have been calculated to be present in an amount of pretest urine containing as much creatinine as the test specimen. It will be noted from Table II that these values are very small. TABLE THE
II
PERCENTAGE
Means f
EXCRETION
OF
VA
AND
VAG
IN THE
3-h
URINE
VAG as y. total dose
S.D.
24 male subjects 6 female subjects 6 pre-test urines (all from male subjects)
24.27 + 3.65 22.71 i_ 4.50 0.123 + 0.029
COLLECTION
VA as % total dose 3.31 2 1.486 3.16 + 1.868 0.041 f 0.007
Note: All percentages represent the amount of PCA from which VA and VAG were derived as a percentage of the administered dose.
RESULTS
The results are shown graphically in Figs. z and 3. In Fig. z, vanillylglycine as a percentage of the administered dose is plotted against urinary pH. In Fig. 3, vanillic acid as a percentage of the administered dose is plotted against urinary pH. The lines in these figures are the regression lines obtained for the 24 male subjects only. The slope of the regression line in Fig. 3, but not in Fig. 2, deviates significantly from zero (see Table III). It will be noted that the slope of the line in Fig. 3 is considerable. At pH 5, the value given by the line for VA x Ioo/totaldose is 1.74; at pH 7, 4.91 * Obtained from Koch-Light
Chemical Company. C&-Z. China. A&,
26 (1969) 413-418
416
PRICE
24.
. 14
I 6.0 Urinary pH
5.0
7.0
Fig. 2. The excretion of vanilloylglycine, plotted against urinary pH. Ordinate: vanillylglycine in a 3-h collection as o/0 of dose of PCA. a, female volunteers; 0, male volunteers.
I
I
5.0
6.0
7.0
pH of urine
Fig. 3. The excretion of vanillic acid, plotted against urinary pH. Ordinate: free vanillic acid in a 3-h urine collection as o/0 of dose of PCA. n , 6 female volunteers; 0, 24 male volunteers. TABLE SOME
III
STATISTICAL
Subjects
24 male
6 female all 30
pH
RESULTS
OBTAINED
IJ~YSUSVA
FROM
THE
DATA
as 0/0total dose
Y
t
0.608 0.873 0.657
3.596 3.583 4.612
pH
versus
VAG
as 0/0total dose
P
1
t
P
0.01 > p > O.OOI 0.05 > p > 0.02 O.OOI >p
-0.209
- I.002 0.795 -0.r94
N.S. N.S. N.S.
0.369
-0.037
It might be expected that the volume of a test solution should be related to its pH (since more dilute urine specimens will tend to be less strongly acidic). The relationship did not attain statistical significance in the present study, although the correlation was in the expected direction (Y = 0.293, t = 1.621, p > 0.1). There was, however, a significant association between VA excretion and urine volume for the total group of 30 subjects. For this reason, partial correlation coefficient+ were calculated: for VA excretion against pH the partial regression coefficient, after adjustClin. Chim.
Acta,
26 (1969)
413-418
URINARY
VANILLIC
ACID AND
pH
417
ment for the effect of volume, was r = 0.616 ($J < 0.001) and for VA excretion against volume, after adjustment for the effect of pH, was Y = 0.295 ($ > 0.1). This confirms that it is pH rather than volume that determines the effect that is here described. DISCUSSION
Weiner and Mudge2>‘2 indicate in their comprehensive studies of the subject under discussion here that an influence of urinary pH on the excretion of an organic acid is generally more likely to occur if this acid is lipid soluble. That VA is more lipid soluble than VAG may be reflected in the high RF (0.78) of the former in the chlorofornl-containing solvent system used compared with the much lower RF (0.10) of the latters. PCA, the excretion of which also appears to be unrelated to urinary pH, has an even lower RF in this solvent system than VAG has. Although VA and PCA have very similar pK, values (4.52 and 4.48 respectively)*3, it is only in the case of VA that excretion is related to urinary pH. These observations are consistent with principles stated by Weiner and Mudge 2~12,namely that both a suitable pK, and a sufficient degree of lipid solubility are necessary if an effect of urinary pH on the excretion of a compound is to occur. Future quantitative studies of phenolic acid excretion under normal and pathological conditions ought to take more account of urinary pH. This is not an impracticable suggestion. In a metabolic ward with accurate collection facilities each specimen of urine making up a 24-h collection could be split immediately into equal parts, half being kept in mineral acid for phenolic acid estimation (and amine analysis, if required), the other half being stored in a preservative, e.g. sodium azide, which will not invalidate the measurement of urinary pH. An example of a recently studied lipid-soluble carboxylic acid to which the above statements may have relevance is 3,4-dimethoxyphenylacetic acid (DMPAA) (refs. 14, IS). DMPAA excretion has been of interest in connection with the disputed excretion of the amine from which DMPAA can derive, 3,4-dimethoxyphenylethylamine (DMPE) in schizophrenic individuals. The judgement that an individual’s DMPAA excretion falls “within the normal range” may need to make some reference to urinary pH. ACKNOWLEDGEMENTS
The author is grateful to the Central Photographic Service, University of Liverpool, and to Mrs. T. Williams for help with the preparation of Figs. 2 and 3. Mr. M. C. K. Tweedie gave valuable statistical advice. This work was initiated while the author was a Bates Research Fellow of the Mental Health Research Fund. The Schizophrenia Research Fund has also provided financial assistance which the author is happy to acknowledge. REFERENCES I M. D. MILNE, B. H. SC~BNER AND M. A. CRAWFORD, Aster. J. Med., 24 (1958) 7og. z I. M. WEINER AND G. H. MUDGE, Amer. J. Med., 36 (1964) 743. 3 C. M. WILLIAMS AND C. C. SWEELEY, in H. A. SZYMANSKI (Ed.), BiocLemicaZApplicatioms Chromalograj$zy, Plenum Press, New York, 1964. C&z. Chim. Acta,
of&s
26 (1969) 413-418
PRICE
4x8 1 C, R. J. RUTHVEN AND M. SANDLER, Cr’ilz. Chiw. A&t, 14 (1~66) 511. H, VA&BY, PvacticaL i%nical ~~~c~~rn~s~~~, 4th ed,, W. Heinemann, London,
5 6 7 8
I. D. P. WOOTTON, Microanalysis ia MedicaE Biochemistry, E. C. BATE-SMITH,The Pharmacology ofPlant Phenolics, J, PRICE,Clin. Chim. Acta, 25 (1969) 31.
g A. N. Boarn, 0. H. EMERSON, F. T.
Churchill, Academic
1967, p. 692. London, ~$4. p. 189. Press, New York, 1959.
JONES AND F. de EDS, J. Biol. Chcm.,
229 (1957) 51. 6 (1961) 484. New York, 1952,p.284. 12 I.M. WEINER, K. C. BLANEH~RD AND G. H. MUDGE, Amer. J. Physiol., 207 (1964)953. 13 R. T. WILLMMS, in E. C. BATE-SMX~K(Ed.), The ~~arrna~~~ogy of P&z& PhsnoKcs, Academic Press, New Yark, r959., p. 13. IO C. I?. SUMERE, F. PARMENTIER AND H. TEUCHY, J. Chromatog., rI L. I-I. C.TIPPETT, The Methods of Stakistics, 4th ed., John Wiley,
r4 F.A. Ku~~~L,R.E.ORMOND AND W.J.A.VANDENHEUVEL,N~~~~,ZII xg A. J. FRIEDRQFFAND K. FURIYA,Natwe, z14(rg67)1127. Cdi%Ckint.b%
26 (1969)413-418
(1966) 606.