Qua nti ta tive Estima ti o n o f p - Hy d r oxyphe ny I py r uvic Acid in Human Urine IRA J. HOLCOMB, D. S. McCANN, and A. J. BOYLE Department o f Chemistry, Wayne Sfate University, Detroit, Mich.
b A rapid, simple method is presented for the estimation of p-hydroxyphenylpyruvic acid (pHPPA) in human urine. A 1 to 2% aliquot of a 24-hour specimen is treated with lead acetate and centrifuged. The supernate i s passed through Dowex 50 W-X4. The effluent is extracted with a mixture of n-butyl alcohol-n-butyl acetate which is followed by extraction with 1 N N a O H . The p-hydroxybenzaldehyde (pHB) formed from pHPPA in base is extracted into butyl acetate where the amount is estimated spectrophotometrically using a wavelength of 2 7 5 mp. Recovery studies show that 9 1 yo of the pHPPA in the urine sample may be accounted for b y this procedure. For quantitative estimation a lower limit of 4 0 pg./ml. of pHPPA must b e present in the initial sample.
T
HE DEVELOPMEST of an analytical method for p-hydro-qphenylpyruvie acid (pHPP.1) in human urine was prompted by an interest in the metabolism of tyrosine in connective tissue diseases. 1 survey of the literature showed that existing methods lack specificity or convenience ( 5 , 8-14 19-21). The average values of pHPP-1 reported range froin 0 to 10 mg./24 hours in normal urines, while average values in liver disease obtained by diffeient laboratoiies are as diverse as 23 to 160 mg. per 24 hours, reyectively (2, 6, 7 , 15).
48202
pHPP.1 is a fairly strong organic acid with a pK, = 2.6: this value is close to that of phenylpyruvic acid. The phenolic pK, = 9.9 is identical lvith the phenol ionization of p-hydroxyphenylacetic. The tautomeric conversion of pHPPA was studied qualitatively by Painter and Zilva (16) and quantitatively by Bucher and Kirberger (3). These morkers established that solid pHPPA is predominantly in the enol form, which on standing in solution slowly undergoes tautomerization to the keto form. The conversion is a first order reaction and the rate is accelerated with increasing pH. Equilibrium ratios of enol to keto forms are given as 0.3, 0.2, 0.05, and 0.05 a t p H 1, 2, 4.5, and 6, respectively. The rate of tautomerization is also increased by divalent nietal , Zn+2 and ions such as C U + ~ Fe+, l\Ig+z. There is a slight difference in the acid strengths of the enol and keto forms, the latter being the stronger acid. The enol forni has a molar absorptivity a t 334 nip of 3.46 X lo6 cm.2/mole in 0.02Ai hydrochloric acid, whereas the keto form absorbs strongly around 278
composition has been observed by Wieland (18), Pitt (17), Doy (4, and Schwartz (22). It is possible that some oxidative decarboxylation takes place so that some p-hydroxyphenylacetic acid may be formed. Schwartz, Wieland, and Pitt believe that the enol form is the participating tautomer undergoing oxidation. Doy suggested a series of hydrolysis reactions for the basic decomposition, but the reaction requires oxygen as evidenced by manometric monitoring in basic solution and stability in a nitrogen atmosphere under alkaline conditions ( 1 ) . Pitt indicates that ultraviolet light accelerates the rate of reaction rvhich would indicate a free radical mechanism.
EXPERIMENTAL
Use was made of the decomposition of pHPP.1 in alkaline solution. -1t room temperature in 1 9 XaOH conversion of pHPPA to pHB is accomplished in 2 to 3 minutes. At lower concentrations of S a O H the reaction is much slower. Figure 1 compares the spectra of converted pHPPA to pHB in base. Reagents. A11 chemicals used were of reagent grade. This stipulation is particularly important with respect t o the butyl acetate. Recoveries from technical grade material of this reagent were poor. Procedure -11 to 2% (between 10 and 25 inl.) aliquot of a 24-hour urine sample is placed in a centrifuge tube and treated with 1 ml. of a
mp.
I n acidic solutions pHPP.1 is quite stable. The absorbance changes only slightly over a 3-week period if the solution is refrigerated. I n basic solution pHPPX undergoes an unusual type of reaction which results in the formation of p-hydroxybenzaldehyde (pHB) and oxalic acid. This type of de-
1.0
Y Z
~
250
300
,
~~
350
I
t
l
300
260
WAVELENGTH, rnp
WAVELENGTH ,mp
Figure 1. Ultraviolet spectra of pHPPA and pHB in 1H sodium hydroxide
Figure 2 . Ultraviolet spectra of converted pHPPA and pHB acid in butyl acetate
pHPP - - - pHB
1.
2.
300 pg. pHPPA 200 pg. PHB
VOL. 37,
NO. 13, DECEMBER 1965
1657
saturated lead acetate solution. Approximately 0.5 ml. of concentrated hydrochloric acid is added to the sample. The tube is then centrifuged a t 2000 r.p.m. for 2 to 3 minutes. The supernate is placed in the reservoir of an ion exchange column containing 6 em. X 1 cm. of Dowex 50 W-X4, (50-100 mesh), prewashed with 25 ml. of 1N KaOH, 25 ml. of H20, 25 ml. of 1N HC1, and 25 ml. of 5N HC1 in that order. The sample is passed through the column and eluted with 0.5N HCl. Eighty milliliters of effluent are collected in 15 to 20 minutes. The effluent is extracted with 1:1 n-butyl acetaten-butyl alcohol in three portions, 40, 30, and 20 ml. These portions are combined and extracted with 20 ml. of 1N NaOH--10, 5, and 5 ml., respectively. The pH of the combined NaOH extracts is adjusted to approximately 7.6 with HC1. This solution is in turn extracted with three successive portions of n-butyl acetate t o make a total of 40 ml. pHB is measured in the butyl acetate extract a t 275 mp using a butyl acetate blank. Standards. Standards containing from 100 to 400 fig. of pHPPA per 25 ml. of water are taken through the same process. Absorbances over this range using a 1-em. light path are shown in Table I. RESULTS A N D DISCUSSION
Samples of 100 to 400 pg. of pHPPA added to 25 ml. of water and taken
Table 1.
Absorption of pHPPA Standards pHPP.4, pg. Absorbance
Table II.
Donor DM
Sex F
DM LL LM
F F M
HK JM HW MC
M F
DJ
M
MM
F
DH
bI
RV
M
1658
F
F
through the process, yield ultraviolet curves in butyl acetate characteristic of pHB in that reagent (Figure 2). These curves exhibit a maximum a t 275 mp and absorbance readings obtained a t this wavelength obey Beer’s law. By using a 5-em. light path the method can be extended to initial concentrations of pHPPA as low as 10 gg./25 ml. It is essential that pHPPA and not pHB is used as a standard and that the standards be taken through the whole process. If equimolar quantities of pHB and p H P P h , respectively, are placed into 1N YaOH which is then neutralized and extracted with butyl acetate, virtually all the pHB is recovered but only 54% of the pHPPA is recovered as pHB, in agreement with some of Pitt’s observations ( 1 7 ) . This recovery can be increased to 65y0 by adding 1 ml. of 1N NaOH saturated with respect to CaC12to the basic phase just before it is extracted with butyl acetate. The precipitation of calcium oxalate obtained through this manner apparently allows more of the pHPPA to dissociate into pHB and oxalic acid. However, the advantage gained did not seem to be sufficient to warrant inclusion of this step in the routine procedure. Table I1 summarizes the results obtained with a series of 24-hour urines both from the point of view of the 24hour excretion of pHPPA and recovery of pHPPA added to a second aliquot of the same specimen. It follows that recoveries of some 15 analyses on 12 different urine specimens ranged from 75 to 116% with an average of 91%. The occurrence of pHPPA in normal urine was verified by paper chromatography. Two aliquots of a urine, one of which was spiked with 300 pg. of pHPPA, were taken through the procedure outlined above. The final butyl
Results Obtained with Series of 24-Hour Urines
Comments Several normal aliquots of same 24-hour specimen Normal Normal Sormal (aged 10) Normal Normal Scleroderma1 Possible rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis Rheumatoid arthritis
ANALYTICAL CHEMISTRY
Milligrams pHPPA Addition of Recovery excreted/ pHPPA of pHPPA 24 hours to sample added, % 50 100 200 300 300 300 300
110 100 100 87 80 87
7.6
300 300 300 300
85 75 97 103
15.0
300
87
21.0
300
77
22.0
300
80
14.5
300
75
8.0
10.4 9.8 5.2
6.0 7.5
9.2
116
Table 111.
Lloyd’s reagent, mg.
Adsorption of pHPPA by Lloyd’s Reagent
pHPPA recovered,
Recovery,
lrg.
70
0
250
100
1000
92
36.8
Varying amounts of Lloyd’s reagent were added t o 250 rg. of pHPPA in 10 ml. of acidified H20 in each instance. Samples were mixed thoroughly by inversion and centrifuged. The supernate was assayed for pHPPA using the Brigg’s reaction.
acetate extract was concentrated a t room temperature by blowing air over it and the concentrated material was spotted on paper, together with a sample of pHB and one of pHPPA converted in KaOH neutralized with HC1 and extracted into butyl acetate. All four spots traveled at the same rate in a butanol-acetic acid-water 4: 1:5 solvent system and developed similar colors when treated with sulfanilic acid. Kone of the following components which might occur in urine interfered with the determination : tyrosine, gentisic, hippuric, p-hydroxyphenylacetic, o-hydroxybenzoic, p-hydroxybenzoic, p-hydroxyphenyllactic, phenylpyruvic, salicyluric, and uric acids. Of these compounds only the p-hydroxybenzoic acid sample showed absorbance in the final extract and this was so small (0.05 absorbance unit for an initial concentration of 250 wg.) as to be considered negligible. Obviously the data shown in Table I1 are very limited and preliminary but it does suggest that the rheumatoid arthritis patient may excrete higher levels of pHPPA than normal controls. Whether this is indicative of an aberrant tyrosine metabolism among these individuals is now under investigation. Critique of a Clinically Accepted Method of Estimation of pHPPA. Most of the methods used currently for p H P P A are based on the Brigg’s reaction. For the sake of comparison the procedure adopted by Woolf (10,21) was chosen as a representative and perhaps the most widely accepted clinical method. The procedure involves preliminary removal of some interfering substances from acidified urine with Lloyd’s reagent, followed by reduction of a phosphomolybdate complex with the pHPPA present in the urine to heteropoly blue. The effect of Lloyd’s reagent on pure standards of pHPP.4 was studied as well as recoveries of pHPP-4 added to urine.
Finally, comparisons were made of pHPP.1 content of several urines run by this method and the one presently described. Table I11 shows the effect of Lloyd’s reagent on pure standards of p H P P h . Table IV compares the procedure outlined above for pHPPA (Method A) with one utilizing Lloyd’s reagent folloiied by Brigg’s reaction (Method B). It is apparent that Method B suffers inherently from two compensating errors. Lloyd’s reagent adsorbs pHPP.1. In most instances a reducing hubstance other than pHPPA remains in the urine even after treatment with the reagent and contributes to the final color. The constitution of any particular urine \Till determine to IThat extent the two errors compensate and a correct answer is largely fortuitous. ACKNOWLEDGMENT
The authors express their appreciation for the technical assibtance rendered by Julia Nitchell. Thanks are due also t o Gloria Senienuk who contributed the paper Chromatographic work.
Table IV. pHPPA in Urine as Determined with Brigg’s Reaction (8) Compared to That Obtained through the Procedure Described in This Paper (A)
Sample DJI HK LL DH HA1
RT’
Millierams DHPPA excYetedli4 hours Method A Method B 10 4 6 0 9 8 15.0 21.0 14.5
41 77 87 72 131 132
LITERATURE CITED
(1) Billek, G Monatsch. 92, 335 (1961). (2) Briggs, 2. H., Harvey, N., Life Sciences 1, 61 (1962). ( 3 ) Bucher. T.. Kirbereer. E.. Biochim. Bzophys. Acta 8 , 401 fi952). ’ (4) Doy, C., Nature 186, 529 (1960). (5) Ewald, W.,Hubener, H., 11~uturu;iss. 48, 720 (1961). (6) Gros, H., Kirberger, E. J., Klin Wochschr. 32, 115 (1954). (7) Henning, T’., Amnon, R., 2. Physiol. Chem. 306, 221 (1957). ( 8 ) Humbel, R., M e d . Lab. 17, 68 (1964).
(9) Knox, W. E., Goswami, hl., J . Biol. Chem. 235, 2662 (1960). (10) Knox, W. E., Pitt, B., Ibid., 225, 675 (1957). (11) Levine, S., hlarples, E., Gordon, H., J . Clin. Invest. 20, 199 (1941). (12) Lin, E., Pitt, B., Civen, &I., Knox, W. E.. J . Biol. Chem. 233. 668 (1958). (13) Nedes, G., Biochem.’ J . 26, 917 (1932). (14) Nishimura, N., Maeda, K., Yasui, S . , Okamoto, H., Natsunaka, hl., Teshina, H., Arch. Derm. 83, 644 (1961). (15) Yonouchi, Y., il’aika Hoken 7, 610 (1960); C. A . 5 5 , 11615f (1961). (16) Painter, H., Zilva, S., Biochem. J. 41, 520 (1947). (17) Pitt, B., Nature 196, 272 (1962). (18) Wieland, H., Ann. Chem. 436, 229 (1924). (19) Williams, C., Anal. Biochem. 4, 423 (1962). (20) Woolf, L. I., “Advances in Clinical Chemistry,” 1-01. 6, p. 187, H.Sobotka
and C. Steward, eds., Academic Press,
New York, 1963. (21) Woolf, L. I., Edmunds, N. E., Biochem. J . 47, 630 (1950). (22) Schwartz, K., Arch. Biochem. Biophys. 92, 168 (1961).
RECEIVED for review December 22, 1964. Resubmitted August 30, 1965. Accepted September 29, 1965. Work supported by C. S. Public Health grant AM-05776.
Direct Radiochemical Determination of Lead-210 in Bone HENRY G. PETROW and ARTHUR COVER lnsfitute o f Environmenfal Medicine, New York University Medical Center, New York,
b A procedure for the radiochemical determination of lead-210 in bone ash is based on the solvent extraction of a lead bromide complex into a quaternary amine. Lead-210 is detected, after a suitable ingrowth period, b y beta-counting its bismuth2 10 daughter. Ash samples up to 30 grams can b e routinely analyzed, and as little as 2 X 1 O - I 2 curie of lead-2 10 can be determined. Interference from other elements, both stable and radioactive, is slight. The procedure is applicable to materials other than bone ash.
B
of the extremely low energy of its beta emission, low concentrations of lead-210 in environmental samples are frequently determined by measurement of polonium-210 ( 1 ) . If this technique is to be reliable, the sample niust first be freed of any polonium-210 initially present. S o lead loss can be tolerated during this separation. After the initial polonium separation, the lead-bearing sample must be set aside for months to allow fresh ingronth of polonium-210 from lead-210 present in ECAUSE
the sample; then a second polonium separation must be performed. Obviously, this is a long, time-consuming process, and is subject to error if the polonium-recovery process is not precise for samples of varying composition. As an alternative to this procedure, lead-210 can be separated and allowed to age for several days, and the 5.0-day bismuth-210 daughter beta-counted. A lead-210 assay can be completed in a few days. Lead recovery can be accurately determined through use of known amounts of carrier lead. If the lead-210bismuth-210 level is too low to permit accurate beta counting, the separated sample can be conveniently stored for polonium-210 ingrowth, since the lead sample, as initially separated, is completely free from polonium present in the original sample. Furthermore, variations in polonium-210 recovery can be minimized, since polonium will always be deposited from an identical matrix, after the lead sample has been dissolved in hydrochloric acid. I n this way, advantage can be taken of the rapidity of the lead-210-bismuth-210 technique and, if necessary, of the ex-
N. Y. treme sensitivity of the polonium method. The procedure is based upon the extraction of a lead bromide complex ion into a quaternary ammonium bromide (.iliquat 336). As an indication of the effectiveness of lead bromide extraction from phosphate solution, 1 gram of lead, as the nitrate, dissolved in 50 ml. of 85%;’,phosphoric acid, is completely extracted into 50 ml. of 30% Aliquat 336 bromide in toluene. EXPERIMENTAL
Apparatus. Omniguard low-background beta counter, Tracerlab, Inc. Aliquat 336 (General Mills Chemical Co.) , RIethyltricaprylammonium chloride, 30 volume % in toluene, washed twice with a n equal volume of 1.53i‘ hydrobromic acid. Procedure. Place up t o 100 grams of bone in a muffle furnace a t 580” t o 600” C. Maximum temperature should not exceed 600”, since higher temperatures result in the loss of lead. Heat for 12 t o 16 hours t o yield a white ash. Keigh up to 30 grams of ash, add 7 ml. of 3 X hydrobromic acid per gram of ash and, 1 ml. of VOL. 37, NO. 13, DECEMBER 1965
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