IT0STUDY PROTEIN METABOLISM with special reference to tyrosine as well as to study the deranged metabolism of aromatic compounds in

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1 A Quantitative Method of Determining Urinary Phenols Lewis A. Barness, William J. Meliman, Thomas Tedesco, Diana G. Young, and Roberf Nocho A quantitative method for the determination of urinary phenols is described. Data obtained in 20 children by this method are similar to averages in adults obtained by others. This is exceeded by the excretion of a phenylketonuric infant when placed on excessive intakes of phenylalanine. IT0STUDY PROTEIN METABOLISM with special reference to tyrosine as well as to study the deranged metabolism of aromatic compounds in phenylketonuria and similar disorders, quantitative determination of urinary phenols is desirable. Volterra (1) measured three phenolic fractions in the urine by distillation. Phenols have been measured quantitatively after ethyl acetate extraction by Bray and Thorpe (2) and after ether extraction by Rogers et a!. (3). A previous report described a method of ether extraction of urine for free, acid, and conjugated phenols (4). A modification of this now offers a rapid method for both quantitative determination of urinary phenols and preparation of phenols for chromatography. Method [rine samples are prepared in triplicate. Two aliquots serve for quantitative measurement of phenols and one for chromatographic identification. Ten milliliters of urine is saturated with 3 gm. of NaCl and ad- From the Department of Pediatrics, Hospital of the University of Pennsylvania, and the University of Pennsylvania School of Medicine, Philadelphia, Pa. Supported in part by U.S.P.H.S. Grants No. A-2231, National Institute of Arthritis and Metabolic Diseases, and No. OG-lO, Division of General Sciences, National Institutes of Health. Received for publication July 30,

2 Vol. 9, No. 5, 1963 URINARY PHENOLS 601 justed top with 2% NaOH. Using all electric stirrer, this mixture and 25 ml. of anhydrous ether are mixed in a 200-ml. centrifuge,jar for 10 mm. The sample is then centrifuged at 2000 rpnl for S mill. The ether layer is pipetted off into a separatory funnel. This extraction is repeated three times, but the mixing period is reduced to 5 miii. for the second and third extractions. The combined ether exti acts are washed three times with 5 ml. of distilled i1o, and this I 12() wash is a(l(led to the residual urine from the pit 8 fraction. At this point the ether extract of the third aliquot is set asi(lo all(l allowed to evaporate to dryness. The other two aliquots are treated separately in the following manner. The washed ether extract is emptied into a 250-mi. round-bottom flask which contains 10 ml. of distilled I LU. [sing a hash evaporator the ether phase is evaporated, leaving the 10 ml. of HO. To measure the amount of phenols extracted, of the aqueous phase is Placed in a 25-mI. graduated cylinder and diluted with H9() to 10 ml. One milliliter of Folin-Ciocalteau reagent and 2 ml. of 20% Na2CO3 are added and the cylinders placed ill boiling 1190 for 1 ruin. The cylinders are allowed to cool, and then 1120 is added to 25 ml. The blue color that develops is read on a photoelectric colorimeter with a red filter (wavelength range of m) and compared to that produced by a phenol standard (10 mg./100 ml.). ruilis fraction rel)resemlts the free phenols, ether extractable at p118 (ph8 fraction). The residual urine combined with the water washings of the previous ether phases are then brought to 1)11 1 with in 119S04 and immediately extracted in the same way. rlihe color is developed and read with p-hydroxy phenylacetic acid (10 mg./l00 ml.) as standard. This fraction consists of the aromatic aci(l phenols H I fraction). The residual urine at ph 1 is then refluxed with an equal volume of in HOSO4 for 1 hr. It is then re-extracted at ph 1 or less. Color is developed and read as for the ph 1 fraction. This fraction contains the conjugated phenols (hydrolyzed fraction). Our fractions ph 1 ali(t hydrolyzed, together, are similar to Volterra s fraction 2, aromatic hydroxy acids. Results Recovery of Phenols by the Described Method Phenol (Baker s reagent, crystalline) freshly opened, and p-hydroxy phenylacetic acid (Nutritional Biochemicals) were added to urine in individual concentrations of 2.5 and 5.0 mg./100 ml. Ilecov-

3 602 BARNESS ft AL. Clinical Chemistry Table 1. RECOVERY OF ADDED PHENOL AND p-hydroxy PHENYL AcETIc AcID p118 ph1 2.5 MG./100 ML. OF EACH 96.5% % MG./100 ML. OF EACH 105.5/( 80.9 % eries of these added substances from five samples of tile same urine were determined, using the ether extraction method described. At ph 8, 103% of added phenol is extracted and at ph 1, 87% (Table 1). When phenols are added separately, it is found that extraction of free phenol is equally effective at ph 8 and H 1, while p-hydroxy phenylacetic and pyruvic acids are not extractable at ph 8 but are maximally extractable at ph 1. Therefore, specificity is obtained by extracting free phenols at ph 8 followed by acid phenols at ph 1. Variations Between Duplicate Determinations of Urines Ten duplicate determinations were analyzed to establish error of the method. These are summarized in Table 2. Most agree within 10%. Table 2. DUPLICATE DETERMINTION s (MG./IL.) ph 8 ph 1 Hydrolyzed Residual

4 Vol. 9, No URINARY PHENOLS 603 Greater variations in the ph 8 fraction may be attributed! to tile very low concentration of phenols extractable at this ph. Differences greater than 15% are repeated as the extraction is considered illadequate. Comparison of Current Method with Steam Distillation Method of Volterra Six urine specimens were analyzed for phenols by the steani ihist ihlation method (A) of Volterra s and by the method described in this report (B). The results are compared in Table 3. Volterra steam-distills urine that is slightly alkaline. This fraction he calls the free volatile phenols. After acidification, the sample is redistihhed and the quailtity of phenols determined. This fraction represents the volatile conjugated phenols. The residue is then ether extracted, amid these I)lIellols Volterra calls the aromatic hydroxy acids. Since this follows sonic hydrolysis, this fraction is similar to our ph 1 (aromatic acid phemiols) and hydrolyzed fractions (conjugated phenols). While the three fractions obtained in each of the two methods cannot be compared directly, the total phenol extraction may be compared. In all instances, more phenols were extracted using the described method. Paper Chromatography of Phenolic Fractions As described in the method section, the ether extracts from the thir(i aliquot are used for chromatographic purposes. After the ether has evaporated, the residue is taken up in 95% ethanol and spotted on Whatman No. 1 paper. An ascending two-way system is used with isopropanol, 200 ml., ammonia, 10 ml., and water, 20 ml. as solvent T Table 3. COMpRIsoN OF EXTRACTION METHOD (A) WITH STEAM DISTILLATION METHOD (B) (LG./AIr.) Urine specimen pit 8 (A) it (ii) ph x (A) Hyd* (A) Jff (B) Reoid (A) lilt (B) *Method A: ph 8: volatile free phenols; p11 1: free phenolic acids; Hyd: conjugated volatile phenols and phenolie acids; Resid: non-ether-soluble. tmethod B yields phenols as follows: Fraction I: volatile phenols and volatile conjugated phenols; Fraction II: phenolic acids and coniugated plienolic acids; Fraction ITT: lioliextractable.

5 604 BARNESS FT AL. Clinical Chemistry and benzene, 125 ml., acetic acid, 72 ml., and water, 3 ml. as solvemit II (5). Considerable variation was noted in the distinction amid relative intensity of spots from the ph 1 fraction. The corresponding hydrolyzed fraction usually showed the same compounds but less intensively. No demonstrable pheiiolic compounds were found in the ph 8 chromatogram when diazotized sulfanilic acid and aroyl-glycine reagent were used as indicators. Phenols excreted by children consisted chiefly of p-oh phemmylacetic acid, p-oh pheiiyl lactic acid, rn-oh benzoic acid, and an unidentified spot near p-oh phenylacetic acid which is yellow with sulfanilic acid. Small amounts of p-oh phenylpyruvic, vanillic, amid ferulic acids were also noted. The ph 1 fraction of urines from the phenylketonuric acid comitained mainly p- and o-hydroxyphenolic compounds. The quantitative variation recorded was not reflected in the apparent intensity of phenolic staining spots. Table 4. PHENOL Case No. Diagnosis Age* (yr.) Wt. (kg.) 1. p 1 8 mg/day vng./kg.,.g./creat. 1 Retardation :y Bronchitis Well Well Retardation Well Cardiac Epilepsy Bronchitis Post polio Scoliosis Pneumonia Neuritis Cerebral palsy Calcinosis Well Post polio , Neuritis Well , Scoliosis MEAN ± S.D. ± 3.1 ± 0.14 ± Adjustei to nearest Kg and year.

6 Vol. 9, No. 5, 1963 URINARY PHENOLS 605 Phenol Excretion in Children (Healthy and Ill) Twenty-four-hour urimies were collected under toluene from 20 children (9 months to 14 years in age). Four were well and the other 16 were hospitalized for varying causes. They received mio unusual diets or drugs. Data from all are similar and are therefore combined. Phenolic acids extracted at ph 1 varied from 5.8 in the 9-month infant to 70.5 mg. per day in a 10 year old (Table 4). The mean daily excretion was 29.5 ± 14.5 mg. per day in the children studied. When expressed on a weight basis, urinary phenolic acids averaged 1.3 ± 0.5 mg./kg. per day, of 0.12 ± 0.06 mg. per milligram of creatinine. The free phenols (ph 8) were excreted in small quamitities (Table 4). They averaged 5.8 ± 3.0 mg./24 hr., or 0.26 ± 0.14 mg./kg. per day, or 0.03 ± 0.03 mg. per milligram creatinine. Conjugated phenols measured after hydrolysis averaged 48.5 ± 31.0 mg. per day, 1.9 ± 0.8 mg./kg. per day, or 0.18 ± 0.09 mg. per milligram of creatinine. EXCRETIO N OF OHILDREN ph 1 Hydrolyzed Total mg/day mg./kg. mg/c real. tag/day mg/kg. mg./creat. mg/day mg/kg. Residual vng./day ±14.5 ±0.5 ±.06 ±31.0 ±0.8 ±0.09 ±45.0 ±1.2 ±18.3

7 606 BARNESS FT AL. Chnical Chemistry Table 5. PHENOL EXCRETION IN PHENYLKEmNURIA Phenylalanine,ng./kg./12 hr. 12-hr. intake period mg/kg/day pit 8 ph 1 fly,l lies 1. A.M P.M i A.M si tog/kg. per day Period Period Total phenols excreted were thus 83.8 ± 45.0 mg. per day or 3.4 ± 1.2 miig./kg. per day. Residual Folin-Ciocaltean reacting material which was not extracted amid is presumal)ly tyrosine-like material averaged 35.0 ± 18.3 mg. per day. Phenol Excretion in Phenylketonuria A 2-year-old male phenylketonuric weighing 12.7 kg., who had been maintained on a limited phenylalanine intake since 2 months of age, was hospitalized. He was fed a measured diet containing increasing amounts of phenylalanimie ( mg. per day). Consecutive 12-hr. urines were collected after 48 hr. of each diet. No phenylalanine was fed during the night collection period. Phenolic fractions for several of these 12-hr. urines are recorded in Table 5. For purposes of comparison, 24-hr. excretions were calculated by combining data from two consecutive 12-hr. collections. The ph 1 fraction, which contains the free phenolic acids, shows a response to the increased phenylalanine intake. The daytime collection appears to be more sensitive to this metabolic manipulation than the total 24-hr. intake. At 360 mg. phenylalanine intake (28.4 mg./kg. body weight) the ph 1 fraction significantly exceeds normal excretion values. Discussion A method for the determination of urinary phenols, using ether extraction, is described. Recovery of added phenol and p-hydroxy phenylacetic acid is at least 79% ; p-hydroxy phenylpyruvic acid, however,

8 Vol. 9, No. 5, 1963 URINARY PHENOLS 607 is very slightly soluble iii ether. Duplicate determhiatiomis agree within 10%. Simultaneous fractions for chromatography are made available. Total iihenolic excretioii in the 20 children studied was 3.4 ± 1.2 rng./kg. per day. Using steam distillation, Volterra detected 2 mg./kg. per day of phenols iii adults. Presumably the steam distillation method does not extract the phenols as thoroughly as ether. Rogers et al. (3) used the method of Schacter, which is closest to that described here. Urine is hydrolyzed first, followed by continuous ether extraction. Fractions are then separated with NaH CO3 and NaOH. Color is developed with Millomi s reagent. if one assumes their patients weighed 70 kg., their excretion rates would be: aromatic hydroxy acids 2 mg./kg., volatile phenols, 3 mg./kg., and ether-insoluble residue, 1.5 mg./kg. per day. Their method does not distinguish between conjugated and nonconjugated phenols. References 1. Volterra, M., Urinary phenols TI-Their significance in normal and pathological conditions. Am. J. Clin. Path. 12, 580 (1942). 2. Bray, H. G., and Thorpe, W. V., Analysis of phenolic compounds of interest in metabolism, in Methods of Biocheinwal Analy8is, vol. 1, D. Glick, ed., Interscience Pub., New York, 1954, p Rogers, W. F.. Burdick, M. P., and Burnett, G. R., The effect of antibiotics on the excretion of phenolic compounds. J. Lab. CUn. Med. 45,87 (1955). 4. Comely, D. A., Bamness, L. A., and Gy#{246}rgy, P., Effect of lactose on nitrogen metabolism and phenol excretion in infants. J. Pediat. 51, 40 (1957). 5. Smith, I., Ed., Uhromatographic and Electrophoretie Techniques, Interscience, London, 1960, p. 300.