Document 3J4q4w36JwE531m11J5v8dj5E

.IFfi The Preservation of Urine Specimens for 6-Aminolevulinic Acid Determination William F. Vincent*1 and William V/. Ullman The effect of light exposure and temperature on urinary 5-aminolevulinic acid recovery was studied. Addition of tartaric acid and storage of urine specimens in the dark results in excellent recovery of 5-aminolevulinic acid, even after several days at room temperature. Proper handling and storage of urine collected during surveys of lead poisoning are emphasized. Additional Keyphrases light exposure storage temperature tartaric acid preservative lead poisoning (0.1 ml/10 ml urine) satisfactorily preserves specimens. However, we found the. use of acetic acid to be rather difficult since the urine specimens were collected by nonprofessionals or parents. The odor of glacial acetic acid in the urine specimen is disagreeable to the patient. More importantly, acetic acid eventually evaporates, even when screw-capped tubes are used. We report here the use of dry tartaric acid as a preservative for urine specimens that are to be examined for a l a and also on the effect of light exposure and refrigeration on al a recovery. e l t a -a min o l e v u l in ic a c id (a l a ) is an inter D mediate metabolite in the synthesis of the henie moiety of hemoglobin, whereby two mole cules of a l a condense to form porphobilinogen. This reaction is catalyzed by the enzyme 8-aminolevulinate dehydratase (5-aminolevulinate hydro lyase, EC 4.2.1.24), which is markedly inhibited by lead ions. Lead in the bones of patients with lead intoxication inhibits this condensation; con sequently, more a l a is excreted in the urine. Measurement of the amount of this metabolite in the urine has been shown to be a suitable screening procedure for the detection of lead intoxication in both children (1) and adults (2, 3). One drawback to this determination, however, is the instability of a l a . Unless the pH of the specimen is decreased and the sample refrigerated, the a l a concentration will rapidly decrease, particularly when the speci men is exposed to light {3,4) - J. R. Davis (personal communication, 1969) has reported that a small amount of glacial acetic acid From the Research Microbiology Laboratory, Laboratory Division, Connecticut State Department of Health, P.O. Box ICS!), Hartford, Conn. 06101. 1 Present address: Division of Laboratories and Research, New York State Department of Health, New Scotland Ave., Albany, N. Y. 12201. Received March 1C, 1070; accepted April 29,1970. Materials and Methods Determination of urinary a l a . This was essen tially the single-column method of Sun el al. (5), in which a l a is adsorbed onto Dowex resin, con verted to a pyrrole, and a colored complex is pro duced when modified Ehrlich's reagent is added; Prepacked, disposable columns for use in this test can be obtained from Bio-Rad Labs, Richmond, Calif. Preservative. One-quarter ml of tartaric acid, 2 mol/liter,' was placed in a 1.5 X 12 cm screwcapped glass tube and evaporated to dryness in an oven at 120 C for 1 to 2 h. The small amount of dried tartaric acid in the bottom of the tube is almost invisible and rapidly goes into solution when the urine sample is added and shaken. This quantity of tartaric acid is sufficient to decrease the pH of 10 to 15 ml of urine to 2.4 to 2.7. For these studies, we used 10-ml aliquots of urine. Urine specime?is. Specimens with increased a l a concentrations were obtained from children suffer ing from lead iutoxication. Results Preserved (acidified) and unpreserved specimens were held either at 4C or 25 aC and stored in the ac 21 re< mi at the are j Dis I; effc1 rec< 612 CLINICAL CHEMISTRY, Vol. 16, No. 7,1970 N36882 ' '^Table 1. Recovery of ^-Aminolevulinic Acid from Urine Specimens Stored at 25C Spoci- Time of men Sampling ^_______% of original ALA remaining Acidified Unprcsenred Light Dark Light Dark i 24 h 48 h 72 h - 2 wk 2 24 h 48 h 72 h 2 wk 3 24 h 48 h 72 h 2 wk 62.3 38.3 28.5 6.2 58.5 42.5 33.5 5.3 54.3 51.5 47.7 5.5 98.2 98.0 98.0 96.5 100.5 98.0 96.4 95.0 98.5 97.0 97.0 91.0 43.7 21.3 15.4 3.4 48.0 19.8 15.0 4.2 50.0 23.0 17.5 6.8 .94.3 82.5 51.0 5.4 90.6 80.6 48.2 7.9 91.0 77.0 48.0 8.0 Initial' a l a concentrations of specimens were: No. 1, 23.4 pg/mt; No. 2, 32.8 ^g/ml; and No. 3, 79.4 /ig/ml. Table 2. Recovery of S-Aminoievtilinic Acid from Urine Specimens Stored at 4C in the Dark" Specimen sampling % of original ALA remaining Acidified Unprescrved i 24 h - 48 h 72 h 2 wk. 2 24 h 48 h 72 h 2 wk 3 24 h 48 h * 22 h 2 wk 99.3 100.0 99.0 96.0 100.0 98.4 97.8 97.5 98.0 98.5 99.0 96.5 93.2 88.4 74.242.5' 96.8 86.2 71.046.5 90.4 86.2 68.451.5 See footnote to Table 1. dark or exposed to artificial light (110 footcandles, achieved with an 100-watt bulb at 12 inches). At 24-h intervals, duplicate 1.0-ml aliquots were removed and the a l a concentration was deter mined. The results obtained with specimens stored at room temperature are shown in Table 1 and those obtained with specimens stored in the cold are shown in Table 2. Discussion The results clearly demonstrate the adverse effects of temperature and light exposure on a l a recovery and the need to decrease the pH of urine specimens if laboratory findings are to be accurate. If a l a is to be estimated immediately after the specimens are collected, the influence of the condi tions is decreased. It was recently reported in a field study that, with tartaric acid as a preserva tive, 48% of the children with increased urinary a l a (10 jug/ml or more) also had increased blood lead (6`). These findings compare favorably with those of Davis and coworkers (1), who assayed for a l a almost immediately after collection of speci mens (J. R. Davis, personal communication, I960). When large numbers of specimens are collected and mailed to a central laboratory, it is imperative that all concerned be instructed in the proper handling and preservation of urine specimens. The necessity for acidifying urine specimens cannot be overemphasized. During the warm July weather, duplicate sets (acidified and unpreserved) of positive urine specimens were mailed on Friday morning and delivered to the laboratory the fol lowing Monday morning, a l a recovery in pre served samples ranged from 92% to 9S%; in un preserved samples, from 42% to 60%. Three of the five unpreserved specimens handled in this manner would have been reported to the physician as hav ing less than 10 jug a l a /iu I when in fact all speci mens contained abnormally high amounts. The authors are grateful to Dr. Joseph "R. Davis for his advice and encouragement during the course of this study and prepara tion of the manuscript. References 1. Davis, J. K., Abrahams, E. H., Fishbein, W. I., and Fabrega, E. A., Urinary delta-aminolevulinic acid (a l a) levels in lead poisoning. II. Correlation of a l a levels with clinical findings in -250 children with suspected lead ingestion. Arch. Environ. Health 17, 164 (1968). 2. Cramer, K., and Selander, S., Control of lead workers by de termination of. urinary delta-aminolevulinic acid. Bril. J. Ind. Med. 24, 283 (1967). 3. Haeger-Aronsen, B., Studies on urinary excretion of deltaaminolevulinic acid and other haem precursors in lead workers and lead-intoxicated rabbits. Scand. J. Clin. Lab. Invest. 12 (Suppl. 47), 1 (1967). 4. Bossenmaier, I., and Cardinal, R., Stability of 5-aminolevulimc acid and porphobilinogen in urine under varying conditions. Cu n . Ch e s i. 14, 610 (196S). 5. Sun, M.-YV., Stein, E., and Gruon, F. W., A single'column method for the determination of urinary fi-aminolevulinic acid. Cl in . Ch e m. 15, 183 (1969). 6. Vincent, W. F., UUmann, W. W., and YVeidner, G. 1,., The measurement of urinary A-aminolevulmic acid in detection of childhood lead poisoning (letter to editor). Amer. J. Clin. Pathol. (accepted for publication). J CLINICAL CHEMISTRY, Voi. 16, No. 7,1970 613 DUP050311838