A Single Column Method for the Determination of Urinary -AminolevuIinic Acid

Transcription

1 A Single Column Method for the Determination of Urinary -AminolevuIinic Acid Mu-Wan Sun, Edward Stein, and Frances W. Gruen A simple method for determination of urinary -aminolevulinic acid is described. Its advantage over procedures now in use is in the utilization of a single ion-exchange column instead of two ion-exchange columns, with resulting saving of time, labor, and material. The method is accurate and useful in screening for lead poisoning. IT HAS BEEN SHOWN that an increased urinary output of 8-aminolevulinic acid (ALA) appears to be correlated closely with exposure to lead (1-3). Blaeger-Aronsen (2) has reported in animal studies that ALA was increased in the urine only with the administration of lead and not with other industrial toxic trace metals. The procedure of Mauzerall and Granick (4) has become an accepted method for the determination of ALA in urine. This procedure is time-consuming, since it requires the use of two ion-exchange columns. The first column is an anion-exchange resin which separates porphobilinogen (PBG) from urine, and the second, a cation-exchange resin which extracts ALA from urine. After the elution from this column, ALA is converted to 2-methyl-3-acetyl-4-(3-propionic acid) pyrrole by heating with acetylacetone. This pyrrole then reacts with dimethylaminobenzaldehyde (DMAB) to produce a cherry-red complex which can be measured spectrophotometrically. #{149} In the routine screening of workers exposed to lead, the determination of PBG is not necessary. This paper will present a single-column modification of the Mauzerall and Granick procedure for the routine determination of urinary ALA. Tn the procedure, it was found that extraction of ALA without prior removal of PBG was found to be satisfactory. Unless the PBG concentration in urine is much higher From the Division of Industrial Hygiene, Nw York State Department of Labor, New York, N. Y We are very grateful to Dr. S. F. MacDonalil of the National Rtse:ire1t Coue,1 in Ottawa, Canada, for kindly supplying u with some of his samples of porphohilinogen. We wish to thank Mrs. Zelda Kessler for her able technical assistance. Received for publication Mar. 29, 1968; accepted for publication July 3,

2 184 SUN ET AL. Clinical Chemistry than the ALA level, as in porphyria, it did iiot interfere with the ALA determination for our purpose. Principle of Method Materials and Method The method consists of removing the ALA from a single cationexchange resin and treating the ALA with acetylacetone to form a pyrrole. This product is then complexed with DMAB (Ehrlich s reagent) to form a red compound. A correction factor is used for PBG and other interfering substances by treating an aliquot of the eluate directly with DI\IIAB. Reagents Ion-e.rclwuqc resin (I)owex 501V-X8) Preparation of resin and column: Wash resin repeatedly with distilled water until the decanted supernate is clear of fine particles. Convert the washed resin to the sodium form by suspending it in twice its volume of 2 N NaOll, allowing it to stand overiiight. Wash the treated resin with distilled water until tile ph of the decanted supernate is neutral. Change the treated resin to tile acid form by washing it with about one volume of 4 N HCI, followed by six volumes of 2 N HC1. Store the resin ill two volumes of 1 N I{Cl in a covered vessel under ref rigeration. A standard curve should be established for each new batch of resin. Chromatographic columns, 1 X 10 cm, with stopcocks are used. Place plug of glass wool at bottom of column. Add ion-exchange resin and let it pack by sedimentation to a level of about cm high. Cut a 1.0-cm disc of filter paper and place it on top of the resin. Sodium acetate, 0.5 M Acetate buffer, ph 4.6 To 700 ml of distilled water, add 57 ml of glacial acetic acid and 136 g of sodium acetate trihydrate. Add distilled water to 1 L. A cetylaceton e Modified Ehrlich s reagent To 35 ml of glacial acetic acid in a 50-mi cylinder, add 1.0 g of p-dimethylamiiiobenzaldehyde and 8.() ml of 70% perchloric acid. Fill cylinder to 50 ml with glacial acetic acid. 8-Am inolevulinic acid standard 6-Amiiiolevulinic acid hydrochloride (Calbiochem), mol. wt , is used to prepare stock solution: 12.8 mg of ALA-IFTC1 is made up to 100 ml with distilled water to give a concentration of 100 jg ALA per milliliter. Approximate dilutions are made from the stock solution to give concentrations of 1-20 g/ml. All solutions are stable under refrigeration.

3 Vol. 15, No. 3, 1969 a-aminolevulinic ACID 185 Procedure Adjust the urine sample to p11 46 with acetic acid. If storage is required, refrigerate at this ph. Using light suction, wash column with ml distilled water. Pipet 1 ml of urine onto tile column. Let it flow out of the column at a rate of six drops per minute. Then, using light suction, wash the column with 30 ml of distilled water followed 1w 4 ml of 0.5 M sodium acetate. ])raiii the column and close stopcock. Add 7 ml of 0.5 M sodium acetate to the column. After 10 mm., collect eluate in a 10-mi volumetric flask. Add approximately 3 ml of acetate buffer to column. Allow it to flow into the volumetric flask till a total of 10 ml is collected. Mix well. Pipet 5 ml of eluate into a screw-capped tube and add 0.1 ml acetylacetone. Transfer remaining eluate into another screw-capped tube; do not add any acetylacetone. Boil both tubes in a water bath for 20 mm. and cool after boiling. i\iix 2 ml of each solution with an equal volume of modified Ehrhich s reagent. In the presence of ALA, a pink to cherry-red color will develop in the tube containing acetylacetone. The tube without acetylacetone is used for correction of errors of absorbance due to PBG and other interfering substances. After 10 mm., read the absorbance at a wavelength of 553 nm. TJse a water blank treated similarly to the urine specimen to adjust the spectrophotometer to zero for samples with and without acetylacetone. Calculation Obtain absorbance (O.D.) of urine with acetylacetone (A) and without acetylacetone (B). Subtract tile 0.1). of B from the 0.1). of A and read the concentration of ALA from the standard curve. Standards were prepared by adding known amounts of ALA-HC1 to human urine and were analyzed by the procedure described. Results The results of duplicate analyses of known amounts of ALA-HC1 added to distilled water and human urine are shown in Tables 1 and 2. In Table 2 the recovery of added ALA by the single-column method is compared with the method of Mauzerall and Granick; the results obtained were in good agreement. A study of the possible interference of PBG on ALA determinations was carried out by using known concentrations of PBG and ALA in aqueous solutions (Table 3). The results show that some of the PBG

4 186 SUN El AL. Clinical Chemistry Table 1. RI.rovEsly of ALA FROM AQrEOUS SOLUTION USING TUE Srsr.u.:-Coi,iiis METHOD addrd A LA bun,! A LA revn,rrr,! (Q) (%) Table 2. COMPARISON OF METHODS FOR ESTIMATION OF ALA ADDED TO NORM.tL llrm.n URINE AL.4 recovered ALA recovered (one-column method) (two-column method ) ALA added (Sc) pg % pg % * Maizeral1 and C.ranick procedure. Table 3. RECOVERY OF ALA WITH ADDED PBG, FROM AQUEOUS SOLUTION ALA found (po) ALA (A - B) PBG added A LA added With acetyl- Without acetyt- by A LA recovered (po) (pg) acetone (A) acetone (B) difference (%) DIRECv ANALYSIS ) () () ONE-COLUMN METHOD () () * Without passing through the ion-exchange column.

5 Vol. IS, No. 3, AMINOLEVULlNIC ACID 187 is elated with the ALA. rfllel.efoi.e we found it necessary to react au aliquot of tile eluate directly with modified Jl1rlicil s reagent iii order to determine a correction factor for PBU. In Table 3 the data show that levels of PBG below 50 g resulted in very little interferencei.e., a small correction factor. however, at 50 g of PB( there is a definite indication of added color iii the filial complex. At the high level of 50 /Lg of PBG, we were able to decrease the added color by increasing the boiling time with acetylacetone to 20 miii. instead of 10 mm., as used in the Mauzerall and Granick procedure. A comparison of tile results of ALA determination in tile urine of workers exposed to lead using the one-column method and tile twocolumn method of Mauzerall and Graiiick is shown in Table 4. The results are in very close agreement, and a statistical analysis of the regression lines gives a regression coefficient of This would indicate that tile one-column method, with a correction for PBG as shown, is a practical and reliable method for the determination of urinary ALA. Table 4. COMPARISON OF METHODS USING 1.0 ML OF URINE SPECIMEN PROM WORKERS EXPOSED TO LEAD ALA (pg/mi) Subject One-column method Two-column method % I)ifference : t.ud Gran ick proce4ure.

6 188 SUN ft AL. Clinical Chemistry Discussion A single ion-exchange column procedure has beeii developed for the determination of ALA in urine. During tile time of preparation of this report, Williams and Few (5) reported a similar modification of the Mauzerall and (iranick procedure. Our results are in agreement with their findings. We made additional studies with PBG and, therefore, Ilave becit able to modify tile procedure iii order to correct for the presence of I BU in tile urine. A discussion ot the various reactions involved in this procedure is presented ill detail by Mauzerall and U ranick (4). Since we are using only the Dowex 50 column, it was important to establish if any of tile PBG is eluted witii tile ALA fraction. rfile initial washing of tile column with distilled water removes tile urea. This is necessary because urea will interfere by producing a yellow color with Ehrlich s reagent. We did not determine if any of the PBG is eluted in this initial wash. Our studies did show that some of the PBG was eluted along with ALA, which resulted in higiler ALA levels (Table 3). Shuster (6) had demonstrated that ALA can be determined in the presence of PBG by reacting the eluate with and without ethyl acetoacetate. We applied tile same principle by using acetylacetone instead of ethyl acetoacetate. In our studies, known mixtures of PBG and ALA were studied (Table 3), and the results showed higher ALA values if not corrected. By determining the correction for PBG, the ALA values showed very good recovery. Our experience has been that urine samples obtained from workers exposed to lead usually have little or no PBG present. We find that our correction blanks are usually low, and the comparison of our method witll the rnetho(1 of Maiizerall and Oranick produced results which are in very good agreement. Previous investigators have demonstrated that lead-exposed workers usually excrete large amounts of ALA with little or no PBG in tile urine (2, 7, 8). Recently Davis and Alidelnlan (.9) described a modification of the Mauzerall and Granick procedure using two disposable ion-exchange columns. Our procedure can be adapted to use one of the two columns, which would shorten the time to carry out this procedure by eliminating the time to prepare the resin column. References 1. Tishkoff, G. H., Granville, N. B., Rosen, B., and Dameshek, W., Excretion of deltaaniiaolevulinic acid in lead intoxication.ada Jlaemalol. 19, 321 (1958L 2. Haeger-Aronsen, B., Studies on urinary excretion of delta-aminolevulinie acid and other haeni precursors in lead workers and lead-intoxicated rabbits. Scand. J. Clin. Lab. Invest., Svppl. 12, 47 (1960).

7 Vol. IS, No. 3, AMINOLEVULINIC ACID Cramer, K., ajul Seiaiider, S., Studies III lead poisoning: Comparison latwec,, different laboratory tests. Brit. J. lad. Med. 22, 311 (1965). 4. Mauzerail, I)., and GraIlick, S., The occurrence and determination of delta-anlinoievulinic acid and porphobilinogen III urine. J. Iliol, Cheat. 219, 435 (1956). 5. Williams, M. K., and Few, J. P., A simplified procedure for the determination of urillal y delta-amjnolaevulinic acid. Brit..1. lad, Med. 24, 294 (1907). 6. S,iuster, L., The detel-nlination of delta-aniinolevuiinic acid. Biocliem. J. 64, 101 (1956). 7. Stich, \V.,Uber einen neuen Biocllenlisehen Lnterscheid zwischen liumaner, akuter Porphyrie und experimenteller Porpliyrie. K/in. Woehsehr, 36, 386 (1958). 8. Tanabe, Y., Investigations of the metabolism of delta-antinoievulinic acid and porpho. bilinogell ji! lead poisoning: I. On tile amounts of delta-aniinolevulinic acid and porphobiiinogen in the urine mid 1)100(1 in lead poisoning. Japan. J. \atwns health 28, 386 (1959). 9. Da is, J. R., and Andclinaa, S. L., Urinary delta-aminolevulinic acid (ALA) levels in lead poisoning: I. A modified method for the rapid determination of urinary deltaalinnoievuiillic acid using disposable ion-exchange chromatography columns. Arch. Environ. Health 15, 53 (1967).