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THE DETECTION OF EARLY LEAD POISONING USING THE DAVIS TEST FOR URINARY ALA A rapid, simplified determination of the increase in urinary delta-aminolevulinic acid (ALA) associated with excessive lead exposure . . . using the Bio-Rad prefilied, disposable SOn exchange columns * Davis, J. R. and Andelman, S. L., Arch. Environ. Health 15 : 53-59 (1967) SENSITIVE - ALA is the only urinary test that correlates closely with blood lead levels during continued lead exposure (r > 0.9). ALA is the only test that shows significantly elevated levels for at least one year after lead insult ceases. Only 0.5 ml of a random urine sample is required for ALA testing. RAPID - 200 Urinary ALA tests can be run by one technician in a normal working day. ... AND ECONOMICAL - Urinary ALA determina tions can be run at a fraction of the cost of running blood leads. ...AND HAS PATIENT ACCEPTANCE - Urine collection avoids the negative attitude towards venipuncture which exists in most adults and almost all children. By far the closest correlation between clinical findings and a biochemical test (for lead poisoning) was the amount of ALA in the urine and the symptom score. Lancet.11 N36923 LEAD POISONING AS A PUBLIC HEALTH PROBLEM AND THE ALA TEST The role of ALA testing in detecting industrial and general environmental lead poisoning has been clearly demonstrated. Cramer and Selander have investigated ALA testing in industry for several years1. They comment on its economy and accuracy and particularly/,on the ability of the urinary ALA test "to detect errors in the prophylactic measures at a very early stage before any symptoms have appeared." They further propose "that the ALA determination should be introduced into the regular statutory examination of lead workers, for it appears to give :definite advantages in all cases." In a survey publication from the State of California Department of Public Health, it was agreed that the concentration of blood lead in the population is i now one-fourth to one-half of that recognized as hazardous to workers in industry2. The margin of safety for workers in battery manu facturing, printing, and lead salvage for example, is therefore, decreased by just this amount. The composite U.S. rural exposure to lead from the air is 0.5 microgram per cubic meter and the composite urban exposure is 1.0 micrograms per cubic meter. However, several large increases from this level have been recently noted. For example, a level of 25 micrograms per cubic meter has been determined over the Los Angeles Freeways8. Inves tigators in this field recommend that ALA in urine be used as a more sensitive test than blood lead. They further propose its use in future studies of lead distribution in the population4. The City of Chicago has paid particular atten tion to pediatric lead poisoning occurring in children who have ingested lead-containing paint and plaster. The Davis test for urinary ALA was developed with the active help of the Chicago Board of Health for the anticipated screening of large numbers of children under 7 years of age living in high-inci dence areas of lead .poisoning5. The major advantage of the urinary ALA test is that it is not just another measure of lead levels in the patient's system. It is rather the earliest measure of lead induced metabolic derangement. HISTORICAL DEVELOPMENT OF DELTA-AMINOLEVULINIC ACID (ALA) MEASUREMENT IN LEAD POISONING 1953 Shemin and Russell demonstrated that ALA was an early intermediate in porphy rin biosynthesis. 1956 Mauzerall and Granick described a method for the determination of ALA in urine employing ion-exchange chromatography for its isolation and p-dimethylaminobenzaldehyde (Ehrlich's reagent) for its colorimetric measurement.7 1960 Haeger-Aronsen found the concentration of urinary ALA to be increased in adult work ers exposed to industrial lead environments. 1965 Bonsignore, Calissano and Cartasegna sug gested that the accumulation of urinary ALA occurring in experimental lead poi soning was the result of an inhibition of the enzyme ALA dehydrase.9 1967 Davis and Andelman modified the existing method for the determination of urinary ALA by introducing disposable, prefilled ion-exchange chromatography columns and using this technique have initiated the first large-scale investigation of urinary ALA levels in children residing in high-incidence areas of lead poisoning.1 2- - DUP050312857 MECHANISM OF DELTA-AMINOLEVULINIC ACID (ALA) ACCUMULATION IN LEAD POISONING GLYCINE 4-. ALA synthetase SUCCINYL-CoA \ / / ALA 'dehydra/e \ --------\----- J>-- Porphobilinogen X Blocked''by / Lead \ /\ Fe Protoporphyrin IX =^HEME THE EVALUATION OF URINARY ALA AND OTHER TESTS FOR DETECTING LEAD POISONING In a leading article, the Lancet has discussed the detection of lead poisoning. Using the clinical symptoms of the disease as the primary criteria, several laboratory tests for lead poisoning are compared.11 The urinary ALA test showed by far the closest correlation with the clinical picture of lead poisoning of all major laboratory tests. Lancet also points out the poor correlation of urinary coproporphyrin and also urinary lead with the same clinical criteria. THE ADVANTAGE OF THE URINARY ALA TEST AS COMPARED WITH BLOOD LEAD MEASUREMENT IN DETECTING EARLY LEAD POISONING. The prolonged increase of urinary ALA due to lead exposure is a major advantage of testing for urinary ALA levels rather than blood lead. ALA levels remain increased for at least one year after lead exposure has been controlled.12 Blood lead levels do not exhibit this constancy since lead leaves the blood to be stored in the skele tal system. A random blood sample taken at this time can show a misleading normal lead level. When the stored lead is released, however, the dangerous effects of lead poisoning reappear. The detection of excessive lead exposure in asymptomatic individuals can be accomplished with a random sampling technique, if the urinary ALA test is used. THE ADVANTAGE OF THE URINARY ALA TEST AS COMPARED WITH URINARY COPROPORPHYRIN MEASUREMENT. Davis and Andelman tested the urine of asymp tomatic children who were positive for lead poison ing by urinary coproporphyrin standards. Eightyfive percent of these coproporphyrin-positive child ren did not have elevated ALA levels and were thus false positive for lead poisoning.10 This high percentage of false positives with the coproporphyria test has been connected with iron deficiency anemia rather than lead poisoning. Urinary ALA levels on the other hand, are not elevated by iron deficiency anemias.13 This lack of correlation between clinical symp toms and urinary eoproporphyrin in adults and children has been noted by other investigators. -3 - DU P 050312858 TEST PROCEDURE STEP 1 Technique The top column con taining anion exchange resin and the bottom col umn containing cation ex change resin arrive fitted together in piggyback fashion. They are placed in the drainage rack in this same position. This two column set is referred to as the column unit. Rationale The piggyback fitting eliminates the need to quantitively transfer the liquid passing through the top column to the bottom column. STEP 3 Technique Apply 0.5 ml of ran dom urine sample to. the top of the column unit. Rationale Urine contains two substances (porphobilino gen and urea) which will interfere with the color imetric determination of ALA. The column unit removes urea and porpho bilinogen. STEP 5 Technique Remove and discard top disposable column from piggyback unit. Rationale Elimination of por phobilinogen retained on top column as an inter fering substance. -4- STEP 2 Technique Apply 10 ml of dis tilled water to the column unit. Rationale Produces uniformity of column flow-rate prior to sample application. STEP 4 Technique Add 3 aliquots of 10 ml each of distilled water to the column unit. Rationale Porphobilinogen is re tained on top column while ALA is retained on bottom column. Urea is washed from both top and bottom columns and is therefore eliminated as an interfering substance. STEP 6 Technique Place bottom dispos able column in a 20 x 150 mm pyrex test tube and add T.O ml of 1.0 M sodi um acetate to the column. Collect the drained liquid in the test tube. Rationale The ALA retained in the bottom column is eluted from the column with sodium acetate and the drained liquid is saved for the colorimetric anal ysis of ALA. DUP050312859 STEP 7 Technique Remove the bottom disposable column from the test tube and discard. Add 0.2 ml of acetylacetone to the contents of the test tube and mix. STEP 8 Technique Place test tube in a boiling water bath for 10 minutes, remove from water bath and allow to cool to room temperature. Rationale ALA is condensed with acetylacetone to form a pyrrole. Rationale A temperature of greater than 90 C allows for completion of conden sation reaction to form the pyrrole in 10 minutes. STEP 9 Technique Add 7.0 ml of freshly prepared Ehrlich's rea gent (p-diinethylaminobenzaldehyde dissolved in a mixture of acetic and perchloric acid) to test tube and mix. Allow to stand for 15 minutes. Rationale The pyrrole formed from ALA condenses with p-dimethylaminobenz alde hyde (DMAB) in acid so lution to form a red-color ed condensation product. STEP 10 Technique Read optical density of final mixture at 553 mu in a standard laboratory colorimeter. r Rationale Normal values of urinary ALA produce a faint yellow color while abnormally high values'of urinary ALA produce a distinct red color. Although elevated values of urinary ALA can easily be detected by the eye, a very accurate quantitation of the urinary ALA level in terms of mg% can readily be obtained by plotting the optical density of the urine sample against known concentrations of pure ALA carried through identical procedure. SUGGESTION FOR LARGE SCALE SCREENING Hand-operated Continuous Syringe Type Pipetters and/or Automatic Pipetting Machines have proved extremely useful for the rapid and easy delivery of all reagents used in the Davis Test for Urinary ALA employing disposable, prefilied ion-exchange chromatography columns. 5- - DUP050312860 COMPARISON OF PRE-TEST SAMPLE COLLECTION METHODS URINARY ALA 1. Only 0.5 ml of a random urine sample is required for the determination of ALA, allowing for ease of sample collection especially from the younger age group. Urine samples can be stored in a dark freezer for several months without loss of the original ALA content. 2. Sample collection can be accomplished by non-medical personnel in a non-hospital envi ronment, allowing for direct sample collection at the home or place of employment. The prob lem involved using collection containers possibly contaminated with traces of lead does not exist for the urinary ALA test. 8. No pain or trauma is associated with urine sample collection. BLOOD LEAD 1. A sample size of 5-10 ml of whole blood is required, this amount being quite difficult to obtain by cubital venipuncture in a small child or infant. 2. Sample collection must be performed by a physician or a highly-trained medical technician, often requiring the aid of a second person when dealing with a small child. 3. Pain and trauma are often associated with blood sample collection, resulting in wide spread adult patient resistance to the test as well as marked parent resistance to the test when deal ing with asymptomatic, apparently-well small children. THE DAVIS TEST FOR URINARY ALA The measurement of Delta-Aminolevulinic Acid (ALA) colorimetrically depends upon the prelimi nary removal of urea and porphobilinogen from the urine. Traditionally this has required the laboratory preparation of two separate ion exchange columns for each determination. Using specially processed resins in piggy-back columns, the Davis Test gives the laboratory a pre pared disposable unit ready for use with simple reagents. With this test now available, screening for excessive lead exposure and early lead poisoning can be accomplished with great reliability and economy. BIBLIOGRAPHY 1. Cramer. K,, and Selander. S., Detection of Industrial Lead Poisoning, Lancet, 1, 544 (1966) Cramer, K., and Selander. S.. Studies in Lead Poisoning. British Journal of Industrial Medicine. 23, 282 (1966) Cramer, K., and Selander, S., Studies in Lead Poisoning: Comparison Between Different Laboratory Tests, British Journal of Industrial Medicine, 22, 311 (1965) 2. Lead Environment and Its Effects on Humans, State of Cali fornia, Department of Public Health, Berkeley, Calif. (1967) 3. Patterson. C.C., Archives of Environmental Health, II, 344 (1965) 4. Thomas, H.V., Milmore, B.K., Heidbreder, G.A. and Kogan. B.A.. Blood Lead of Persons Living Near Freeways, Ar chives of Environmental Health, 15, 695 (1967) 5. Chicago Board of Health News Letter, 7. No. 4. December (1967) 6. Shemin, D.. and Russell. C.S.. Delta-Aminolevulinic Acid. Its Role in the Biosynthesis of Porphyrins and Purines, J. Am. Chem. Soc,, 75, 4873 (1953) 7. Mauzerall, D. and Granick, S., The Occurrence and Deter mination of Delta-Aminolevulinic Acid and Porphobilinogen in Urine /. Biol. Chem., 219, 435 (1956) 8. 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 (1960) 9. Bonsignore, D., Calissano, P. and Cartasegna, C.. Behavior of Delta-Aminolevulinic Acid Dehydrase in Experimental Lead Poironing, Bollettino della Societa Itaiiana di Biologia Sperimentale, 41. 443 (1965) 10. Davis J. R., and Andelman, S. L.. Urinary Delta-Amino levulinic Acid (ALA) Levels in Lead Poisoning I. A Modi fied Method for the Rapid Determination of Urinary DeltaAminolevulinic Acid Using Disposable Ion-Exchange Chro matography Columns, Arch. Environ. Health, 15, 53 (1967) 11. Editorial (Leading Article), Lancet, 1, 191 (1966) 12. Saita, G. and Moreo, L.. Determination of Blood and Uri nary Delta-Aminolevulinic Acid in the Diagnosis of Past Lead Poisoning, Med. Lavoro, 55, 357 (1964) 13. Griggs, R.S.. Lead Poisoning. Hematologic Aspects. Prog. Hemat.. 4, 117 (1964) 6- - DUP050312861 PREPARATION OF A STANDARD CALIBRATION CURVE 1. Six aliquots of the ALA hydrochloride stock solution (0.01 ml, 0.05 ml, 0.10 ml, 0.20 ml, 0.S0 ml and 0.50 ml) are used as working standards. Volume of standard slock solution ={1 ml 100 pg} 0.01 ml 0.05 ml 0.10 ml 0.20 ml 0.30 ml 0.50 ml jjg of ALA par total reaction volume 1 wr 5 Jig 10 jig 20 jig 30 jig 50 jig Corresponding mg% of vrlnorf ALA for a 0.5 ml sample of urine applied to the column 0.20 mg% 1.00 mg% 2.00 mg% 4.00 mg% 6.00 mg% 10.00 mg% 2. Dilute each of the six working standards to 7.0 ml mark with 1.0 M sodium acetate using graduated test tubes. 3. Pipet 7.0 ml of 1.0 M sodium acetate into an empty test tube to be used as a reagent blank. 4. Add 0.2 ml of aeetylacetone to each tube and mix. 5. Place tubes in a boiling water bath for 10 minutes, remove and cool to room temperature. 6. Add 7.0 ml of Ehrlich's reagent to each tube, mix and let stand for 15 minutes for complete color development. 7. Set a colorimeter to 100% transmission at 553 mji using the reagent blank. 8. Read the optical density of the standards and unknowns. 9. Prepare a standard curve by plotting the con centrations of the standards against optical density. 10. Read the concentration of urinary ALA in the unknowns (0.5 ml urine sample applied to the column unit) in terms of mg% from the sample calibration curve shown below: INTERPRETATION OF URINARY ALA VALUES , URINARY ALA RANGE H %) URINARY ALA CODE 0.00 - 0.54 0.55 - 0.99 1.00 - 1.49 1.50 - 1.99 2.00 - 2.99 3.00 - 5.99 6.00 -10.00 NORMAL TRACE 1 PLUS 2 PLUS 3 PLUS 4 PLUS 5 PLUS 7- - AVERAGE COMPLETION TIME FOR ALA DETERMINATION 20 MEASUREMENTS REQUIRE 1 HOUR 40 MEASUREMENTS REQUIRE 2 HOURS 80 MEASUREMENTS REQUIRE 3 HOURS 200 MEASUREMENTS REQUIRE 5 HOURS DUP050312862 ORDERING INFORMATION Catalog No. Description Pricing TEST KITS 95002 20 Piggy-back Column Units (Complete with reagents* and standard) 95004 40 Piggy-back Column Units (Complete with reagents* and standard) 95020 200 Piggy-back Column Units (Complete with reagents* and standard) 95501 * Note-. Perchloric acid (72%) is not supplied with the kit but is available in one pound bottles sufficient for 200 tests. $ 34.50 60.00 207.00 4.00 SUPPORT RACK FOR HOLDING DISPOSABLE COLUMNS DURING TEST PROCEDURES 95502 Support rack for 40 Column Units with drain tray and numbered pipet guide as shown on page 1. 13.75 COLUMNS ONLY 95108 80 Piggy-back Column Units 95120 200 Piggy-back Column Units Prices will be quoted on larger amounts. 94.00 190.00 REPIPETS FOR EXTRA CONVENIENCE 95801 1 ml, with 950 ml amber bottle 47.50 95802 10 ml, with 950 ml amber bottle V All prices are FOB shipping point, either Richmond, California, or New York City. Terms: Net 30 days. 47.50 ' '..ill:" ' Main Office and Laboratories 32nd & Griffin Avenue Richmond, California 94804 Telephone: (415) 234-4130 Telex: 033-658 Eastern Sales and Warehouse 22 Jones Street New York, N.Y. 10014 Telephone: (212) WA 4-4232 Telex: 01-26108 European Distribution Munchen-Grafelfing Am Kirchenholzl 6 West Germany Telephones: 833698 Telex: 528014 833699 DUP050312863