acidity is always increased in both the excited singlet state and the triplet state. However, the KT usually lies much closer to the ground state value than does the excited singlet state acidity constant. The excited state acidity constants in the triplet state showed a much smaller dependence on the type of compound under investigation. The values obtained were always within two pK units of the values measured for the ground state, which is an agreement with the results ob-
tained by other authors (1, 17). For the singlet excited state acidity constants, variations of from 5 to 20 pK units from the ground state are known (18).
RECEIVEDfor review February 19,1974. Accepted June 17, 1974. (17) S . G. Schulman, L. B. Sanders, and J. D. Winefordner, Photochem. Photobiol., 13, 381 (1971). (18) H. H. Jaffe. D. L. Beveridge, and L. Jones, J. Amer. Chem. SOC., 86, 2932 (1964).
Potential Sources of Analytical Error in the Erythrocytic 6-Aminolevulinic Acid Dehydratase Test for Lead Exposure Theo. J. Kneip, Norman Cohen, and Virginia Rulon New York University Medical Center, 550 First Avenue, New York, N. Y. 100 16
The determination of 6-aminolevulinic acid dehydratase activity (ALA-D) in erythrocytes is one of the most sensitive tests proposed for screening large populations of children potentially exposed to lead. Despite the excellent sensitivity of ALA-D to lead exposures as shown by Hernberg (1, 2 ) for normal and elevated levels of blood lead concentration, and the relatively simple spectrophotometric equipment required for its analysis, the test has not been widely adopted for routine use. The occurrence of erratic and widely variable ALA-D values, as observed in this as well as other laboratories ( 3 ) , has been one cause for the unpopularity of this test. A second factor for the lack of acceptance is the unavailability of the purified enzyme for the determination of quantitative calibration curves. Present studies employing this test are part of a broad research program designed to elucidate dose-response relationships for lead exposures for controlled ingestion of leaded compounds by infant baboons ( 4 ) . Baboons are exposed to lead by intravenous injection of lead chloride or by forced feeding of various doses of lead acetate, paint pigment (lead hydroxycarbonate), powdered paint containing a lead drier compound (lead octoate), or lead octoate dissolved in olive oil. Several clinical and biochemical indices of lead exposure are routinely monitored and evaluated with respect to the duration and magnitude of the lead dose rate as well as to the accumulated lead body burden.
EXPERIMENTAL Activity of ALA-D is determined in hemolyzed whole blood by the method of Granick ( 5 ) with two modifications. Hemolysis is achieved by immersing the sample tube in a Dry Ice-acetone mixture for 10 minutes, eliminating the use of the saponin. Mercuric chloride is not used in the Erhlich’s reagent, and is replaced by Nethylmaleimide (6) in the trichloroacetic acid reagent to prevent sulfhydryl interference in the subsequent color development step. The porphobilinogen (PBG) formed by condensation of two moleS . Hernberg and J. Nikkanen, Lancet, 1, 64 (1970). S. Hernberg, S . Tola, J. Nikkanen, and S . Valkonen. Arch. Environ. Health, 25, 109 (1973). B. G. King, Yo Dr. Dale Ruthig, Environmental Health Services Division, Center for Disease Control, Atlanta, Ga. 30333, personal communication, 1974. N. Cohen. T. J. Kneip, V. Rulon, and D. H. Goldstein, “Environmental Health Perspectives.” Experimental Issue 7, DHEW Pub. NIH 74-218, U.S. Government Printing Office, Washington, D.C., 1974, p 161 S. Granick, S. Sassa, J. L. Granick, R. D. Levere, and A. Kappas, Proc. Nat. Acad. Sci. US,69, 2285 (1972). ti. 6 . Burch, and A. L. Siegel, Clin. Chem., 17, 1038 (1971).
cules of 6-aminolevulinic acid (&ALA) through the catalytic action of ALA-D is measured spectrophotometrically with a Beckman Model DB spectrophotometer and expressed as nmol PBG/ mlRBC/hr.
RESULTS AND DISCUSSION The ALA-D test was difficult to control throughout the early history of the lead toxicity study. Early values for erythrocytic ALA-D in infant baboons ingesting lead compounds over a wide range of dose rates, showed considerable scatter and almost no correlation to the magnitude of their lead exposures. When it was noted that ALA-D values were more variable in blood collected in heparinized 5-ml glass Vacutainer tubes than in blood collected in 10-ml glass Vacutainer tubes, a study of the sampling procedure was initiated. (All the glass Vacutainer tubes are products of Becton-Dickinson. The polystyrene tubes are products of Falcon Plastics. Both producers are Divisions of BectonDickinson and Co., Rutherford, N.J.). Three types of tubes and two types of stoppers were subsequently employed for blood collection: polystyrene tubes, 10-ml glass tubes with amber or green stoppers, and 5-ml glass tubes with amber stoppers. The amber rubber stoppers are used for those tubes certified to contain no more than 1 pg lead per tube-ie., “minimal lead.” Tubes with green rubber stoppers are not certified as “minimal lead.” Blood samples were collected from animals exposed to lead at three different dose levels. For example, B-31 (a 4I/~-yrold male weighing 13.3 kg) and B-330 (a 5-yr old female weighing’6.3 kg) had been ingesting lead as lead acetate as the dose rate of 16.1 mg Pb/kg/day over a period of 7 to 8 months. The animal B-556 (a 1-yr old female weighing 2.3 kg) had been ingesting lead in the form of lead octoate drier in dried paint solids at the rate of 100 pg Pb/ kg/day over a period of 3Ih months. The blood lead concentrations of the samples drawn during this investigation were extremely high for B-330 and B-31, and in the range of normal human values for B-556 and B-650 (a 1%yr old female weighing 4 kg) which was not exposed to any lead compound.) Values for ALA-D were determined in aliquots of blood transferred from a single plastic syringe into the different types of collection tubes. To prevent initial blood-stopper contact, the stopper was removed from the tube before the blood was transferred from the syringe. The blood was allowed to flow down the inside wall of the tube and was
A N A L Y T I C A L CHEMISTRY, VOL. 46, NO. 12, OCTOBER 1974
1863
Table I. Sample Container Effects on Measured ALA-D Values in Baboon Blood (Values in nmol PBG/ml RBC/hr) Animal No.
Date
B-31 B-330
10-ml Glass tube
Polystyrene tube
5/9
Amber stopper
Error,
NO
Error,
5%”
stopper
%“
32 33
359 352
122 141
30 47 235 330
39 30 406 437
1020 970 995 30 - 36
280 330 305 0 - 36 6 0.3 7.4
Average error, % B-31 B-330 B-556 B-650
5/16
73 32 25
Average error, % a
Error,
‘Z=
30 30 250 331
Green stopper
Error,
779 1024
2330 3000 2665 1350 777 115 52 573
No stopper
70”
435 412 506 503
54 75 30 30 287 376
Error,
7oa
69 127 98 0 - 36 22 14 0
(ALA-Dglnsr - A L A - D p o l y s t y r e n e ) / A L A - D ~ ~ l y ~Xt ~100. *~~~
‘Oo0:
I
2
1
1
i
2o 0 40
XI
0
X,
40
60
80 100 20
40
60
80
200
20
40
60
80
Days Pre Cays Post Exposure
Figure 1. 6-Aminolevulinic acid dehydratase
variations for control animals
Animals: 8-131 (0);B-145 (A).(1) Routine use of 10-ml “minimal lead” tubes without stopper contact. (2) Routine use of polystyrene tubes sealed with parafilm
mixed by manually rotating the tube in an upright position for 15-20 seconds to dissolve the heparin. In this way, contact was avoided between the blood and the lubricated area where the stopper had originally been. After a volume of blood necessary to run the assay was taken from the sample, the stopper was replaced. The tube was then inverted for a period of three minutes to allow the blood to contact the stopper. While a totally consistent pattern was not obtained, it is clear from the data in Table I that stopper contact often resulted in values with a positive bias-Le., values greater than those obtained without stopper contact. Continued evaluation of the difference between 5-ml and 10-ml amber-stoppered tubes confirmed the results. During these intercomparisons, however, clotting was observed in some of the 5-ml tubes, so that a further modification of the sampling procedure was necessary. The walls of the plastic syringe into which the blood sample is drawn are now rinsed with a solution of sodium heparin (“Liquaemin Sodium ‘lo’,” lcm3 = 1000 USP units, Organon, Inc., West Orange, N.J.) before the needle is inserted into the vein. No clotting problem has been observed when blood taken in this manner is transferred to a “minimal lead” tube from which the stopper has been removed or to a polystyrene tube, and is mixed by rotating as previously described. The tubes are then covered with parafilm for transfer to the laboratory. As can be seen in Figures 1 through 3, the ALA-D activity of animals having normal or elevated blood lead levels has been strongly affected by this sampling problem. The sporadic nature of the effect is seen initially by the im1864
provement observed for some time while using 10-ml “minimal lead” tubes without stopper contact, and subsequent interference as shown a t day 182 in Figure 2 and a t day 64 in Figure 3. The animals represented in Figure 3 had begun exposure after the original change in the sampling procedure. As it was believed that a new problem had developed, a complete recheck of the ALA substrate and repreparation of all reagents was performed. Only one lot of 10-ml “minimal lead” tubes had been used since refinement of the sampling procedure. It was finally determined by the further intercomparisons shown in Table I1 that the 10-ml tubes were variable from box to box within the same lot. Subsequent to this discovery, polystyrene tubes have been used for all blood sample collections after ascertaining that this change would not interfere in the blood lead determination. The change to polystyrene tubes resulted in a return to ALA-D values representative of those expected for both exposed and unexposed animals. The per cent error due to blood collection in the glass tubes is plotted in Figure 4 and can be seen to be a function of the actual enzyme activity-ie., that observed in blood collected in polystyrene tubes. (The dashed line shows a theoretical curve calculated from an assumed hypothetical value in polystyrene of 300, 200, 100, 50, and 25 nmol PBG/ml RBC/hr and a hypothetical constant value of 400 nmol PBG/ml RBC/hr. for glass sample tubes.) The excellent fit of the curve to the actual experimental data is indicative of the complete restoration of ALA-D activity regardless of the magnitude of the original inhibition due to lead.
ANALYTICAL CHEMISTRY, VOL. 46, NO. 12, OCTOBER 1974
T a b l e 11. S a m p l e C o n t a i n e r E f f e c t s on M e a s u r e d ( V a l u e s in n m o l PBG/ml R B C / h r ) Animal No.
Polystyrene tube
Date
6/12 6/12 6/12 6/12 6/12
B-31 B-330 B-159 B-179 B-199 B-642 B-652 B-191 B-191 B-191 B-654 B-654 B-656 B-656 B-656
ALA-D
6/12 9/12 9/14 9/18 9/12 9/18 9/12 9/14 9/13
'- t
1
