Document x1Yw9rMNL7voD519qp1wqKdqm

Report No. 335-72 August 24, 1972 &0 EVIDENCE AS TO WHETHER DEPRESSION OF RED CELL 6-AMINOLEVULINIC ACID DEHXDRASE BY LEAD IN DOGS OCCURS "IN VIVO" OR IS AN "IN VITRO" ARTIFACT INHERENT IN METHODS USED TO MEASURE ITS ACTIVITY______________________________________________________________ ?. Medical Research Project No. 1QA11 r' ttt.Uf.MT.eK HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE N40744 Distribution List EVIDENCE AS TO WHETHER DEPRESSION OF RED CELL 6-AMINOLEVULINIC ACID DEHXDRASE BY LEAD IN DOGS OCCURS "IN VIVO" OR IS AN "IN VITRO" ARTIFACT INHERENT IN METHODS USED TO MEASURE ITS ___________________________ ACTIVITY ___________ Medical Research Project No. 1QA11 Haskell Laboratory Report No, 335-72 C. F. Reinhardt/ J, A. Zapp, Jr. P. E. Smith, Jr./H. J. Trochimowicz/A. Azat J. R. Barnes/j. 6. Aftosmis/R. S . Waritz J. F. Morgan D. B. Hood H. Sherman C. S. Hornberger F. D. Griffith \ Haskell Laboratory File Donald R. Diggs, Organic Chemicals Department (3) Robert C. Butler, Organic Chemicals Department C. W. Maynard, Organic Chemicals Department Physiology Section File M. E. Maxfield N. W. Henry, III DUP050058434 INTRODUCTION The depression of red cell 6-aminolevulinic acid dehydrase (ALAD) activity, a phenomenon subsequent to absorption of lead, is assuming importance as a possible indicator of lead exposure (1-6), This indirect assay of lead absorption is based on the demonstration that the logarithm of ALAD activity has an inverse linear relation to blood lead concentration within the range of 5 fig-90 pg Pb/100 ml blood (3, 4, 7). Such a relation ship predicts depression of red cell ALAD activity in association with very low concentrations of lead in blood, e.g. below 5 p.g/100 ml (3, 7), and precludes a "no-effect11 level for blood lead with respect to red cell ALAD, For the individual exposed to lead, the significance of the depres sion of his red cell ALAD activity is dependent, in the final analysis, upon whether the depression occurred "in vivo", as is generally assumed (1-9), or whether it occurred after his blood was withdrawn for analysis, i.e. "in vitro". Since circulating lead is bound to the red blood cells, and since analyses of ALAD activity are performed on hemolysates of these cells (10), the possibility exists that the depression of ALAD activity as determined for such hemolysates may occur subsequent to the hemolysis. In support of the contention that the depression of red cell ALAD activity in individuals exposed to lead occurs "in vivo", is the demonstration that the depression of ALAD activity effected by addition DUP050058435 EVIDENCE AS TO WHETHER DEPRESSION OF RED CELL 5-AMINOLEVULINIC ACID DEHYDRASE BY LEAD IN DOGS OCCURS "IN VIVO" OR IS AN "IN VITRO" ARTIFACT INHERENT IN METHODS USED TO MEASURE ITS _______________________________ ACTIVITY__________________ Medical Research Project No. 10A11 Haskell Laboratory Report No. 335-72 Report by: And: n . Mary E. MaxfieId Research Physiologist ay. .^rgh: JM- Norman We Henrryy, II# Chemist Approved for Biochemistry: hn R. Barnes Chieef7~ml<ochemistry Section Approved by: f NB 185, pp. 100-115 (Biochem.) MEM/HWH/ jtd Date: August 22, 1972 DUP050058436 -2- of inorganic lead to hemolysates of "normal" blood differs from the former in at least two respects (5) ; (1) The optimum pH, 6.4, for red cell ALAD activity following addition of inorganic lead to hemolysate is the same as for "normal" blood, whereas the optimum pH for ALAD activity of blood from lead-exposed individuals has shifted to a lower pH, 5.8. (2) The depression of red cell AlAD activity of the blood from lead-exposed individuals is reversed by heat-denaturation, whereas the depression due to addition of inorganic lead to hemolysates of "normal11 blood is not so reversed* However convincing as these differences may be, they do not unequivocally prove that the depression of red cell ALAD activity observed for blood from exposed individuals did occur "in vivo." Rather, these differences may reflect differences between an "in vitro" depressant action due to lead ions, and that due to a lead complex in which form the lead may exist in its attachment to circulating red blood cells. The experiments herein reported were designed to test whether the depression of the red ceil AlAD activity of the blood from lead-exposed dogs, as measured by a commonly used method, is an "In vivo", i.e. occurred in the animal, or an "in vitro", i.e. occurred during the analys is, phenomenon. In design, the experiments are simple* Whole blood from a leadexposed dog, hence containing lead in whatever form it exists in $ DUP050058437 circulating blood, was added, in varying proportions, to whole blood from a "normal" (control) dog* The mixed samples were then analysed for resultant red cell ALAD activity and lead concentration. If the depression of ALAD activity of the blood of the lead-exposed dog occurred "in vivo", addition of this lead-containing blood should not affect the ALAD activity of "normal" blood other than by simple, dilution. On the other hand, if the depression occurs during the analysis, i.e. "in vitro", the enzymes from both bloods should be affected, and the logarithm of the resultant ALAD activity should have an inverse linear relation to the resultant lead concentration. METHODS Ten male beagle dogs, available from a previous study (11), supplied the blood used in the present experiments. Five of these dogs*, fed the basic diet of ground Purina dog meal, provided normal (N) blood, with lead concentrations ranging from "0" (not detectable) to 7 pg/100 ml and red cell ALAD activities ranging from 24 units to 39 units/ml RBG. After approximately one month on the basic diet to which 1000 ppm lead, as the acetate, was added, the other five dogs provided blood (L) with lead concentrations ranging from 52 jjbg to 70 pg./100 ml and red cell ALAD activities ranging from "0" to 3 units/ml RBG. For each experiment, duplicate series of mixtures of whole blood (as withdrawn, i.e. not hemolysates) were prepared by mixing blood, N, from one of the normal dogs with blood, L, from one of the lead*-fed dogs, to provide mixtures containing the following proportions of N and L bloods : *Three of these dogs were controls (no lead added to diet) in the previous study and the other two dogs (100 ppm lead) had been on lead-free diets for at least62 weeks before starting the present experiments. DUP050058438 N Blood; 100 L Blood: 0 Percentage 80 60 50 40 20 0 20 40 50 60 80 100 The mixtures were analysed for resultant blood lead concentration, using an atomic absorption spectrophotometer (Model 303, Perkin-Elmer Corp., Norwalk, Conn.), and for red cell AIAD activity, using the method of Bonsignore et al (12) . Hematocrit ratios were determined by the microhematocrit method. Five such experiments were performed, each dog serving only once as a donor. RESULTS \ The determination of the accuracy of the measurements, including mixing of blood samples, was based on the differences between the two values obtained for duplicate mixtures, and was calculated according to the equation* Standard Deviation * + /'Uijmj-mg) ^ 2x N where m^ and m2 are the values for the duplicate mixtures, and N is the number of pairs of duplicates. The standard deviations so calculated are:. Blood lead concentration: + 2.8 |xg, AIAD activity: + 2.2 units. The data presented in the figures are the averages of the values obtained for the duplicate determinations. Recommended by Dr* R* D. Snee, Engineering Department, E. X. Du Pont Be Nemours and Company. DUP050058439 -5- AlAD Activity of Samples of Nonnal Blood Diluted With Plasma Preliminary to the main experiments, the effect of simple dilution of whole blood on red cell ALAD activity was determined for two series of dilutions. In each series, whole blood (not hemolysates) withdrawn from a normal dog was diluted with the dog's own plasma to provide samples consisting of 80%, 60%, 50%, 40%, and 20% of normal blood (N). The effect ) of dilution of tbe blood on red cell AlAD activity is demonstrated by the results obtained for the first series of dilutions in Figures 1, A and B* in which the ordinate scale (Optical Density*) is linear (Figure 1A) or logarithmic (Figure IB). The data show that when whole blood is diluted with plasma, the relationship between optical density (y) and the per centage of blood in the sample (x) conforms to the equation y *= a + bx (Figure 1A) rather than to the equation log y * a + bx (Figure LB). The regression, calculated by the method of least Squares, for the data in Figure 1A has a correlation coefficient (r) of 0.9980, and accounts for 99*6% of the variance in optical densities* The regression calculated for the data of the second dilution series, has a correlation coefficient of 0.9986, and accounts for 99.7% of the variance. The difference between the two equations is important for in human blood, the relation between red cell ALAD activity and the blood lead concentration appears to conform to the equation log y - a + bx (3,4,7). That this same relationship holds for dog blood is shown by the data in Figure 2, obtained on female dogs** in an earlier study (11). *0ptical densities, rather than units of ALAD activity, are plotted against percentages of blood because ALAD activity, expressed as units/ml RBC, would be the same for all dilutions. In Figures 2-5, ALAD activity, as units /ml RBC, is used since the red cell volumes were essentially equal in all blood samples of a given series. **For unknown reasons, the female dogs provided a more even distribution of blood lead concentrations than the males. DUP050058440 -6- Effect of Lead in Blood on ALAD Activity of Normal Blood The effect of adding lead-containing blood on the red cell ALAD activity of normal blood is shown by the data in Figures 3, A and B, in which units (Figure 3A) or log units (Figure 3B) of A1AD activity are plotted against the percentage of normal blood in each mixture. As when normal blood was diluted with plasma, the relationship between ALAD activity (y) and the percent of normal blood in the mixture (x) is expressed by the equation y = a + bx, rather than by log y = a + bx. "I The effect of adding the lead-containing blood appears to be that of simple dilution. On the assumption that the reduction in ALAD activity of the normal blood was due to simple dilution by blood with low AIAD activity, the resultant activity for each blood mixture was predicted. The agree < '5 ment between predicted (x) and observed values (y) are shown by the regression, y = a + bx, with its + 95% confidence limits, in Figure 4. u The relatively close agreement between observed and predicted values strongly supports the contention that the only significant effect of mixing lead-containing blood With normal blood was to dilute the ALAD activity of the latter. The relationship between the red cell ALAD activities of the mixtures and the corresponding lead concentrations is shown in Figures 5, A and B. It is obvious from these graphs that the inverse linear relationship between the logarithm of the ALAD activity (y) and blood lead concentration (x) as usually described, does not apply to these mixtures of normal and lead-containing bloods (Figure 5B). Rather, the I DUP050058441 -7- relationship conforms to the equation: y = a + bx, for which in this case, the slope b has a negative value (Figure 5A). DISCUSSION If lead acts to depress red cell ALAD activity "in vitro" rather than "in vivo," all samples of blood as withdrawn (i.e. prior to hemo lysis) would presumably have normal ALAD activities. The ALAD activity (y) of samples of mixed normal and lead-containing bloods would have the same relationship to resultant lead concentration (x) as has been shown to occur for unmixed samples obtained from lead-exposed Individuals, i.e. log y a + (-b)x. The present data demonstrate, however, that the ALAD activities of the samples of mixed blood are not predictable by this relationship, but rather by y = a + (-b)x which indicates that a different mechanism is responsible for the observed reductions in AlAD activities. The data presented in Figures 3, A and B, and in Figure 4 provide evidence that the reduction in AlAD activities of the samples of mixed normal and lead-containing bloods resulted from simple dilution of the ALAD activity contributed by the portion of normal blood in the mixture. . Lead, presumably present in the form in which it exists in circulating blood, did not have a measurable depressant action on the activity of this "normal" ALAD. DUP050058442 -8- Within the limits of the resultant lead concentrations of the mixtures, which ranged from 12 pg to 57 (Xg/100 ml, the present data support the generally - held concept that the red cell ALAS depression observed in lead-exposed individuals, as measured by a widely accepted method (12), occurred "in vivo," and is not an "in vitro" artifact inherent in the method. SUMMARY * The reduction in red cell A1AD activity of normal blood, when mixed with lead-containing blood from a lead-exposed dog, may be attributed to simple dilution, rather than to a depressant action of the lead during or subsequent to the preparation of the hemolysates. This is interpreted as indicating that the depressant action of lead on the red cell ALAP of lead-exposed individuals occurs "in vivo," and is not an "in vitro" artifact inherent in the method of measurement of ALAP activity. MEM/j td 8/22/72 DUP050058443 REFERENCES 1. de Bruin, A., /rad G. -T. de Jong-Heisterkamp. IE BLOCAGE ENZYMATIQUE DE LA A-ALA DESHYDRASE PAR LE PLGMB. Ann. Biol. Clin,, 26: 717-723, 1968. 2. Nakao, K., 0. Wada, and Y. Yano. 6-AMINOLEVULINIC ACID DEHYDRATASE ACTIVITY IN ERYTHROCYTES FOR THE EVALUATION OF LEAD POISONING. Clin. China. Acta, 19: 319-325, 1968, 3. Hernberg, S., J. Nikkanen, G. Mellin, and H. Lilius. 6-AMINOLEVULINIC ACID DEHXDRASE AS A MEASURE OF LEAD EXPOSURE. Arch. Environ. Health. 21: 140-145, Aug. 1970. 4. Weissberg, J. B., F. Lipschutz, and F. A. Oski. 6-AMINOLEVULINIC ACID DEHYDRATASE ACTIVITY IN CIRCULATING BLOOD CELLS. New Eng. J. Med. 284: 565-569, Mar. 18, 1971. 5. Nikkanen, J,, S. Hernberg, and S. Tola. MODIFICATIONS OF THE 6-AMINOLEVULINIC ACID DEHYDRATASE TEST AND THEIR SIGNIFICANCE FOR ASSESSING DIFFERENT INTENSITIES OF LEAD EXPOSURE. Work Environ. Health V: 46-52, 1972. 6. Hernberg, S. EFFECT OF LEAD ON 6-AMINOLEVULINIC ACID DEHYDRATASE. A SELECTIVE REVIEW. Pracov. Lek 24: 77-83, 1972. 7. Millar, J. A., V. Battistini, R. L. C. Gumming, F. Carswell, and A. Goldberg. LEAD AND 6-AMINOLAEVULINIC ACID DEHYDRATASE LEVELS IN MENTALLY RETARDED CHILDREN AND IN LEAD-POISONED SUCKLING RATS. The Lancet 2: 695-698, Oct. 3, 1970. 8. Chisolm, J. J., Jr. LEAD POISONING. Scientific American 224: 15-23, February 1971. 9. Prerovska, I. and J. Teisinger. EXCRETION OF LEAD AND ITS BIOLOGICAL ACTIVITY SEVERAL YEARS AFTER TERMINATION OF EXPOSURE. Brit. J. Industr. Med. 27.: 352-355, Oct. 1970. 10. Quoted in BIOLOGICAL EFFECTS OF ATMOSPHERIC POLLUTANTS. LEAD: AIRBORNE LEAD IN PERSPECTIVE. Committee on Biological Effects of Atmospheric Pollutants, Division of Medical Sciences, National Research Council, 1972. National Academy of Sciences, 2101 Constitution Ave., Washington, D.C. 20418, pp. 103, 106, 11, Maxfield, Mary E., G. J. Stopps, J. R. Barnes, R. D. Snee, and A. Azar. EFFECT OF LEAD ON BLOOD REGENERATION FOLLOWING ACUTE HEMORRHAGE IN DOGS. Accepted for publication in Am. Industr. Hyg. Ass. J., May 1972. DUP050058444 REFERENCES CONT'D. 12, Bonsignore, D., P. Calissano, and E. Cartasegna. A SIMPLE METHOD FOR THE DETERMINATION OF BLOOD 6-AMINOLEVULINIC ACID DEHYDRASE. Med. D. Lavoro 56 (3): 199, March 1965. MEM/jtd 8/7/72 DUP050058445 Figures 1, A and B The effect of whole blood dilution with plasma upon the normal red cell ALAD activity* A: Optical density (O.D.), on linear scale, vs* percent of blood contained in sample* Calculated Regression: y a + bx + 95% confidence limits: B: Log optical density vs. percent of blood contained in sample. Curve fitted to data "by eye.*1 DUP050058446 DUP050058447 I reel Q 80 60 40 20 "1 % BLOOD IN SAMPLE DUP050058448 Figure 2 Relationship between log red cell ALAD activity and blood lead concentration, female dogs. Data obtained in previous study (11). Calculated regression: log y ~ a ~ bx mmmmmmm #: ALAD activity not measurable. These points, arbitrarily placed below the limit of sensitivity ^ were omitted in the calculation of the regression. DUP050058449 Figures 3, A and B The effect of lead-containing blood on the red cell ALAD activity of normal blood. A: Units of ALAD activity, or linear scale, versus percent of normal blood contained in sample. Calculated regression, y= a + bx mmmmmmmmmmm : ALAD activity not measurable. Not included in calculation of regression. B. Log units ALAD activity versus percent of normal blood contained in samples. Curve fitted to data "by eye." ; ALAD activity not measurable. Arbitrarily placed below limit of sensitivity " DUP050058451 DUP050058452 DUP050058453 Figure 4 Observed ALAD activity of mixtures of normal and lead-containing bloods versus predicted values. Calculated regression y= a + bx + 95% confidence limits - -> DUP050058454 DUP050058455 Figures 5, A and B Red cell ALAD activity of mixtures of normal and lead-containing bloods versus resultant lead concentration of the mixtures. A. Units of ALAD activity, a linear scale, versus blood lead concentration, Calculation regression .mmmmmmmmmmmmm t; ALAD activity not measurable. Arbitrarily positioned below sensitivity limit ............ "ii. Data not included in calculation of regression. B. Log units of ALAD activity versus blood lead con*centration. #: ALAD activity not measurable. Arbitrarily positioned below sensitivity limit -------- ~ Curve fitted to data "by eye." DUP050058456 DUP050058457 DUP050058458