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Dose-effect and dose-response relationships for lead in children
1152
December 1975 The JournaI o f P E D I A T R I C S
Dose-effect and dose-response relationships for lead in children Lead absorption and prevention of the serious effects of lead poisoning are re-examined from the viewpoints of the critical organ and critical effect concepts and the associated dose-effect and doseresponse relationships. I f the critical organ is the first affected and the critical effect is the first measurable adverse effect, intervention on this basis should prevent the occurrence of later, more serious effects. In the range of lead absorption of greatest current pediatric concern (blood lead in the range of 50 to 80 l~g/dl), blood lead values are not a good predictor o f critical effect, whereas' chelatable lead is significantly and linearly related to evidence of critical effect on hemoglobin synthesis in the bone marrow. Erythrocyte protoporphyrin and &aminolevulinic acid and coproporphyrin in urine are indicators of this effect. The dose-response concept provides a better way of viewing the relationship between blood lead and measures of adverse effect than do the classifications of "'sensitivity," "specificity," "false negatives," and "false positives, "" which are often employed in the evaluation of screening tests'. The doseresponse concept recognizes the uniqueness of the individual and the presence of susceptible and resistant individuals in heterogeneous population groups. With the dose-response concept, individuals may be identified as reactors or nonreactors, according to whether they exhibit a particular effect. Among the various indicators of lead's critical (or first) effect on hemoglobin synthesis, erythrocyte protoporFhyrin potentially is the most practical for monitoring children at high risk for plumbism.
J. Julian Chisolm, Jr., M.D., Maureen B. Barrett, A.B., and E. David Mellits, Sc.D., Baltimore, Md.
BLOOD LEAD MEASUREMENTS are used widely in epidemiologic studies as a biologic indicator o f exposure From the Department of Pediatrics, Johns Hopkins University School of Medicine, the Department of Pediatrics, Baltimore City Hospitals, and The John F. Kennedy Institute. Support for this work was provided, in part, by grants from the United States Public Health Service, National Institute for Occupational Safety and Health (8-RO1 OH 00307), and United States Public Health Service, Maternal and Child Health Project No. 464, Grant (RR-52) from the General Clinical Research Centers Program of the Division o f Research Resources, National Institutes of Health; United States Public Health Service (5 MO1) Clinical l~esearch Center, Grant (RR-35), International Lead Zinc Research Organization, Inc., New York, N.Y. Maternal and Child Health Project 917 and Department of Health, Education, and Welfare, Health Services and Mental Health Administration Contract No. H S M 99-72-23. Reprint address: Baltimore City Hospitals, 4940 Eastern Ave., Baltimore, Md. 21224.
Vol. 87, No. 6, part 2, pp. 1152-1160
to leadD" Difficulties arise, however, when one attempts to use this indicator of group exposures as a diagnostic indicator of toxicity. There is a lively debate in the current literature concerning the choice and interpretation of various diagnostic tests for monitoring individuals who are Abbreviations used Pb-B: blood lead (/~g Pb/dl whole blood) Pb-U: lead in urine ALA-D: . &aminolevulinic acid dehydratase ALA-U: &aminolevulinic acid in urine CP-U: coproporphyrin in urine free erythrocyte protoporphyrin FEP: CaEDTA: edathamil calcium disodium EDTA: ethylenediaminetetra-acetic acid AAS: atomic absorption spectrophotometry ASV: anodic stripping voltammetry at high risk for plumbism. It may be useful to restate the classical concepts of toxicology as they relate to lead exposure, absorption, and effect in h u m a n populations> ~ In human studies the dose of a metal can rarely be defined as it is in experimental animal studies. Because dose at the
Volume 87 Number 6, part 2
Dose-effect and dose-response relationships for lead
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Fig. l. Theoretical form of dose-effect and dose-response curves. When the law of biologic random variation applies, this same curve may be used to plot both frequency of response (as a percentage of the population) and degree of effect against dose. Area between A' and B' shows a curvilinear portion of the curve and the area between B' and C' the linear portion. (Adapted from Kjellstr0m and Eng. '~) site of action is not precisely known, application of these concepts to epidemiologic studies in h u m a n beings differs somewhat from their customary use in experimental toxicology. 5 New definitions of the terms "critical effect" and "critical site" were recently agreed upon by the Subcommittee on the Toxicology of Metals? They were developed for nonradioactive toxic metals and differ from the definitions of these terms used in radiation biology. For metals such as lead, the "critical effect" is the most sensitive and specific biologic change beyond acceptable physiologic variation which is caused by the metal. ~ Although different effects may be produced by a metal, the critical effect is thefirst measurable adverse effect, not necessarily the most serious one. "Subcritical effects" are measurable biologic changes which do not impair cellular function. The "critical site" is the location in t h e body where the critical effect occurs; it may be a system, organ, cell type, or cell component. The internal dose m a y be defined in this context as the a m o u n t of a toxic metal present at the critical site. The critical organ is the first to reach its critical concentration (ie, the internal dose sufficient to produce the effect). This approach has advantages from the viewpoint of preventive medicine. Intervention on the basis of first or critical effects at an early reversible stage should prevent the occurrence of later adverse effects that may have serious consequences from the clinical viewpoint. For inorganic lead, derangement of hemoglobin synthesis in the erythroid cells o f the bone marrow is the critical effect. 2' 5 W a d a and associates 7
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,ugPb/lOOml WHOLE BLOOD Fig. 2. Relationship between blood lead concentration and 8-aminolevulinic acid excretion. With Pb-B as indicator of dose and ALA-U as indicator of effect, a dose-effect relationship is apparent. Pb, B range is 25-800/Lg/dl. ALA-U range is 0.6 to 52 mg/mV24 hr. Data are replotted from previously published reports.,,, ,1 Logarithmic scales for Pb-B and ALA-U are used to conserve space. have demonstrated a substantial reduction in the activities of 8-aminolevulinic acid dehydratase and heme synthetase, and a parallel reduction in the incorporation of I~Cglycine into heme and globin in erythroid cells' obtained by aspiration from the bone marrow of occupationally exposed workmen with blood lead in the 40-90 /~g/dl range. The indicators of this critical effect, as measured in peripheral blood and urine, are increased urinary 8aminolevulinc acid, increased urinary coproporphyrin Ill, increased "free" erythrocyte protoporphyrin, and anemia. Decreased ALA-D activity, as measured in vitro in mature red cells in peripheral blood, is considered a subcritical effect, 5 though it is a highly useful indirect indicator of exposure. 8 Although t h e combination of increased ALA-U, CP-U, and FEP, together with decreased ALA-D (peripheral blood) is diagnostically pathognomonic for lead poisoning, A L A - U is, in practice, the single most specific indicator of this critical effect, especially in children. Increases in FEP and CP-U may
1154
Chisolm, Barrett, and Mellits
The Journal of Pediatrics December 1975
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Fig. 3. Relationship between daily ALA-U and blood lead. Range in blood lead concentration in 51 preschool children encompasses the normal and somewhat elevated range (25 to 75 t~gPb-B). This line depicts this relationship which is best fitted by the following quadratic equation: mg ALA-U/m~/24 hr = 2.4135-0.07500 (Pb-B) + 0.001265 (Pb-B)2. The standard error about this fitted line is _+ 0.6 and p < 0.01. Details of this study are reported elsewhere.~1 The curvilinear nature of the relationship between Pb-B in this range and ALA-U is apparent. also be caused by iron deficiency and hepatocellular injury, respectively; only acute alcoholic intoxication and acute intermittent porphyria, an uncommon disorder, cause significant elevation in ALA-U. Since derangement in hemoglobin synthesis due to lead is reversible and precedes significant renal injury and clinically evident neurologic manifestations, the critical effect concept should serve well for prevention of plumbism. There are, however, at present no biologic measures of comparable sensitivity and specificity which can be related to lead's effect on the nervous system. Nevertheless, the available evidence suggests that derangement in hemoglobin synthesis probably precedes functional impairment of the nervous system. DOSE-EFFECT
RELATIONSHIPS
The dose-effect relationship is one in which a quantitative change in a function or concentration of a metabolite (effect) is related to a quantitative change in the concentration of a toxic substance (dose)? Many studies ~ support the assumption that toxicologic responses follow the law of biologic random variation. The classical dose-effect curve (Fig. 1) is a cumulative normal distribution curve based on this assumption. Typically, dose is plotted on the abscissa against effect on the ordinate. In Fig. 2, Pb-B is taken as the indicator of dose and ALA-U as the indicator of critical effect. ALA-U is expressed throughout as ALAU/m~/24 hr, since a statistically significant (p < 0.01)
relationship between surface area and ALA-U has been found in young children?~ 1~The distribution of the data in Fig. 2 resembles the distribution predicted by the theoretical dose-effect curve (Fig. 1). This suggests that Pb-B over a very broad range in concentration is useful as a general approximation of dose. Kjellstr6m and Eng 12 call attention to the fact that the quantitative relationship between dose and effect may differ according to the dose interval. This is especially important in empirical studies, such as those in human populations in which dose, particularly internal dose, is not known. If, for example, the dose interval in the population under study falls in the area in Fig. 1 bounded by A' and B', nonlinear relationships between dose and effect are to be expected. If, on the other hand, dosage in the population tinder study happens to fall in the area bounded by B' and C' in Fig. 1, linear relationships are to be expected, since this is the linear portion of the doseeffect curve. Studies in two groups of asymptomatic children falling in these two segments of the curve have been carried out. In the first study reported in detail elsewhere, 11 analysis of the data from 51 preschool children with Pb-B in the rather broad range of 25 to 75 /~g indicates that, as a group, they fall in the curvilinear portion of the curve. Polynomial regression analyses showed that the relationship between the indicator of dose (Pb-B) and effect (ALA-U) is curvilinear and best fitted by a quadratic equation, as shown in Fig. 3. Chelatable lead was also used in this group as an indicator of internal dose. Here a statistically significant linear relationship was found between ALA-U and chelatable lead (r = 0.78, p < 0.001). In this group, both Pb-B and chelatable lead were found equally good for predicting ALA-U, since the standard deviations about each of the fitted regression fines were equivalent. However, the linear relationship between chelatable lead and ALA-U allows for greater ease of interpretation than the curvilinear relationship between blood lead and ALA-U." The relationship in children is similar to the curvilinear relationship between Pb-B and ALA-U reported by Selander and Cram6r 13 in adults having the same broad range of blood lead. In this group the curvilinear relationship between Pb-B and chelatable lead is such that an increase in Pb-B from 25 to 75/~g is associated, on the average, with a seven- to eightfold increase in chelatable lead. Inspection of the data in Fig. 2 suggests that the apparent linear portion of this dose-effect curve in children lies in the area in which Pb-B varies between 50 and 80/~g and ALA-U between 2 and 10 mg/m2/24 hr. This is comparable to the area bounded by B' and C' in Fig. 1. To test this hypothesis, various indicators of dose have been compared recently in a new group of children with Pb-B
Volume 87 Number 6, part 2
Dose-effect and dose-response relationships for lead
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Fig. 4. Apparent linear portion of dose-effect curve: Relationship between Pb-B and ALA-U. ALA-U is significantly related to surface area 11 and so is plotted as mg ALA-U/m2/24 hr to compensate for differences in body size. No significant relationship between Pb-B and ALA-U is found in this group. Other relationships for these same ten children are presented in Figs. 5, 6, and 7.
Fig. 5. Apparent linear portion of dose-effect curve: Relationship between chelatable lead and ALA-U. Chelatable lead is expressed as t~M Pb excreted/mM EDTA administered to compensate for differences in amount of CaEDTA administered. Dosage of CaEDTA is 50 mg/kg/day given intramuscularly in divided dose at 12-hour intervals. Other relationships for these same ten children are presented in Figs. 4, 6, and 7.
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in this 50 to 80/zg range. To date, the n u m b e r of children studied is small, but does permit preliminary interpretation. In this group, blood for Pb-B and erythrocyte protoporphyrin 13 and quantitative 24-hour collections of urine for Pb-U and A L A - U were obtained prior to chelation therapy. Chelatable lead was measured quantitatively in 24-hour collections of urine for the next three days. During these three days, C a E D T A was given intramuscularly in a dose of 25 m g / k g at 12-hour intervals or a total dose of 50 m g / k g / d a y . Pb-B ranged between 48 and 68 /zg. Chelatable lead, spontaneous urinary lead output, and Pb-B were tested as indicators of dose against ALA-U and erythrocyte protoporphyrin as indicators of critical effects. Complete collections of urine on all four days were obtained from ten children. The results of linear regression analysis of these data are summarized in Table I and Figs. 4 and 5. Within this small group, chelatable lead and Pb-U were significantly and linearly related to effect, as indicated by A L A - U (p < 0.001), but Pb-B was not predictive o f A L A - U (p > 0.10). In the first group of 51 children (Fig. 3), 18 had Pb-B in the same 48 to 68/~g range. Similar statistical analyses of the data from this subgroup of 18 children gave comparable results; namely, a significant linear relationship between chelatable lead and ALA-U but no significant linear relationship between Pb-B and ALA-U. It is of interest that
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Fig. 6. Apparent linear portion of dose-effect curve: Relationship between erythrocyte protoporphyrin and ALA-U. Erythrocyte protoporphyrin is measured by a partial extraction screening procedure (normal value = 4.21 _+ 1.96 /~g PROTO/dl whole blood). Other relationships for these same ten children are presented in Figs. 4, 5, and 7.
statistically significant linear relationships were found between erythrocyte protoporphyrin and A L A - U (Fig. 6) and erythrocyte protoporphyrin and chelatable lead (Fig. 7), though these relationships were not as close as those in which ALA-U was used as the indicator of effect (Fig. 5). Because the n u m b e r of children in the 50 to 80 Pb-B range studied to date is small, these results should be considered
1 156
Chisolm, Barrett, and Mellits
The Journal of Pediatrics December 1975
Table I. Statistical summary of comparisons between different indicators of dose and effect in ten children with increased lead absorption*
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Indicators of effect
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*Standard linear regression analysis with indicators of dose as the independent yariable. D F = 8 (ie, N-2 = 8). t l /~M P b / m M E D T A = 1.8/~g P b / m g C a E D T A . ~Mean = 57.6/~g P b / d l (range = 48 to 68/Lg P b / d l whole blood). w by three-column technique to exclude interference due to amino acetone.'"" '~, ~ IlMeasured by simplified partial extraction screening technique. TM
Fig. 7. Apparent linear portion of dose-effect curve: Relationship between erythrocyte protoporphyrin and chelatable lead. Other relationships for these same ten children are presented in Figs. 4, 5, and 6.
tentative. Larger numbers will be required to support firmly the hypothesis that, for lead, the 50 to 80/~g Pb-B range represents the linear portion of the dose-effect curve (Fig. 1). When groups are studied which cover a broad range in Pb-B, including the normal segment, curvilinear relationships between Pb-B and ALA-U and between Pb-B and chelatable lead are evident from most published studies. ~, 11, " On the other hand, there is today concern about the more narrow, somewhat elevated 50 to 80 ~g Pb-B range; this is the range in which many are asymptomatic whereas some have symptoms suggestive of mild plumbism. There are also conflicting reports concerning the occurrence of adverse nervous system effects in this group. The data just summarized strongly suggest that this 50 to 80/~g Pb-B range falls in the linear portion of the dose-effect curve for lead as related to deranged heme synthesis (but not necessarily to other types of effect). They further indicate that Pb-B in this 50 to 80/~g range is the least reliable indicator of internal lead dose at the site of action in the bone marrow. As reviewed elsewhere, TM Pb-B can fluctuate rather widely in relation to alimentary intake. Furthermore, differences in diet can affect the fraction absorbed. ~ The half-life of Pb-B is short. In ratg, for example, after a single injection, lead disappears very rapidly from blood but persists much longer within the cells of the liver and kidney? ~ The present state of the art of analysis is such that the 95% confidence limit for a single blood lead value is _+ 6/~g at best; interlaboratory
studies reveal even wider discrepancies. By itself, a single Pb-B measurement is therefore not a very useful diagnostic test, though a series of Pb-B values over a period of time is useful in plotting trends. These limitations impose severe restrictions on the use of Pb-B as the only indicator of internal dose in studies concerning the various acute and chronic effects of lead. The close relationship between spontaneous daily urinary lead output and ALA-U indicates that Pb-U may be an excellent predictor of effect and, by inference, of internal dose. These findings are in agreement with some of the results reported in adults. TM ~9However, the work of Emmerson2~and previous work in children 2l indicate that renal injury can limit spontaneous urinary excretion of lead, but not the diuresis of lead which follows administration of CaEDTA. On the other hand, CP-U and ALAU are grossly elevated in acutely ill patients even when Pb-U is normal. The use of Pb-U as an indicator of internal dose is probably limited to those with normal renal function. The highly statistically significant linear relationship between chelatable lead and ALA-U indicates that it is the best indicator of internal dose. The relationship i s linear over a range in Pb-B of 25 to 75/~g. Some studies in adults1,. 19suggest that total chelatable lead as determined during several days of CaEDTA administration might provide a better indicator than a single injection. The results summarized in Table I suggest that the first day's output and the three-day total output following EDTA
I~gPROTO/IOOml WHOLEBLOOD
Volume 87 Number 6, part 2
therapy are equivalent for predicting ALA-U, at least in children with normal renal function. Past work in this laboratory suggests that diuresis of lead following' a single injection of CaEDTA is not uniformly complete in less than six hours, and that injection of CaEDTA at 50 mg/ kg/day in divided doses at 12-hour intervals may be preferable to a single injection of 25 mg/kg. During the past decade, a number of CaEDTA mobilization tests at both dosage levels have been carried out in asymptomatic children without untoward incident. The principal hazard probably lies in the use of a single diagnostic injection of CaEDTA in a child with highly elevated soft tissue lead levels in whom therapeutic amounts of chelating agents given for three to five days are indicated. A preceding measurement of FEP (or CP-U or ALA-U) would identify patients needing full therapeutic amounts of the drug, particularly those with suspicious symptoms. All of the above studies require quantitative 24-hour urine collections for ALA-U and Pb-U. In addition, the method used for ALA-U to exclude interferences due to other aminoketones is more cumbersome than the usual methods for ALA which may be susceptible to these interferences. TM 1~ MeasUrement of ALA-U in random voidings does not provide useful data. 11 It is therefore of considerable interest that erythrocyte protoporphyrin is significantly related to both ALA-U (Fig. 6) and chelatable lead (Fig. 7), even when measured by a simple micro field screening technique. With this method, about oneseventh of the total FEP is extracted. Total extraction of either FEP or zinc protoporphyrin 22 might provide an even better estimate of dose (chelatable lead) and effect (ALA-U), though this is not certain in view of the influence of iron status on erythrocyte protoporphyrin concentration. These findings a r e i n agreement with the conclusion Of Sassa and associates ~3 that FEP measurements provide a better reflection than Pb-B of steady-state conditions in the bone marrow. Further studies of the type summarized in Table I are needed to confirm the hypothesis that the 50 to 80/~g Pb-B range represents the linear portion of the dose-effect curve for lead. If supported by further study on larger numbers of children, the data in Figs. 6 and 7 have important implications for clinical practice. Whereas quantitative measurement of chelatable lead and ALA-U are not readily adapted to clinical practice, rather precise measurements of erythrocyte protoporphyrin can be made on capillary samples of blood; this may have the potential to serve as an indicator of both internal dose and critical effect. All of the preceding comments concern the interrelationships between indicators of acute effects and dose in prospectively tested children, including some actively assimilating excess lead. The critical effect of lead on
Dose-effect and dose-response relationships for lead
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/~g Pb/lOOml WHOLE BLOOD Fig. 8. Dose-responserelationship. Pb-B is plotted on the abscissa as the indicator of dose. Frequency of response, according to Pb-B groups, is plotted on the ordinate. Frequency of response in each Pb-B group is based on the proportion in each group with erythrocyte protoporphyrin greater than normal range (mean plus 2 SD) as stated in Fig. 6. The total group (N = 115) is divided according to hematocrit to compensate at least partially tbr concurrent iron deficiency and to illustrate the background response due to this factor. Data are plotted from previously published reports?....
hemoglobin synthesis is reversible and diminishes after excessive intake of lead abates. Comparable measurements of ALA-U, chelatable lead, Pb-U, and Pb-B have been made in adolescents known to have had severe acute lead poisoning 11 to 17 years earlier. 11 This group of 55 included 36 with well-documented records of acute encephalopathy during the preschool years. In all patients, values for Pb-B, Pb-U, chelatable lead, and ALA-U fell within the expected normal ranges '~ so ~hat no retrospective diagnosis of plumbism in the remote past would have been possible on the basis of these tests. DOSE-RESPONSE
RELATIONSHIPS
This relationship is one in which the percentage (response rate) of the population having a specific effect is related to the concentration (dose) of a toxic substance. The dose-response relationship specifies the percentage of "reactors" and "nonreactors" in a population at each of several dose levels? When the law of biologic random variation applies, the dose-response and dose-effect curves have the same general form (Fig. 1), but the response curve shows the-frequency of response for a particular effect in a population rather than the degree of effect. Dose-response relationships should be determined in epidemiologically suitable samples using an indicator
1I 58
Chisotrn, Barrett, and Mellits
of effect to measure the change in response rate with increasing dose. "Background response" is the percentage of the population having a specific effect caused by factors other than the particular agent under consideration. If background response is present, the curve will not reach "zero" response, even at low dose. This background factor is well illustrated when erythrocyte pro toporphyrin, which is also raised in iron deficiency states, is used to measure response rates in relation to Pb-B (Fig. 8). Clearly, hemoglobin, hematocrit, or other tests relevant to iron status, as well as tests for lead, have to be m a d e to compensate for this background response. " Even so, the identification of reactors and nonreactors in the population is highly useful from the viewpoint o f health care delivery. Previously, we have classified screening test results ~ as false negative or false positive, according to the percentage of positive or negative results in relation to a specific Pb-B value such as 40 /~g. Such a classification assumes, erroneously, that all will react uniformly at the same Pb-B interval.* It does not take into account the presence of highly susceptible and highly resistant individuals in a heterogeneous population; the concept of reactors and nonreactors does. In children with hematocrit > 36% and Pb-B < 30 /zg, we have found that erythrocyte protoporphyrin levels are low and independ e n t o f Pb-B concentration. If a mean normal value is established for this group, a value greater than the m e a n plus 2 SD may be used to identify positive reactors. Studies in which erythrocyte protoporphyrin is used as the indicator of effect show that _> 90% of children with Pb-B _> 50 /~g are positive reactors and that the 50% response rate is associated with Pb-B in the 40 to 50 /~g range. Erythrocyte protoporphyrin measurements which can be made in capillary samples of blood can be used in population studies of dose-response relationships, and probably in identified high-risk groups, to determine the degree of effect in individuals. In vitro assay for A L A - D activity in peripheral blood has proved quite useful for dose-response rate studies in population groups; however, A L A - D activity is reduced to a low and relatively constant value at Pb-B _> 50 to 60/~g so that increasing severity of effect at higher levels is not readily detected as it is with ALA-U, CP-U, or F E P measurements. For the purposes of preventive pediatrics, erythrocyte protoporphyrin, if used in conjunction with some simple measure to detect anemia (such as hematocrit) can serve as the primary *Classifications based on a specific Pb-B value or a very narrow Pb-B range are also based on the assumption that Pb-B is a reliable indicator of internal dose. Though it is possible that this assumption may be of some use under carefully controlled steady-state conditions, it is increasingly evident that it is not valid in the highly unstable epidemiologic circumstances under which childhood plumbism actually occurs.
The Journal o f Pediatrics December 1975
screening test in children at high risk for plumbism. It should be repeated at appropriate intervals during the early preschool years. With this approach, a lead test such as Pb-B is needed in positive reactors, but not in nonreactors.
REFERENCES
1. Waldron HA: The blood lead threshold, Arch Environ Health 29:271, 1974. 2. Zielhuis RL: Dose-response relationship for inorganic lead, Report to Direction Health Protection, Europ Econ Comm, Jtme, 1974. 3. Roberts TM, Hutchinson TC, Paciga J, Chattopadhyay A, Jervis RE, and Van Loon J: Lead contamination around secondary smelters: Estimation of dispersal and accumulation by humans, Science 186:1120, 1974. 4. Landrigan PJ, Gehlbach SH, Rosenblum BF, Shoults JM, Candelaria RM, Barthel WF, Liddle JA, Smrek AL, Staehling NW, and Sanders JF: Epidemic lead absorption near an ore smelter: The role of particulate lead, N Engl J Med 292:123, 1975. 5. Nordberg G, editor: Effects and dose-response relationships of toxic metals, Amsterdam, 1975, Elsevier Publishing Company (in press). 6. Nordberg G, and Norseth T: Critical organ concept and indicators of early effects in evaluating and establishing dose-response relationships for toxic metals, in Reference No. 5 cited above. 7. Wada O, Yano Y, Toyokawa K, Suzuki T, Suzuki S, and Katsunuma H: Human responses to lead; in special references to porphyrin metabolism in bone marrow erythroid cells, and clinical and laboratory study, lnd Health 10:84, 1972. 8. Tola S, Hernberg S, Asp S, and Nikkanen J: Parameters indicative of absorption and biological effect in new lead exposure: A prospective study, Br J Industr Med 30:134, 1973. 9. Burn JH, Finney D J, and Goodwin CG: Biological standardization, London, 1950, Oxford University Press. 10. Chisolm JJ Jr: The use of chelating agents in the treatment of acute and chronic lead intoxication in childhood, J. PZI~IATR73:1, 1968. 11. Chisolm JJ Jr, Mellits ED, and Barrett MB: Interrelationships among blood lead concentration, quantitative daily ALA-U and urinary lea d output following calcium EDTA, in Reference No. 5 cited above. 12. Kjellstr6m T, and Eng M: Mathematical and statistical approaches in evaluating dose-response relationships for metals, in Reference No. 5 cited above. 13. Selander S,and Cram6r K: Interrelationships between lead in blood, lead in urine, and ALA in urine during lead work, Br J Industr Med 27:28, 1970. 14. Chisolm JJ Jr, Mellits ED, Keil JE, and Barrett MB: A simple protoporphyrin assay-microhematocrit procedure as a screening technique for increased lead absorption in young children, J PEOIATR84:490, 1974. 15. Schlenker FS, Taylor NA, and Kiehn BP: The chromatographic separation, determination, and daily excretion of urinary porphobilinogen, amino acetone, and &aminolevulinic acid, Am J Clin Pathol 42:349, 1964.
Volume 87 Number 6, part 2 16. Chisolm JJ Jr, Mellits ED, Keil JE, and Barrett MB: Variations in hematologic responses to increased lead absorption in young children, Environmental Health Perspectives, Exp. Issue no. 7, May, 1974 pp 7-12. 17. Castellino N, and Aloj S: Intracellular distribution of lead in the fiver and kidney o f the rat, Br J Industr Med 26:139, 1969. 18. Ellis, R.W.: Urinary screening tests to detect excessive lead absorption, Part I: A comparison, Br J Industr Med 23:263, 1966. 19. Selander S, Cramtr K, and Hallberg L: Studies in lead poisoning, oral therapy with peniciltamine: Relationship between lead in blood and other laboratory tests, Br J Industr Med 23:282, 1966. 20. Emmerson BT: Chronic lead nephropathy: The diagnostic use of calcium EDTA and the association with gout, Australas. Ann Med 12:310, 1963. 21. Byers RK, MaloofCA, and Cushman M: Urinary excretion of lead in children. Diagnostic application, Am J Dis Child 87:548, 1954. 22. Lamola AA, and Yamane T: Zinc protoporphyrin in the erythrocytes of patients with lead intoxication and iron deficiency anemia, Science 186:936, 1974. 23. Sassa S, Granick JL, Granick S, Kappas A, and Levere RD: Studies in lead poisoning, I. Microanalysis of erythrocyte protoporphyrin levels by spectrofluorometry in the detection of chronic lead intoxication in the subclinical range, Biochem Med 8:135, 1973.
APPENDIX Laboratory methods. Data from this laboratory included in this report encompass studies carried out over the past 12 years. Most have been reported in greater detail elsewhere? ~ 11.... ~6 Since 1962, ALA-U has been measured in acetate-buffered aliquots o f urine passed successively through Dowex 2 x 8, IRC-50, and Dowex 50 x 8 ion-exchange resin columns. ALA-U is measured colorimetricatly in the final eluate from Dowex 50 x 8. The method is similar to that of Schlenker and associates. '5 How these methods differ from the more commonly used methods, as well as the statistical basis for expressing daily urinary output as ALAU/m~/day, are discussed in detail elsewhere. 11 Data in Figs. 2, 3, 4, and 6 are based on this method. Erythrocyte protoporphyrin values in Figs. 6, 7, and 8 are according to a field-screening technique?~, 1~ In this method, zinc protoporphyrin is partially extracted and measured fluorometrically against protoporphyrin IX standards in the same solvent at 635 nm. The values found are about one-seventh of the values found with a standard micro technique for FEP. Two methods have been used for blood lead. Between 1935 and 1972, all Pb-B tests have been done on 10 ml samples in laboratories at the Baltimore City Health Department under the direction of Emanuel Kaplan, Sc.D., by a standard mixed-color, double extraction, colorimetric dithizone method? 1 Pb-B values in Figs. 2 and 3 are based on this method. Pb-B values in Figs. 4 and 8 are based on an HCIO,-CHCI~ filtrate atomic absorption (AAS) method developed in this laboratory, which briefly is as follows: Mix 2 ml each of CHC13, ultrasonically homogenized whole heparinized blood, and 0.9 molar HCIO,, centrifuge, and
Dose-effect and dose-response relationships for lead
1 15 9
filter. Measure Pb in filtrate by flame AAS (283.3 nm, simultaneous deuterium background correction) against similarly treated spiked whole blood reference standards. Duplicate 2 ml aliquots are measured in triplicate. Recovery of added Pb in a range of 10 to 150/~g/dl is _+ 2/~g P b / d l of the target amounts. Least squares regression analysis of results on venous samples from 113 children split for analysis for both dithizone and this AAS method showed no significant proportional error (slope = 1.022), small constant error (intercept = 2 t~g Pb/dl), and random error (using N-2) of +_+_3.7 I~g P b / d l (S.D. y/x). TM Lead in urine was measured by both AAS and the anodic stripping voltammetry on 2 ml aliquots of ultrasonically homogenized urine, following wet digestion with nitric a n d perchloric acids and trace amounts o f sulphuric acid in 50% hydrogen peroxide. The final digest (at 204 ~ C) is further treated at lower temperature to convert the residual salts to hydrated crystals, which are readily soluble and are taken up in 2 ml of 0.015M HCIO4. Recovery o f added lead With and without an added tenfold excess of CaEDTA (10 gm/1) is 95-105%. Samples from children receiving CaEDTA generally contained 0.4 to 4 ~tg Pb/ ml and were measured directly in this dilute HCIO, solution by flame AAS without further concentration or extraction. Spontaneous Pb-U (control day) was measured by ASV in 1 ml aliquots because concentration of lead was generally less than 0.10/~g/ml which is below the useful and analytical range for the above AAS method. For matrix compensation, all measurements were run against pooled normal urine spiked at several levels with added inorganic lead. (For details, see Chisolm JJ Jr, Barrett MB, and Harrison HV: Johns Hopkins Med J 137:6, 1975. Clinical study groups. Informed written consent was obtained prior to study. All inpatient studies (quantitative 24-hour urine collections, including CaEDTA mobilization test) were carried out in the Clinical Research Centers in The Johns Hopkins Hospital under protocols approved by the Committee on Clinical Research, Johns Hopkins Medical School. Data (Figs. 2 and 3) on asymptomatic children are taken from a prospective screening study of 542 inner city preschool children without known history of prior plumbism in the Comprehensive Child Care Clinic at The Johns Hopkins Hospital during 1969 and 1970. Each week, two or three children were admitted for studies reported elsewhere." Most of the children with Pb-B < 40/~g were siblings of children with Pb-B > 40/zg. In all, 83 were admitted. Statistical relationships between ALA-U and age, height, and surface area were determined in this group, as well as interrelationships among ALA-U, chelatable lead, and PbB. Similar studies were carried out in a separate group of 55 adolescents '~ known to have had prior plumbism. In all cases, polynomial regression analyses showed that correction of Pb-B to a derived value for a constant hematocrit resulted in uniformly lowered correlation coefficients and, in some comparisons, lesser statistical significance for fitted regression lines involving Pb-B; therefore, only the actual Pb-B values were used. Although the concentration of ALA in random voidings may vary in the same child up to 1,000 to 2,000%, daily output on two successive days agreed within 15%. This explains, at least in part, the relative uselessness, from the diagnostic viewpoint, of measuring ALA concentration in random voidings from young children. Still another factor is the variable positive
1 16 0
Chisolm, Barrett, and Mellits
contribution of interfering aminoketones unrelated to lead to results obtained with the usual simpler methods for ALA. :1 Data in Table I and Figs. 4 to 7 are based on similar inpatient studies in a separate and much smaller group of asymptomatic children drawn from a group being prospectively screened at neighborhood locations through The John F. Kennedy Institute (Baltimore). These children were selected on the basis of a screening
The Journal of Pediatrics December 1975
Pb-B _> 50/~g. The Pb-B value used for statistical analyses are those measured just prior to the first injection of CaEDTA, which in some cases differed from the preceding screening test value by up to 10 ~g Pb. The clinical characteristics of the prospectively screened group of 115 preschool children on which the doseresponse curves in Fig. 8 are based are described elsewhere.'" TM
December 1975 The JournaI o f P E D I A T R I C S
Dose-effect and dose-response relationships for lead in children Lead absorption and prevention of the serious effects of lead poisoning are re-examined from the viewpoints of the critical organ and critical effect concepts and the associated dose-effect and doseresponse relationships. I f the critical organ is the first affected and the critical effect is the first measurable adverse effect, intervention on this basis should prevent the occurrence of later, more serious effects. In the range of lead absorption of greatest current pediatric concern (blood lead in the range of 50 to 80 l~g/dl), blood lead values are not a good predictor o f critical effect, whereas' chelatable lead is significantly and linearly related to evidence of critical effect on hemoglobin synthesis in the bone marrow. Erythrocyte protoporphyrin and &aminolevulinic acid and coproporphyrin in urine are indicators of this effect. The dose-response concept provides a better way of viewing the relationship between blood lead and measures of adverse effect than do the classifications of "'sensitivity," "specificity," "false negatives," and "false positives, "" which are often employed in the evaluation of screening tests'. The doseresponse concept recognizes the uniqueness of the individual and the presence of susceptible and resistant individuals in heterogeneous population groups. With the dose-response concept, individuals may be identified as reactors or nonreactors, according to whether they exhibit a particular effect. Among the various indicators of lead's critical (or first) effect on hemoglobin synthesis, erythrocyte protoporFhyrin potentially is the most practical for monitoring children at high risk for plumbism.
J. Julian Chisolm, Jr., M.D., Maureen B. Barrett, A.B., and E. David Mellits, Sc.D., Baltimore, Md.
BLOOD LEAD MEASUREMENTS are used widely in epidemiologic studies as a biologic indicator o f exposure From the Department of Pediatrics, Johns Hopkins University School of Medicine, the Department of Pediatrics, Baltimore City Hospitals, and The John F. Kennedy Institute. Support for this work was provided, in part, by grants from the United States Public Health Service, National Institute for Occupational Safety and Health (8-RO1 OH 00307), and United States Public Health Service, Maternal and Child Health Project No. 464, Grant (RR-52) from the General Clinical Research Centers Program of the Division o f Research Resources, National Institutes of Health; United States Public Health Service (5 MO1) Clinical l~esearch Center, Grant (RR-35), International Lead Zinc Research Organization, Inc., New York, N.Y. Maternal and Child Health Project 917 and Department of Health, Education, and Welfare, Health Services and Mental Health Administration Contract No. H S M 99-72-23. Reprint address: Baltimore City Hospitals, 4940 Eastern Ave., Baltimore, Md. 21224.
Vol. 87, No. 6, part 2, pp. 1152-1160
to leadD" Difficulties arise, however, when one attempts to use this indicator of group exposures as a diagnostic indicator of toxicity. There is a lively debate in the current literature concerning the choice and interpretation of various diagnostic tests for monitoring individuals who are Abbreviations used Pb-B: blood lead (/~g Pb/dl whole blood) Pb-U: lead in urine ALA-D: . &aminolevulinic acid dehydratase ALA-U: &aminolevulinic acid in urine CP-U: coproporphyrin in urine free erythrocyte protoporphyrin FEP: CaEDTA: edathamil calcium disodium EDTA: ethylenediaminetetra-acetic acid AAS: atomic absorption spectrophotometry ASV: anodic stripping voltammetry at high risk for plumbism. It may be useful to restate the classical concepts of toxicology as they relate to lead exposure, absorption, and effect in h u m a n populations> ~ In human studies the dose of a metal can rarely be defined as it is in experimental animal studies. Because dose at the
Volume 87 Number 6, part 2
Dose-effect and dose-response relationships for lead
1 15 3
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Fig. l. Theoretical form of dose-effect and dose-response curves. When the law of biologic random variation applies, this same curve may be used to plot both frequency of response (as a percentage of the population) and degree of effect against dose. Area between A' and B' shows a curvilinear portion of the curve and the area between B' and C' the linear portion. (Adapted from Kjellstr0m and Eng. '~) site of action is not precisely known, application of these concepts to epidemiologic studies in h u m a n beings differs somewhat from their customary use in experimental toxicology. 5 New definitions of the terms "critical effect" and "critical site" were recently agreed upon by the Subcommittee on the Toxicology of Metals? They were developed for nonradioactive toxic metals and differ from the definitions of these terms used in radiation biology. For metals such as lead, the "critical effect" is the most sensitive and specific biologic change beyond acceptable physiologic variation which is caused by the metal. ~ Although different effects may be produced by a metal, the critical effect is thefirst measurable adverse effect, not necessarily the most serious one. "Subcritical effects" are measurable biologic changes which do not impair cellular function. The "critical site" is the location in t h e body where the critical effect occurs; it may be a system, organ, cell type, or cell component. The internal dose m a y be defined in this context as the a m o u n t of a toxic metal present at the critical site. The critical organ is the first to reach its critical concentration (ie, the internal dose sufficient to produce the effect). This approach has advantages from the viewpoint of preventive medicine. Intervention on the basis of first or critical effects at an early reversible stage should prevent the occurrence of later adverse effects that may have serious consequences from the clinical viewpoint. For inorganic lead, derangement of hemoglobin synthesis in the erythroid cells o f the bone marrow is the critical effect. 2' 5 W a d a and associates 7
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,ugPb/lOOml WHOLE BLOOD Fig. 2. Relationship between blood lead concentration and 8-aminolevulinic acid excretion. With Pb-B as indicator of dose and ALA-U as indicator of effect, a dose-effect relationship is apparent. Pb, B range is 25-800/Lg/dl. ALA-U range is 0.6 to 52 mg/mV24 hr. Data are replotted from previously published reports.,,, ,1 Logarithmic scales for Pb-B and ALA-U are used to conserve space. have demonstrated a substantial reduction in the activities of 8-aminolevulinic acid dehydratase and heme synthetase, and a parallel reduction in the incorporation of I~Cglycine into heme and globin in erythroid cells' obtained by aspiration from the bone marrow of occupationally exposed workmen with blood lead in the 40-90 /~g/dl range. The indicators of this critical effect, as measured in peripheral blood and urine, are increased urinary 8aminolevulinc acid, increased urinary coproporphyrin Ill, increased "free" erythrocyte protoporphyrin, and anemia. Decreased ALA-D activity, as measured in vitro in mature red cells in peripheral blood, is considered a subcritical effect, 5 though it is a highly useful indirect indicator of exposure. 8 Although t h e combination of increased ALA-U, CP-U, and FEP, together with decreased ALA-D (peripheral blood) is diagnostically pathognomonic for lead poisoning, A L A - U is, in practice, the single most specific indicator of this critical effect, especially in children. Increases in FEP and CP-U may
1154
Chisolm, Barrett, and Mellits
The Journal of Pediatrics December 1975
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Fig. 3. Relationship between daily ALA-U and blood lead. Range in blood lead concentration in 51 preschool children encompasses the normal and somewhat elevated range (25 to 75 t~gPb-B). This line depicts this relationship which is best fitted by the following quadratic equation: mg ALA-U/m~/24 hr = 2.4135-0.07500 (Pb-B) + 0.001265 (Pb-B)2. The standard error about this fitted line is _+ 0.6 and p < 0.01. Details of this study are reported elsewhere.~1 The curvilinear nature of the relationship between Pb-B in this range and ALA-U is apparent. also be caused by iron deficiency and hepatocellular injury, respectively; only acute alcoholic intoxication and acute intermittent porphyria, an uncommon disorder, cause significant elevation in ALA-U. Since derangement in hemoglobin synthesis due to lead is reversible and precedes significant renal injury and clinically evident neurologic manifestations, the critical effect concept should serve well for prevention of plumbism. There are, however, at present no biologic measures of comparable sensitivity and specificity which can be related to lead's effect on the nervous system. Nevertheless, the available evidence suggests that derangement in hemoglobin synthesis probably precedes functional impairment of the nervous system. DOSE-EFFECT
RELATIONSHIPS
The dose-effect relationship is one in which a quantitative change in a function or concentration of a metabolite (effect) is related to a quantitative change in the concentration of a toxic substance (dose)? Many studies ~ support the assumption that toxicologic responses follow the law of biologic random variation. The classical dose-effect curve (Fig. 1) is a cumulative normal distribution curve based on this assumption. Typically, dose is plotted on the abscissa against effect on the ordinate. In Fig. 2, Pb-B is taken as the indicator of dose and ALA-U as the indicator of critical effect. ALA-U is expressed throughout as ALAU/m~/24 hr, since a statistically significant (p < 0.01)
relationship between surface area and ALA-U has been found in young children?~ 1~The distribution of the data in Fig. 2 resembles the distribution predicted by the theoretical dose-effect curve (Fig. 1). This suggests that Pb-B over a very broad range in concentration is useful as a general approximation of dose. Kjellstr6m and Eng 12 call attention to the fact that the quantitative relationship between dose and effect may differ according to the dose interval. This is especially important in empirical studies, such as those in human populations in which dose, particularly internal dose, is not known. If, for example, the dose interval in the population under study falls in the area in Fig. 1 bounded by A' and B', nonlinear relationships between dose and effect are to be expected. If, on the other hand, dosage in the population tinder study happens to fall in the area bounded by B' and C' in Fig. 1, linear relationships are to be expected, since this is the linear portion of the doseeffect curve. Studies in two groups of asymptomatic children falling in these two segments of the curve have been carried out. In the first study reported in detail elsewhere, 11 analysis of the data from 51 preschool children with Pb-B in the rather broad range of 25 to 75 /~g indicates that, as a group, they fall in the curvilinear portion of the curve. Polynomial regression analyses showed that the relationship between the indicator of dose (Pb-B) and effect (ALA-U) is curvilinear and best fitted by a quadratic equation, as shown in Fig. 3. Chelatable lead was also used in this group as an indicator of internal dose. Here a statistically significant linear relationship was found between ALA-U and chelatable lead (r = 0.78, p < 0.001). In this group, both Pb-B and chelatable lead were found equally good for predicting ALA-U, since the standard deviations about each of the fitted regression fines were equivalent. However, the linear relationship between chelatable lead and ALA-U allows for greater ease of interpretation than the curvilinear relationship between blood lead and ALA-U." The relationship in children is similar to the curvilinear relationship between Pb-B and ALA-U reported by Selander and Cram6r 13 in adults having the same broad range of blood lead. In this group the curvilinear relationship between Pb-B and chelatable lead is such that an increase in Pb-B from 25 to 75/~g is associated, on the average, with a seven- to eightfold increase in chelatable lead. Inspection of the data in Fig. 2 suggests that the apparent linear portion of this dose-effect curve in children lies in the area in which Pb-B varies between 50 and 80/~g and ALA-U between 2 and 10 mg/m2/24 hr. This is comparable to the area bounded by B' and C' in Fig. 1. To test this hypothesis, various indicators of dose have been compared recently in a new group of children with Pb-B
Volume 87 Number 6, part 2
Dose-effect and dose-response relationships for lead
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Fig. 4. Apparent linear portion of dose-effect curve: Relationship between Pb-B and ALA-U. ALA-U is significantly related to surface area 11 and so is plotted as mg ALA-U/m2/24 hr to compensate for differences in body size. No significant relationship between Pb-B and ALA-U is found in this group. Other relationships for these same ten children are presented in Figs. 5, 6, and 7.
Fig. 5. Apparent linear portion of dose-effect curve: Relationship between chelatable lead and ALA-U. Chelatable lead is expressed as t~M Pb excreted/mM EDTA administered to compensate for differences in amount of CaEDTA administered. Dosage of CaEDTA is 50 mg/kg/day given intramuscularly in divided dose at 12-hour intervals. Other relationships for these same ten children are presented in Figs. 4, 6, and 7.
10,0 r =0,83 p ~ 0.01
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in this 50 to 80/zg range. To date, the n u m b e r of children studied is small, but does permit preliminary interpretation. In this group, blood for Pb-B and erythrocyte protoporphyrin 13 and quantitative 24-hour collections of urine for Pb-U and A L A - U were obtained prior to chelation therapy. Chelatable lead was measured quantitatively in 24-hour collections of urine for the next three days. During these three days, C a E D T A was given intramuscularly in a dose of 25 m g / k g at 12-hour intervals or a total dose of 50 m g / k g / d a y . Pb-B ranged between 48 and 68 /zg. Chelatable lead, spontaneous urinary lead output, and Pb-B were tested as indicators of dose against ALA-U and erythrocyte protoporphyrin as indicators of critical effects. Complete collections of urine on all four days were obtained from ten children. The results of linear regression analysis of these data are summarized in Table I and Figs. 4 and 5. Within this small group, chelatable lead and Pb-U were significantly and linearly related to effect, as indicated by A L A - U (p < 0.001), but Pb-B was not predictive o f A L A - U (p > 0.10). In the first group of 51 children (Fig. 3), 18 had Pb-B in the same 48 to 68/~g range. Similar statistical analyses of the data from this subgroup of 18 children gave comparable results; namely, a significant linear relationship between chelatable lead and ALA-U but no significant linear relationship between Pb-B and ALA-U. It is of interest that
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Fig. 6. Apparent linear portion of dose-effect curve: Relationship between erythrocyte protoporphyrin and ALA-U. Erythrocyte protoporphyrin is measured by a partial extraction screening procedure (normal value = 4.21 _+ 1.96 /~g PROTO/dl whole blood). Other relationships for these same ten children are presented in Figs. 4, 5, and 7.
statistically significant linear relationships were found between erythrocyte protoporphyrin and A L A - U (Fig. 6) and erythrocyte protoporphyrin and chelatable lead (Fig. 7), though these relationships were not as close as those in which ALA-U was used as the indicator of effect (Fig. 5). Because the n u m b e r of children in the 50 to 80 Pb-B range studied to date is small, these results should be considered
1 156
Chisolm, Barrett, and Mellits
The Journal of Pediatrics December 1975
Table I. Statistical summary of comparisons between different indicators of dose and effect in ten children with increased lead absorption*
6~ -
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Indicators of dose Chelatable lead (t~M Pb/mM EDTA)t
Indicators of effect
ALA-Uw (mg/m'-'/24 hr) PROTOI] (/zg/dl wb)
First day only
0.91 r p < 0.001 0.75 4 r < 0.02
"Three- Pb - U day (1~g/24 pblB* total hr) O*g/ dl)
0.91 0.88 < 0.001 < 0.001 > 0.78 0.70 < 0.01 < 0.05 <
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*Standard linear regression analysis with indicators of dose as the independent yariable. D F = 8 (ie, N-2 = 8). t l /~M P b / m M E D T A = 1.8/~g P b / m g C a E D T A . ~Mean = 57.6/~g P b / d l (range = 48 to 68/Lg P b / d l whole blood). w by three-column technique to exclude interference due to amino acetone.'"" '~, ~ IlMeasured by simplified partial extraction screening technique. TM
Fig. 7. Apparent linear portion of dose-effect curve: Relationship between erythrocyte protoporphyrin and chelatable lead. Other relationships for these same ten children are presented in Figs. 4, 5, and 6.
tentative. Larger numbers will be required to support firmly the hypothesis that, for lead, the 50 to 80/~g Pb-B range represents the linear portion of the dose-effect curve (Fig. 1). When groups are studied which cover a broad range in Pb-B, including the normal segment, curvilinear relationships between Pb-B and ALA-U and between Pb-B and chelatable lead are evident from most published studies. ~, 11, " On the other hand, there is today concern about the more narrow, somewhat elevated 50 to 80 ~g Pb-B range; this is the range in which many are asymptomatic whereas some have symptoms suggestive of mild plumbism. There are also conflicting reports concerning the occurrence of adverse nervous system effects in this group. The data just summarized strongly suggest that this 50 to 80/~g Pb-B range falls in the linear portion of the dose-effect curve for lead as related to deranged heme synthesis (but not necessarily to other types of effect). They further indicate that Pb-B in this 50 to 80/~g range is the least reliable indicator of internal lead dose at the site of action in the bone marrow. As reviewed elsewhere, TM Pb-B can fluctuate rather widely in relation to alimentary intake. Furthermore, differences in diet can affect the fraction absorbed. ~ The half-life of Pb-B is short. In ratg, for example, after a single injection, lead disappears very rapidly from blood but persists much longer within the cells of the liver and kidney? ~ The present state of the art of analysis is such that the 95% confidence limit for a single blood lead value is _+ 6/~g at best; interlaboratory
studies reveal even wider discrepancies. By itself, a single Pb-B measurement is therefore not a very useful diagnostic test, though a series of Pb-B values over a period of time is useful in plotting trends. These limitations impose severe restrictions on the use of Pb-B as the only indicator of internal dose in studies concerning the various acute and chronic effects of lead. The close relationship between spontaneous daily urinary lead output and ALA-U indicates that Pb-U may be an excellent predictor of effect and, by inference, of internal dose. These findings are in agreement with some of the results reported in adults. TM ~9However, the work of Emmerson2~and previous work in children 2l indicate that renal injury can limit spontaneous urinary excretion of lead, but not the diuresis of lead which follows administration of CaEDTA. On the other hand, CP-U and ALAU are grossly elevated in acutely ill patients even when Pb-U is normal. The use of Pb-U as an indicator of internal dose is probably limited to those with normal renal function. The highly statistically significant linear relationship between chelatable lead and ALA-U indicates that it is the best indicator of internal dose. The relationship i s linear over a range in Pb-B of 25 to 75/~g. Some studies in adults1,. 19suggest that total chelatable lead as determined during several days of CaEDTA administration might provide a better indicator than a single injection. The results summarized in Table I suggest that the first day's output and the three-day total output following EDTA
I~gPROTO/IOOml WHOLEBLOOD
Volume 87 Number 6, part 2
therapy are equivalent for predicting ALA-U, at least in children with normal renal function. Past work in this laboratory suggests that diuresis of lead following' a single injection of CaEDTA is not uniformly complete in less than six hours, and that injection of CaEDTA at 50 mg/ kg/day in divided doses at 12-hour intervals may be preferable to a single injection of 25 mg/kg. During the past decade, a number of CaEDTA mobilization tests at both dosage levels have been carried out in asymptomatic children without untoward incident. The principal hazard probably lies in the use of a single diagnostic injection of CaEDTA in a child with highly elevated soft tissue lead levels in whom therapeutic amounts of chelating agents given for three to five days are indicated. A preceding measurement of FEP (or CP-U or ALA-U) would identify patients needing full therapeutic amounts of the drug, particularly those with suspicious symptoms. All of the above studies require quantitative 24-hour urine collections for ALA-U and Pb-U. In addition, the method used for ALA-U to exclude interferences due to other aminoketones is more cumbersome than the usual methods for ALA which may be susceptible to these interferences. TM 1~ MeasUrement of ALA-U in random voidings does not provide useful data. 11 It is therefore of considerable interest that erythrocyte protoporphyrin is significantly related to both ALA-U (Fig. 6) and chelatable lead (Fig. 7), even when measured by a simple micro field screening technique. With this method, about oneseventh of the total FEP is extracted. Total extraction of either FEP or zinc protoporphyrin 22 might provide an even better estimate of dose (chelatable lead) and effect (ALA-U), though this is not certain in view of the influence of iron status on erythrocyte protoporphyrin concentration. These findings a r e i n agreement with the conclusion Of Sassa and associates ~3 that FEP measurements provide a better reflection than Pb-B of steady-state conditions in the bone marrow. Further studies of the type summarized in Table I are needed to confirm the hypothesis that the 50 to 80/~g Pb-B range represents the linear portion of the dose-effect curve for lead. If supported by further study on larger numbers of children, the data in Figs. 6 and 7 have important implications for clinical practice. Whereas quantitative measurement of chelatable lead and ALA-U are not readily adapted to clinical practice, rather precise measurements of erythrocyte protoporphyrin can be made on capillary samples of blood; this may have the potential to serve as an indicator of both internal dose and critical effect. All of the preceding comments concern the interrelationships between indicators of acute effects and dose in prospectively tested children, including some actively assimilating excess lead. The critical effect of lead on
Dose-effect and dose-response relationships for lead
lOC
~ -~
....
;-57
ac /
/
/
/# I /
/
3c
LQ I.~ 2C ~-
1 157
oc
/
1C I
20-29
,T 3 0 - 3 5 %
N=40 HEMATOGRIT ->36% N=75 I
30-39
I
40-49
50-159
I :='60
/~g Pb/lOOml WHOLE BLOOD Fig. 8. Dose-responserelationship. Pb-B is plotted on the abscissa as the indicator of dose. Frequency of response, according to Pb-B groups, is plotted on the ordinate. Frequency of response in each Pb-B group is based on the proportion in each group with erythrocyte protoporphyrin greater than normal range (mean plus 2 SD) as stated in Fig. 6. The total group (N = 115) is divided according to hematocrit to compensate at least partially tbr concurrent iron deficiency and to illustrate the background response due to this factor. Data are plotted from previously published reports?....
hemoglobin synthesis is reversible and diminishes after excessive intake of lead abates. Comparable measurements of ALA-U, chelatable lead, Pb-U, and Pb-B have been made in adolescents known to have had severe acute lead poisoning 11 to 17 years earlier. 11 This group of 55 included 36 with well-documented records of acute encephalopathy during the preschool years. In all patients, values for Pb-B, Pb-U, chelatable lead, and ALA-U fell within the expected normal ranges '~ so ~hat no retrospective diagnosis of plumbism in the remote past would have been possible on the basis of these tests. DOSE-RESPONSE
RELATIONSHIPS
This relationship is one in which the percentage (response rate) of the population having a specific effect is related to the concentration (dose) of a toxic substance. The dose-response relationship specifies the percentage of "reactors" and "nonreactors" in a population at each of several dose levels? When the law of biologic random variation applies, the dose-response and dose-effect curves have the same general form (Fig. 1), but the response curve shows the-frequency of response for a particular effect in a population rather than the degree of effect. Dose-response relationships should be determined in epidemiologically suitable samples using an indicator
1I 58
Chisotrn, Barrett, and Mellits
of effect to measure the change in response rate with increasing dose. "Background response" is the percentage of the population having a specific effect caused by factors other than the particular agent under consideration. If background response is present, the curve will not reach "zero" response, even at low dose. This background factor is well illustrated when erythrocyte pro toporphyrin, which is also raised in iron deficiency states, is used to measure response rates in relation to Pb-B (Fig. 8). Clearly, hemoglobin, hematocrit, or other tests relevant to iron status, as well as tests for lead, have to be m a d e to compensate for this background response. " Even so, the identification of reactors and nonreactors in the population is highly useful from the viewpoint o f health care delivery. Previously, we have classified screening test results ~ as false negative or false positive, according to the percentage of positive or negative results in relation to a specific Pb-B value such as 40 /~g. Such a classification assumes, erroneously, that all will react uniformly at the same Pb-B interval.* It does not take into account the presence of highly susceptible and highly resistant individuals in a heterogeneous population; the concept of reactors and nonreactors does. In children with hematocrit > 36% and Pb-B < 30 /zg, we have found that erythrocyte protoporphyrin levels are low and independ e n t o f Pb-B concentration. If a mean normal value is established for this group, a value greater than the m e a n plus 2 SD may be used to identify positive reactors. Studies in which erythrocyte protoporphyrin is used as the indicator of effect show that _> 90% of children with Pb-B _> 50 /~g are positive reactors and that the 50% response rate is associated with Pb-B in the 40 to 50 /~g range. Erythrocyte protoporphyrin measurements which can be made in capillary samples of blood can be used in population studies of dose-response relationships, and probably in identified high-risk groups, to determine the degree of effect in individuals. In vitro assay for A L A - D activity in peripheral blood has proved quite useful for dose-response rate studies in population groups; however, A L A - D activity is reduced to a low and relatively constant value at Pb-B _> 50 to 60/~g so that increasing severity of effect at higher levels is not readily detected as it is with ALA-U, CP-U, or F E P measurements. For the purposes of preventive pediatrics, erythrocyte protoporphyrin, if used in conjunction with some simple measure to detect anemia (such as hematocrit) can serve as the primary *Classifications based on a specific Pb-B value or a very narrow Pb-B range are also based on the assumption that Pb-B is a reliable indicator of internal dose. Though it is possible that this assumption may be of some use under carefully controlled steady-state conditions, it is increasingly evident that it is not valid in the highly unstable epidemiologic circumstances under which childhood plumbism actually occurs.
The Journal o f Pediatrics December 1975
screening test in children at high risk for plumbism. It should be repeated at appropriate intervals during the early preschool years. With this approach, a lead test such as Pb-B is needed in positive reactors, but not in nonreactors.
REFERENCES
1. Waldron HA: The blood lead threshold, Arch Environ Health 29:271, 1974. 2. Zielhuis RL: Dose-response relationship for inorganic lead, Report to Direction Health Protection, Europ Econ Comm, Jtme, 1974. 3. Roberts TM, Hutchinson TC, Paciga J, Chattopadhyay A, Jervis RE, and Van Loon J: Lead contamination around secondary smelters: Estimation of dispersal and accumulation by humans, Science 186:1120, 1974. 4. Landrigan PJ, Gehlbach SH, Rosenblum BF, Shoults JM, Candelaria RM, Barthel WF, Liddle JA, Smrek AL, Staehling NW, and Sanders JF: Epidemic lead absorption near an ore smelter: The role of particulate lead, N Engl J Med 292:123, 1975. 5. Nordberg G, editor: Effects and dose-response relationships of toxic metals, Amsterdam, 1975, Elsevier Publishing Company (in press). 6. Nordberg G, and Norseth T: Critical organ concept and indicators of early effects in evaluating and establishing dose-response relationships for toxic metals, in Reference No. 5 cited above. 7. Wada O, Yano Y, Toyokawa K, Suzuki T, Suzuki S, and Katsunuma H: Human responses to lead; in special references to porphyrin metabolism in bone marrow erythroid cells, and clinical and laboratory study, lnd Health 10:84, 1972. 8. Tola S, Hernberg S, Asp S, and Nikkanen J: Parameters indicative of absorption and biological effect in new lead exposure: A prospective study, Br J Industr Med 30:134, 1973. 9. Burn JH, Finney D J, and Goodwin CG: Biological standardization, London, 1950, Oxford University Press. 10. Chisolm JJ Jr: The use of chelating agents in the treatment of acute and chronic lead intoxication in childhood, J. PZI~IATR73:1, 1968. 11. Chisolm JJ Jr, Mellits ED, and Barrett MB: Interrelationships among blood lead concentration, quantitative daily ALA-U and urinary lea d output following calcium EDTA, in Reference No. 5 cited above. 12. Kjellstr6m T, and Eng M: Mathematical and statistical approaches in evaluating dose-response relationships for metals, in Reference No. 5 cited above. 13. Selander S,and Cram6r K: Interrelationships between lead in blood, lead in urine, and ALA in urine during lead work, Br J Industr Med 27:28, 1970. 14. Chisolm JJ Jr, Mellits ED, Keil JE, and Barrett MB: A simple protoporphyrin assay-microhematocrit procedure as a screening technique for increased lead absorption in young children, J PEOIATR84:490, 1974. 15. Schlenker FS, Taylor NA, and Kiehn BP: The chromatographic separation, determination, and daily excretion of urinary porphobilinogen, amino acetone, and &aminolevulinic acid, Am J Clin Pathol 42:349, 1964.
Volume 87 Number 6, part 2 16. Chisolm JJ Jr, Mellits ED, Keil JE, and Barrett MB: Variations in hematologic responses to increased lead absorption in young children, Environmental Health Perspectives, Exp. Issue no. 7, May, 1974 pp 7-12. 17. Castellino N, and Aloj S: Intracellular distribution of lead in the fiver and kidney o f the rat, Br J Industr Med 26:139, 1969. 18. Ellis, R.W.: Urinary screening tests to detect excessive lead absorption, Part I: A comparison, Br J Industr Med 23:263, 1966. 19. Selander S, Cramtr K, and Hallberg L: Studies in lead poisoning, oral therapy with peniciltamine: Relationship between lead in blood and other laboratory tests, Br J Industr Med 23:282, 1966. 20. Emmerson BT: Chronic lead nephropathy: The diagnostic use of calcium EDTA and the association with gout, Australas. Ann Med 12:310, 1963. 21. Byers RK, MaloofCA, and Cushman M: Urinary excretion of lead in children. Diagnostic application, Am J Dis Child 87:548, 1954. 22. Lamola AA, and Yamane T: Zinc protoporphyrin in the erythrocytes of patients with lead intoxication and iron deficiency anemia, Science 186:936, 1974. 23. Sassa S, Granick JL, Granick S, Kappas A, and Levere RD: Studies in lead poisoning, I. Microanalysis of erythrocyte protoporphyrin levels by spectrofluorometry in the detection of chronic lead intoxication in the subclinical range, Biochem Med 8:135, 1973.
APPENDIX Laboratory methods. Data from this laboratory included in this report encompass studies carried out over the past 12 years. Most have been reported in greater detail elsewhere? ~ 11.... ~6 Since 1962, ALA-U has been measured in acetate-buffered aliquots o f urine passed successively through Dowex 2 x 8, IRC-50, and Dowex 50 x 8 ion-exchange resin columns. ALA-U is measured colorimetricatly in the final eluate from Dowex 50 x 8. The method is similar to that of Schlenker and associates. '5 How these methods differ from the more commonly used methods, as well as the statistical basis for expressing daily urinary output as ALAU/m~/day, are discussed in detail elsewhere. 11 Data in Figs. 2, 3, 4, and 6 are based on this method. Erythrocyte protoporphyrin values in Figs. 6, 7, and 8 are according to a field-screening technique?~, 1~ In this method, zinc protoporphyrin is partially extracted and measured fluorometrically against protoporphyrin IX standards in the same solvent at 635 nm. The values found are about one-seventh of the values found with a standard micro technique for FEP. Two methods have been used for blood lead. Between 1935 and 1972, all Pb-B tests have been done on 10 ml samples in laboratories at the Baltimore City Health Department under the direction of Emanuel Kaplan, Sc.D., by a standard mixed-color, double extraction, colorimetric dithizone method? 1 Pb-B values in Figs. 2 and 3 are based on this method. Pb-B values in Figs. 4 and 8 are based on an HCIO,-CHCI~ filtrate atomic absorption (AAS) method developed in this laboratory, which briefly is as follows: Mix 2 ml each of CHC13, ultrasonically homogenized whole heparinized blood, and 0.9 molar HCIO,, centrifuge, and
Dose-effect and dose-response relationships for lead
1 15 9
filter. Measure Pb in filtrate by flame AAS (283.3 nm, simultaneous deuterium background correction) against similarly treated spiked whole blood reference standards. Duplicate 2 ml aliquots are measured in triplicate. Recovery of added Pb in a range of 10 to 150/~g/dl is _+ 2/~g P b / d l of the target amounts. Least squares regression analysis of results on venous samples from 113 children split for analysis for both dithizone and this AAS method showed no significant proportional error (slope = 1.022), small constant error (intercept = 2 t~g Pb/dl), and random error (using N-2) of +_+_3.7 I~g P b / d l (S.D. y/x). TM Lead in urine was measured by both AAS and the anodic stripping voltammetry on 2 ml aliquots of ultrasonically homogenized urine, following wet digestion with nitric a n d perchloric acids and trace amounts o f sulphuric acid in 50% hydrogen peroxide. The final digest (at 204 ~ C) is further treated at lower temperature to convert the residual salts to hydrated crystals, which are readily soluble and are taken up in 2 ml of 0.015M HCIO4. Recovery o f added lead With and without an added tenfold excess of CaEDTA (10 gm/1) is 95-105%. Samples from children receiving CaEDTA generally contained 0.4 to 4 ~tg Pb/ ml and were measured directly in this dilute HCIO, solution by flame AAS without further concentration or extraction. Spontaneous Pb-U (control day) was measured by ASV in 1 ml aliquots because concentration of lead was generally less than 0.10/~g/ml which is below the useful and analytical range for the above AAS method. For matrix compensation, all measurements were run against pooled normal urine spiked at several levels with added inorganic lead. (For details, see Chisolm JJ Jr, Barrett MB, and Harrison HV: Johns Hopkins Med J 137:6, 1975. Clinical study groups. Informed written consent was obtained prior to study. All inpatient studies (quantitative 24-hour urine collections, including CaEDTA mobilization test) were carried out in the Clinical Research Centers in The Johns Hopkins Hospital under protocols approved by the Committee on Clinical Research, Johns Hopkins Medical School. Data (Figs. 2 and 3) on asymptomatic children are taken from a prospective screening study of 542 inner city preschool children without known history of prior plumbism in the Comprehensive Child Care Clinic at The Johns Hopkins Hospital during 1969 and 1970. Each week, two or three children were admitted for studies reported elsewhere." Most of the children with Pb-B < 40/~g were siblings of children with Pb-B > 40/zg. In all, 83 were admitted. Statistical relationships between ALA-U and age, height, and surface area were determined in this group, as well as interrelationships among ALA-U, chelatable lead, and PbB. Similar studies were carried out in a separate group of 55 adolescents '~ known to have had prior plumbism. In all cases, polynomial regression analyses showed that correction of Pb-B to a derived value for a constant hematocrit resulted in uniformly lowered correlation coefficients and, in some comparisons, lesser statistical significance for fitted regression lines involving Pb-B; therefore, only the actual Pb-B values were used. Although the concentration of ALA in random voidings may vary in the same child up to 1,000 to 2,000%, daily output on two successive days agreed within 15%. This explains, at least in part, the relative uselessness, from the diagnostic viewpoint, of measuring ALA concentration in random voidings from young children. Still another factor is the variable positive
1 16 0
Chisolm, Barrett, and Mellits
contribution of interfering aminoketones unrelated to lead to results obtained with the usual simpler methods for ALA. :1 Data in Table I and Figs. 4 to 7 are based on similar inpatient studies in a separate and much smaller group of asymptomatic children drawn from a group being prospectively screened at neighborhood locations through The John F. Kennedy Institute (Baltimore). These children were selected on the basis of a screening
The Journal of Pediatrics December 1975
Pb-B _> 50/~g. The Pb-B value used for statistical analyses are those measured just prior to the first injection of CaEDTA, which in some cases differed from the preceding screening test value by up to 10 ~g Pb. The clinical characteristics of the prospectively screened group of 115 preschool children on which the doseresponse curves in Fig. 8 are based are described elsewhere.'" TM