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Archives of Environmental Health: An International Journal Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/vzeh20
Depressed Serum Erythropoietin in Pregnant Women with Elevated Blood Lead a
a
a
Joseph H. Graziano Ph.D. , Vesna Slavkovic M.S. , Pam Factor-Litvak M. Phil. , b
b
Dusan Popovac M.D. , Xemail Ahmedi M.D. & Ali Mehmeti M.D.
c
a
Department of Pharmacology Columbia , University College of Physicians & Surgeons New York , New York, USA b
University of Pristina Pristina , Yugoslavia
c
Medicinski Centar Titova Mitrovica , Yugoslavia Published online: 03 Aug 2010.
To cite this article: Joseph H. Graziano Ph.D. , Vesna Slavkovic M.S. , Pam Factor-Litvak M. Phil. , Dusan Popovac M.D. , Xemail Ahmedi M.D. & Ali Mehmeti M.D. (1991) Depressed Serum Erythropoietin in Pregnant Women with Elevated Blood Lead, Archives of Environmental Health: An International Journal, 46:6, 347-350, DOI: 10.1080/00039896.1991.9934401 To link to this article: http://dx.doi.org/10.1080/00039896.1991.9934401
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Depressed Serum Etythropoietin in Pregnant Women
Downloaded by [New York University] at 05:37 12 May 2015
with Elevated Blood Lead
JOSEPHH. CRAZIANO, Ph.D. VESNA SLAVKOVIC, M.S. PAM FACTOR-LITVAK, &Phil. Department of Pharmacology Columbia University College of Physicians & Surgeons New York, New York DUSAN POPOVAC, M.D. XEMAIL AHMEDI, M.D. University of Pristina Pristina, Yugoslavia A l l MEHMETI, M.D. Medicinski Centar Titova Mitrovica, Yugoslavia
ABSTRACT. During the course of a prospective study of lead exposure and pregnancy outcome in 1502 women, we tested the hypothesis that environmental lead exposure is associated with depressed serum etythropoietin concentration. At mid-pregnancy and at delivery, blood samples were stratified by hemoglobin concentration; within each hemoglobin stratum, sera of women with the lowest and highest whole blood lead concentrations were selected for serum erythropoietin analysis. Analysis of variance revealed that women with higher blood lead lewls had inappropriately low serum erythropoietin at both mid-pregnancy and at deliiry. Thus, depressed serum erythropoietin appears to indicate lead nephrotoxicity, and it may also be responsible for the anemia associated with lead poisoning.
ASSOCIATIONS between lead (Pb) exposure and symptoms of anemia have been known for centuries.’ Most modern investigators have ascribed the anemia of Pb poisoning to its well-known adverse effects on heme synthesis’ or to other known effects of Pb, including shortened red cell sur~ival,~ineffective erythropoieand inhibition of erythrocyte pyrimidine-5’ -nudeotidase activity.’ Grandjean et a1.6 recently described delayed blood regeneration capacity in Pb-exposed workers who had experienced normal hemoglobin (Hgb) concentrations and hematocrits prior to blood donation. That report led us to hypothesize that Pb NovembedDecember 1991 [Vol. 46 (No. 6)]
may inhibit the production of erythropoietin (EPO), a glycoprotein hormone that regulates both steady-state and accelerated erythrocyte production. This hypothesis was tested in this study by measuring circulating EPO concentrations of selected pregnant women who were enrolled in a large epidemiologic study of Pb exposure and pregnancy outcome.
Materials and methods This prospective study included 1 502 pregnant women who lived in two towns in the Autonomous Province of Kosovo, Yugoslavia, a mountainous farm 347
Downloaded by [New York University] at 05:37 12 May 2015
region currently in the process of industrialization. A lead smelter, refinery, and battery factory are major elements of the economy of the city of Titova Mitrovica. Approximately 40 km to the south lies Pristina, the capitol city of Kosovo. Residents of Pristina do not experience lead exposure. Details of the pregnancy outcome study have been presented in earlier reports that have described the determinants of elevated blood lead (BPb)' and the past reproductive histories' of women in this cohort. Briefly, between May 1985 and December 1986, women who were on average 18 wk pregnant were recruited from the governmentfunded prenatal clinics in T. Mitrovica and Pristina. After informed consent was obtained, a total of 900 women in Pristina and 602 women in T. Mitrovica agreed to participate; the disparity in sample sizes reflects the different sizes of the clinics. Venous whole blood and serum samples were obtained at mid-pregnancy (N = 1502) and at delivery (both maternal and umbilical cord blood, N = 1 061) for the measurement of whole blood Pb (BPb),' erythrocyte protoporphyrin (EP),'' Hgb, and serum ferritin concentrations." Blood and sera samples were stored at 4 "C, transported approximately monthly to Columbia University on wet ice, and stored either at 4 "C (whole blood) or -20 "C (sera) until analyzed. All four blood measures, including EPO," are remarkably stable when stored appropriately. Selected serum samples were analyzed in duplicate for EPO; a commercially available enzyme immunoassay (AMGEN, Thousand Oaks, CAI was used. In our laboratory, the limit of detection of the assay was 4 mUlml, and the coefficient of variation for duplicate measures was 6.3%. EPO analyses are costly; therefore, a strategy for sample selection was devised that allowed us to control for the known effect of Hgb level on EPO c~ncentration.'~ First, using samples obtained at midpregnancy, we defined four strata based upon the observed Hgb concentrations: 9.0-9.9, 10.0-1 0.9, 1 1.O-11.9, and 12.0-1 2.9 g/dl (conversion factor to mmolll = 0.6206). Next, within each Hgb stratum, the available cases were ranked according to the observed BPb. Then, within each Hgb stratum, we selected the 6 cases with the highest and lowest BPbs, respectively, for EPO analysis. Thus, 48 mid-pregnancy sera were assayed for EPO. (Because we were unsure of the effects of hemolysis on the EPO assay, several hemolyzed sera were excluded a priori in favor of the next ranked nonhemolyzed case.) The same strategy for sample selection was then used to choose 48 cases for EPO analysis from the 1 061 available maternal serum samples obtained at the time of delivery; only 5 of these 48 cases had been selected for the mid-pregnancy study.
Results The mean mid-pregnancy Hgb, serum ferritin, BPb, EP, and EPO concentrations of each group are presented in Table 1. It is apparent that women in the lower Hgb strata tended to have lower serum ferritin and higher EP concentrations, indicating that iron deficiency was the most likely cause of the low Hgbs; however, 348
we cannot rule out additional causes of anemia. Among women in the "high" BPb groups, it might appear that the mean BPb fell as the mean Hgb fell. We suspect that this observation was not a biological phenomenon; more likely, it relates to the geographic distributions of Pb exposure and iron deficiency in T. Mitrovica: high BPb is more common near the center of the city7 where air Pb concentrations are highest, whereas iron deficiency is more prevalent as one moves away from the city into the surrounding countryside. We searched for an effect of mid-pregnancy BPb on mid-pregnancy serum EPO by conducting an analysis of variance (ANOVA)I4 that treated BPb as a discrete variable (i.e., high versus low). The ANOVA showed a significant Hgb effect (p = .OO01) and a significant BPb effect (p = .049). The Hgb by BPb interaction term was not significant (p = .26). The mean maternal delivery Hgb, serum ferritin, BPb, EP, and EPO concentrations of each of the groups are presented in Table 2. Again, the serum ferritin and EP concentrations suggest that iron deficiency contrib uted to the low Hgb concentrations among women in the lower Hgb strata. An ANOVA of the EPO concentrations observed at delivery (Table 2) again revealed significant Hgb and BPb effects (p = .009 and .055, respectively); the interaction term was not significant @ = .201). For illustrative purposes we present the combined data from mid-pregnancy and delivery in Figure 1. Compared with the groups with low BPb, the serum EPO was inappropriately low in the groups with high BPb in each Hgb stratum, except the 12.0-12.9 gldl st ratum .
Discussion These findings are consistent with and may partially explain the "delayed blood regeneration capacity" o b served by Grandjean et aL6 in lead workers. In that study, groups of lead-exposed workers and non-leadexposed workers with mean BPbs of approximately 44.3 and 7.2 pgldl (conversion factor to pmolll = .207), respectively, had virtually identical pre-study mean Hgbs. Following the donation of a unit of blood, however, the lead-exposed workers lagged in their ability to generate reticulocytes and to reconstitute red cell mass. In the current study, women with Hgb deficits and moderately elevated BPbs were unable to respond with appropriate increments in serum EPO. More than 90% of EPO is produced in the proximal renal tubule^,'^,'^ an anatomical site where Pb acc u m u l a t e ~ .In ~ ~response to hypoxia, the initial step leading to renal EPO biosynthesis appears to be increased calcium entry, which leads to phospholipase A, activation and, ultimately, to adenylate cyclase activation." We speculate that Pb may interfere with the calcium "trigger" to EPO biosynthesis. The inhibition of EPO production by Pb appears to be an early and relatively sensitive indicator of Pb nephrotoxicity. We aim to determine whether there i s a threshold for the effect of Pb on serum EPO concentration; therefore, the full set of samples in our serum bank will be analyzed. For example, with regard to the effects of Archives of Environmental Health
I
I-Low BPb I-High BPb
6 6
2-Low BPb &High BPb
6 6 6 6
10.6 10.7 11.6 11.6
6 6
12.3 f 0.1 12.4 f 0.1
3-LOW BPb 3-High BPb QLow BPb 4-High EiPb
-
Downloaded by [New York University] at 05:37 12 May 2015
I
Table 1. Hematologic Findings at Mid-Pregnancy
9.6 f 0.11/ 9.6 f 0.1 f 0.1 f 0.1 f 0.1 f 0.1
-
3.5 f 0.3 16.9 f 2.5
54.5 f 8.0 68.7 f 17.8
2.2 f 0.2 4.7 f 2.3
73.2 f 10.5 60.2 f 8.0
3.5 28.1 2.5 28.5
35.4 f 7.1 80.6 f 26.5
4.5 f 2.0 19.4 f 9.1
37.2 f 6.2 18.0 f 4.1
24.2 62.6 28.3 40.6
10.6 13.3 15.5 19.5
10.4 11.4 15.8 14.0
f 0.3 f 4.1 f 0.1 f 3.3
2.4 f 0.2 33.8 f 1.0
-
f 3.0 f 28.3
f 3.2 f 12.9
f 2.7 f 2.8
f 4.6 f 5.8
-
3.1 0.6 f 2.9 f 3.4 f f
-
Notes: Hgb hemoglobin, BPb blood lead, EP erythrocyte protoporphyrin, and €PO erythropoietin. *Analysis of variance for EPO revealed a significant Hgb effect (p .oOOl) and a significant BPb effect (p .049). tconversion factor to mmolll 0.6206. *Conversion factor to pmolll 0.207. tjconversion factor to pmolll 0.01 77. #Conversion factor to rs/l 1.O. //Standard error of the mean.
I
---
-
Table 2. Hematologic Findings at Delivery ~~
n
Group 1-Low BPb 1-High BPb 2-LOWBPb 2-High BPb 3-LOW BPb 3-High BPb 4Low BPb 4-High BPb Notes: Hgb
Hb (gldllt 9.4 9.6 10.5 10.5 11.3 11.6 12.4 12.5
-
-
f 0.211 f 0.1
BPb (rg/dl)*
f 0.1
3.5 24.9 4.2 29.4 3.5 37.8
f 0.2 f 0.1
3.6 f 0.1 38.6 f 2.1
f 0.1
f 0.1 f 0.1
Serum ferritin (ng/ml)#
EP WdM
f 0.4 f 1.6
49.0 72.4 39.0 107.8 30.5 135.0
f 0.2 f 2.6
f 0.4 f 0.7
-
~
f 6.8
f 12.9 f 12.9 f 29.1 f 5.9 f 22.1
27.3 f 4.4 119.6 f 42.4
EPO' (mU1ml)
8.5 f 3.4 3.9 f 1.0 5.8 f 1.5 5.2 f 1.1 38.5 f 12.5 5.9 f 1.0 8.8 f 3.5 23.6 f 8.0
-
68.3 36.9 35.5 37.2 52.4 27.2 9.8 10.8
f 11.0 f 10.2 f
9.0
f 11.1 f 16.2
6.3 1.9 f 3.4 f
f
-
hemoglobin, BPb blood lead, EP erythrocyte protoporphyrin, and EPO erythropoietin. .055). *Analysis of variance for EPO revealed a significant Hgb effect (p .oooS)and a significant BPb effect (p tconversion factor to mmolll 0.6206. *Conversion factor to pmolll 0.207. §Conversion factor to pmolll 0.01 77. #Conversion factor to rs/l 1.O. //Standard error of the mean.
-
---
Pb on enzymes of the heme synthetic pathway, there is no apparent threshold for the effect of Pb on d-aminolevulinic acid deh~dratase'~; in contrast, a threshold BPb of approximately 17 pg/dl is required for the inhibition of ferrochelatase activity, which leads to a rise in red cell porphyrins.M In addition, except for Hgb, little is known about other factors that might influence serum EPO concentration, e.g., age, weighdheight, ethnicity, smoking, occupation. Further analyses from this ongoing prospective study will aim to fully describe the determinants of serum EPO. We suspect that these may account for some of the variation in EPO observed at any given Hgb concentration Cables 1 and 2). In the absence of iron deficiency, anemia resulting from Pb toxicity typically occurs only when the BPb exNovembedDecember 1991 [Vol. 46 (No. 6)]
-
ceeds 40 pg/dl in children and 50 pg/dl in adults.z'-zz Even though the mean BPbs of our "high" BPb groups ranged only from 23.1 to 36.2 pg/dl (Fig. 11, with an overall high BPb mean (across mid-pregnancy and delivery) of 29.7 pg/dl, depressed serum EPO was apparent. The consequences of deranged heme synthesis have been extraordinarily useful for screening populations for evidence of undue Pb absorption. However, the inhibition of heme synthesis by Pb cannot adequately explain the anemia of Pb toxicity because adequate quantities of heme are synthesized, even in the presence of extraordinarily high Thus, it appears likely that the anemia of Pb poisoning may be due, in part, to the inhibition by Pb of renal EPO production. 349
80 -"LOW"
(4 4)
Blood Lead
T
701
I
6Z -"High" ( )
Downloaded by [New York University] at 05:37 12 May 2015
9.0-9.9
10.0- 10.9
-
11.0-11.9
Blood Lead
Mean 8Pb (pg/dl)
12.0- 12.9
Hemoglobin concentration (g/dl) strata
Fig. 1. Combined EPO findings of independent experiments conducted at mid-pregnancy flable 1) and at delivery (Table 2). Each bar represents the mean ( 2 SD) serum EPO concentration of a group of 12 women (6 at mid-pregnancy and 6 at delivery) whose concurrent hemoglobin concentrations were within the indicated stratum. The mean blood lead (BPb) of each group is shown in parentheses.
********** This study was supported by grant R 0 1 ES03460 and the Lucille B. Markey Charitable Trust. Submitted for publication December 17, 1990; revised; accepted for publication April 16, 1991. Requests for reprints should be sent to: Joseph H. Graziano, Ph.D., Department of Pharmacology, Columbia University, College of Physicians & Surgeons, 630 West 168th Street, New York, NY 10032.
********** References 1. Nriagu 10. Lead and lead poisoning in antiquity. New York: John Wiley and Sons, 1983. 2. Moore MR, Meredith PA, Goldberg A. Lead and heme biosynthesis. In: Singhal RL, Thomas JA, Eds. Lead toxicity. Baltimore, MD: Urban & Schwarzenberg, 1980; 79-117. 3. Hernberg S, Nurminen M, Hasan J. Non-random shortening of red cell survival times in men exposed to lead. Environ Res 1967; 11247-61. 4. Berk PD, Tschudy DP, Shepley LA, Waggoner JG, Berlin NI. Hematologic and biochemical studies in a case of lead poisoning. Am J Med 1970; 48:137-44. 5. Paglia DE, Valentine WN, Dahlgren JG. Effects of low-level lead exposure on pyrimidine-5' -nucleotidase and other erythrocyte enzymes. Possible role of pyrimidine-5' -nucleotidase in the pathogenesis of lead-induced anemia. J Clin Invest 1975; 56: 1164-69. 6. Grandjean P, Jensen BM, Sando HS, Jorgensen PJ, Antonsen S. Delayed blood regeneration in lead exposure: an effect on reserve capacity. Am J Public Health 1989; 79:1385-88. 7. Graziano JH, Popovac D, Factor-Litvak P, et al. Determinants of elevated blood lead during pregnancy in a population surrounding a lead smelter in Kosovo, Yugoslavia. Environ Health Perspect 199O;89:95-100. 8. Murphy MJ,Graziano JH, Popovac D, et al. Past pregnancy outcomes among women living in the vicinity of a lead smelter in Kosovo, Yugoslavia. Am J Public Health 1% 80:33-35. 9. Fernandez F, Hilligoss D. An improved graphite furnace method for the determination of lead in blood using matrix modification and the L'vov platform. Atomic Spectr 1982; 3:130-31. 10. Piomelli S. A micromethod for free erythrocyte porphyrins: the FEP test. J Lab Clin Med 1973; 81:932-40.
350
11. Miles LEM, Lipschitz DA, Bieter CP, Cook JP. Measurement of serum ferritin by a 2-site immunoradiometric assay. Anal Chem 1974; 61:209-24. 12. Eckhart KU, Kurtz A, Hirth P, Scigalla P, Wieczorek L, Bauer C. Evaluation of stability of human erythropoietin in samples for radioirnmunoassay. Klin Wochenschr 1988; 66:241-45. 13. Wide L, Bengtsson C, Birgegard C. Circadian rhythm of erythropoietin in human serum. Br Haematol 1989; 72:85-90. 14. Kleinbaum DG, Kupper LL. Applied regression analysis and other multivariable methods. North Scituate, MA: Duxbury Press, 1978. 15. Car0 J, Erslev AJ. Biologic and immunologic erythropoietin in extracts from hypoxic whole rate kidneys and their glomerular and tubular fractions. J Lab Clin Med 1984; 103:922-31. 16. Erslev AJ, Car0 J. Physiologic and molecular biology of erythropoietin. Med Oncol Tumor Pharmacother 1986; 3:159-
64. 17. Coyer RA. Renal changes associated with lead exposure. In: Mahaffey KR, Ed. Dietary and environmental lead: human health effects. Amsterdam: Elsevier Scientific Publishers, 1985; 315-38. 18. Fisher JW, Nelson PK, Beckman B, Burdowski A. Kidney control of erythropoietin production. In: Dunn MJ, Ed. Renal endocrinology. Baltimore, MD: Williams and Wilkins, 1983; 14280. 19. Hernberg S, Nikkanen G. Enzyme inhibition by lead under normal conditions. Lancet 1970 i:63-65. 20. Piomelli 5, Seaman C, Zullow D, Curran A, Davidow B. Threshold for lead damage to heme synthesis in urban children. Proc Natl Acad Sci 1982; 79:3335-39. 21. World Head Organization, Geneva. Environmental Health Criteria. 3. Lead 1977; 16-17. 22. Landrigan PI, Froines JR, Mahaffey KR. Blood lead burden: summary of epidemiological data on its relation to environmental sources and toxic effects. In: Mahaffey KR, Ed. Dietary and environmental lead: human health effects. Amsterdam: Elsevier Scientific Publishers, 1985; 421-51, 23. Lichtman HC, Feldman F. In vitro pyrrol and porphyrin synthesis in lead poisoning and iron deficiency. J Clin Invest 1963; 42: 830-39. 24. Piomelli S, Lamola AA, Poh-Fitzpatrick MB, Seaman C, Harber LC. Erythropoietic protoporphyria and lead intoxication: the molecular basis for difference in cutaneous photosensitivity. I. Different rates of disappearance of protoporphyrin from the erythrocytes. J Clin Invest 1975; 56:1519-27.
Archives of Environmental Health
Archives of Environmental Health: An International Journal Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/vzeh20
Depressed Serum Erythropoietin in Pregnant Women with Elevated Blood Lead a
a
a
Joseph H. Graziano Ph.D. , Vesna Slavkovic M.S. , Pam Factor-Litvak M. Phil. , b
b
Dusan Popovac M.D. , Xemail Ahmedi M.D. & Ali Mehmeti M.D.
c
a
Department of Pharmacology Columbia , University College of Physicians & Surgeons New York , New York, USA b
University of Pristina Pristina , Yugoslavia
c
Medicinski Centar Titova Mitrovica , Yugoslavia Published online: 03 Aug 2010.
To cite this article: Joseph H. Graziano Ph.D. , Vesna Slavkovic M.S. , Pam Factor-Litvak M. Phil. , Dusan Popovac M.D. , Xemail Ahmedi M.D. & Ali Mehmeti M.D. (1991) Depressed Serum Erythropoietin in Pregnant Women with Elevated Blood Lead, Archives of Environmental Health: An International Journal, 46:6, 347-350, DOI: 10.1080/00039896.1991.9934401 To link to this article: http://dx.doi.org/10.1080/00039896.1991.9934401
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Depressed Serum Etythropoietin in Pregnant Women
Downloaded by [New York University] at 05:37 12 May 2015
with Elevated Blood Lead
JOSEPHH. CRAZIANO, Ph.D. VESNA SLAVKOVIC, M.S. PAM FACTOR-LITVAK, &Phil. Department of Pharmacology Columbia University College of Physicians & Surgeons New York, New York DUSAN POPOVAC, M.D. XEMAIL AHMEDI, M.D. University of Pristina Pristina, Yugoslavia A l l MEHMETI, M.D. Medicinski Centar Titova Mitrovica, Yugoslavia
ABSTRACT. During the course of a prospective study of lead exposure and pregnancy outcome in 1502 women, we tested the hypothesis that environmental lead exposure is associated with depressed serum etythropoietin concentration. At mid-pregnancy and at delivery, blood samples were stratified by hemoglobin concentration; within each hemoglobin stratum, sera of women with the lowest and highest whole blood lead concentrations were selected for serum erythropoietin analysis. Analysis of variance revealed that women with higher blood lead lewls had inappropriately low serum erythropoietin at both mid-pregnancy and at deliiry. Thus, depressed serum erythropoietin appears to indicate lead nephrotoxicity, and it may also be responsible for the anemia associated with lead poisoning.
ASSOCIATIONS between lead (Pb) exposure and symptoms of anemia have been known for centuries.’ Most modern investigators have ascribed the anemia of Pb poisoning to its well-known adverse effects on heme synthesis’ or to other known effects of Pb, including shortened red cell sur~ival,~ineffective erythropoieand inhibition of erythrocyte pyrimidine-5’ -nudeotidase activity.’ Grandjean et a1.6 recently described delayed blood regeneration capacity in Pb-exposed workers who had experienced normal hemoglobin (Hgb) concentrations and hematocrits prior to blood donation. That report led us to hypothesize that Pb NovembedDecember 1991 [Vol. 46 (No. 6)]
may inhibit the production of erythropoietin (EPO), a glycoprotein hormone that regulates both steady-state and accelerated erythrocyte production. This hypothesis was tested in this study by measuring circulating EPO concentrations of selected pregnant women who were enrolled in a large epidemiologic study of Pb exposure and pregnancy outcome.
Materials and methods This prospective study included 1 502 pregnant women who lived in two towns in the Autonomous Province of Kosovo, Yugoslavia, a mountainous farm 347
Downloaded by [New York University] at 05:37 12 May 2015
region currently in the process of industrialization. A lead smelter, refinery, and battery factory are major elements of the economy of the city of Titova Mitrovica. Approximately 40 km to the south lies Pristina, the capitol city of Kosovo. Residents of Pristina do not experience lead exposure. Details of the pregnancy outcome study have been presented in earlier reports that have described the determinants of elevated blood lead (BPb)' and the past reproductive histories' of women in this cohort. Briefly, between May 1985 and December 1986, women who were on average 18 wk pregnant were recruited from the governmentfunded prenatal clinics in T. Mitrovica and Pristina. After informed consent was obtained, a total of 900 women in Pristina and 602 women in T. Mitrovica agreed to participate; the disparity in sample sizes reflects the different sizes of the clinics. Venous whole blood and serum samples were obtained at mid-pregnancy (N = 1502) and at delivery (both maternal and umbilical cord blood, N = 1 061) for the measurement of whole blood Pb (BPb),' erythrocyte protoporphyrin (EP),'' Hgb, and serum ferritin concentrations." Blood and sera samples were stored at 4 "C, transported approximately monthly to Columbia University on wet ice, and stored either at 4 "C (whole blood) or -20 "C (sera) until analyzed. All four blood measures, including EPO," are remarkably stable when stored appropriately. Selected serum samples were analyzed in duplicate for EPO; a commercially available enzyme immunoassay (AMGEN, Thousand Oaks, CAI was used. In our laboratory, the limit of detection of the assay was 4 mUlml, and the coefficient of variation for duplicate measures was 6.3%. EPO analyses are costly; therefore, a strategy for sample selection was devised that allowed us to control for the known effect of Hgb level on EPO c~ncentration.'~ First, using samples obtained at midpregnancy, we defined four strata based upon the observed Hgb concentrations: 9.0-9.9, 10.0-1 0.9, 1 1.O-11.9, and 12.0-1 2.9 g/dl (conversion factor to mmolll = 0.6206). Next, within each Hgb stratum, the available cases were ranked according to the observed BPb. Then, within each Hgb stratum, we selected the 6 cases with the highest and lowest BPbs, respectively, for EPO analysis. Thus, 48 mid-pregnancy sera were assayed for EPO. (Because we were unsure of the effects of hemolysis on the EPO assay, several hemolyzed sera were excluded a priori in favor of the next ranked nonhemolyzed case.) The same strategy for sample selection was then used to choose 48 cases for EPO analysis from the 1 061 available maternal serum samples obtained at the time of delivery; only 5 of these 48 cases had been selected for the mid-pregnancy study.
Results The mean mid-pregnancy Hgb, serum ferritin, BPb, EP, and EPO concentrations of each group are presented in Table 1. It is apparent that women in the lower Hgb strata tended to have lower serum ferritin and higher EP concentrations, indicating that iron deficiency was the most likely cause of the low Hgbs; however, 348
we cannot rule out additional causes of anemia. Among women in the "high" BPb groups, it might appear that the mean BPb fell as the mean Hgb fell. We suspect that this observation was not a biological phenomenon; more likely, it relates to the geographic distributions of Pb exposure and iron deficiency in T. Mitrovica: high BPb is more common near the center of the city7 where air Pb concentrations are highest, whereas iron deficiency is more prevalent as one moves away from the city into the surrounding countryside. We searched for an effect of mid-pregnancy BPb on mid-pregnancy serum EPO by conducting an analysis of variance (ANOVA)I4 that treated BPb as a discrete variable (i.e., high versus low). The ANOVA showed a significant Hgb effect (p = .OO01) and a significant BPb effect (p = .049). The Hgb by BPb interaction term was not significant (p = .26). The mean maternal delivery Hgb, serum ferritin, BPb, EP, and EPO concentrations of each of the groups are presented in Table 2. Again, the serum ferritin and EP concentrations suggest that iron deficiency contrib uted to the low Hgb concentrations among women in the lower Hgb strata. An ANOVA of the EPO concentrations observed at delivery (Table 2) again revealed significant Hgb and BPb effects (p = .009 and .055, respectively); the interaction term was not significant @ = .201). For illustrative purposes we present the combined data from mid-pregnancy and delivery in Figure 1. Compared with the groups with low BPb, the serum EPO was inappropriately low in the groups with high BPb in each Hgb stratum, except the 12.0-12.9 gldl st ratum .
Discussion These findings are consistent with and may partially explain the "delayed blood regeneration capacity" o b served by Grandjean et aL6 in lead workers. In that study, groups of lead-exposed workers and non-leadexposed workers with mean BPbs of approximately 44.3 and 7.2 pgldl (conversion factor to pmolll = .207), respectively, had virtually identical pre-study mean Hgbs. Following the donation of a unit of blood, however, the lead-exposed workers lagged in their ability to generate reticulocytes and to reconstitute red cell mass. In the current study, women with Hgb deficits and moderately elevated BPbs were unable to respond with appropriate increments in serum EPO. More than 90% of EPO is produced in the proximal renal tubule^,'^,'^ an anatomical site where Pb acc u m u l a t e ~ .In ~ ~response to hypoxia, the initial step leading to renal EPO biosynthesis appears to be increased calcium entry, which leads to phospholipase A, activation and, ultimately, to adenylate cyclase activation." We speculate that Pb may interfere with the calcium "trigger" to EPO biosynthesis. The inhibition of EPO production by Pb appears to be an early and relatively sensitive indicator of Pb nephrotoxicity. We aim to determine whether there i s a threshold for the effect of Pb on serum EPO concentration; therefore, the full set of samples in our serum bank will be analyzed. For example, with regard to the effects of Archives of Environmental Health
I
I-Low BPb I-High BPb
6 6
2-Low BPb &High BPb
6 6 6 6
10.6 10.7 11.6 11.6
6 6
12.3 f 0.1 12.4 f 0.1
3-LOW BPb 3-High BPb QLow BPb 4-High EiPb
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I
Table 1. Hematologic Findings at Mid-Pregnancy
9.6 f 0.11/ 9.6 f 0.1 f 0.1 f 0.1 f 0.1 f 0.1
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3.5 f 0.3 16.9 f 2.5
54.5 f 8.0 68.7 f 17.8
2.2 f 0.2 4.7 f 2.3
73.2 f 10.5 60.2 f 8.0
3.5 28.1 2.5 28.5
35.4 f 7.1 80.6 f 26.5
4.5 f 2.0 19.4 f 9.1
37.2 f 6.2 18.0 f 4.1
24.2 62.6 28.3 40.6
10.6 13.3 15.5 19.5
10.4 11.4 15.8 14.0
f 0.3 f 4.1 f 0.1 f 3.3
2.4 f 0.2 33.8 f 1.0
-
f 3.0 f 28.3
f 3.2 f 12.9
f 2.7 f 2.8
f 4.6 f 5.8
-
3.1 0.6 f 2.9 f 3.4 f f
-
Notes: Hgb hemoglobin, BPb blood lead, EP erythrocyte protoporphyrin, and €PO erythropoietin. *Analysis of variance for EPO revealed a significant Hgb effect (p .oOOl) and a significant BPb effect (p .049). tconversion factor to mmolll 0.6206. *Conversion factor to pmolll 0.207. tjconversion factor to pmolll 0.01 77. #Conversion factor to rs/l 1.O. //Standard error of the mean.
I
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Table 2. Hematologic Findings at Delivery ~~
n
Group 1-Low BPb 1-High BPb 2-LOWBPb 2-High BPb 3-LOW BPb 3-High BPb 4Low BPb 4-High BPb Notes: Hgb
Hb (gldllt 9.4 9.6 10.5 10.5 11.3 11.6 12.4 12.5
-
-
f 0.211 f 0.1
BPb (rg/dl)*
f 0.1
3.5 24.9 4.2 29.4 3.5 37.8
f 0.2 f 0.1
3.6 f 0.1 38.6 f 2.1
f 0.1
f 0.1 f 0.1
Serum ferritin (ng/ml)#
EP WdM
f 0.4 f 1.6
49.0 72.4 39.0 107.8 30.5 135.0
f 0.2 f 2.6
f 0.4 f 0.7
-
~
f 6.8
f 12.9 f 12.9 f 29.1 f 5.9 f 22.1
27.3 f 4.4 119.6 f 42.4
EPO' (mU1ml)
8.5 f 3.4 3.9 f 1.0 5.8 f 1.5 5.2 f 1.1 38.5 f 12.5 5.9 f 1.0 8.8 f 3.5 23.6 f 8.0
-
68.3 36.9 35.5 37.2 52.4 27.2 9.8 10.8
f 11.0 f 10.2 f
9.0
f 11.1 f 16.2
6.3 1.9 f 3.4 f
f
-
hemoglobin, BPb blood lead, EP erythrocyte protoporphyrin, and EPO erythropoietin. .055). *Analysis of variance for EPO revealed a significant Hgb effect (p .oooS)and a significant BPb effect (p tconversion factor to mmolll 0.6206. *Conversion factor to pmolll 0.207. §Conversion factor to pmolll 0.01 77. #Conversion factor to rs/l 1.O. //Standard error of the mean.
-
---
Pb on enzymes of the heme synthetic pathway, there is no apparent threshold for the effect of Pb on d-aminolevulinic acid deh~dratase'~; in contrast, a threshold BPb of approximately 17 pg/dl is required for the inhibition of ferrochelatase activity, which leads to a rise in red cell porphyrins.M In addition, except for Hgb, little is known about other factors that might influence serum EPO concentration, e.g., age, weighdheight, ethnicity, smoking, occupation. Further analyses from this ongoing prospective study will aim to fully describe the determinants of serum EPO. We suspect that these may account for some of the variation in EPO observed at any given Hgb concentration Cables 1 and 2). In the absence of iron deficiency, anemia resulting from Pb toxicity typically occurs only when the BPb exNovembedDecember 1991 [Vol. 46 (No. 6)]
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ceeds 40 pg/dl in children and 50 pg/dl in adults.z'-zz Even though the mean BPbs of our "high" BPb groups ranged only from 23.1 to 36.2 pg/dl (Fig. 11, with an overall high BPb mean (across mid-pregnancy and delivery) of 29.7 pg/dl, depressed serum EPO was apparent. The consequences of deranged heme synthesis have been extraordinarily useful for screening populations for evidence of undue Pb absorption. However, the inhibition of heme synthesis by Pb cannot adequately explain the anemia of Pb toxicity because adequate quantities of heme are synthesized, even in the presence of extraordinarily high Thus, it appears likely that the anemia of Pb poisoning may be due, in part, to the inhibition by Pb of renal EPO production. 349
80 -"LOW"
(4 4)
Blood Lead
T
701
I
6Z -"High" ( )
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9.0-9.9
10.0- 10.9
-
11.0-11.9
Blood Lead
Mean 8Pb (pg/dl)
12.0- 12.9
Hemoglobin concentration (g/dl) strata
Fig. 1. Combined EPO findings of independent experiments conducted at mid-pregnancy flable 1) and at delivery (Table 2). Each bar represents the mean ( 2 SD) serum EPO concentration of a group of 12 women (6 at mid-pregnancy and 6 at delivery) whose concurrent hemoglobin concentrations were within the indicated stratum. The mean blood lead (BPb) of each group is shown in parentheses.
********** This study was supported by grant R 0 1 ES03460 and the Lucille B. Markey Charitable Trust. Submitted for publication December 17, 1990; revised; accepted for publication April 16, 1991. Requests for reprints should be sent to: Joseph H. Graziano, Ph.D., Department of Pharmacology, Columbia University, College of Physicians & Surgeons, 630 West 168th Street, New York, NY 10032.
********** References 1. Nriagu 10. Lead and lead poisoning in antiquity. New York: John Wiley and Sons, 1983. 2. Moore MR, Meredith PA, Goldberg A. Lead and heme biosynthesis. In: Singhal RL, Thomas JA, Eds. Lead toxicity. Baltimore, MD: Urban & Schwarzenberg, 1980; 79-117. 3. Hernberg S, Nurminen M, Hasan J. Non-random shortening of red cell survival times in men exposed to lead. Environ Res 1967; 11247-61. 4. Berk PD, Tschudy DP, Shepley LA, Waggoner JG, Berlin NI. Hematologic and biochemical studies in a case of lead poisoning. Am J Med 1970; 48:137-44. 5. Paglia DE, Valentine WN, Dahlgren JG. Effects of low-level lead exposure on pyrimidine-5' -nucleotidase and other erythrocyte enzymes. Possible role of pyrimidine-5' -nucleotidase in the pathogenesis of lead-induced anemia. J Clin Invest 1975; 56: 1164-69. 6. Grandjean P, Jensen BM, Sando HS, Jorgensen PJ, Antonsen S. Delayed blood regeneration in lead exposure: an effect on reserve capacity. Am J Public Health 1989; 79:1385-88. 7. Graziano JH, Popovac D, Factor-Litvak P, et al. Determinants of elevated blood lead during pregnancy in a population surrounding a lead smelter in Kosovo, Yugoslavia. Environ Health Perspect 199O;89:95-100. 8. Murphy MJ,Graziano JH, Popovac D, et al. Past pregnancy outcomes among women living in the vicinity of a lead smelter in Kosovo, Yugoslavia. Am J Public Health 1% 80:33-35. 9. Fernandez F, Hilligoss D. An improved graphite furnace method for the determination of lead in blood using matrix modification and the L'vov platform. Atomic Spectr 1982; 3:130-31. 10. Piomelli S. A micromethod for free erythrocyte porphyrins: the FEP test. J Lab Clin Med 1973; 81:932-40.
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11. Miles LEM, Lipschitz DA, Bieter CP, Cook JP. Measurement of serum ferritin by a 2-site immunoradiometric assay. Anal Chem 1974; 61:209-24. 12. Eckhart KU, Kurtz A, Hirth P, Scigalla P, Wieczorek L, Bauer C. Evaluation of stability of human erythropoietin in samples for radioirnmunoassay. Klin Wochenschr 1988; 66:241-45. 13. Wide L, Bengtsson C, Birgegard C. Circadian rhythm of erythropoietin in human serum. Br Haematol 1989; 72:85-90. 14. Kleinbaum DG, Kupper LL. Applied regression analysis and other multivariable methods. North Scituate, MA: Duxbury Press, 1978. 15. Car0 J, Erslev AJ. Biologic and immunologic erythropoietin in extracts from hypoxic whole rate kidneys and their glomerular and tubular fractions. J Lab Clin Med 1984; 103:922-31. 16. Erslev AJ, Car0 J. Physiologic and molecular biology of erythropoietin. Med Oncol Tumor Pharmacother 1986; 3:159-
64. 17. Coyer RA. Renal changes associated with lead exposure. In: Mahaffey KR, Ed. Dietary and environmental lead: human health effects. Amsterdam: Elsevier Scientific Publishers, 1985; 315-38. 18. Fisher JW, Nelson PK, Beckman B, Burdowski A. Kidney control of erythropoietin production. In: Dunn MJ, Ed. Renal endocrinology. Baltimore, MD: Williams and Wilkins, 1983; 14280. 19. Hernberg S, Nikkanen G. Enzyme inhibition by lead under normal conditions. Lancet 1970 i:63-65. 20. Piomelli 5, Seaman C, Zullow D, Curran A, Davidow B. Threshold for lead damage to heme synthesis in urban children. Proc Natl Acad Sci 1982; 79:3335-39. 21. World Head Organization, Geneva. Environmental Health Criteria. 3. Lead 1977; 16-17. 22. Landrigan PI, Froines JR, Mahaffey KR. Blood lead burden: summary of epidemiological data on its relation to environmental sources and toxic effects. In: Mahaffey KR, Ed. Dietary and environmental lead: human health effects. Amsterdam: Elsevier Scientific Publishers, 1985; 421-51, 23. Lichtman HC, Feldman F. In vitro pyrrol and porphyrin synthesis in lead poisoning and iron deficiency. J Clin Invest 1963; 42: 830-39. 24. Piomelli S, Lamola AA, Poh-Fitzpatrick MB, Seaman C, Harber LC. Erythropoietic protoporphyria and lead intoxication: the molecular basis for difference in cutaneous photosensitivity. I. Different rates of disappearance of protoporphyrin from the erythrocytes. J Clin Invest 1975; 56:1519-27.
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