International Archives of
Int Arch Occup Environ Health 43, 177-182 (1979)
O
utpaona Il
and Evronn © Springer-Verlag 1979
An Effect of Erythrocyte Protoporphyrin on Blood Manganese in Lead-Exposed Children and Adults* A A E Wibowo, H J A Salle, P del Castilho, and R L Zielhuis Coronel Laboratory for Occupational and Environmental Health, Faculty of Medicine, University of Amsterdam, st Constantijn Huygensstraat 20, Amsterdam, The Netherlands
Summary In one study in 2 and 3-year-old children (geom average 143 Pg Pb/l) and in two studies in workers (geom average 483 lg Pb/l and 323 lg Pb/1) manganese in blood levels increased with blood lead and with free erythrocyte porphyrin (FEP) The zero-order correlation between PbB and Mn B disappeared when FEP was kept constant The conclusion is drawn that the increase of MnB with increasing PbB is probably not due to simultaneous exposure, neither to direct interaction between Pb and Mn, but mediated through a relationship between Mn and porphyrin in erythrocytes. Key words: Blood lead
Blood manganese
Erythrocyte porphyrin
Recently we published data on a relationship between blood lead (PbB) and blood manganese (Mn B) concentrations, as suggestive evidence of a biological interaction (Zielhuis et al , 1978) In epidemiological studies in children and in male workers we observed an increase of Mn B with increasing Pb B-concentrations These relationships were observed under widely different conditions of age and exposure; the possibility of simultaneous exposure appeared to be minimal, but could not be ruled out These studies confirmed a similar relation as found by Delves et al (1973) in 189 children (2 months to 15 years of age), suspected of having lead poisoning and by Joselow et al (1978) in 98 healthy children ( 1-5 years of age) Those authors ascribed the increased levels of manganese and lead to simultaneous exposure, either through pica or traffic exhaust. We have reevaluated our data, particularly analyzing a possible association between erythrocyte porphyrin (EP), measured as free erythrocyte porphyrin (FEP), and blood manganese Both EP and Mn are present in the mitochondria of erythrocyte precursors; in addition a binding of Mn to porphyrins has been described (Borg and Cotzias, 1958 ; Norris and Klein, 1961 ; Mahoney and Sargent, 1967 ; NRC, 1973 ; Hancock and Fritze, 1973 ; Prasad, 1976). * This study was supported by a grant from the Dutch Prevention Fund Offprint requests to: A A E Wibowo, M D (address see above)
0304-0131/79/0043/0177/$ 1 20
178
A A E Wibowo et al.
We examined the relationships between PbB, Mn B and FEP in three studies, one in children, two in adults, for which data on all three parameters were available.
Material and Methods Groups Examined Group I consisted of 108 children, two and three years of age, living 0 5-8 km away from a secondary lead smeltery In 1976 we examined PbB, MnB, FEP, hemoglobin (Hb), and hematocrit (Ht) A relationship between PbB and FEP could be established (Zielhuis et al , 1979). Group II consisted of 118 male workers from the same secondary lead smeltery At examination in 1977 intravenous (i v ) blood (15 ml) was sampled at Monday morning, always in the day shift We measured PbB, MnB, FEP, Hb, Ht, and serum iron (FeS). Group III consisted of a small group of 13 male workers from the painting department of a car factory, examined in 1976, We measured PbB, Mn B, and FEP. The data of groups I and II, each numbering more than 100 subjects, will be used for statistical analysis; the data of the small group III will only be taken into account as supporting evidence.
Analysis of Blood Blood collection and analysis was performed in sequence according to a randomly assigned number (double blind) Metals were measured in whole blood by electrothermal atomic absorption spectrometry, using the carbon tube atomizer Model CRA 63 with the AA 6 spectrometer, equipped with a simultaneous background correction system (Model BC-6), all from Varian Techtron, according to the method published by del Castilho and Herber (1977); the micromethod for blood manganese will be the subject of a forthcoming paper The precision (1 x s d , day to day variation included) of PbB-analysis was PbB ± 30 l g/l, of MnB-analysis + 2 Ag/l. Fe-S was measured according to the modified Giovaniello method (1967), using an Auto Analyzer (Techtron), in the Central Clinical Laboratory, Wilhelmina Gasthuis, University Hospital, Amsterdam. Hb was measured spectrophotometrically after conversion to cyanmethemoglobin and Ht with an Adams Autocrite Centrifuge. FEP was measured with the micromethod of Piomelli (1973), in a simple one-step extraction and subsequent measurement of FEP by a fluorometer (Fluorespec Model SF-1, Baird Atomic Inc ) The standard was coproporphyrin-I (preweighted vials, Sigma). All measurements were in triplicate. For some of the parameters the SI units have been used For PbB and MnB we have maintained the old unit of pg/l Conversion of PbB in jtg/l to the SI unit (jrmol/l) can be done - 2. by multiplication with 4 83 x 10 - 3, for Mn B with 1 82 x 10
StatisticalAnalysis Logarithmic transformation was used for all parameters Results are given as geometric means and standard deviations. In order to assess the relationship between two parameters, Pearson correlation coefficients are used Significance (P) is given one-sided For the PbB-Mn B-FEP relationship a partial correlation coefficient is calculated under control of either PbB or FEP.
Results Tables 1, 2, and 3 present the data for groups I, II, and III Within all three groups there exists an increased lead exposure, with a larger range in Pb B-values in
Table 1 Result of blood analysis of 2 to 3-year-old children Blood indices
Number of cases
Geometric mean
Range of standard deviation
Lead in blood (g/l) Manganese in blood (g/l) Free erythrocyte porphyrin (lg/100 ml rbc) Hemoglobin (mmol/l) Hematocrit (%)
107 101 105 105 105
143 70 37 1 7 97 37 3
93 -218 51 96 24 6 55 8 7 40 8 59 34 6 40 3
Partial correlation of the logarithm of PbB, MnB and FEP in 2 to 3-year-old children, when either PbB or FEP is kept constant; degrees of freedom between brackets; P indicates level of significance Zero order partial PbB PbB
FEP
PbB constant
FEP
Mn B
FEP
FEP constant Mn B
Pb B
MnB
-
0 33 (97) P< 0 001
Mn B
0 16
-
(97)
P = 0 06
0 35
(97)
0 32
P< 0 001
(96)
0 05
P< 0 001
(96)
-
P> 0 1
Table 2 Results of blood analysis of adults occupationally exposed to lead Blood indices
Number of cases
Geometric mean
Range of standard deviation
Lead in blood (lg/l) Manganese in blood (Lg/l) Serum iron mol/l) Free erythrocyte porphyrin (pg/100 ml rbc) Hemoglobin (mmol/l) Hematocrit (%)
115 110 109 113 110 113
483 96 24 5 152 9 9 61 46 6
349 -667 64 14 3 18 5 32 4 65 5 -356 8 8 89 10 38 43 5 50 0
Partial correlation of the logarithm of PbB, MnB and FEP in adults occupationally exposed to lead, when either PbB or FEP is kept constant; degrees of freedom between brackets; P indicates level of significance Zero order partial PbB PbB
FEP Mn B
FEP
PbB constant Mn B
FEP
FEP constant Mn B
PbB
-
0 67 (101) P< 0 001 0 27 (101)
P< 0 01
0 45 (101)
P O1
adults than in children' There was no evidence of any effect on Hb and Ht; subjectively the subjects were reported to be healthy. The tables also present data on various interrelationships In both group I and group II the zero order correlation between FEP and Mn B is not much different from the partial correlation, when keeping PbB constant However, the zero order correlation between Pb B and Mn B disappears when FEP is kept constant. The same is observed in group III. There also exists a positive relationship between PbB and Hb or Ht in group II (r= 0 29, P < 0 01 and r=0 24, P
Int Arch Occup Environ Health 43, 177-182 (1979)
O
utpaona Il
and Evronn © Springer-Verlag 1979
An Effect of Erythrocyte Protoporphyrin on Blood Manganese in Lead-Exposed Children and Adults* A A E Wibowo, H J A Salle, P del Castilho, and R L Zielhuis Coronel Laboratory for Occupational and Environmental Health, Faculty of Medicine, University of Amsterdam, st Constantijn Huygensstraat 20, Amsterdam, The Netherlands
Summary In one study in 2 and 3-year-old children (geom average 143 Pg Pb/l) and in two studies in workers (geom average 483 lg Pb/l and 323 lg Pb/1) manganese in blood levels increased with blood lead and with free erythrocyte porphyrin (FEP) The zero-order correlation between PbB and Mn B disappeared when FEP was kept constant The conclusion is drawn that the increase of MnB with increasing PbB is probably not due to simultaneous exposure, neither to direct interaction between Pb and Mn, but mediated through a relationship between Mn and porphyrin in erythrocytes. Key words: Blood lead
Blood manganese
Erythrocyte porphyrin
Recently we published data on a relationship between blood lead (PbB) and blood manganese (Mn B) concentrations, as suggestive evidence of a biological interaction (Zielhuis et al , 1978) In epidemiological studies in children and in male workers we observed an increase of Mn B with increasing Pb B-concentrations These relationships were observed under widely different conditions of age and exposure; the possibility of simultaneous exposure appeared to be minimal, but could not be ruled out These studies confirmed a similar relation as found by Delves et al (1973) in 189 children (2 months to 15 years of age), suspected of having lead poisoning and by Joselow et al (1978) in 98 healthy children ( 1-5 years of age) Those authors ascribed the increased levels of manganese and lead to simultaneous exposure, either through pica or traffic exhaust. We have reevaluated our data, particularly analyzing a possible association between erythrocyte porphyrin (EP), measured as free erythrocyte porphyrin (FEP), and blood manganese Both EP and Mn are present in the mitochondria of erythrocyte precursors; in addition a binding of Mn to porphyrins has been described (Borg and Cotzias, 1958 ; Norris and Klein, 1961 ; Mahoney and Sargent, 1967 ; NRC, 1973 ; Hancock and Fritze, 1973 ; Prasad, 1976). * This study was supported by a grant from the Dutch Prevention Fund Offprint requests to: A A E Wibowo, M D (address see above)
0304-0131/79/0043/0177/$ 1 20
178
A A E Wibowo et al.
We examined the relationships between PbB, Mn B and FEP in three studies, one in children, two in adults, for which data on all three parameters were available.
Material and Methods Groups Examined Group I consisted of 108 children, two and three years of age, living 0 5-8 km away from a secondary lead smeltery In 1976 we examined PbB, MnB, FEP, hemoglobin (Hb), and hematocrit (Ht) A relationship between PbB and FEP could be established (Zielhuis et al , 1979). Group II consisted of 118 male workers from the same secondary lead smeltery At examination in 1977 intravenous (i v ) blood (15 ml) was sampled at Monday morning, always in the day shift We measured PbB, MnB, FEP, Hb, Ht, and serum iron (FeS). Group III consisted of a small group of 13 male workers from the painting department of a car factory, examined in 1976, We measured PbB, Mn B, and FEP. The data of groups I and II, each numbering more than 100 subjects, will be used for statistical analysis; the data of the small group III will only be taken into account as supporting evidence.
Analysis of Blood Blood collection and analysis was performed in sequence according to a randomly assigned number (double blind) Metals were measured in whole blood by electrothermal atomic absorption spectrometry, using the carbon tube atomizer Model CRA 63 with the AA 6 spectrometer, equipped with a simultaneous background correction system (Model BC-6), all from Varian Techtron, according to the method published by del Castilho and Herber (1977); the micromethod for blood manganese will be the subject of a forthcoming paper The precision (1 x s d , day to day variation included) of PbB-analysis was PbB ± 30 l g/l, of MnB-analysis + 2 Ag/l. Fe-S was measured according to the modified Giovaniello method (1967), using an Auto Analyzer (Techtron), in the Central Clinical Laboratory, Wilhelmina Gasthuis, University Hospital, Amsterdam. Hb was measured spectrophotometrically after conversion to cyanmethemoglobin and Ht with an Adams Autocrite Centrifuge. FEP was measured with the micromethod of Piomelli (1973), in a simple one-step extraction and subsequent measurement of FEP by a fluorometer (Fluorespec Model SF-1, Baird Atomic Inc ) The standard was coproporphyrin-I (preweighted vials, Sigma). All measurements were in triplicate. For some of the parameters the SI units have been used For PbB and MnB we have maintained the old unit of pg/l Conversion of PbB in jtg/l to the SI unit (jrmol/l) can be done - 2. by multiplication with 4 83 x 10 - 3, for Mn B with 1 82 x 10
StatisticalAnalysis Logarithmic transformation was used for all parameters Results are given as geometric means and standard deviations. In order to assess the relationship between two parameters, Pearson correlation coefficients are used Significance (P) is given one-sided For the PbB-Mn B-FEP relationship a partial correlation coefficient is calculated under control of either PbB or FEP.
Results Tables 1, 2, and 3 present the data for groups I, II, and III Within all three groups there exists an increased lead exposure, with a larger range in Pb B-values in
Table 1 Result of blood analysis of 2 to 3-year-old children Blood indices
Number of cases
Geometric mean
Range of standard deviation
Lead in blood (g/l) Manganese in blood (g/l) Free erythrocyte porphyrin (lg/100 ml rbc) Hemoglobin (mmol/l) Hematocrit (%)
107 101 105 105 105
143 70 37 1 7 97 37 3
93 -218 51 96 24 6 55 8 7 40 8 59 34 6 40 3
Partial correlation of the logarithm of PbB, MnB and FEP in 2 to 3-year-old children, when either PbB or FEP is kept constant; degrees of freedom between brackets; P indicates level of significance Zero order partial PbB PbB
FEP
PbB constant
FEP
Mn B
FEP
FEP constant Mn B
Pb B
MnB
-
0 33 (97) P< 0 001
Mn B
0 16
-
(97)
P = 0 06
0 35
(97)
0 32
P< 0 001
(96)
0 05
P< 0 001
(96)
-
P> 0 1
Table 2 Results of blood analysis of adults occupationally exposed to lead Blood indices
Number of cases
Geometric mean
Range of standard deviation
Lead in blood (lg/l) Manganese in blood (Lg/l) Serum iron mol/l) Free erythrocyte porphyrin (pg/100 ml rbc) Hemoglobin (mmol/l) Hematocrit (%)
115 110 109 113 110 113
483 96 24 5 152 9 9 61 46 6
349 -667 64 14 3 18 5 32 4 65 5 -356 8 8 89 10 38 43 5 50 0
Partial correlation of the logarithm of PbB, MnB and FEP in adults occupationally exposed to lead, when either PbB or FEP is kept constant; degrees of freedom between brackets; P indicates level of significance Zero order partial PbB PbB
FEP Mn B
FEP
PbB constant Mn B
FEP
FEP constant Mn B
PbB
-
0 67 (101) P< 0 001 0 27 (101)
P< 0 01
0 45 (101)
P O1
adults than in children' There was no evidence of any effect on Hb and Ht; subjectively the subjects were reported to be healthy. The tables also present data on various interrelationships In both group I and group II the zero order correlation between FEP and Mn B is not much different from the partial correlation, when keeping PbB constant However, the zero order correlation between Pb B and Mn B disappears when FEP is kept constant. The same is observed in group III. There also exists a positive relationship between PbB and Hb or Ht in group II (r= 0 29, P < 0 01 and r=0 24, P
