International Archives of
(kculutkHll
Int Arch Occup Environ Hlth 37,73-88(1976)
mnul Elviiimmnilelui Health ©
by Springer-Verlag 1976
Free Erythrocyte Protoporphyrin as an Indicator of the Biological Effect of Lead inAdult Males I Relationship between Free Erythrocyte Protoporphyrin and Indicators of Internal Dose of Lead L ALESSIO, P A BERTAZZI, F TOFFOLETTO, and V FOA Clinica del Lavoro "L Devoto", Universit A di Milano, Via S Barnaba 8, I-20122 Milano, Italy
Summary The relationship between FEP and the indicators of an internal dose of lead (PbB, Pb U, PbU-EDTA) was considered in a group of adult male sub150 g/ jects with varying lead exposure, whose PbB values ranged from 15 100 ml; a highly significant correlation was found between FEP and PbB, PbU, and PbU-EDTA (r = 0 904 ; r = 0 889 ; r = 0 894, respectively). The regression curves representing the relationship between the erythrocyte metabolite and the indicators of internal dose are uniform and are of nonlinear (logistic) type In the first stage, within the range of normal values (up to 46 ig/100 ml RBC), FEP increases moderately with the rise of internal dose; subsequently it increases exponentially and tends finally to 300 g/100 ml RBC. an asymptotic value in the range 250 The data obtained in our investigation suggest that FEP is a useful test to assess metabolic damage in adult subjects arising from an "abnormal" lead absorption and to evaluate the amount of "active deposit" of the metal present in the body. Validity analysis showed that the erythrocyte metabolite can reliably be used as a screening test for monitoring occupationally exposed subjects and can also be applied in general population studies, since FEP has a good val70 g/100 and in the Pb U-EDTA range of 500 idity in the PbB range of 40 2000
2
g/ 4 h.
Key words: urine
Protoporphyrin in erythrocytes
Lead
Chelatable lead
Abbreviations
Correlation
Pb B = blood lead
Lead in blood
Nonlinear regression
(g/OO ml);
Lead in
Validity.
Pb U = lead in urine
(g/l);
Pb U-EDTA = amount of chelatable lead excreted with 24 h urine after administration of Ca Na 2 EDTA (1 g intravenously); FEP = free erythrocyte protoporphyrin (g/100
ml RBC); CPU = urinary coproporphyrin (g/l);
ALAU =
urinary delta-aminolevulinic acid (mg/l); B-TLV = biological threshold limit value
73
INTRODUCTION It is now common knowledge that lead can inhibit some enzymatic activities of heme biosynthesis Enzymatic inhibition produces an accumulation of the precursory metabolites of heme in blood and urine, the measurement of which can be used to reveal the existence or to assess the entity of metabolic damage caused by an "abnormal" absorption of the metal. Heme synthetase is extremely sensitive to the action of lead and the inhibition of this enzymatic activity causes in vivo an accumulation of protoporphyrin IX in erythrocytes, which is related to the fact that this mitochondrial enzyme regulates the incorporation of iron in the protoporphyrin molecule (Rimington, 1937 ; Dresel and Falk, 1956 ; Boyett and Butterworth, 1962 ; Gibson and Goldberg, 1970) In occupationally exposed subjects the concentration of this erythrocyte metabolite rises and can reach levels from 10 to 50 times higher than the values found in subjects not occupationally exposed to lead, such an increase preceding a rise in urinary coproporphyrin Moreover, FEP remains high over a long period in subjects who have suffered from lead intoxication even when exposure to lead has ceased for a number of years (Vigliani and Angeleri, 1935 ; Watson, 1950 ; Saita et al., 1954 ; Rubino et al , 1958 ; Sano et al , 1960 ; Vigliani and Saita, 1963 ; de Bruin, 1971 ; Albahary, 1972). The usefulness of FEP measurement has made considerable advances in pediatrics in the past 2 years as a result of studies carried out using microanalytical methods It was shown that in children there exists a highly significant correlation between blood lead and PEP (Kommolz et al , 1972 ; Sassa et al , 1973 ; Piomelli, 1973 ; Piomelli et al , 1973 ; Chisolm et al , 1974). The measurement of the metabolite proved particularly useful in identifying infant populations with PbB levels above 40 jg/100 ml; beyond such values FEP undergoes an exponential type increase. However, this diagnostic test has not been currently used in the last 10 15 years for monitoring subjects occupationally exposed to lead; it is used almost exclusively to verify persistence of the biological effects of lead in cases of nonrecent exposure (Secchi and Alessio, 1972) Some authors have even questioned the usefulness of FEP in monitoring adult subjects According to Albahary (1968), FEP is less sensitive than ALAU and therefore its application has no use in public health monitoring Moreover, Haeger-Aronsen (1971) maintains that this test, like the search for stippled cells, is less indicative than urine tests for assessing occupational exposure Although Zielhuis in 1973 emphasized the necessity of undertaking more profound research on FEP in adult subjects, only a limited number of studies have been done recently.
74
The aim of the present research is to ascertain whether FEP in adult male subjects is a useful and valid test for assessing lead exposure, by establishing the relationship that exists in adults between FEP and the internal lead load.
MATERIALS AND METHODS The study was carried out on a group of male subjects aged between 25 and 45 years who had Pb B levels in the range 15 150 vg/100 ml This group was made up of the following subgroups: 1 Inhabitants of the city of Milan, not occupationally exposed to lead, with only environmental exposure to the metal; 2 Lead workers with varying degrees of occupational exposure (hand type-setters, linotype-setters, accumulator production workers, pottery workers, lead smelters). The occupationally exposed subjects had worked continuously with lead for at least 1 year We excluded from our research females workers exposed to organic lead, individuals with a daily wine consumption of more than 1000 ml, subjects with liver or blood disorders. The relationship between FEP and Pb B and PbU was considered in about 200 subjects examined as outpatients Heparinized blood samples for measurement of PbB and FEP were obtained by venipuncture PbU was measured on spot samples of urine; urine samples with density below 1010 were not analyzed. In 92 inpatients of our clinic who had been absent from work for between 2 and 10 days, PbU was measured in 24-h urine collections after i v administration of Ca Na2 EDTA (1 g diluted in 250 cc of 5% glucosate solution), after which the relationship between FEP and Pb U-EDTA was studied. The blood and urine analyses were performed on samples of blood and urine collected on the same day, except for the PbUEDTA test, which was usually performed after ;an interval of 3 4 days. Lead in blood and urine was measured by atomic absorption spectrophotometry, using the method of Andreoletti et al (1972) for PbB, and the method of Zurlo et al (1969) for Pb U; FEP was measured using the method of Schwartz and Wikoff (1952). Table 1 gives, for each of the analytical tests considered in this study, the "normal" values applied in our institute, drawn from the study of healthy adult male subjects, aged between 20 and 45 years, residing in Milan, and not occupationally exposed to lead. Statistical analysis was performed to examine the functional relationships between FEP and the indicators of dose (PbB, PbU, Pb U-EDTA) We evaluated the correlation in order to study the degree of connection between these parameters, and regression to study how the erythrocyte metabolite varies with the indicator
75
Table 1 "Normal" values of FEP and indicators of internal dose applied at the Clinica del Lavoro No
Test
Mean value
cases
Normal limit (mean + 2 SD)
Pb B 161
32
49
256
29
46
102
39
80
26
430
620
(pg/100 ml) FEPa (ig/100
ml RBC)
Pb U (ig/l) spot samples Pb U-EDTA Grisler/24 h) (From From Grisler et al
Table 2
(1976)
Calculation of validity (validity = sensitivity + specificity) Indicator of effect
Indicator of internal load
Positive
Negative
Total
Positive
a
b
a+b
Negative
c
d
c +d
_____________________________________________
a Sensitivity = a + b a+b
d Specificity =
c+d
of internal load The analysis was done on a Univac 1106 computer at the Centro di Calcolo of the University of Milan. The corresponding values of the two variables were plotted on a diagram and the function which best fitted the data was sought using the least squares method Several different functions were calculated For the linear and exponential equations we used the Statjob Regan 2 program, and for the nonlinear logistic equations we used the program described by Marubini and Resele (1971) The determination coefficient (r2 ) was calculated to select the function giving the best estimate of the data An approximate estimation of the 95 % confidence limits of the predicted mean value of y (FEP) was obtained by calculating the standard error (SE) of the predicted value of y. We also examined the validity of FEP as an indicator of effect due to internal lead load Validity indicates the degree to which a situation, as observed, reflects the "true" situ-
76
Table 3 Results of statistical analysis of relationship between FEP and indicators of dose Tests
No cases
Correlation coefficient
P value
Regression function
b = 0 079 c = 280
a= Pb B v
FEP
201
0 904
50 g/100 ml RBC cutoff was fairly high, giving 17 % false negatives and only 2% false positives. When FEP is used in screening occupationally exposed subjects, the values which appear most discriminating are 75 and ~100 g/100 ml RBC With a FEP cutoff of 75 g/100 ml RBC, at a PbB level of 60 pg/100 ml, we obtained only 3% false negatives and 1% false positives Also at PbB concentrations of >70 pg/100 ml good validity was obtained with no false negatives and 12% false positives A FEP cutoff of 100 pg/100 ml RBC was also highly discriminating: at PbB 60 pg/100 ml our data gave 6% false negatives and 1% false positives; with PbB > 70 pg/100 ml, they gave 2% false negatives and 10 % false positives. 83
There also proved to be a good correlation between FEP and the quantity of lead excreted in 24 h urine after administration of 1 g i v CaNa 2 EDTA Regression analysis showed that FEP is elevated beyond the upper normal limit when Pb U-EDTA exceeds 750 pg/24 h (Fig 3) and attains values of 180 ig/100 ml RBC when the amount of chelatable lead excreted is 2000 pg/ 2 4 h. Teisinger (1971) indicates this level as "critical", beyond which workers should be transferred from jobs exposing them to lead It should, however, be noted that Teisinger used a dose of 2 g CaNa 2 EDTA. Validity of FEP was quite high at Pb U-EDTA levels of 1000 and > 1500 pg/24 h At such PbU-EDTA levels, a discriminating value of FEP of 100 or >,75 g/100 ml RBC appears suitable. With FEP cutoff 100 pg/100 ml RBC, at a PbU-EDTA level of >1000 g/24 h, we obtained 13 % false negatives and no false positives At > 1500 g/24 h we had 4% false negatives and 3% false positives Taking a FEP cutoff of 75 g/100 ml RBC, at Pb U-EDTA 1000 g/24 h, we had 8% false negatives and no false positives At > 1500 pg/ 2 4 h, all the positives, i e , the subjects with Pb U-EDTA greater than 1500, were correctly classified according to the FEP value used; but 6% of the negative subjects, i e , with Pb U-EDTA lower than 1500 pg/24 h, were classified as positive. At PbU-EDTA values of >2000, fair validity with high specificity was, however, obtained taking a FEP value of 150 g/ 100 ml RBC. Our study suggests therefore that FEP permits a fairly accurate prediction of the amount of chelatable lead Since chelatable lead is strictly dependent on the active deposit of the metal in the soft tissues of the body, including the trabecular bone (Teisinger et al , 1969), this test is a useful indicator as to whether a worker should be transferred from a lead-exposing job and whether therapy with chelating agents is advisable. The relationship which was found between FEP and chelatable lead seems particularly interesting since it is very likely that, as an indicator of biologically effective internal dose, Pb U-EDTA is more relevant than PbB In fact, the former is an expression of the lead loosely deposited, and the latter is a function of the quantity of lead absorbed from the environment, minus the lead deposited in the bone cortex and soft tissues and the lead excreted with urine and feces (Waldron, 1971), and it is a well-known fact that occupational exposure may be intermittent. There was an equally good correlation between PbU and FEP. This confirms the behavior of FEP according to the internal lead load, further supported by the type of regression function, which was the same as the previous regressions Nevertheless, we do not think any particular assumptions can be made about
84
this relationship; insofar as PbU was measured on spot samples it was not possible to collect urine samples at prefixed times because the subjects under study were at work.
CONCLUSIONS The data obtained in our study suggest that FEP is a useful test for the assessment of the metabolic hazard caused in adult males by an "abnormal" lead absorption, and for indirect evaluation of the quantity of the biologically effective internal load of the metal in the body From our results it also appears that the erythrocyte metabolite can be used as a screening test both for monitoring occupationally exposed groups and for general population studies, since it is closely correlated to the indicators of internal dose and has good predictive validity in the 40 70 g/100 ml range for PbB and in the 500 2000 g/24 h range for Pb U-EDTA Besides cases of "abnormal" lead absorption, increases in the erythrocyte metabolite are found in a limited number of pathologic conditions FEP levels as high as those occurring in severe lead poisoning are found in cases of erythropoietic protoporphyria, a rare congenital disease, and in thalassemia major Modest increases have been found in cases of iron deficiency, serious liver disease, and in tumors (Saita et al , 1966 ; Wintrobe, 1967 ; Chisolm, 1971 ; Baloh, 1974) Therefore, if we exclude the aforementioned causes which, moreover, apart from iron deficiency, are rarely found in workers, high levels of FEP are without doubt evidence of metabolic damage due to an "abnormal" lead absorption. In using this test, an important problem may arise from the fact that normalization of FEP after cessation of exposure is slower than in other indicators of effect currently used The reason for this is that there is an excess of FEP in the erythrocytes which remains within the RBCs until they are destroyed (Albahary, 1972) However, high values of FEP found even many years after cessation of lead exposure (Saita et al , 1954 ; Rubino et al , 1958) call for further studies Such studies are already underway in our laboratory and the aim is to investigate whether persistence of the increased metabolite level can be considered "unimportant" for the health of the worker, or whether it should instead be considered "alarming", since it represents an inhibition of heme synthetase which is perpetuated by the lead released from the body deposit Finally, account must be taken of the fact that there is a latency period between formation of FEP in the maturing erythrocytes of the bone marrow and appearance of the metabolite in the circulating erythrocytes (Sassa et al , 1973): in fact, in an experiment on a volunteer group Stuik (1974) demonstrated that after cessation of administration of lead, FEP continues to increase even
85
when Pb B falls The existence of this time lag between increase in FEP and beginning of "abnormal" lead absorption (evaluated 3 weeks) should as 3 months and by Stuik as 2 by Sassa et al whose exposure subjects be taken into due accou? t when examining lead workers who and to lead is of only recent commencement, concentrations. lead exposed to fluctuating have been Our study was made on male subjects occupationally exposed to inorganic lead We do not think, therefore, that the considerations made can also be extended to female subjects and to In fact, a higher "susceptisubjects exposed to organic lead pathway in females has biosynthesis of the heme to lead bility" (1975), Roels et al ( 1974) and by Stuik been observed recently and it is also a wellknown fact (Waldron and Stofen, 1974) that porphyrin metabolism is altered to a lesser degree in organic lead intoxication than in inorganic lead intoxication. Acknowledgements The authors express their appreciation to Prof E C Vigliani and Prof R L Zielhuis for helpful discussions, to Dr Maria Angela May Manara for assistance in data processing, and to Mrs Maria Rosa Castoldi for technical assistance.
REFERENCES Albahary, C : Views on actual occupational lead poisoning, Report Work Conf Inorg Lead, Amsterdam (1968); cited by Zielhuis (1972) Albahary, C : Lead and hemopoiesis The mechanism and consequences of erythropathy of occupational lead poisoning Amer J Med 52, 367-378 (1972) Andreoletti, F , Griffini, A , Colombo, G , Zurlo, N : Dtermination du Plomb dans le sang par analyse spectrophotometrique a absorption atomique. In: Environmental health aspects of lead Int Symposium Amsterdam (1972), p.1071 Luxembourg: Commission European Communities 1973 Baloh, R W : Laboratory diagnosis of increased lead absorption Arch environm Hlth 28, 198-208 (1974) Boyett, J D , Butterworth, C E : Lead poisoning and haemoglobin synthesis. Report of a study of fifteen patients with chronic lead intoxication. Amer J Med 32, 884-890 (1962) de Bruin, A : Certain biological effects of lead upon the animal organism. Arch environm Hlth 23, 249-264 (1971) Chisolm, J J , jr : Screening techniques for undue lead exposure in children: Biological and practical considerations J Pediat 79, 719-725 (1971) Chisolm, J J , jr , Mellits, E D , Keil, J E , Barret, M B : A simple protoMicrohematocrit procedure as a screening technique for porphyrin assay increased lead absorption in young children Pediatrics 84, 490-496 (1974) Dresel, E I B , Falk, J E : Studies on the biosynthesis of blood pigments. 2 Haem and porphyrin formation in intact chicken erythrocytes Biochem J. 63, 72-79 (1956)
86
Gibson, S L M , Goldberg, A : Defects in haem synthesis in mammalian tissues in experimental lead poisoning and experimental porphyria 38, 63-72 (1970)
Clin Sci.
Grisler, R , Gobbi, A , Colombo, G , Colombo, R : Valori normali di protoporfirina IX Libera eritrocitaria rilevati in 256 individui adulti di sessomaschileabitanti in Lombardia Med d Lavoro 67, 158-162 (1976) Haeger-Aronsen, B : An assessment of laboratory tests used to monitor the exposure of lead workers Brit J industr Med 28, 52-58 (1971) Kehoe, R A : The habern lectures 1960: The metabolism of lead in man in health and disease 3 Present hygienic problems relating to the absorption of lead J roy Inst publ Hlth 24, 177-203 (1961) Kommolz, L , Thatcher, L G , Blodgett, F M , Good, T A : Rapid protoporphyrin quantitation for detection of lead poisoning Pediatrics 50, 625-631 (1972) Lane, R E , Hunter, D , Malcolm, D , Williams, M K , Hudson, T G F , Browne, R.C , McCallum, R I , Thompson, A R , de Kretser, A J , Zielhuis, R L , Cramer, K , Barry, P S I , Goldberg, A , Beritic, T , Vigliani, E C , Truhaut, R , Kehoe, R A , King, E : Diagnosis of inorganic lead poisoning: A statement Brit med J 4, 501 (1968) MacMahon, B , Pugh, T F Boston: Little, Brown Marubini, E , Resele, L the Gompertz function 2, 16-23 (1971)
: Epidemiology Principles and methods, p 261. & Co 1970 F : Computer program for fitting the logistic and to growth data Computer Programs in Biomedicine
Piomelli, S : A micromethod for free erythrocyte porphyrins: The FEP test. J.Lab clin Med 81, 932-940 (1973) Piomelli, S , Davidow, B , Guinee, V F , Young, P , Gay, G : The FEP (Free Erythrocyte Porphyrins) test: A screening micromethod for lead poisoning. Pediatrics 51, 254-259 (1973) Rimington, C : An enzymatic theory of haemopoiesis C R Lab Carlsberg 22, 454 (1937) Roels, H A , Lauwerys, R R , Buchet, J P , Vrelust, M Th : Response of free erythrocyte porphyrin and urinary 6-aminolevulinic acid in men and women moderately exposed to lead Int Arch Arbeitsmed 34, 97-108 (1975) Rubino, G F , Prato, V , Fiorato, L : Erythrocyte copper and porphyrins in the lead poisoning Brit J Haemat 4, 103-107 (1958) Saita, G , Moreo, L , Croce, G : Il ricambio porfirinico nel saturnismo cronico e in anemie e epatopatie non saturnine Med d Lavoro 57, 167-174 (1966) Saita, G , Moreo, L , Fabiani, A : Studio sulle porfirine nel sangue midollare enel sangue periferico nel saturnismo Med d Lavoro 45, 293-299 (1954) Sano, S , Inoue, S , Harada, A , Orita, Z : Relation between free protoporphyrin and haemoglobin in red blood cells in lead poisoning Acta Sch Med. Univ Kyoto 36, 182-185 (1960) Sassa, S , Granick, J L , Granick, S , Kappas, A , Levere, R D : Studies in lead poisoning I Analysis of erythrocyte protoporphyrin levels by spectrofluorimetry in the detection of chronic lead intoxication in the subclinical range Biochem Med 8, 135-148 (1973) Schwartz, S , Wikoff, H M : The relation of erythrocyte coproporphyrin and protoporphyrin to erythropoiesis J biol Chem 194, 563-573 (1952)
87
Secchi, G C , Alessio, L : Valutazione critica del significato dei tests biologici nell'accertamento dell'assorbimento e dell'intossicazione da piombo Med d Lavoro 63, 102-108 (1972) Stuik, E J : Biological response of female volunteers to inorganic lead. Int Arch Arbeitsmed 33, 83-97 (1974) Teisinger, J : Biochemical responses to provocative chelation by edetate disodium calcium Arch Environm Hlth 23, 280-283 (1971) Teisinger, J , Prerovsk&, I , Sedivec, V , Flek, J , Roth, Z : Attempt on determination of biologically active lead in organism in experimental poisoning Int Arch Gewerbepath Gewerbehyg 25, 240-255 (1969) Vigliani, E C , Angeleri, C : Ricerche sulla porfirinemia Clin Med Ital. 66, 5-12 (1935) Vigliani, E C , Saita, G : Malattia da Piombo In: La Medicina del Lavoro (S Caccuri, ed ), Vol I, p 139 Milano: Wasserman 1963 Waldron, H A : Correlation between some parameters of lead absorption and lead intoxication Brit J industr Med 28, 195-199 (1971) Waldron, H A , St6fen, D : Subclinical lead poisoning London New York: Academic Press 1974 Watson, C J : The erythrocyte coproporphyrin: Variation in respect to erythrocyte protoporphyrin and reticulocytes in certain of the anemias. Arch Int Med 86, 797-809 (1950) Wintrobe, M : Clinical hematology, 6th ed , p 7 61 Philadelphia, Pennsylvania: Lea & Febiger 1967 Zielhuis, R L : Permissible limits for inorganic lead in industry Proc. 16th Int Congr Occup Hlth, p 510-512, Tokyo (1969) Zielhuis, R : Effects of small doses of lead in adult male subjects. Report to directorate social affairs Direction Health Protection, Commission of European Communities (1973) Zielhuis, R L : Lead absorption and public health: An appraisal of hazards. In: Environmental health aspects of lead Int Symp Amsterdam (1972), p.631 Luxembourg: Comm Europ Commun 1973 Zielhuis, R L , Verberk, M M : Validity of biological tests in epidemiological toxicology Int Arch Arbeitsmed 32, 167-190 (1974) Zurlo, N , Griffini, A M , Colombo, G : Determination of lead in urine by atomic absorption spectrophotometry after coprecipitation with thorium. Analytica Chimica Acta 47, 203-208 (1969)
Received February 23, 1976 / Accepted March 2, 1976
88
(kculutkHll
Int Arch Occup Environ Hlth 37,73-88(1976)
mnul Elviiimmnilelui Health ©
by Springer-Verlag 1976
Free Erythrocyte Protoporphyrin as an Indicator of the Biological Effect of Lead inAdult Males I Relationship between Free Erythrocyte Protoporphyrin and Indicators of Internal Dose of Lead L ALESSIO, P A BERTAZZI, F TOFFOLETTO, and V FOA Clinica del Lavoro "L Devoto", Universit A di Milano, Via S Barnaba 8, I-20122 Milano, Italy
Summary The relationship between FEP and the indicators of an internal dose of lead (PbB, Pb U, PbU-EDTA) was considered in a group of adult male sub150 g/ jects with varying lead exposure, whose PbB values ranged from 15 100 ml; a highly significant correlation was found between FEP and PbB, PbU, and PbU-EDTA (r = 0 904 ; r = 0 889 ; r = 0 894, respectively). The regression curves representing the relationship between the erythrocyte metabolite and the indicators of internal dose are uniform and are of nonlinear (logistic) type In the first stage, within the range of normal values (up to 46 ig/100 ml RBC), FEP increases moderately with the rise of internal dose; subsequently it increases exponentially and tends finally to 300 g/100 ml RBC. an asymptotic value in the range 250 The data obtained in our investigation suggest that FEP is a useful test to assess metabolic damage in adult subjects arising from an "abnormal" lead absorption and to evaluate the amount of "active deposit" of the metal present in the body. Validity analysis showed that the erythrocyte metabolite can reliably be used as a screening test for monitoring occupationally exposed subjects and can also be applied in general population studies, since FEP has a good val70 g/100 and in the Pb U-EDTA range of 500 idity in the PbB range of 40 2000
2
g/ 4 h.
Key words: urine
Protoporphyrin in erythrocytes
Lead
Chelatable lead
Abbreviations
Correlation
Pb B = blood lead
Lead in blood
Nonlinear regression
(g/OO ml);
Lead in
Validity.
Pb U = lead in urine
(g/l);
Pb U-EDTA = amount of chelatable lead excreted with 24 h urine after administration of Ca Na 2 EDTA (1 g intravenously); FEP = free erythrocyte protoporphyrin (g/100
ml RBC); CPU = urinary coproporphyrin (g/l);
ALAU =
urinary delta-aminolevulinic acid (mg/l); B-TLV = biological threshold limit value
73
INTRODUCTION It is now common knowledge that lead can inhibit some enzymatic activities of heme biosynthesis Enzymatic inhibition produces an accumulation of the precursory metabolites of heme in blood and urine, the measurement of which can be used to reveal the existence or to assess the entity of metabolic damage caused by an "abnormal" absorption of the metal. Heme synthetase is extremely sensitive to the action of lead and the inhibition of this enzymatic activity causes in vivo an accumulation of protoporphyrin IX in erythrocytes, which is related to the fact that this mitochondrial enzyme regulates the incorporation of iron in the protoporphyrin molecule (Rimington, 1937 ; Dresel and Falk, 1956 ; Boyett and Butterworth, 1962 ; Gibson and Goldberg, 1970) In occupationally exposed subjects the concentration of this erythrocyte metabolite rises and can reach levels from 10 to 50 times higher than the values found in subjects not occupationally exposed to lead, such an increase preceding a rise in urinary coproporphyrin Moreover, FEP remains high over a long period in subjects who have suffered from lead intoxication even when exposure to lead has ceased for a number of years (Vigliani and Angeleri, 1935 ; Watson, 1950 ; Saita et al., 1954 ; Rubino et al , 1958 ; Sano et al , 1960 ; Vigliani and Saita, 1963 ; de Bruin, 1971 ; Albahary, 1972). The usefulness of FEP measurement has made considerable advances in pediatrics in the past 2 years as a result of studies carried out using microanalytical methods It was shown that in children there exists a highly significant correlation between blood lead and PEP (Kommolz et al , 1972 ; Sassa et al , 1973 ; Piomelli, 1973 ; Piomelli et al , 1973 ; Chisolm et al , 1974). The measurement of the metabolite proved particularly useful in identifying infant populations with PbB levels above 40 jg/100 ml; beyond such values FEP undergoes an exponential type increase. However, this diagnostic test has not been currently used in the last 10 15 years for monitoring subjects occupationally exposed to lead; it is used almost exclusively to verify persistence of the biological effects of lead in cases of nonrecent exposure (Secchi and Alessio, 1972) Some authors have even questioned the usefulness of FEP in monitoring adult subjects According to Albahary (1968), FEP is less sensitive than ALAU and therefore its application has no use in public health monitoring Moreover, Haeger-Aronsen (1971) maintains that this test, like the search for stippled cells, is less indicative than urine tests for assessing occupational exposure Although Zielhuis in 1973 emphasized the necessity of undertaking more profound research on FEP in adult subjects, only a limited number of studies have been done recently.
74
The aim of the present research is to ascertain whether FEP in adult male subjects is a useful and valid test for assessing lead exposure, by establishing the relationship that exists in adults between FEP and the internal lead load.
MATERIALS AND METHODS The study was carried out on a group of male subjects aged between 25 and 45 years who had Pb B levels in the range 15 150 vg/100 ml This group was made up of the following subgroups: 1 Inhabitants of the city of Milan, not occupationally exposed to lead, with only environmental exposure to the metal; 2 Lead workers with varying degrees of occupational exposure (hand type-setters, linotype-setters, accumulator production workers, pottery workers, lead smelters). The occupationally exposed subjects had worked continuously with lead for at least 1 year We excluded from our research females workers exposed to organic lead, individuals with a daily wine consumption of more than 1000 ml, subjects with liver or blood disorders. The relationship between FEP and Pb B and PbU was considered in about 200 subjects examined as outpatients Heparinized blood samples for measurement of PbB and FEP were obtained by venipuncture PbU was measured on spot samples of urine; urine samples with density below 1010 were not analyzed. In 92 inpatients of our clinic who had been absent from work for between 2 and 10 days, PbU was measured in 24-h urine collections after i v administration of Ca Na2 EDTA (1 g diluted in 250 cc of 5% glucosate solution), after which the relationship between FEP and Pb U-EDTA was studied. The blood and urine analyses were performed on samples of blood and urine collected on the same day, except for the PbUEDTA test, which was usually performed after ;an interval of 3 4 days. Lead in blood and urine was measured by atomic absorption spectrophotometry, using the method of Andreoletti et al (1972) for PbB, and the method of Zurlo et al (1969) for Pb U; FEP was measured using the method of Schwartz and Wikoff (1952). Table 1 gives, for each of the analytical tests considered in this study, the "normal" values applied in our institute, drawn from the study of healthy adult male subjects, aged between 20 and 45 years, residing in Milan, and not occupationally exposed to lead. Statistical analysis was performed to examine the functional relationships between FEP and the indicators of dose (PbB, PbU, Pb U-EDTA) We evaluated the correlation in order to study the degree of connection between these parameters, and regression to study how the erythrocyte metabolite varies with the indicator
75
Table 1 "Normal" values of FEP and indicators of internal dose applied at the Clinica del Lavoro No
Test
Mean value
cases
Normal limit (mean + 2 SD)
Pb B 161
32
49
256
29
46
102
39
80
26
430
620
(pg/100 ml) FEPa (ig/100
ml RBC)
Pb U (ig/l) spot samples Pb U-EDTA Grisler/24 h) (From From Grisler et al
Table 2
(1976)
Calculation of validity (validity = sensitivity + specificity) Indicator of effect
Indicator of internal load
Positive
Negative
Total
Positive
a
b
a+b
Negative
c
d
c +d
_____________________________________________
a Sensitivity = a + b a+b
d Specificity =
c+d
of internal load The analysis was done on a Univac 1106 computer at the Centro di Calcolo of the University of Milan. The corresponding values of the two variables were plotted on a diagram and the function which best fitted the data was sought using the least squares method Several different functions were calculated For the linear and exponential equations we used the Statjob Regan 2 program, and for the nonlinear logistic equations we used the program described by Marubini and Resele (1971) The determination coefficient (r2 ) was calculated to select the function giving the best estimate of the data An approximate estimation of the 95 % confidence limits of the predicted mean value of y (FEP) was obtained by calculating the standard error (SE) of the predicted value of y. We also examined the validity of FEP as an indicator of effect due to internal lead load Validity indicates the degree to which a situation, as observed, reflects the "true" situ-
76
Table 3 Results of statistical analysis of relationship between FEP and indicators of dose Tests
No cases
Correlation coefficient
P value
Regression function
b = 0 079 c = 280
a= Pb B v
FEP
201
0 904
50 g/100 ml RBC cutoff was fairly high, giving 17 % false negatives and only 2% false positives. When FEP is used in screening occupationally exposed subjects, the values which appear most discriminating are 75 and ~100 g/100 ml RBC With a FEP cutoff of 75 g/100 ml RBC, at a PbB level of 60 pg/100 ml, we obtained only 3% false negatives and 1% false positives Also at PbB concentrations of >70 pg/100 ml good validity was obtained with no false negatives and 12% false positives A FEP cutoff of 100 pg/100 ml RBC was also highly discriminating: at PbB 60 pg/100 ml our data gave 6% false negatives and 1% false positives; with PbB > 70 pg/100 ml, they gave 2% false negatives and 10 % false positives. 83
There also proved to be a good correlation between FEP and the quantity of lead excreted in 24 h urine after administration of 1 g i v CaNa 2 EDTA Regression analysis showed that FEP is elevated beyond the upper normal limit when Pb U-EDTA exceeds 750 pg/24 h (Fig 3) and attains values of 180 ig/100 ml RBC when the amount of chelatable lead excreted is 2000 pg/ 2 4 h. Teisinger (1971) indicates this level as "critical", beyond which workers should be transferred from jobs exposing them to lead It should, however, be noted that Teisinger used a dose of 2 g CaNa 2 EDTA. Validity of FEP was quite high at Pb U-EDTA levels of 1000 and > 1500 pg/24 h At such PbU-EDTA levels, a discriminating value of FEP of 100 or >,75 g/100 ml RBC appears suitable. With FEP cutoff 100 pg/100 ml RBC, at a PbU-EDTA level of >1000 g/24 h, we obtained 13 % false negatives and no false positives At > 1500 g/24 h we had 4% false negatives and 3% false positives Taking a FEP cutoff of 75 g/100 ml RBC, at Pb U-EDTA 1000 g/24 h, we had 8% false negatives and no false positives At > 1500 pg/ 2 4 h, all the positives, i e , the subjects with Pb U-EDTA greater than 1500, were correctly classified according to the FEP value used; but 6% of the negative subjects, i e , with Pb U-EDTA lower than 1500 pg/24 h, were classified as positive. At PbU-EDTA values of >2000, fair validity with high specificity was, however, obtained taking a FEP value of 150 g/ 100 ml RBC. Our study suggests therefore that FEP permits a fairly accurate prediction of the amount of chelatable lead Since chelatable lead is strictly dependent on the active deposit of the metal in the soft tissues of the body, including the trabecular bone (Teisinger et al , 1969), this test is a useful indicator as to whether a worker should be transferred from a lead-exposing job and whether therapy with chelating agents is advisable. The relationship which was found between FEP and chelatable lead seems particularly interesting since it is very likely that, as an indicator of biologically effective internal dose, Pb U-EDTA is more relevant than PbB In fact, the former is an expression of the lead loosely deposited, and the latter is a function of the quantity of lead absorbed from the environment, minus the lead deposited in the bone cortex and soft tissues and the lead excreted with urine and feces (Waldron, 1971), and it is a well-known fact that occupational exposure may be intermittent. There was an equally good correlation between PbU and FEP. This confirms the behavior of FEP according to the internal lead load, further supported by the type of regression function, which was the same as the previous regressions Nevertheless, we do not think any particular assumptions can be made about
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this relationship; insofar as PbU was measured on spot samples it was not possible to collect urine samples at prefixed times because the subjects under study were at work.
CONCLUSIONS The data obtained in our study suggest that FEP is a useful test for the assessment of the metabolic hazard caused in adult males by an "abnormal" lead absorption, and for indirect evaluation of the quantity of the biologically effective internal load of the metal in the body From our results it also appears that the erythrocyte metabolite can be used as a screening test both for monitoring occupationally exposed groups and for general population studies, since it is closely correlated to the indicators of internal dose and has good predictive validity in the 40 70 g/100 ml range for PbB and in the 500 2000 g/24 h range for Pb U-EDTA Besides cases of "abnormal" lead absorption, increases in the erythrocyte metabolite are found in a limited number of pathologic conditions FEP levels as high as those occurring in severe lead poisoning are found in cases of erythropoietic protoporphyria, a rare congenital disease, and in thalassemia major Modest increases have been found in cases of iron deficiency, serious liver disease, and in tumors (Saita et al , 1966 ; Wintrobe, 1967 ; Chisolm, 1971 ; Baloh, 1974) Therefore, if we exclude the aforementioned causes which, moreover, apart from iron deficiency, are rarely found in workers, high levels of FEP are without doubt evidence of metabolic damage due to an "abnormal" lead absorption. In using this test, an important problem may arise from the fact that normalization of FEP after cessation of exposure is slower than in other indicators of effect currently used The reason for this is that there is an excess of FEP in the erythrocytes which remains within the RBCs until they are destroyed (Albahary, 1972) However, high values of FEP found even many years after cessation of lead exposure (Saita et al , 1954 ; Rubino et al , 1958) call for further studies Such studies are already underway in our laboratory and the aim is to investigate whether persistence of the increased metabolite level can be considered "unimportant" for the health of the worker, or whether it should instead be considered "alarming", since it represents an inhibition of heme synthetase which is perpetuated by the lead released from the body deposit Finally, account must be taken of the fact that there is a latency period between formation of FEP in the maturing erythrocytes of the bone marrow and appearance of the metabolite in the circulating erythrocytes (Sassa et al , 1973): in fact, in an experiment on a volunteer group Stuik (1974) demonstrated that after cessation of administration of lead, FEP continues to increase even
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when Pb B falls The existence of this time lag between increase in FEP and beginning of "abnormal" lead absorption (evaluated 3 weeks) should as 3 months and by Stuik as 2 by Sassa et al whose exposure subjects be taken into due accou? t when examining lead workers who and to lead is of only recent commencement, concentrations. lead exposed to fluctuating have been Our study was made on male subjects occupationally exposed to inorganic lead We do not think, therefore, that the considerations made can also be extended to female subjects and to In fact, a higher "susceptisubjects exposed to organic lead pathway in females has biosynthesis of the heme to lead bility" (1975), Roels et al ( 1974) and by Stuik been observed recently and it is also a wellknown fact (Waldron and Stofen, 1974) that porphyrin metabolism is altered to a lesser degree in organic lead intoxication than in inorganic lead intoxication. Acknowledgements The authors express their appreciation to Prof E C Vigliani and Prof R L Zielhuis for helpful discussions, to Dr Maria Angela May Manara for assistance in data processing, and to Mrs Maria Rosa Castoldi for technical assistance.
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Secchi, G C , Alessio, L : Valutazione critica del significato dei tests biologici nell'accertamento dell'assorbimento e dell'intossicazione da piombo Med d Lavoro 63, 102-108 (1972) Stuik, E J : Biological response of female volunteers to inorganic lead. Int Arch Arbeitsmed 33, 83-97 (1974) Teisinger, J : Biochemical responses to provocative chelation by edetate disodium calcium Arch Environm Hlth 23, 280-283 (1971) Teisinger, J , Prerovsk&, I , Sedivec, V , Flek, J , Roth, Z : Attempt on determination of biologically active lead in organism in experimental poisoning Int Arch Gewerbepath Gewerbehyg 25, 240-255 (1969) Vigliani, E C , Angeleri, C : Ricerche sulla porfirinemia Clin Med Ital. 66, 5-12 (1935) Vigliani, E C , Saita, G : Malattia da Piombo In: La Medicina del Lavoro (S Caccuri, ed ), Vol I, p 139 Milano: Wasserman 1963 Waldron, H A : Correlation between some parameters of lead absorption and lead intoxication Brit J industr Med 28, 195-199 (1971) Waldron, H A , St6fen, D : Subclinical lead poisoning London New York: Academic Press 1974 Watson, C J : The erythrocyte coproporphyrin: Variation in respect to erythrocyte protoporphyrin and reticulocytes in certain of the anemias. Arch Int Med 86, 797-809 (1950) Wintrobe, M : Clinical hematology, 6th ed , p 7 61 Philadelphia, Pennsylvania: Lea & Febiger 1967 Zielhuis, R L : Permissible limits for inorganic lead in industry Proc. 16th Int Congr Occup Hlth, p 510-512, Tokyo (1969) Zielhuis, R : Effects of small doses of lead in adult male subjects. Report to directorate social affairs Direction Health Protection, Commission of European Communities (1973) Zielhuis, R L : Lead absorption and public health: An appraisal of hazards. In: Environmental health aspects of lead Int Symp Amsterdam (1972), p.631 Luxembourg: Comm Europ Commun 1973 Zielhuis, R L , Verberk, M M : Validity of biological tests in epidemiological toxicology Int Arch Arbeitsmed 32, 167-190 (1974) Zurlo, N , Griffini, A M , Colombo, G : Determination of lead in urine by atomic absorption spectrophotometry after coprecipitation with thorium. Analytica Chimica Acta 47, 203-208 (1969)
Received February 23, 1976 / Accepted March 2, 1976
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