Document gb72ppvwjD6Q4YZqk9D23qOgJ

N40687 DUP050056049 DUP050056050 'v a a m tm TRANSLATION: Basecqz, J.-M., R. Lauwerys and J.-P. Buchet: COMPARATIVE STUDY OF VARIOUS BIOLOGICAL LEAD-EXPOSURE TESTS. (Etude comparative de divers tests biologiques d'exposition au plomb.) Arch, Mai. Prof., Vol. 32, No, 6, pp. 453-463, 1971, |Industrial Toxicology Unit, Catholic university of Louvain, and National Sci entific Research Foundation, Brussels (Belgium). i i ! ABSTRACT. This study confirms the validity of determining ? : urinary 5-aminolevulinic acid for monitoring lead^exposed workers. Its correlation with urine lead is definitely U J superior to that of coproporpjhyrinuria. Stippled cell studies offer no interest, ijhis study demonstrates, how ever, that significant inhibition of the globular enzyme, S-aminolevulinic dehydratase, can occur without an eleva tion of the urinary 6 -amino 1 evulinic acid level. Determine at ion of the activity of this; enzyme is therefore a very sensitive and very early test of lead exposure. Even in ! subjects who are not occupationally exposed to lead, enzy matic activity is a function of the amount of circulating blood. This observation confirms the role of lead as an environmental pollutant. Although lead has been one of the most extensively studied industrial i # toxins, early diagnosis of lead accumulation before the appearance of clinical! ! i I signs of poisoning remains a difficult problem. Blood lead and urinary lead are generally considered as two valid indices; for estimating the degree of exposure (2, 21, 22, 35], but certain technical > j ! j ^ j difficulties limit their generalization for the routine monitoring of workers.! , ;5; j......... . | j ' ` j Over the last ten years, several authors [5, 14, 31] have emphasized the j f : '' J importance of determining the urinary concentration of 6-aminolevulinic acid j j (ALA) for evaluating the metabolic response of the organism. Nevertheless, J stippled cell studies, in the majority1 of cases, still constitute the only : ,! H analysis performed by industrial medical services. j Some authors, however, Vi ' recommend semi-quantitative evaluation of coproporphyrinuria [29]. j | ? We thought it would be of interest to conduct a comparative study of DUP050056053 various biological tests for monitoring lead exposure. In addition, we also i carried out a detailed study of the action of lead on an erythrocyte enzyme, 6-aminolevulinic acid dehydratase (ALA-d). Several recent studies [4, 7, 8, |18] indicate that this enzyme is readily Inhibited by lead. j 's | MATERIAL AND METHODS I I. Description of Measurement Techniques ! ...................... r i (1) Determination of Lead in Air ;; Lead dust was collected on filters using a TFIA-2 Staplex High^-Vplume Air Sampler equipped with TFA No. 41 filters. After mineralizing the sample, lead was determined by polarography [24]. j (2) Urinary lead and blood lead were determined by the dithizon colori- 1 : j metric method described by Keenan et al. [19] \ (3) Stippled cells were counted according to the standard Manson-Schwarzj technique. (4) The creatinine level was determined by JaffdVs method [16]. (5) Determination of Urinary Coproporphyrins Quantitative determination of urihary copropprphyrins was performed by fluorometry according to Talmanf s method [32]. 1 for semi-quantitative determination. Donath fs method [9] was used (6) Determination of Urinary ^-Aminolevulinic Acid (ALA) f This was performed according to a modification of Grabecki1 s method [13].j >: This method consists in directly complexing ALA with acetylacetone to form il ' l j pyrrole, which yields a colored complex with Ehrlich's reagent. We noted that 2 DUP050056054 certain urinary metabolites interfered with the development of the colorimetric reaction, and that this interference varied from one urine specimen to the other. This phenomenon therefore decreased the accuracy of readings made In reference to a standard curve. The addition of an internal ALA standard to ! m:i > the urine specimen enabled ns to remedy this shortcoming. Three colorimetric 1 V.) ] | | i; measurements were thus performed for e4ch urine specimen: \ Ui (1) urine specimen treated in the standard manner according to Grabecki; M| | ! > (2) a specimen to which acetylaedtone is not added (blank); 14 ] r (3) a specimen to which 20 pg of ALA is added. ] After subtracting the blank, it is easy to determine the urinary ALA con-1 ,;>o ! I centration by a simple rule of three [proportion -- Tr.]. jI The technique is described below. j Acetylacetone, acetate buffer (1 Af, pH 4.6) containing, per liter of j water, 136 g of sodium acetate SH^O and 57 ml of glacial acetic acid, and an j ALA stock solution: 10 mg of ALA per 100 ml of acetate buffer; this solution j j: JI is stored at 0C out of contact with light; a standard ALA solution (20 yg/ml)| \ 'i is prepared by diluting the stock solution five times with the acetate buffer \n y? Ehrlich1 s reagent: 1 g of dimethylb enzaldehyde and approximately 30 ml of 46 ii glacial acetic acid are placed in a 50lml flask; 8 ml of 70% perchloric acid I-..;' 43 is then added and the remaining volume! is filled with glacial acetic acid. 44 i 4% These last two solutions are prepared before use. 46 47 48 Procedure Three tubes are prepared for each urine specimen. Tube A contains, 1 ml DUP050056055 of urine, 0.2 ml of acetylacetone, and 5.8 ml of acetate buffer. Tube B (blank) contains 1 ml of urine and 6 ml of buffer. One milliliter of urine, 0.2 ml of : ; acetylacetone, 1 ml of standard ALA solution, and 4.8 ml of buffer are placed I in lube C. The tubes are thoroughly shaken and placed in a water bath at 100cj - for 10 minutes. After cooling, 7 ml of Ehrlich's reagent is added. The solu- : 1 ! jtions are mixed again and absorbency at) 555 my against water is read after 15 I ' * jminutes. The amount of ALA expressed iji yg per ml of urine is equal to: DGa -- DOj, DOe -- DO* X 2Q. The results are then calculated in mg/g of creatinine. i We were able to demonstrate the existence of an excellent correlation between the results provided by this method and those obtained by the technique ,/s I; v ?1 described by Mauzerall and Granick [25] (Figure 1). Urinary ALA (mg/g creatinine) (modified Grabedki method) Figure 1. Determination of urinary ALA. Correlation between Mauzerall-Granick method (y) and modified Grabecki method (a?). 4 DUP050056056 (7) Red Blood Cell Activity of 6-ALA Dehydrate8e (ALA-D) ALA-D activity was determined by colorimetric measurement of the amount of porphobilinogen formed when a preparation of hemolyzed red blood cells is incu-4 jbated at 37C for a predetermined time in the presence of ALA [4] Blood is iI ^collected over heparin, and centrifuged: at 0C and 3,000 rpm in a Sorvall ] centrifuge. After eliminating the plasma, the red blood cells are washed by j |j 1? H Iredispersion in an isotonic NaCl solution at 0C and centrifuged again, this j }j I | A {process is repeated two times. The redi blood cells are then resuspended in 1 rl j 14 saline solution, in which the hematocrit is read before hemolysis of the red J 19 20 1 [blood cells by 4 rapid and successive ; freezings and thawings. j This preparatioij 2i i ; i \-u of hemolyzed red blood cells is diluted! 3 or 4 times (according to whether lead -exposed subjects are involved or not) with an Na^HPO^-NaHgPO^ buffer (0.05 M, j 40 IpH 7) and an aliquot (generally 0.5 ml, equivalent to 0.125-0.166 ml of total | |.................... '............................... ..........\.......... ' " ......'..... ......... : ........( {blood) is incubated at 37C for 75 minujtes in the presence of 0.5 ml of an {aqueous ALA solution (27 mH) and 1 ml of phosphate buffer. We then followed I Bonsignore's procedure: at the end of the incubation period, 2 ml of a 10% I j j [trichloroacetic acid solution containing 0.1 M HgC^ is added. After centri- j fugation, 2 ml of supernatant is mixed with 2 ml of Ehrlich's reagent. 21 After 15 minutes, the absorbency of the colored solution is read at 555 mp j . V) 40 with the aid of a Zeiss PMQ-Il spectrophotometer. I A tissue blank test is per-j | 4i formed for each analysis. It consists of the standard incubation mixture in 44 44 j 44 which the substrate has been replaced by i an equivalent volume of buffer. The 45 incubation time and blood volume are selected on the basis of preliminary exper Ah A? 44 iments (Figures 2 and 3) conducted to determine the conditions under which the 11 jamount of porphobilinogen formed is linearly proportional to the volume of blood and the incubation time. DUP050056057 12,5 / //I1 /7,5 m 2,S : \ A:i L .. M 0 40S Ctl 0,15 J$,2 425 0,3 ] 11 Amount of blood (ml) 4' r Figure 2. influence of amount of blood on amount of por ir. phobilinogen formed from ALA. 74 20 4! 44 44 Incubation time (minutes) :' ; ;.( Figure 3. Influence of incubation time on amount of por 1 > phobilinogen formed from ALA (an amount of hemolyzed red M> blood cells equivalent to 6.125 ml of total blood was V selected for this experiment). \H 44 In addition to the "conventional" [method described above, enzyme activity 4! 44 also was determined in two samples of tfhe same blood, one pre^incubated for 5 44 44 minutes at 60C and the other pre-incubated for 20 minutes at 37C in the pres 41 44 ence of 10 mM of glutathione (GSH) * Because of the considerable enzyme acti 47 %$\ vation by heat and GSH, the volume of lj>lood used was reduced respectively to V-: 0.2 and 0.1 ml of blood diluted 3 or 4 times. DUP050056058 The results expressed in enzyme units per ml of red blood cells and per hour are calculated by applying the following formula: Uh,l000 hematocrit (%) x blood ivolume (ml) Where is the absorbency after 60 minutes of incubation and P0n0 is that of i * the blank* 1 1> Red blood cell ALA-D activity was measured during the 12 hours following i 4 {blood sampling* Urinary coproporphyrin and ALA were determined during the 24 hours following urine collection. The brine specimens used for ALA determina^ 14 41 tion were maintained acid by adding tarj:ric acid [ 34], whereas those used for lit coproporphyrin determination were stored under J.\ [16]. The bipod and urine samples were kept at 4C until the moment of analysis* > II. Description ok Populations (1) Control Population | )i The control population consisted of 22 malesubjects who had not been 0004' :j 4' u pationally exposed to lead and whose agh distribution was as follows: the U youngest subject was 20 years old and the oldest was 53 years old. The mean j U, and the median were identical: 33 years. iii 40 (2) Lead-Exposedj Population 41 44 44 This population consisted of 53 workers from a storage-battery factory fo^ 4.4 j 44 whom only urine studies and stippled cejLl counts were performed, and 25 print >4 -r ing-plant workers for whom blood studies and some urine tests could be per 4A 4-` formed. For certain technical reasons (unfresh urine, sample lost during analysisi DUP050056059 etc.), all the analyses anticipated were not performed for all the selected workers (see results)* RESULTS V I. Control Population to i. The averages and standard deviations of various parameters measured for ; ; subjects who had not been occupationally exposed to lead are presented below: blood lead ....... 34.1 1.8 yg/100 ml of blood 77.8 4.3 yg/100 ml of red blood cells V) urinary lead . . . . . . . . 35.2 2.8 yg/g creatinine 20 ':! urinary ALA . . , . . . , . 4.6 0.3 g/g creatinine urinary coproporphyrins . . 23 2.2 yg/g creatinine hematocrit ....................................44.5 0.5% red blood cell ALA-D (standard method without activation) . 59 4 U/ml RBC/hour. As was carefully pointed out by Van Houte and Code (33), it is more logij- |cal to express the blood lead level by the volume of red blood cells, rather than by the volume of total blood. Indeed, since more than 95% of the circu lating lead is fixed to red blood cells, this form of expression accounts for it w, hematocrit differences from subject to Subject. On the other hand, we express y< i 4ft the concentration of various urinary metabolites per gram of creatinine, A 4! 44 correction is then introduced for the Variable degree of dilution of urine j 44 specimens collected at any time during jthe day. Comparison of the degree of i', 4i) scatter of the results expressed per volume of urine and per gram of creatinines ! (Figure 4) confirms the significance of this correction, this conclusion ] agrees with that made by Gibson [11]. ; 8 DUP050056060 Urinary 6-ALA mg/l mg/gm creat. 20 20 to 10 r-- V* .V O (a) Ptomb urines pg/t pg/gm creat. wo r------- nr- 100 * !! , so .--.V. so *** , ., . .ft n A *... . '.... >1 Copro urines J*0 pg/gm creat. 100 100 SO >v> L_u vV. Figure 4. Comparison of urinary ALA, lead, and copropprphyrins expressed per liter cjf urine and per gram of urin ary creatinine (control subj dcSsX Key: (a) urinary lead; (b) urinary coproporphyrins. | II. Storage-Battery Plant Workers The average values of the lead concentrations in the air determined monthly for three months at different working areas in the storage-battery plant .are j presented below: ] I3 plate stacking........................... j........ . 0.321 mg/m plate pasting and breaking . 0.257 mg/m3 soaking 0.244 mg/ni plate casting . . . . . . . I. . . . . . . . . 0.232 mg/m" mixer tipping . .. . . . 0.125 mg/m lead oxide storage .............. 0.097 mg/m DUP050056061 Lead exposure is thus relatively considerable since 4 working areas where two-thirds of the selected workers are employed exhibit air lead levels that are above the tolerable limit (0.2 mg Pb/m^ air, ACGIB 1969) [1]. ] The significance of this exposure is reflected in a distinct elevation of ithe average values of the various parameters of lead accumulation (Figure 5) . | ;i :; j ; ] * |Out of 47 blood lead levels measured, none was within normal limits (< 50 Rg/gj ii 1>< ' 11 i creatinine) and 23 (50%) exceeded the permissible limit (150 yg/g creatinine). ': i i! !? ri j Thirty-five out of 51 (68%) exhibited urinary ALA levels above the upper limit j i of the norm (< mg/g creatinine) and 16 subjects (31%) excreted an amount of ALA }'4 Vi above the tolerable limit (20 mg/g creatinine) , .V! i .4 ; j Figure 5. Comparison of urinary lead, coproporphyrins, and ALA in control subjects and storage-battery plant workers (the vertical line represents the standard devia tion) > Key: (a) urinary lead (ug/g creatinine); (b) urinary coproporphyrins (ug/g creatinine); (c) urinary 6-ALA (mg/g creatinine); (d)lcontrols; (e) exposed. i x:i 44 Urinary coproporphyrins are abnormally increased (concentration greater 44 44 than 100 yg/g of creatinine) in 20 out of 53 selected workers (38%), but the 44 | 47 maximum permissible value (250 yg/g of creatinine), however, is exceeded in >4 ionly 11 subjects (21%). Finally, an elevation of stippled cells (over 4/40 fields) could be observed in only 13 subjects (25%). 10 DUP050056062 We looked for the existence of a possible correlation between these vari ous parameters and the air lead concentration. There is no Significant corre lation between the degree of exposure and stippled cells, ALA, and urinary ' coproporphylins (Figures 6, 7 and 8). The relationship between atmospheric i !lead and blood lead is better (r = 0.364), but it is not significant (p > 0.10) j ij j(Figure 9). r li ' Ii h j We also studied the relationship between urinary lead (which expresses the i> j ;i | jamount of absorbed lead) and the tests (ALA, coproporphyrins, stippled cells) | '! > j ! ] which should, in principle, reflect the significance of metabolic alterations j i? \produced by lead (Figures 10, 11 and 12). Only the urinary ALA level exhibits! j' 1 ] r, lasignificantcorrelation withthe urinary lead level (p < 0.01). j !j ! Moreover, we noted that the accuracy of the s emi-quantitative method for j ,, j i; I \ ; i \evaluating urinary coproporphyrins was jfar from being excellent. Figure 13 ] I | illustrates the relationship between the results of semi-quantitative determinf '! ] j J I ation performed by the same person and those of a quantitative determination ' ' : ! )i h ! carriedoutwith the same urinespecimens, The transverse barrepresents the j ! average quantitative determination for each category of semi-quantitative ! determinations. It is noted that although these averages correspond to visual j ' >1- t estimation, there is considerable overlapping of individual values. 4 ) III. Printing-plant Workers 42 As Figure 14 indicates, the degree! of lead accumulation is definitely les3 !4 I 44 than that among the storage-battery pldnt workers. 4b I The average blood lead level (50 wg/100 ml of blood or 115 pg/100 ml BBC), l however, Is above the norm (34.1 ng/100 ml of blood or 77.8 pg/100 ml BBC), but it is nevertheless within tolerable limits (70 pg/100 ml of blood or 11 DUP050056063 160 yg/100 ml SBC). Urinary excretion of ALA is not significantly increased. I ; `Measurement of the activity of the globular enzyme ALA-D, however, indicates ja definite reduction in enzyme activity^ among the exposed subjects (Figure 15)j (p < 0.01). This inhibition is not totally irreversible. Indeed, enzyme acti-r lI vation by heat and glutathione permits a restoration of enzyme activity. I \:> o r - 0,006 n * u-- *ao! M 17 in r<Hu 5 ' W U14 27? &. M _y. : M iii ---- * Jl-..... CO ;m .2 1 Lead in air (mg/ia3 ) Figure 6. (per 40 5 Correlation between number of stippled cells and air lead j coneentration. r . o,oot .** K n ,4> SBT % 1 H S't1DH4 60 60 m 4 \___i- S; `* i x J__:_L 40 0,100 0,200 0,300 41 42 Lead in air (mg/m3 ) 44 'i 44 Figure 7. Correlation between urinary ALA and air lead 4:'. concentration. ! 4(> 47 44 12 DUP050056064 CO au jcs cs 1400 1200 1.000 --- 1 r - 0,057 n (BOO aR UQ*8<UU 8 60 Htd 60 d H 600 400 200 0 1 .M _JL* 0,100 .a 16 ' .* 1 i1 l a. M * 0,200 (1300 JL. J 0,400 . J3 Lead In aijr (mg/in'3) .f Figure 8. Correlation between coproporphyrinuria and air lead concentration. 1 Figure 9. Correlation between urinary blood and air lead concentration. Under these conditions, the difference between the control subjects and exposed subjects is definitely reduced.; It is, however, significant (p < 0.05). I The degree of ALA-D inhibition is proportional to blood lead. There is actu- | ally an excellent negative correlation l(r - - 0.820) between blood lead and the \ logarithm of enzyme activity (Figure Id). It should be emphasized that even > ; :among subjects who were not occupationally exposed to lead, enzyme activity is! * inversely proportional to blood lead. 13 DUP050056065 Urinary lead (yg/g creatinine Figure 10. Correlation between urinary ALA and urinary- lead. \ Urinary lead (yjg/g creatinine) Figure 11. Correlation between number of stippled cells (per 40 fields) and urinary lead, | : 14 DUP050056066 woo Urinary lead (yg/g creatinine) Figure 12. Correlation between coproporphyrinuria and urin ary lead. coproporphyrinuria Figure 13. Correlation between quantitative and semiquantitative determination of urinary coproporphyrins. i 15 DUP050056067 s I9 ? -< *v 9 Figure 14. Comparison of blood lead, urinary ALA, and hemat ocrit in control subjects and;printers. Key: (a) blood lead (jig/100 ml BBC) ; (b) controls | (c) exposed; (d) hematocrit (%); (e) urinary 6-ALA (mg/g creatinine). i i--____ ___J i i (e)chaleur G.S.H. Figure 15. Comparison of ALAj dehydratase activity in control subjects and printers. Key : i (a) d-ALA-D (units/ml RBC/hr); .(b) controls; (c) exposed; (dj) normal; (e) heat. i i I j 16 DUP050056068 H T$ Blood lead (yjg/100 ml BBC) M r; Figure 16. Correlation between blood lead (logarithmic scale) H, and ALA dehydratase activity. Key: (a) controls; (b) exposed. r; 20 DISCUSSION i 2! In spite of the very strict preventive measures employed in the storage- :!j ! j-battery plant, the toxic risk is still! very considerable. It is inherent to V j j? | |the amount of lead handled in a form that readily releases lead dust. ! I The absence of a valid correlation' between the air lead concentration in j (the working area and the various biological tests is not surprising. Indeed, | 'during a study conducted in England among workers at a storage-battery plant, IWilliams [36] noted, by using individual samplers, that the exposure of worker^ \ \ ; J performing the same task could vary in a 1 to 4 ratio. These variations could I in probably be attributed to differences in personal working habits* j no ] 1 no In contrast to urinary excretion of ALA, coproporphyrinuria and the number 4i 44 of stippled cells are not a valid indication of the amount of lead absorbed. 45 ! 44 This agrees with the observations of other authors [2, 3, 6, 7, 20, 23, 33] who on 4b have emphasized the absence of specificity of these two tests and especially X:{ the lack of an early elevation in the number of stippled cells. Because of the numerous studies that confirm this conclusion, stippled cell studies for rou tine monitoring of lead workers should be abandoned in our opinion, The per- 17 DUP050056069 foraance of this insufficiently sensitive test provides a false sense of secur- ' ity. In Sweden, the law requires urinary ALA studies for regular monitoring of ,workers exposed to the risk of lead poisoning 15]. The analyses performed among the printers indicate, however, that a mod'- i ;erate exposure that is still insufficient for producing an exaggerated excre- j j tion of 6-aminolevulinic acid nevertheless can produce a biological alteration; in the form of an inhibition of the erythrocyte enzyme, 5-ALA dehydratase. ;j This enzyme is so sensitive to the action of lead that even subjects who are j not occupationally exposed exhibit an enzyme activity that is inversely propor tional to their blood lead. This observation, which confirms that made by Hemberg and Nikkanen [17], suggests that the determination of this enzyme activity perhaps may be a test that is too sensitive for monitoring lead-exposed workers. "I j This discovery, however, presents a significant problem: can it be con- j eluded that individuals could be exposed to a contaminant that produces inhi- I bition of enzyme activity even if the possible pathological consequences of i this inhibition are ignored? It should be emphasized that this problem falls | within the framework of industrial medicine. Lead is indeed a contaminant of \ our environment [12]. In addition, a toxic effect of lead among "normally exposed" individuals should not be excluded since this study demonstrates that a moderate amount of circulating lead is capable of inhibiting the synthesis of heme, i.e., a molecule that is indispensable for the production of numerous enzymes (catalase, tryptophan pyrrolase, etc.). How can we immediately fail to conclude that these very biochemical alter- | ations or others that are still unspecified are not reflected at the level of J various organs by certain functional disorders? Some recent studies conducted 18 DUP050056070 with individuals who were not occupationally exposed, and children in particu lar, tend to confirm the substantial foundation of such a hypothesis. Although ;injury to the central nervous system consecutive to acute lead poisoning in I children has been known for a long time, the possible deleterious effect of lop |lead concentrations on brain development has been recently pointed out, speci-j Vi> i } fically in reference to the observation of a slight elevation of blood lead inj H 1 4 mentally retarded children [27, 28] M I 3jt is known, moreover, that lead can be j i| transmitted from the mother to the fetus via the placenta and later to the j ii child via the maternal milk [10, 30], In addition, recent animal experiments j ; j 14 have demonstrated that the administration of lead to lactating mothers may prof i i' .( 2 i duce an inhibition of ALA-D in various tissues of newborn infants, including - ) -) | the brain. 'M 'f; u >4 48 > 4-0 41 44 44 44 4r> 4f. 47 19 DUP050056071 REFERENCES X. A.C.G.I.H.: Threshold limit values of airborne contaminants adopted by A.C.G.I.H. for 1969. .2 Albahary, C.: Porphyrin disorders in lead poisoning. Ardh. Mat, Prof,, Vol. 25, p. 495, 1964. 3. Basin, B.: Determination of delta-aminolevulinic acid in lead poisoning. Ardh, Mat. Prof., Vol. 24, p. 638, 1963. 4. Bonsignore, D., P. Calissano and 0. Cartasegna: A simple method for deter;) mining 6-aminolevulinic dehydratase in the blood: enzyme behavior in lead poisoning. Med. Lavoro., Vol. 56, p. 199, 1965. j 5, Danieli, G., M. Gadjos-Torok and A. Gadjos: A new biological test of lead ij poisoning: increase of urinary :and plasma levels of delta-aminolevulinif c acid. Pathol. Biol., Vol.9, p. 1481,1961. j .6 Davis, J. R. and S. L. Andelman: iOrinarydelta-aminolevulinic acid (ALA)I levels in lead poisoning. Ardh. Environ. Health, Vol. 15, p, 53, 1967. 7, De Bruin, A. and H. Hoolboom: Early signs of lead-exposure: a compata- j tive study of laboratory tests. Brit. J. Indust. 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Granick: The occurence and determination of delta- -aminolevulinic acid and porphobilinogen in urine. J. Biol. Chem., Vol. 219, p. 435, 1956. Meek, S. F., T. Mooney and G. G. Harold: Urinary porphyrins in lead poisoning. Induet, Med. Surg., Vol. 17, p. 469, 1948. j 1 :: y-i) 27. :i Millar, J. A., V. Battistini, R. L. C, Gumming, F. Carswell and A. Gold- j berg: Lead and delta-aminoluvulinic acid dehydratase levels in mentally retarded children and in lead-poisoned suckling rats. The Lanoet October, p. 695, 1970. i'. 28. Moncrieff, A. A. , 0. P. Roumides, IB. E. Clayton, A. D. Patrick, A, G. C. $7 Ml 4(1 4 ; 29. 44 44 -14 44 30. 4b r 7*y> 1 Renwick and G. F. Roberts: Lead poisoning in children. Arch. Die. ChUdh. , Vol. 39, p. 1, 1964. Ornosky, M.: Coproporphyrinuria and urine-lead findings: fifteen years of experience. Amer. Indust* Hyg, Assoc,, Vol. 29, p. 228, 1968. i Rosenblum, W. F. and M. G. Johnsoi: Neuropathologic changes produced in suckling mice by adding lead to the maternal diet, Avoh. Path., Vol. | 85, p. 640, 1968. 22 DUP050056074 31. Selander, S. and K. Cramer: Interrelationship between lead in blood, lead in urine and ALA in urine during lead work. Brit, J. Indust. Med,, Vol. 27, p. 28, 1970. Talman, E.L.: Standard Methods of Clinical Chemistry. Academic Press, I !> 33. ^i I Mj Ed. Seligson, New York, Vol. 2, 1958, p. 137. J' ! } Van Houte, G. and P. Code: Proposal of a new, simple, and more reliable test for determining lead poisoning. Arch. Mai. Prof. , Vol. 30, p. 675, 1969. r 34. Vincent, W. F. and W. W. Ullman.: The preservation of urine specimens for delta^-aminolevulinic acid determination. Clinical Chemistry, Vol. 16, .H p. 612, 1970. 35. Williams, M, K., E. King and J. Walford: Method for estimating objectively \ the comparative merits of biological tests of lead exposure. Bpit. Med4 r j.........'.......................... \ . : ? <J, > p 618 , 1968 '' ' J '. " \i 36. Williams, M. K., E. King and J. Walford: " " ' .... ' ' ' ......... . ' j I i An investigation of lead absorp-f | tion in an electric accumulator factory with the use of personal samplers, jj ' Brit. J. Indust. Med. , Vol. 26, p. 202, 1969. j 4> >n V) 4; J 41 >,; 44 14 4f:> V uil 4* 23 DUP050056075 ing about wornon-hazardous '>rosi$ stage, as mid be avoided. stone pneumoe is very short ways of evolunn developing, be. emphasized. olysis in vitro, iaUlies of dust hr bearing the erf with others, Archives des Maladies Profcss ionnelles, de Midecine du Travail et de Stcuriti Sociale (Paris), 1971, T. 32, n 6, Juin (pp. 453-464). comparative de divers tests biologiques d'exposition au plomb. Par'" |i-M. BASECQZ, R. LAUWERYS et J.-P. BUCHET (*) (Unite de Toxicologic Industricllc, UniversiU Calholiquc de Louvain, avenue Ghai>elle-aiix-Chgmps, 4, 1200 Bruxelles, Belgique.) Bien que le plomb ait 6U Tun des toxiques industrials les plus 6tudi6s, le diag nostic prScoce d'une impregnation saturnine avant rapparition des manifestations cllniques d'mtoxication reste un problfcme difficile. La plombdime et la plomburie sont g6nralement consid6r6es commc deux indices valables permettant d'apprdcier le degr d'expositioii [2, 21, 22, 35], mais ccartaines diffieultds techniques limitent leur generalisation pour le contrdle de routine des travailleurs. Depuis une dizaine d'anndes, plusieurs auteurs (5, 14, 31] ont souligne Tint4rt de la determination de la concentration urinaire de Yacide B-aminolduulinique (ALA) afin d'apprecier la rdponse metabolique de Torganismc. Ndanmoins, la recherche des hematies ponctuees constitue encore, la plupart du temps, la seule analyse pratiquec par les services medicaux du travail. Certains auteurs, toutefois, pr<5conisent revaluation semi-quantitative de la coproporphyrinurie [29]. Nous avons jugd interessant d'entreprendre une etude comparative des divers tests biologiques de contrdie d'exposition au plomb. Nous avons, en outre, etudie, de manure ddtaillSe, Taction du plomb sur une enzyme erythrocytaire, Yacide S-amimlivulinique di&ydratase (ALA-D). Plusieurs travaux rdeents [4, 7, 8, 18] indiquent en e&et que cette enzyme est facilement inhibee par le plomb. Mat&riel et m&thodes I- -- Description des techniques de mesuie. --> 1) Do s ag e d u p l o mb d a n s l 'a ir . -- Le pr6Ivement ` des poussi&res de plomb a ete pratiqud >ur filtres a l'nide d'un appareil Staplex High Volume Air Sampler xnod&le TFIA-2, muni de filtres TFA n 41. Apr6s mineralisation de rechuntillon, on eflectua le dosage du plomb par poiarographie [24]. 2) La p l o mb u b d b et la p l o mbmie furent determines par la methode colorimdtrique h la dithizone de Keenan et al. [19], 3) La n u mfir a t io n d e s H#.MATtF.s p o n c t u e e s fut pratfquge selon la technique classique de MansonSchwarz. 4) Lc t a u x d e c r e a t in in e fut determine par la methods de JalTd [10]. 5) ' Do s a g e d e s c o p r o p o r p h y r in e s u r in mr es . -- La recherche quantitative des coproporphyrincs urinaires a ete effectuge par fluorimetrie selon la methode de Taluuin [32]. La methode de Donatli [9] a etd utilise pour 1/e dosage semi-quantitatif. {*) Charge dc Rccherches, Foods National de la Recherche ScienLiquc, Ar c h . Ma l . Pr o f ., 1971, 32, n 6, Juin. 30 DUP050056076 454 J.-M. ASCQZ, rt. LAUWERYS ET j.~P, BUCKET 6) Do s a g e d e l 'a c id e 5-amt n 'OIJ^v u u n iq u e (ALA) d a n s l e s u r in e s . -- II tut realist scion unc modilien lion de la methodc de Crabccki (131. Cette nuSthodc consistc h cotnplexer dircctement 1'ALA avee l'ncdtylac^tone pour former du pyrrole lequel donne un complexe colord avee le rdactif d'EhrHch. Nous avons constntd qitc certains metabolites uriiutircs interferaient avee le dtfveloppcmcnt de la reaction colorltnctrique et cc d'unc mnniere variable d'unc urine ft.rmitrp. Cc phdnomenc cllmlmuilt alnsi la precision des lectures faltes par reference ii une courbc standard. L'udilitlon a I'urine il'un standard interne d'ALA nous a perinis de pulHcr cct Inconvenient. Pour clmque oclumtillon d'urine, 3 mesttres colorimetrlquos furent done prutiqutfes : 1 sur un dchantillon d'urine traltc normalcment scion Grabecki; 2 sur un 6chnntillcm auquei on n'ajoute pas d'acdtylncdtono (blanc) ; 3 sur un echnntillon auquei on ajoute 20 gg d'ALA. Apris soustraction du blanc, ii est aisd, par une simple risgle de trols* do determiner la concentration urlnulre en ALA. La technique est dicrite ci-nprds. Ractlfs: ac6tylac<6lone, tampon acdtnte (1 M, pH 4,6) contenant par litre d'eau 136 g d'acStatc de sodium 311,0 et 57 nil d'ucidcacdtique glacial, solution stock d'ALA : 10 mg d'ALA par 100 ml de tam pon aedtate; cctte solution est conservcc a 0 C, & l'nbri do In lumidre, solution standard d'ALA (20 gg/ml) prdparde en dilitant 5 fois la solution stock avee du tampon acetate, rdactif d'Jihrlich : dans tin ballon jaugd de 50 ml, on inlroduit 1 g de dimcthylbenzalddliycle et environ 30 ml d'acidc aedlique glacial; on ajoute ensuite 8 ml d'acide perchlorique h 70 p. 100 ct on portc a volume avee do racide ac.6t.ique glacial. Ces deux dernidres solutions sont prdparecs avant l'emploi. Procidi .* Pour cheque dehantillon d'urine, on prepare 3 tubes. Le tube A contlent 1 mi d'urinc, 0,2 ml d'acdtylacdtonc et 5,8 ml de tampon aedtate. Le tube B (blanc) contient 1 ml d'urine et 6 ml de tampon. Dans le tube C sont ajoutdsl ml d'urinc, 0,2 ml d'acdtylncetone, i ml de solution standard d'ALA et 4,8 ml de tampon. Lcs tubes sont bien ngitds et placds au bain-marie 5 100 C pendant 10 minutes. Aprfcs refroidlssemcnt, on ajoute 7 ml de rOactif d'Ehrlieh. Oil mdlange a nouveau et aprds 15 mn, on lit I'ubsorbunoe & 555 mix contre de l'eau. Lu qunntite d'ALA exprimoo en ug/ml d'urine est cgole 5 ooA - POB DOc -- POg x 20. Les rdsultats sont ensuile calculus en mg/g de erdatinine. Nous avons pu demontrer resistance d'une excellente correlation entro les rdsultats fournis par cette mdthode et ceux obtenus par la technique de Ma-uzernll et Granick [25] (fig. 1). 0 # 10 20 30 40 50 60 $ ALA urines (mg/gm erfeot.) >- (G modifie) . .Fig. 1 -- IWrrniiimtbn dr VAL.l itrfnntrc Correlaitimt entre la mdthodc de Maurcrall et (Iranick (y) et cello de (Vrubeeki mod Kiev ( x). 7) Ac t iv it y d e l 'e n z y me 8ALA-d ^iiy d na t a s f (ALA-D) d e s g l o b u l e s r o u g e s . -- L'activitd do l'enzymc 6rythrocytalrc ALA-D fut dctcrrtilnec en mesurant colorim6triquement la quantise de porphobillnogenc formic quand une prepani':<m de globules rouges h6molys6s est incubde h 37 F, pendant un temps d^tcnulmS en presence d'ALA (4). Le sang est recucllH sur hdparhic, centrirtigd & 0 C a 3 000 t/nm dans une centrifugcusc Sorvall BC,. Aprds dlimination du plasma, les globules rouges son t lav<6s par red ispeedon dims une solution isotonique do NaCl ii 0 C et centri fuges 5 nouvemt. Co proedde est r<Speto dcux fois; Les globules rouges sont ensuite resuspendus dans In solution saline dont on lit l'hdnmlnrr.de avant I'luimolysc des globules rouges purl couj;e- iationset d^cong^latlons mpidcssucccssives. C!cte prcpuriiHon de glolmles rouges hemolyses cst di lute 3 ou l fois (scion qu'il s'ngit de suje's expo ses au plomb ou non) avee du tampon INajHPD,- Nnll.PO*, 0,05 M. pH7 et un aliquot (en genera! 0.5 mlt'quivnJcnt a 0.125-0,160 ml dc sang total* est incUbd 37 C pendant 75 mn on presence dc 0,5 nil d'une solution aqueuse d'A .A ('JZ mV* et de 1 ml de tamoon phospliaic. Xous av->rj> ensuite suivi le pror.ede.de I'.onsigsi -re Ic --m de la periodc . t'fnei'ibation sont ajo'des '2 mi (Time solution d'ucide triehioraee*ijue a 10 p. !*> contenant du IfgCl3 0,1 M. Apres ce?0':f*ga?>on, 2 ml du surnageant sont melanges 5 2 ml du r(*actif d'Khrlith. Apres 15 nm. Vab.sorhanee de la solution coloree cst hte a 535 mg a 1'aUle d'uu spcctrofdiotonu'trc Zeiss P.MQ 11. Vn bbmc-tvssu est oRitiqitt' pour phaque analyse. II est eonstitue du melange dMarah 'tlrin DUP050056077 tr TESTS BIOLOG1QVES D'EXPOSITION AU PLOMB 455 selon une modiVLA iivcc race- Nous mms 'i^n color! mE- oivcision dcs -v|,r,,cs i habitue! danslcquel ic substmt a etE remplucE par un 'volume Equivalent de tampon* La selection dii temps d'lnctibation et du volume dc sang a ete eJTec tuEo sur lu base d'cxpErlences prElimlmiircs (fig, 2 et 3) deter minant les conditions dans Icsquelics la qumititE do porphobilinogenc formEo Etuit Iin&iircment proportionnello ait volume do sang et ft la durEe do la periode d'incubation. 12,5 M0,0 1 concentration > r7%icEty te de ' ,v ai de tarn- lA '20 ag'-ml.? y `- o.n t ',1 i rp i 4^0 0.0 r*>? ? a !? >{ >n. y ml " s <roi'* rvy* A 6 *05 v 0,15 (U $25 0,3 Quontftfc dt song (ml) 0 15 30 45 50 75 ,fc (f Ttmp* d'Incubation {minutes) / Fig. 2. -- Influence de la quantify de sang sur la quantity dc porphoblllnogEne XormEe ft purtir de FALA. 90 vr -ette "'R.-vr \\\ do ' .r '-:Rr*c : rl.A `A- <r-'Kvuv* . ' les : .....V'**iS. S..V J.1' .(ipoH'c ; - - . on*v ev>p- ' 'it iicnvrn! * -rV'CTHM' tic ' \ \*7 n*\r * \ < ;. tv r*s 'vu'^.rc 'aU->n Fig. 3. -- Influence du temps cTincubation sur la qitnntite dc prophobiliuogEne formEo ft partir de l'ALA (une qinmtlte do globules rouges hemolyses Eqiuvalcnte ft 0,125 ml de sang total fut sElectlonnEc pour Cette experience). En plus de la mdthode ordinaire decrite ci-dcssus, PactivitC enzymatique fut aussl dEterminEe sur deux Echnntillons du mEme sang. Tun preincubE 5 mn uCO0 C, t'autre prEincttbE 20 nm ft37C en prEsence de glutnthion (GSfl) 10 nvM. Vu l'iniportante activation de l'enzymc par la chaleur et le GSH, 1c volume de sang utilise Cut reduit rcspcctivement ft 0,2 et 0,1 ml do sang diluE 3 ou 4 fois. Les resultnts cxprimds en unitEs d'enzyme par ml de globules rouges et par heure sont calculEs en appliqiumt la formulc suivante : (DO,, --DO,,)X 1 000 JZSjk hEmatocrite (%) x volume sang (ml) oil DO,, est Tabsorbance apres 60 mn d'incubution et DO, eelle du blanc. L'activit6 de la ALA-D dcs globules rouges fut mesurde dans les 12 heures sulvant la prise de sang. La determination de TALA et dcs coproporphyrines urinaires fut pratiquEe dans les 24 heures suivant la collcctc dcs urines. Les urines servant au dosage d*ALA furent maintenues acidcs par addition d'acidc tartrique [34], ceiles servant au dosage des coproporphyrines furent conservees sous %a8C03 [16]. Les echantillons de sang et d'urine furent gardes h 4 C jusqu'au moment de 1'analyse. II. -- Description des populations. -- 1) Po p u i.w io 'n c o n t *i6i.k . -- Rile comporte 22 sujets mnsculins non professionnellcment exposEs au piomb dent la distribution en tonclioo de Tftge est lu suivante : ie plus jeunc ft 20 ans et le plus age .k * a ns. La moyenne et la mediune sont identiques : 33 ans. 2) Poi*vr..\TtoN Kx"osr-.r-: av * p l o u ii. -- File est composer de 53 ouvriers d'ane usinc d'uccumulateurs oft seuls ont pu etre pratiques les exameiisd*urinc et lu numeration dcs hEmalies ponctuees et de 25 ouvriers d'une impriiuerie oft nous avtms ju prutiquer les exnntcns s inguins et certains tests urinuircs. Pour certoines raisons techniques (urines non fratehes, perte de rEchantiilon durunt l*nnnlyse...), toutes les nnalyses prevaes n'ont pus EtE pratUmEes che/. tous les ouvriers selectionnOs (voir resultats). DUP050056078 456 jr.-JVA BASECQZ, .JR, LAUWERYS JET J.-P, BUCKET Resultats. I. -- Population temoiri. -- Les inoyenmes et erreurs standard des divers parametres mesurds chez les sujets non professionncllement exposes au plomb sont pr4sentds ci-dessous : plombdniie ................................. ........... plomburlc ........................................ ALA urinnire ........................................................... coproporphyrincs urlnalres.................... hematocrite,................................ .............................. ALA-D dcs globules rouges (iwcthode ordinaire sans activation) .................................................. 34.1 1,8 gg/100 ml de sang 77,8 4,3 jxg/100 ml de globules rouges 35.2 2,8 ixg/g creatinine 4,6 0,3 g/g crdatininc 23 2,2 jxg/g creatinine 44,5 0,5 % 59 4 U/nvl G.R./heurc. $ ALA urines mg/t rng/gm c 20 20 10 10 v 0 -- .l --:-------- p 0 VL-Jl #_% /Plomb urines jig/l pg/gm creot. 100 r~ 100 r 50 50 00 Copro urines , pg/l pg/gm erect. 100 - ----------------- 100 50 so 0L 0 Fig. 4. Comparateon <fes (mix nrfrmfres de VALAt de plomb et de coproporphyrities exprini& par litre d'urine et par g de creatinine uruudre (sujets contrOles). tassage des plaques . ........ *. tarttnage et cassage dcs plaques encuvage ..................................... coulee dcs plaques ........... basculage des mnlaxcurs ........... stockagc des oxydes de plomb . Ainsi que Tont judicicusement sou* lignO Van Houle et Code [33], il est plus logique d'exprimer le taux de plombdmie par volume de globules rouges plutOt que par volume de sang total. En effet, comrae plus dc 95 p. 100 du plomb cireulant sont fixds aux globules rouges, cettc forme d'expression tient compte des diffe rences d'hdmatocrite d'un sujet k i'autre. Nous exprimons d-autre part la concentration de divers metabo lites urinaires par g de creatinine. Une correction est ainsi introduitc pour le degre variable de dilution des urines recueillies k un moment quelconque de la journec. La compa* raison du degre de dispersion des resultats exprimes par volume d'urine et par g de creatinine (fig. 4) continue VinterSt d'une telle correction. Cette conclusion rejoint celle de Gibson [11]. II. -- Ouvriers de rusine d'accumulateurs. --r Les vaieurs moyennes des taux de plomb dans Tair dose mensuellement durani 3 mois k differents postes dc travail de rusine d'accumulateurs sont prdsentes ddessous : 0,321 mg/m1 0,257 mg/m' 0,244 mg/ma 0,232 mg/m1 0,125 mg/m* 0,097 mg/in* DUP050056079 divers paraib sont pre- s rouges isemcnt sou [33], il est le taux de de globules volume de nnc plus de ev'-'vit sont forme des dtffe' sujet A `XGre oart rs mdtaboc'vtinme. ;7`roduitc ' dilution ' moment La compa re rsion des me d'uriue t) couflrme etiou. Cette Gibson [11], sine tFacou> moyennes ; I*air dose nols a difYd- (lc Fusine vsentes ei- ni ;n* n>ii m1 ui^, - u' : nig/m* * mg nf TESTS BIOLOQIQUES D>EXPOSITION AtJ PLOMB 457 ^exposition au plomb est done relativement Importante pulsque 4 postes de travail occupant deux-tiers des ouvriers sdiectionnds prdscutent des taux dc plomb dans Fair supericurs A la llmite tolerable (0,2 nig Pb/m* air, A.C.G.I.H. 1969) [1J* L'importance de cctte exposition sc reflete dans tine nette dldvation des valours moyennes des divers paramdtres d'imprcgnation saturnine (fig. 5). Des 47 plomburics mesurdcs, aucune n'dtait dans les limites de la . normale (< 50 pg/g creatinine) et 23 (50 p, 100) ddpassaient la llmite permissible (150 pg/g creatinine). 35 sujets sur 51 (68 p. 100) prdsen* talent des taux urinaires dc TALA superieurs A la llmite sypdrieure de la normale (< 8 mg/g erdatinine) et 16 sujets (31 p. 100) exerdtalent une quantite d'ALA supdricure A la limite toldrable (20 mg/g erdatinine). X Les coproporphyrines urinaires se trouvaient anormalement augmentdes Fig. 5. *-- Comparalson des taux urinaires de plomb, coproporphfjrines el de ALA chez lea sujets contrdlcs et les ouvriers d*une uslne d'nccumulatears (In ligno vcrticale represente l'erreur standard). (taux supdrieur A 100 pg/g de erda tinine) chez 20 des 53 ouvriers sdlec- tionnes (38 p. 100), mais la valcur maximalc permissible (250 pg/g de erdatinine) n'dtait cependant ddpassde que chez 11 sujets (21 p. 100). Enfm, une dldvation des hdmaties a ponctuations basophiles (supdrieur A 4/40, champs) ne fut observde que chez 13 sujets (25 p. 100). Nous avons recherchd Fexisteiice d'une correlation dventuelle entre ces divers paramdtres et le taux de plomb dans Fair. II n'existe aucune correlation significative entre le degrd d'exposition et les punctuations basophiles, FALA et les coproporphy- rines urinaires (fig. 6, 7 et 8). La relation entre plomb atmosphdrique et plomburie est meilleur (r = 0,364), mais non significative (p > 0,10) (fig. 9). Nous avons dgalement dtudid la relation entre le plomb urinaire (re fidtant la quantitd de plomb absorbee) et les tests (ALA, coproporphyrines, ponctuations basophiles) qui deyraient, en principe, refldter Fimportance des alterations mdtabo- liques produites par le plomb (fig. 10, 11 et 12). Seul le taux de FALA urinaire prd- sentc une corrdlation significative avec le taux dc plomb urinaire (p < 0,01). Nous avons, de plus, constate que la precision dc la mdthode semi-quantitative devaluation des coproporphyrines urinaires dtait loin d'dtre excellente. La figure 13 filustre la relation entre les rdsultats du dosage semi-quantitatif pratiqud par la meme personne et ceux du dosage quantitatif exdcutd sur les indines urines. La barre transvcrsale reprdsetitc la moyenne des dosages quantitatifs pour chaque categoric des dosages semi-quantitatifs. On constate que, si ces moyennes corres pondent A ['appreciation visuellc, ii existe un important chevauchement des valeurs individucllcs. III. -- Ouvriers d'imprimerle. --vComme Findique la figure 14, le degrd d'imprdgnation par le plomb est nettement moindre quo celui des ouvriers dc Fusine d'accumulateurs. La plombcmic moyenne (50 p.g/100 mi sang ou 115 jxg/100 ml O. H.) est cepen dant supdricurc A la normale (34,1 |xg/100 nil sang ou 77,8 jxg/100 ml G.R.), mais reste ndanmoins dans les limites tolerables (70 j^g/100 ml sang ou 160 |xg/10Q ml G.R.). L'excretion urinaire de FALA n'est pas significativement augmentde. Cependant, la mesure dc Factivitd de Fcnzyme globulairo ALA-D met en dvidencc une nette reduc- DUP050056080 458 J.-M. BASECQZ, R. LAUWERYS ET J.-P. BUCIIET r 0,006 20 '.* IS wo r iTM i- l> to |~ 10 .(-- t 5- K * 8 so Im 0 - :m * -JUS nit J *i 20 0,100 JL 0,200 0,300 0J,400 ^ 0 flf Ptomb dons olr (mg/m) r o,ooi i" *1 M M Lj l s 0,100 0,200 0,300 g Ptomto dons olr (mg/m?) * rJ 0,400 7 q iioo-h t -0,057 - E 1.000 H 600 ft Fig. 6. -- Correlation entre 16 nombrc d'h6- <00 n ' inaties ti ponctuations bnsophtlcs (par 40 champs) et le tmpC do plonib dans Fair. ft ' "1 i! (- c * * Fig. 7. -- Correlation entre FA-LA itrlnaire ;:i 0,100 0,200 GpOO 0,400 y et le taux de plonib dans Fair. , ;ii PUmb dons sir (mg/in3) i< Fig, 8, -- Correlation entre la copreporpliy-. rlnurle et le taux de plonib dims Fair. Fig. 9. --* Correlation entre la plomburle et le taux d.e plonib dans Fair. tion de l'activit^ enzymatique des sujets exposes (fig. 15) (p <: 0,01). Cette inhibi tion n'est pas totalcment irreversible. En efTet, 1*activation dc 1'enzymc par la chaleur et le glutathion perrnet une restauration de ractivity enzymatique. X Dans ces conditions, la difference sujets contrdles -- sujets exposes est nettcment rdduite. Ellc rcste cepcndant signifleative(p < 0,05). Le dcgrd d'inlvibition de la ALA-D est proportionncl h la plomb<$mie. n existe en efTet une.excellence correlation nega tive (r * -- 0,820) entre la plombdrnie ct le logarithme de l1activity enzymatique (fig. 16). II importe de souligncr que, mCme chcz I.es sujets non professionnellcmcnt exposes au plomb, l'activite enzymatique est inversement proportionnellc h la plombdmie. DUP050056081  1* K i -4---- 1 0,400 7 nombrc d*h6phUc;s (par 4Q mb clans Voir. '.MiA urlnulre u fair. ruproporphy* * dans l'air. a ptomburlc ns Fair. (*tte iniiibi'mc par la ue. nettcment elaALA-D '* tiim negaizymatique imeUcment a la plom- TESTS BIOLOGIQUES B*EXPOSITION AU PLOMJB * w' i > eo t 5 *- to 0 209 300 ^ Ptomfe urine* (jjg/gm ertat) 459 Fig. tO. * -- Correlation entre 1*ALA urinaire et la ploniburle. ^ Fig. 11. -- Correlation entre le nombre d'h6mattes & ponctuations basophileS (par 40 champs) et !u ptomburlc. ' Fig. 12. -- Correlation entre la coproporphyrlnurie et la plomburJe. Fig. 13. --- Correlation entre le dosage quan- tttatif et semi-quantltatif ties copropor- phyrlnes urtnnircs. t Fig. 14*-----Comparalson de la plomhtmic, dc l*ALA urtnairc et de Vlicmatocrilc chez lep sujets contrdles et les ixnprimeurs. |u % 1.000 * 400 f 200 13 [ 11__ Sk 4t: -J1 r _U / Copreporphyrinurla mi-quantitative 0 100 200 300 400 Plomb urines (jig/gm ertat.) 500 11 0 100 200 300 400 Plomb urines (jig/gm ertat.) 500 12 s 6. mi -I m lyi/tfi 5 tx'.j i. n m cm 14 DUP050056082 460 J.-M. BASECQZ, R. LAUWERYS ET J.-P. BUCHET L. J-----~----J---____ --I. -J 25 50 75 WQ 125 ISO Js^ \q M 6 Ptombtm}* (jig /WO ml 0.(1.) "Fig. 15. -- ComparaUton de tacltoiU de , Venzyme irylhrocytalrc ALA-ddliurtratas 16 ordinaire iSm I---- ----G.S.H. chez Jcs sujets contrOles et lcs imprimcurs. AFig. 16. -- Correlation entre la plonib<5mie (dchelle logaritlunlquc) et I'actlvitd de renzym odrytliroeytaire ALA-cUHiyciratnsc. 15 Y. Discussion. Malgrd les mesures de prevention trds rlgoureuses appliqudes dans Fusine d'accumulateurs, le risque toxique reste encore trds important. II est inherent k la quantitd de plomb mauipuld sous une forme libdrant facilement des poussidres plombbfdres, Vabsence de correlation valable entre la concentration de plomb dans Fair au niveau du poste de travail et les divers tests biologiques n'est pas surprenantc. En efFet, lors d'une dtude pratiqude en Anglotcrre chez des ouvriers d'une usine d'accumulateurs, Williams [36] a constate, par Fempioi d'dchantillonneurs individuels, que Fexposition des ouvriers effectuant le mdme travail pouvait varler dans un rapport de 1 k 4. Ces variations Slant probabiement attribuables k des differences dans les habitudes personnelles de travail. Contrairement k Fexcretion urinaire de FALA, la coproporphyrhmrie et le taux d'hdmaties ponctudcs ne sont pas un reflet valable de la quantity de plomb absorbee. Ceci rejoint les constatations d'autres auteurs [2, 3, 6, 7, 20, 23, 33] qiti out soulignd Fabsencc de spdcificite de ces 2 tests ct surtout le manque de prdcocitd de Fdldvation du taux d'hdmaties ponctudcs. Yu les nombreux travaux qui conflrmcnt cette conclu sion, la recherche des hematics ponctudcs pour le contrOle de routine des travailleurs du plomb dcvrait, il notre avis, etre abarulonnde. L'exdcution de ce test trop peu sensible donne une fausse sdcuritd. En SuMe, la Idgislation exige la recherche de FALA urinaire pour le eontrOle regulier des travailleurs exposes au risque saturnin [15]. Les analyses pratiqudes chez les imprimcurs indiquent cependant qu'une expo sition moderde encore insufFisante pour provoquer une exerdtion'exagdrde d'acide S-aminoldvuliniquc peut ndanmoins produirc une altdration biologique sous la forme i DUP050056083 b'r Ar-aildMiiuydmraiaHse 16 imprimeur&. plombemie 'itcUrile dc ieliydrakise. 'usine d'accun t k la quanJieres plomhidans Fair au (irenante. En usine d'accus individuels, trier dans un es differences ie et le taux mb absorbde. ont sbulignd dc rdldvation cette condu it travailieurs est trop pea recherche de risque satur- ^u'une expo se (Facide ">us la forme 1^,'* TESTS BIOLOGIQUES D'EXPOSITION AU PLOMB 461 d'une inhibition de Fenzyme drythrocytaire $-ALA-ddhydratase. Cette enzyme est teliement sensible a Faction du plomb que mime les sujets non professionnellement exposes prdsentent unc activity enzymatique inversement proportionnelle k leur plombdmie. Cette observation, qui confirme celle de Hernberg et Nikkanen [17] sugglre que la determination de cette activity enzymatique constitue peut-etre un test trop sensible pour le contrite des travailieurs exposes au plomb. Cette ddcouverte pose cependant un probllme important : peut-on toldrer que des individus soient exposes k un contaminant qui entratnd une inhibition d'une activite enzymatique meme si on ignore encore les consequences pathologiques dventuelles de cette inhibition ? II faut souligner que le probllme ainsi souleve ddborde le cadre de la Mddecine du travail. Le plomb est en effet un contaminant de iiotre enyironnement [12]. Aussi une action toxique du plomb chez les individus normalement exposes n'est pas k exclure puisque la presente etude ddmontre qu'une quantity module de plomb circulant est ddjl capable d'entraver la synthase de I'hlme, molecule indispensable k Felaboration de nombreuses enzymes (catalase, tryptophane pyrrolase...). Comment, d^s lors, ne pas dmettre l'hypothlse que ces mimes alterations biochimiques ou d'autres encore imprdcisdes ne se traduisent au niveau de divers organes par certains troubles fonctionnels ? Quelques etudes recentes pratiqudes chez des personnes non professionnellement exposdes, notamment des enfants, tentent de confirmer le bien fondd d'une telle hypothlse. Si Fatteinte du systlme nerveux cen tral consecutive k l'intoxication saturnine infantile aigul est connue depute longtemps, l'influence ddldtire possible de faibtes concentrations de plomb sur le ddveloppement du cerveau a dtd rdeemment soulevde, suite notamment k l'observatioh d'une ldglre ellvation de la plombdmie chez des enfants arridrds mentaux [27, 28}. On salt, en outre, que le plomb peut Itre transmis de la mire au foetus via le placenta et plus tard k l'enfant via le.lait maternel [10, 30]. De plus, des expdrtences animates rdeentes out ddmontri que Fadministration de plomb a des mires allaitantes pouvaient entratner, chez les nouveau-nes, une inhibition de la ALA-D de divers tissus dont le cerveau. Rlsuhil, Notre etude confirme la validity dK la determination de Facide S-aminolevuli- nlque urinaire pour le contrdle des travailteurs exposes au plomb. Sa correlation avec la plomburie est nettement supdrieure k celle de la coproporphyrinurie. La recherche des hematies k ponptiiatibns basopjriles rt'offre aucun intdret. / \' . 0 Notre travail ddmontre cependant qu'uneMnhibition significative de l'enzyme globulaire acide 8-aminbfdvulinique dehydratase, pent survenir sans eldvation du taux urinaire de Facide 8-aminoldvulinique. La ddtermination de Factivite de cette enzyme constitue done un test trls sensible et trls precoce d'exposition au plomb. Mime chez les sujets non professionnellement exposds au plomb, Factlvltd enzy matique est fonction de la quantitd de plomb circulant. Cette observation confirme le rdle du plomb comme agent polluant de notre environnement. DUP050056084 462 J.-ikT. BASECQZ, i?. LAUWERYS ET 7.-P. BUCHET Bibliographic. Til A.C.G.I.H. : Threshold limit values of airborne contaminants adopted by A.C.G.I.H., for 1909. ' *12] Al b a h a r y , C. : Les troubles porphyriquesdans le saturnisme. Arch. Mai. Prof., 1964, 25, 495. MS] Ba s in , IS. : Le dosage de 1 ncide delta-amino-l6vulinique dans le saturnisme. Arch. Mai, Prof.. 1903, 24, 638. v t x [4] Bo n s ig n o r e, D., Ca l is s a n o , P. e. Ca r t a s e g n a , C. : Un semplice metodo per la determinazione della 8-aminolevulinico deidratasl ncl sangue : comportamento delPenzima nelPintossicazione saturnine. Med. Lauoro, 1965^50, 199. x [5] Da n ie l i, G., GApjos-ToROk, M. eFGADjos, A.: Un nouveau test biologique du saturnisme.: augmen- tation^du taUX urinaire et plasnmtique de Vacide delta-amino-16vuIinique. PalhoL Biol.t 1961, [6] Da v is , J. R. and An d e l ma n , S. L. : Urinary delta-aminolevulinic ncid (ALA) levels in lead poison ing. Arch. Environ. Health, 1967, 16, 53, [7] p e Br u in , A. and Ho o l bo o m, H.; Early signs of lead-exposure : a comparative study of laboratory . " tests. Brit. J. Indust. Med.. 1967, 24, 203. -- [8] d e Br u in , A. : Effect of lead exposure on the level of delta-aminolevulinic dehydratase activity. Med. Lavoro, 1968, 69, 411. [9] Do n a t ii, W. F. : A simple portable apparatus for the semi-quantitative determination of the copro porphyrin content in urine. Arh. gig. tada, 1956, 7, 77. [10] Fe r n , V. and Ca r p e n t e r , S. J. ; Developmental malformations resulting from the administration of lead salts. Expl. Mol. Path., 1967, 7, 208. [11] Gib s o n , S. L.M., Ma c k e n zie , J. C. and Go l d b er g , A. : The diagnosis of industrial lead poisoning. Brit. J. Indust. Med., 1968, 25, 40. [12] Go l d s mit h , J, R. and He x t e r , A. C. : Respiratory exposure to lead : epidemiological and experi mental dose. Response relationship. Science, 1967, 158, 132. '--[IS] Gr a b e c k i, I., IIa d u c h , T. 4Ml Ur b a n o w ic z, H. : Die elnfachen Bestimmungsmethoden der Delta- Aminoiavulins&ure im Ham. Irit. Arch. Gewerbepath. Gctoerbehyg., 1967, 23, 226. . A [14] Ha e g e r -Ar o n s ex , B. : Studies on urinary excretion of delta-aminolevulinic acid and othemiaem precursors in lead workers and lead Intoxicated rabbits. Scand. J. Clin. Lab. Invest1969, 12, suppl. 47. [15] Ha e g e r -Ar o n s en , B. : Evaluation of two methods for measuring delta-aminolevulinic acid in urine. Scand. J. Clin. Lab. Inu., 1970, 25, 19. fl6] He n r y , R. J. : Clinical chemistry--Principles and Technics, Harper and Row Publishers, 1065. [17] He r n b e r g , S. and Nik k a n e n , J. : Enzyme inhibition by lead under normal urban conditions. The Lancet, January 1970, 63. [18] He r b n e r g , S., Nik k a n e n , J., Mf .l l in , G. and Lil iu s , H. : Delta-aminolevulinic acid dehydrase as a measure of lead exposure. Arch. Enoiron. Heaith, 1970, 21, 140. [19] Ke e n a n , R. G., By 'e r s , D. I-L, Sa l t z ma n , B. E. and Hy s l o p, F. L. : The USPHS method for determining lead in air and in biological materials. Amer. Indust. Hgg. Assoc. J., 1963, 24, 481. [20] Ke h o e, R. A.: Industrial lead poisoning, in Industrial Hygiene and Toxicology, volume II, p. 941. Interscience Publishers, 1963. 21] Ke h o e, R. A. : Normal metabolism of lead. Arch. Environ. Health, 1964, 8, 232* 122] Keh o r , R. A. : Metabolism of lead under abnormal conditions. Arch. Environ. Health, 1964, 8, 235. 23] Kin o , E. and Th o mps o n , A, R.: The measurement of lead absorption in industry. Ann. Occup. Hug., 1961. 3, 247. .24T Ko l t h o f f , L. M. and Lix g a n e, J. J.: Polarography, 2nd ed... Interscience Publishers Inc., New York. f25j Ma u z e r a l , D. and Gr a x ic k , S.: The occurence and determination of delta-aniinolevuiinic acid and porphobilinogen in urine. J. Biol. Chem1956, 219, 435. [26] Me e k , S. F., Mo o n e y , T. and Ha r o l d , G. C.: Urinary porphyrins in lead poisoning. Indust. Med. Surg., 1948, 17, 469. [27] Mil l a r , J. A., Ba t t is t in i, V., Cu mmin g , R. L. C,, Ca r s w e l l , F. and Go l d b e r g , A.: Lead and delta- aminolaevulinic acid dehydratase levels in mentally retarded children and in lead-poi$oned suck ling rats. The Lancet, October 1970, 695. [28] Mo n c r t f f f , A. A., Ko u mid l s , O. P., Cl a Yt o n , B. E., Pa t r ic k , A. D., Re x w ic k , A. G. C. and Ro ber t s , G. E. : Lead poisoning in children. Arch. Dis. Childh., 196-1, 39, 1. [29] Or n o s k y , M.:: Conroporphyrinuria and urine-lead findings: fifteen years of experience. Amer. Indust. Hua. Assoc., 1968, 29, 228. [30] Ro s e n b l u m, \V. F. and Jo h n s o n , M. G. : Neuropathologic changes produced in suckling mice by adding lead to the maternal diet. Arch. Path., 1968, 85, 640. [31] Se l a n d e r , S. and Cr a mer , K. : Interrelationship between lend in blood, lead in urine and ALA in Urine during lead work. Brit. J. Indust. Med.. 1970, 27. 28. / [32] Ta l ma n , E.^ L. :^Standnrd^methods of clinical cneuiislry.-^d.)SeligsonT)Acaaemic Press, New York, ^ [33] Va n Ho u t e, G.^Co c l e, P. : Proposition d*un nouveau test simple plus fidele de depistage du satur nisme. Arch. Mal. Prof., 1969, 30, 675. [34] Vin c e n t , W. F. and Ul l ma n , \Y. W.: The preservation of urine specimens for delta-aminolevulinic acid determination. Clinical Chemistry, 1970, 16, 612. [35] Wil l ia ms , M. K,, Kin g * E. and Wa l f o r d , J. : Method for estimating objectively the comparative merits of biological tests of tend exposure. Brit. Med. J., 1968, 618. [36] Wil l ia ms , 31. K., Kin g , E. and Wa l f o r d , .1., An investigation of lead absorption in an electric accumulator factory with the use of personal samplers. Brit. J. Indust. Med., 1969, 26, 202. DUP050056085 /. 'J * i.. / -girti'rim,il _ aj^o^ss-igL.-arf^ . n:r fi.I.H.. for I960. 1901, 25, 493. U. Mai. Prof., 1963, * la deUTinhitizione ndl'inlossicnzione turnismo : augmen ts/W. liiol., 1961, vHs in lead poison* mdy of laboratory hydrntase activity. at ion of the coproUu` administration rial lead poisoning. logical and experi- r(1ih. mien der Delta* id and otherhuem Invest., 1909, 12, iiiuii* acid In urine. `ublinhers, 1965. u conditions. The arid dehydrasc as J*riS method for ./.. 1903. 24, 4gt . volume iI, p. 941. "M. 1901, 8, 235. .Vnn. Ckcup. Jfyg., 'y/m\, N*ew York. l*v ntinic acid and hulust. Med. : I.*a<l and dcltaad-poisoned suck* x. A. G. C. and e, Anier. Indust. Mtcldintf mice by trine ami ALA in l Vy-w. New York, pida^e <|a sntiir*'..n.iinolevulir.ic the comparative l in an26e, le2c0t2r.ic TESTS BIOLOGIQUES D'EXI'OSITIOS AU PLOMB 463 Summary. Comparative study df various, biological lead exposure tests by J**M. BASECQZ, R. LAUVVERYS and J.-P. BUCHET (Bruxelles, Belgique). //\\ ./ \ Our study, confirms the validity of the'^determination of urinary 3-aminolevulinic acid for the control of lead-cxpbsed workers. Us correlation with urine-lead is definitely superior to that of coproporjmyrinuria. Stippled cells research offers no interest. Our work shows howeydr that a signi ficant inhibition of the globular enzyme $-aminolevulinic dehydratase,'can occur without increase in the urinary 3*aminolevulinic acid level. Thereforeytnc determination of the activity of this enzyme proves to be a very sensitive and veruearly test of lead exposure. Even in subjects non occupationally exposed to lead, the enzyme activity depends on the amount of circulaling lead. This observation confirms the part of lead as a factor of pollution. (Arch. Mat. Prof.t 1971, $2, 453-463)> i \ DUP050056086