- Email: [email protected]
Polarographic determination of lead in whole blood using the anodic stripping polarography technique
BIOCHEMICAL
MEDICINE
Polarographic Using
the
4, 89-96
( 1970)
Determination Anodic
Stripping
D. ROOSELS Fund of Occupational
of
Lead
Polarography AND
in Whole
Blood
Technique
L. HEULENS
Disease, Ministry for Social Care, Brussels 1, Belgium Received
January
7, 1970
The determination of lead in blood is one of the most valuable criteria for the determination of an exposure to lead poisoning, because it is a specific indication of the presence of the toxic agent in the circulatory tract ( 1). Many methods have been proposed to determine the lead content of whole blood (2-7). All of them must resolve two main problems: ( 1) the extraction of lead from the red blood cells ( RBC ) and (2) accurate and specific determination of lead ions among many interfering substances. An attractive method was described by Nylander and Holmquist (4) but the reproducibility of their results remains problematic. Detailing the problems related to the determination of the lead content of whole blood we distinguish three main steps: ( 1) the mineralisation of blood; (2) the extraction of lead ions from the mineralisate into a polarographic medium; (3) the study of an appropriate polarographic medium. We found a simple and reliable method whose principles may also be applied to the determination of lead in other biological materials. METHOD
Minmalisation Fifteen milliliters of nitric acid (D = 1.4, treated with dithizone) are added to 5 gm of whole blood ( taken on versenate ) in an Erlenmeyer flask of 200 ml with a wide neck. The flask is covered with a watch-glass, and the mixture is heated gently on a hotplate until a clear and transparent solution is obtained. After cooling, 15 ml of perhydrol are added in 3ml portions. After each addition the solution is heated until no more foam is produced and, after cooling, a new fraction of perhydrol is added after which diluted nitric acid (l/5 with water) and 5 ml of the ammonium citrate solution are added. 89
Extraction The resulting solution is neutralised with ammonia ( 1~ .I: 091 ) ~ using phenol red as indicator, and transferred quantitatively into a separatory funnel of 250 ml. The Erlenmeyer flask is rinsed with 25 ml buffer solution. These are quantitatively transferred in the separator): funnel. The solution is then immediately shaken with 10 ml of 0.003% dithizone solution (in chloroform) for at least 30 seconds. The chloroform layer is transferred to another separatory funnel, and the first extraction is rcpeated twice. The combined chloroform layers are shaken with 10 ml 1 N HCl. The pH of this HCl layer must remain acid after extraction; if not, add a few drops of concentrated HCl and shake again. The HCl layer is transferred to a volumetric flask of 50 ml and brought to volume with KC1 0.125 N. Polarogra ph y Twenty-five milliliters of this solution are transferred to a polarographic cell and degassed with nitrogen for 15 minutes. After the anodic stripping polarography technique, the hanging mercury drop is saturated with lead ions (for 3 minutes under stirring and 1 min without stirring) at a constant negative voltage (-0.75 V starting voltage) and a constant temperature ( 25” ) . The anodic stripping current is rneasured into a linearly increasing voltage range of +l V (from -0.75 to +0.25 V) at a sensitivity of 2. lo-” A/mm. The lead wave appears at -0.35 V. Apparatus
and Products
All the products are of the analytical grade. All glass material is of the Pyrex or jenaer type, treated with dithizone to remove all traces of lead contamination. Double-distilled water is always used. Blood is taken on a polystyrene container on versenate 1 mg/ml. Ammonium citrate solution: 20 gm 100 ml water, treated with dithizone as explained for the buffer solution. Bufier solution. The solution consists of 236 gm of ammonium citrate dissolved in 150 ml of concentrated ammonia (D = 0.91) and diluted to 500 ml with water. Add 10 gm KCN and 5 mg Na$O, to this solution. The solution is transferred to a separatory funnel and shaken with two 20-ml portions of dithizone in chloroform (30 mg/hter) to remove any lead which may be present in the reagents, Excess dithizone is washed out with two 30-ml portions of chloroform. To this solution 1 liter of concentrated ammonia is added. Perhydrol: 30% solution of hydrogen peroxide. Polarograph Methrom-Herisau type E 261, anodic stripping attachment. Standard curves are made with lead nitrate.
POLAROGRAPHIC
DETFBMINA'ITON
91
LEAD IN BLOOD
OF
DISCUSSION
Minerahation Searching for a simple and reliable method which could be used in routine examination we tested different methods described in the toxicological literature. Direct hemolysis of R3C by hydrochloric acid, as proposed by Teisinger (3), was readily abandoned because it may have technical imperfections, such as the impossibility to become a constant extraction volume, the presence of many interfering substances in the polarographic solution, and the unquantitative recovery of lead components retained on the protein precipitate. Consequently, we obtained systematic low values. And, as may be seen from Figure 1, the distribution of these normal values is asymmetric. A study of these results revealed them to be not distributed normally. The further assertion of Teisinger that saturation of whole blood with oxygen prevents the liberation of porphyrins and the presence of these interfering substances in the polarographic solution when acid is added, seems to be unfounded. We did not find any differences between the two polarograms of the same blood with and without saturation with oxygen. In the dry-ashing techniques the lack of a perfectly located temperature control, the possibility of local overheating, and the incomplete
1
i
,
0
L5
T L-1--1
10
15
20
l-l
25 30 35 40 45 Concentrat~an lead py/lOO
'71
;I
ri
I
50 55 ml blood
60
I
I
65
70
_ 75
FIG. 1. Frequency distribution of the lead content of whole blood in micrograms per/100 ml after a direct hemolysis method as proposed by Teisinger (3). The blood samples are taken from men who do not have occupational contact with lead.
92
AOOSE:LS
AND
HEULENS
dissolution of ashed material may cause partial loss of lead components. For this reason we searched for a method of mineralisation after a wetashing technique without concentration by evaporation. The mineralisation is carried out until1 a clear discoloured solution is obtained. A criterion for complete mineralisation is the complete destruction of lead-EDTA complexes. Lead-EDTA complexes may, indeed, be present in whole blood for diagnostic, therapeutic, preventive, or evaluation reasons. Because lead is more strongly bound to EDTA than to dithizone (8)) it cannot be extracted from lead-EDTA complexes by dithizone alone. The quantitative recovery of lead is thus submitted to the destruction of lead-EDTA complexes. As can be seen from Table 1, this is realised with the procedure outlined above. The addition of ammonium citrate is indispensable for the optimisation of the action of the buffer solution.
The combined action of strong ionic activity and the presence of many interfering ions hinder the direct polarography of the mineralisation. The specific extraction of lead from this mineralisate into a convenient polarographic medium is necessary. Tbe extraction of lead from aqueous solution is optimal only in a small pH range. Therefore, the solution must be buffered (9). The excess of acid present is neutralised by addition of ammonia. This prevents also the destruction of dithizone by oxidising agents. The solution must be treated immediately with dithizone. Indeed, when the buffered solution is allowed to stand before adding the dithizone-chloroform solution, the extraction is incomplete. Probably, competition takes place between lead and iron ions in the reaction with dithizone. The spectrophotometic determination of lead-d&zone complexes cannot be applied here because of the small amount of lead present. POLAROCRAPHIC WAVE LEAD-NITRATE
TABLE 1 HEIGHTS OBTAINED ON ANALYSIS AND LEAD-EDTA SOLUTIONS KNOWN AMOUNTS OF LEALI Wave
Lead content (P!imO !M 50 100 150
Lead
nitrate 41 76 112.5
heights
OF SAMPLES WITK
(nm) Lead-EDTA 38 83 114.5
0~
POLAROGBAPHIC
DETERMINATION
OF
LEAD
IN
93
BLOOD
of lead ions from the chloroform solution to a polarois done by a second extraction. Lead is extracted from the lead-dithizone complexes in acid medium. For this reason the extract must remain acid after contact with the dithizone solution. The transfer
graphic medium
Poikography The anodic stripping polarography technique is a very sensitive method ( 10e8 mole/liter) as compared with the classical polarography (lo-” mole/liter). It is strongly indicated for the study of the lead content of blood. Following Nylander and Holmquist the lack of reproducibility of their results is due to the presence of small carbontetrachloride particles in the acid phase, We polarographed lead solutions in the presence and in the absence of chloroform and did not find any difference in the polarographic result. In accordance with Duyckaert and Cosemans ( lo), who studied experimentally the action of various factors on the peak current we found that small variation of the HCl concentration of the polarographic solution influences greatly the polarographic result. We suspect the disturbances found by Nylander and Holmquist to be due to variations in the HCl concentration of their resulting solutions. Figure 2 shows the variations of the polarographic result with the concentration of HCl of the solution. The curve presents maxima and mm t 150
50.
0
FIG. 2. Variations of the heights (deviation) in function of the variation solution at constant lead concentration.
0.5 Concentration
in
N
1
of HCI
millimeters of the of HCl concentration
polarographic wave in the polarographic
94
* KCI 0.1 N + HCI (’ HCI ~-~--.--_..-----.~ 0
__ 010 Concentration
005
015
~
0.20
of HCI
FIG. 3. Variation of the heights (in millimeters) of the polarographic wave (deviation) in function of the concentration of HCl in the polarographic solution in absence (a) and in presence ( 0) of KC1 0.1 N.
Standard
curve
50 Concentratmn
lead
,ug/lOO
100 g blood
FIG. 4. Standard curve of lead determination in whole blood following the method described. Polarographic wave heights in millimeters in function of the ancentration of lead nitrate added in pg/lOO gm of blood.
POLAROGRAPJSIC
DETERMINATION
OF
TABLE LEAD
____
N 1 2
CONTENT
LEAD
95
2
OF BLOOD SAMPLES OF MEN OCCVPATIONAL CONTACT WITH
WHO
Do NOT HAVE
LEAD”
N
LC
N
LC
N
LC
N
LC
35.0 s.0
12 13
32.0
2s
.33. <‘,
24
7.0
:<4 35 36 :;7 3X 39
36.0 45. 0 45.0 34.0 26.0 82.0
43 46 47 4s 49 50
23.5 24.0 ‘4.0 46.0 10.0
15.0 23.0 18.0 33.0
.51 32 111
7 27.0
37.5 14 32.0 15 s:;. 5 17 28.0 16 20.0
2.5
10.0
26 28 27
18.0 17.0 -50.0
29
17.0
11.0
18 19
a’2.6 37.5
9 x2.0
20
3.5.3
30 27.0 31 lti.0
42.0
21
29 .,>-
32
14.-T
40 41 42 43
26.0
22
29.0
33
37.0
44 :
10 11
BLOOD
LC
:i 16.0 4 2Y.5 ir 24.0 29.0
8
IN
41.0 13.0 SO..5
27.9 s 12.x Al - 2s 2. ::
UNL = dl + 2s = 53.5 5 The lead content in fig/100 gm of blood (LC), the mean value (M), the sigma value (s), the 95y0 limit values (M f as), and the upper normal limit (UNL) are given. N represents the case number.
minima which are difficult to explain. In order to avoid these variations, we studied the influence of KC1 on the answer of the polarograph. We found that a concentration of KC1 0.1 N greatly stabilises the result of the polarograph over a range of HCl concentrations from 0.01-0.18 N.
Concentrotian
lead pq/loOg
blood
FIG. 5. Frequency distribution of the lead content of whole blood found on studying the lead content of whole blood of men who did not have occupational contact with lead.
These are the limits of the HCI concentrations solution in this technique (Fig. 3). Nornial
Values
of Lead
of the polarographic
in. BlootI
Standard curve. The standard curve was obtained by adding variable amounts of lead nitrate to a constant weight of blood (Fig. 4). Normal valuues. We determined the lead content of the blood of 52 men who did not have occupational contact with lead. Table 2 gives the results and the M -+ 2s values of this group. Figure 5 gives the frequency distribution of these values. As can be seen the distribution occurs after a perfect Gaussian curve. Examination of the normal blood samples shows values of less than 60 pg/lOO gm blood and 80% of the results are situated between 1S and 40 /lg/lOO gm of blood. WC cannot confirm the assertion of Berman (6) that there exists a difference between normal values with and without any contact with lead she found among children. SUMMARY
Anodic stripping polarography is used to determine the lead content of whole blood. The mineralisation of blood samples takes place under the consecutive action of nitric acid, perhydrol, and gentle heating. Lead is extracted from the mineralisate with a dithizone-chloroform solution. The extraction of this dithizone solution with HCl 1 N permits the transfer of lead ions into the polarographic medium. The influence of the concentration of HCl on the polarographic behavior of lead ions is studied. A HCl-KC1 solution is found to be the best polarographic medium Normal values have been determined on blood samples of workers who were not exposed occupationally to lead contamination. 1. KEHOE,
REFERENCES R. A., Symp. Environmental Lead Contamination
2. DICK, J. M., ELLIS, 3. TEISINGER, J., in
R. W.,
AND STECK, J., Brit.
J. Znd.
Med.
1965. 18, 2&286
( 1961).
“Polarography in Medicine, Biochemistry and Pharmacy.” Brezina, M. and Zuman, P., Wiley (Interscience), New York (1958). 4. NXANDER, A. L., AND HOLMQUIST, C. E., Arch. Znd. Hyg. Occup. Med. 19, 188-191 (1954). 5. F--m, R., TRU=~ R., AND Ihmm, C., C. R. H. Acad. Sci. 624-627 ( 1956). 6. BERMAN, P., Pedid. C&n. 5, 287-291 (1968). 7’. JONES, J. C. H., An&St, 93, 214-218 (1968). 8. Roosns, D., AND VANDERKEEL, J., At. Absorption Newslett. 7, 9-10 (1968). 9. CLAES, J. H., ‘Xbische Chemie” Wouters, Leuven, Belgium (1964). 10. DUYCKAERT, G., AND COSEMANS, J., Ind. Chim. Beige. 31, 333-345 (19w).
MEDICINE
Polarographic Using
the
4, 89-96
( 1970)
Determination Anodic
Stripping
D. ROOSELS Fund of Occupational
of
Lead
Polarography AND
in Whole
Blood
Technique
L. HEULENS
Disease, Ministry for Social Care, Brussels 1, Belgium Received
January
7, 1970
The determination of lead in blood is one of the most valuable criteria for the determination of an exposure to lead poisoning, because it is a specific indication of the presence of the toxic agent in the circulatory tract ( 1). Many methods have been proposed to determine the lead content of whole blood (2-7). All of them must resolve two main problems: ( 1) the extraction of lead from the red blood cells ( RBC ) and (2) accurate and specific determination of lead ions among many interfering substances. An attractive method was described by Nylander and Holmquist (4) but the reproducibility of their results remains problematic. Detailing the problems related to the determination of the lead content of whole blood we distinguish three main steps: ( 1) the mineralisation of blood; (2) the extraction of lead ions from the mineralisate into a polarographic medium; (3) the study of an appropriate polarographic medium. We found a simple and reliable method whose principles may also be applied to the determination of lead in other biological materials. METHOD
Minmalisation Fifteen milliliters of nitric acid (D = 1.4, treated with dithizone) are added to 5 gm of whole blood ( taken on versenate ) in an Erlenmeyer flask of 200 ml with a wide neck. The flask is covered with a watch-glass, and the mixture is heated gently on a hotplate until a clear and transparent solution is obtained. After cooling, 15 ml of perhydrol are added in 3ml portions. After each addition the solution is heated until no more foam is produced and, after cooling, a new fraction of perhydrol is added after which diluted nitric acid (l/5 with water) and 5 ml of the ammonium citrate solution are added. 89
Extraction The resulting solution is neutralised with ammonia ( 1~ .I: 091 ) ~ using phenol red as indicator, and transferred quantitatively into a separatory funnel of 250 ml. The Erlenmeyer flask is rinsed with 25 ml buffer solution. These are quantitatively transferred in the separator): funnel. The solution is then immediately shaken with 10 ml of 0.003% dithizone solution (in chloroform) for at least 30 seconds. The chloroform layer is transferred to another separatory funnel, and the first extraction is rcpeated twice. The combined chloroform layers are shaken with 10 ml 1 N HCl. The pH of this HCl layer must remain acid after extraction; if not, add a few drops of concentrated HCl and shake again. The HCl layer is transferred to a volumetric flask of 50 ml and brought to volume with KC1 0.125 N. Polarogra ph y Twenty-five milliliters of this solution are transferred to a polarographic cell and degassed with nitrogen for 15 minutes. After the anodic stripping polarography technique, the hanging mercury drop is saturated with lead ions (for 3 minutes under stirring and 1 min without stirring) at a constant negative voltage (-0.75 V starting voltage) and a constant temperature ( 25” ) . The anodic stripping current is rneasured into a linearly increasing voltage range of +l V (from -0.75 to +0.25 V) at a sensitivity of 2. lo-” A/mm. The lead wave appears at -0.35 V. Apparatus
and Products
All the products are of the analytical grade. All glass material is of the Pyrex or jenaer type, treated with dithizone to remove all traces of lead contamination. Double-distilled water is always used. Blood is taken on a polystyrene container on versenate 1 mg/ml. Ammonium citrate solution: 20 gm 100 ml water, treated with dithizone as explained for the buffer solution. Bufier solution. The solution consists of 236 gm of ammonium citrate dissolved in 150 ml of concentrated ammonia (D = 0.91) and diluted to 500 ml with water. Add 10 gm KCN and 5 mg Na$O, to this solution. The solution is transferred to a separatory funnel and shaken with two 20-ml portions of dithizone in chloroform (30 mg/hter) to remove any lead which may be present in the reagents, Excess dithizone is washed out with two 30-ml portions of chloroform. To this solution 1 liter of concentrated ammonia is added. Perhydrol: 30% solution of hydrogen peroxide. Polarograph Methrom-Herisau type E 261, anodic stripping attachment. Standard curves are made with lead nitrate.
POLAROGRAPHIC
DETFBMINA'ITON
91
LEAD IN BLOOD
OF
DISCUSSION
Minerahation Searching for a simple and reliable method which could be used in routine examination we tested different methods described in the toxicological literature. Direct hemolysis of R3C by hydrochloric acid, as proposed by Teisinger (3), was readily abandoned because it may have technical imperfections, such as the impossibility to become a constant extraction volume, the presence of many interfering substances in the polarographic solution, and the unquantitative recovery of lead components retained on the protein precipitate. Consequently, we obtained systematic low values. And, as may be seen from Figure 1, the distribution of these normal values is asymmetric. A study of these results revealed them to be not distributed normally. The further assertion of Teisinger that saturation of whole blood with oxygen prevents the liberation of porphyrins and the presence of these interfering substances in the polarographic solution when acid is added, seems to be unfounded. We did not find any differences between the two polarograms of the same blood with and without saturation with oxygen. In the dry-ashing techniques the lack of a perfectly located temperature control, the possibility of local overheating, and the incomplete
1
i
,
0
L5
T L-1--1
10
15
20
l-l
25 30 35 40 45 Concentrat~an lead py/lOO
'71
;I
ri
I
50 55 ml blood
60
I
I
65
70
_ 75
FIG. 1. Frequency distribution of the lead content of whole blood in micrograms per/100 ml after a direct hemolysis method as proposed by Teisinger (3). The blood samples are taken from men who do not have occupational contact with lead.
92
AOOSE:LS
AND
HEULENS
dissolution of ashed material may cause partial loss of lead components. For this reason we searched for a method of mineralisation after a wetashing technique without concentration by evaporation. The mineralisation is carried out until1 a clear discoloured solution is obtained. A criterion for complete mineralisation is the complete destruction of lead-EDTA complexes. Lead-EDTA complexes may, indeed, be present in whole blood for diagnostic, therapeutic, preventive, or evaluation reasons. Because lead is more strongly bound to EDTA than to dithizone (8)) it cannot be extracted from lead-EDTA complexes by dithizone alone. The quantitative recovery of lead is thus submitted to the destruction of lead-EDTA complexes. As can be seen from Table 1, this is realised with the procedure outlined above. The addition of ammonium citrate is indispensable for the optimisation of the action of the buffer solution.
The combined action of strong ionic activity and the presence of many interfering ions hinder the direct polarography of the mineralisation. The specific extraction of lead from this mineralisate into a convenient polarographic medium is necessary. Tbe extraction of lead from aqueous solution is optimal only in a small pH range. Therefore, the solution must be buffered (9). The excess of acid present is neutralised by addition of ammonia. This prevents also the destruction of dithizone by oxidising agents. The solution must be treated immediately with dithizone. Indeed, when the buffered solution is allowed to stand before adding the dithizone-chloroform solution, the extraction is incomplete. Probably, competition takes place between lead and iron ions in the reaction with dithizone. The spectrophotometic determination of lead-d&zone complexes cannot be applied here because of the small amount of lead present. POLAROCRAPHIC WAVE LEAD-NITRATE
TABLE 1 HEIGHTS OBTAINED ON ANALYSIS AND LEAD-EDTA SOLUTIONS KNOWN AMOUNTS OF LEALI Wave
Lead content (P!imO !M 50 100 150
Lead
nitrate 41 76 112.5
heights
OF SAMPLES WITK
(nm) Lead-EDTA 38 83 114.5
0~
POLAROGBAPHIC
DETERMINATION
OF
LEAD
IN
93
BLOOD
of lead ions from the chloroform solution to a polarois done by a second extraction. Lead is extracted from the lead-dithizone complexes in acid medium. For this reason the extract must remain acid after contact with the dithizone solution. The transfer
graphic medium
Poikography The anodic stripping polarography technique is a very sensitive method ( 10e8 mole/liter) as compared with the classical polarography (lo-” mole/liter). It is strongly indicated for the study of the lead content of blood. Following Nylander and Holmquist the lack of reproducibility of their results is due to the presence of small carbontetrachloride particles in the acid phase, We polarographed lead solutions in the presence and in the absence of chloroform and did not find any difference in the polarographic result. In accordance with Duyckaert and Cosemans ( lo), who studied experimentally the action of various factors on the peak current we found that small variation of the HCl concentration of the polarographic solution influences greatly the polarographic result. We suspect the disturbances found by Nylander and Holmquist to be due to variations in the HCl concentration of their resulting solutions. Figure 2 shows the variations of the polarographic result with the concentration of HCl of the solution. The curve presents maxima and mm t 150
50.
0
FIG. 2. Variations of the heights (deviation) in function of the variation solution at constant lead concentration.
0.5 Concentration
in
N
1
of HCI
millimeters of the of HCl concentration
polarographic wave in the polarographic
94
* KCI 0.1 N + HCI (’ HCI ~-~--.--_..-----.~ 0
__ 010 Concentration
005
015
~
0.20
of HCI
FIG. 3. Variation of the heights (in millimeters) of the polarographic wave (deviation) in function of the concentration of HCl in the polarographic solution in absence (a) and in presence ( 0) of KC1 0.1 N.
Standard
curve
50 Concentratmn
lead
,ug/lOO
100 g blood
FIG. 4. Standard curve of lead determination in whole blood following the method described. Polarographic wave heights in millimeters in function of the ancentration of lead nitrate added in pg/lOO gm of blood.
POLAROGRAPJSIC
DETERMINATION
OF
TABLE LEAD
____
N 1 2
CONTENT
LEAD
95
2
OF BLOOD SAMPLES OF MEN OCCVPATIONAL CONTACT WITH
WHO
Do NOT HAVE
LEAD”
N
LC
N
LC
N
LC
N
LC
35.0 s.0
12 13
32.0
2s
.33. <‘,
24
7.0
:<4 35 36 :;7 3X 39
36.0 45. 0 45.0 34.0 26.0 82.0
43 46 47 4s 49 50
23.5 24.0 ‘4.0 46.0 10.0
15.0 23.0 18.0 33.0
.51 32 111
7 27.0
37.5 14 32.0 15 s:;. 5 17 28.0 16 20.0
2.5
10.0
26 28 27
18.0 17.0 -50.0
29
17.0
11.0
18 19
a’2.6 37.5
9 x2.0
20
3.5.3
30 27.0 31 lti.0
42.0
21
29 .,>-
32
14.-T
40 41 42 43
26.0
22
29.0
33
37.0
44 :
10 11
BLOOD
LC
:i 16.0 4 2Y.5 ir 24.0 29.0
8
IN
41.0 13.0 SO..5
27.9 s 12.x Al - 2s 2. ::
UNL = dl + 2s = 53.5 5 The lead content in fig/100 gm of blood (LC), the mean value (M), the sigma value (s), the 95y0 limit values (M f as), and the upper normal limit (UNL) are given. N represents the case number.
minima which are difficult to explain. In order to avoid these variations, we studied the influence of KC1 on the answer of the polarograph. We found that a concentration of KC1 0.1 N greatly stabilises the result of the polarograph over a range of HCl concentrations from 0.01-0.18 N.
Concentrotian
lead pq/loOg
blood
FIG. 5. Frequency distribution of the lead content of whole blood found on studying the lead content of whole blood of men who did not have occupational contact with lead.
These are the limits of the HCI concentrations solution in this technique (Fig. 3). Nornial
Values
of Lead
of the polarographic
in. BlootI
Standard curve. The standard curve was obtained by adding variable amounts of lead nitrate to a constant weight of blood (Fig. 4). Normal valuues. We determined the lead content of the blood of 52 men who did not have occupational contact with lead. Table 2 gives the results and the M -+ 2s values of this group. Figure 5 gives the frequency distribution of these values. As can be seen the distribution occurs after a perfect Gaussian curve. Examination of the normal blood samples shows values of less than 60 pg/lOO gm blood and 80% of the results are situated between 1S and 40 /lg/lOO gm of blood. WC cannot confirm the assertion of Berman (6) that there exists a difference between normal values with and without any contact with lead she found among children. SUMMARY
Anodic stripping polarography is used to determine the lead content of whole blood. The mineralisation of blood samples takes place under the consecutive action of nitric acid, perhydrol, and gentle heating. Lead is extracted from the mineralisate with a dithizone-chloroform solution. The extraction of this dithizone solution with HCl 1 N permits the transfer of lead ions into the polarographic medium. The influence of the concentration of HCl on the polarographic behavior of lead ions is studied. A HCl-KC1 solution is found to be the best polarographic medium Normal values have been determined on blood samples of workers who were not exposed occupationally to lead contamination. 1. KEHOE,
REFERENCES R. A., Symp. Environmental Lead Contamination
2. DICK, J. M., ELLIS, 3. TEISINGER, J., in
R. W.,
AND STECK, J., Brit.
J. Znd.
Med.
1965. 18, 2&286
( 1961).
“Polarography in Medicine, Biochemistry and Pharmacy.” Brezina, M. and Zuman, P., Wiley (Interscience), New York (1958). 4. NXANDER, A. L., AND HOLMQUIST, C. E., Arch. Znd. Hyg. Occup. Med. 19, 188-191 (1954). 5. F--m, R., TRU=~ R., AND Ihmm, C., C. R. H. Acad. Sci. 624-627 ( 1956). 6. BERMAN, P., Pedid. C&n. 5, 287-291 (1968). 7’. JONES, J. C. H., An&St, 93, 214-218 (1968). 8. Roosns, D., AND VANDERKEEL, J., At. Absorption Newslett. 7, 9-10 (1968). 9. CLAES, J. H., ‘Xbische Chemie” Wouters, Leuven, Belgium (1964). 10. DUYCKAERT, G., AND COSEMANS, J., Ind. Chim. Beige. 31, 333-345 (19w).