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The Content of Arsenic in the Hair in a Case of Acute Lethal Arsenic Poisoning
T h e Content of Arsenic in the H a i r in a Case of Acute Lethal Arsenic Poisoning A. WYTTEKBACH Eidg. Institztt fur Realztorforschung, Wiirenlingen
P. BARTI-IE U n i t of renal diseases, Universitatsklinik Base1 E. P. MARTIN I
Introduction I t is generally accepted that the intake of arsenic will manifest itself by an increased content of arsenic in the hair; thus the concentration and the distribution of arsenic along a hair can serve as proof for chronic or acute arsenic poisoning (Smith and Lenihan, 1964). However, information on the relation between ingested arsenic and arsenic excreted into the hair, which is of great significance to the forensic application of the phenomenon, is still scant. Two cases involving sublethal doses have been reported (Smith, 1963), and experiments with guinea pigs involving acute and sub-acute doses are mentioned by Jervis (Jervis, 1967). The present work reports on the findings in a case of acute, lethal poisoning with a known amount (6g) of arsenic. Since work of this kind involves arsenic concentration as low as 1 ppm and hair samples of 0.5 cm length (weighing approximately 3 x 10-5 g) an analytical method capable of measuring as little as 3 x 10-11 g of arsenic is of prime importance. Activation analysis does meet this requirement and is the method adopted by us. Its principles and applications to forensic investigations are covered by Gibbons (Gibbons, 1963). Case history
The patient was an 18-year-old male; he took 8 grams of arsenic dissolved in warm water in order to commit suicide. 60 minutes later onset of characteristic symptoms: heavy vomiting which increased in intensity, and severe muscle spasms especially of the lower limbs. 10 hours after the intake the patient was admitted a t the Emergency Unit, Dept. of Internal Medicine of the University Clinic a t Basle, where the following findings were made: Shock (blood pressure 75/20 mm Hg); heart rate 122/min; general dehydration, severe muscle spasms; subcutaneous emphysema on chin, neck and left side of chest. The emergency treatment consisted of infusions of plasma, sodium chloride, glucose, and plasma-expanders, and of injections of BAL as an antidote to the poison. One day later, after a transient reduction of urinary output, there was a slow recovery of the general condition. However, 80 hours after intake, acute hallucinosis made necessary a transfer to the Psychiatric Dept. of the University Clinic. The treatment was there continued with infusions and BAI,.
The next day the patient showed high fever and progressive loss of consciousness, whereupon he was transferred back to the Dept. of Internal Medicine. He was still reacting to pain and had continual convulsions of the whole body muscles. On his back appeared big blisters. Therapy was continued with BAL and Diazepam (ValiumR). 122 hours after the intake this respiration became irregular (Cheynes-Stokes) and changed finally to gasping. Death 158 hours after the intake was due to respiratory insufficiency and terminal blood aspiration. The main autopsy findings were: edema of the brain, degeneration of ganglion cells in the cerebrum and the cerebellum; bleeding ulcers of the gastric mucosa with terminal blood aspiration; congestion of the internal organs. 5 hairs were pulled from the right parietal side of the skull 150 hours after the intake, that is 8 hours before death.
Experimental Treatment of the samples prior to irradiation: One hair a t a time was weighed and then cut with a stainless steel scalpel into pieces of 0.5 to 1.6 cm length. These pieces were washed individually in acetone and water (for 5 minutes in each solvent), dried and slipped into envelopes of polyethylene for irradiation. All handling was done with platinum tipped tweezers. -
Irradiation: All samples from one hair were irradiated together with As-standards to an integrated flux of 1017 n/cm2. Radiochemical isolation of 76As: Work on the samples started one to two hours after the end of the irradiation. The hair sections were dissolved in a mixture of 1ml H2S04, 1 ml HN03 and a few drops of H202after addition of 30 mg As and 5 mg each of Cu and Sb. Upon completion of the dissolution the mixture was made 8 N in HC1 and As& precipitated with thioacetamid a t 0°C. The precipitate was centrifuged, washed and dissolved in 112 ml each of HzS04 and HzO2. The resulting solution was made 11 T\i in HC1 and As111 was extracted into benzene after addition of NaI and ascorbic acid (Green and Kafalas, 1954). The organic phase was washed and then back extracted with water. Finally, As-metal was precipitated with NaH2P02from 6 N HC1, collected on a disk of filter paper, washed, dried, weighed and mounted for counting. Recovery of As was usually about 70%. Measurement of the samples: Under the irradiation conditions chosen by us, most samples showed a rather weak activity, thus ruling out y-spectroscopy. The samples were, therefore, counted on a p-counter with anticoincidence-shielding. 3 x 10-11 g As resulted in 10 cpm. The background of the counter was 1.8 cpm. Samples were measured for four days, and the decay curves analysed by a least-square computer program. -411 samples except one decayed with the established half-life of 26.5 h, thus proving that they were free from contamination. Results and discussion Table 1 and 2 summarize the pertinent data of our samples and give the results of analyses for arsenic. The weight per cm of the hair analysed by us (Table 1) fluctuated between 26 and 63 pglcm, which was roughly consistent with the numbers given by Kerr (Kerr, 1964). Kerr, weighed 40 hairs from one head, finding a mean of 54 pglcrn and a range of 37 to 73 pg/cm. 195
As Kerr has noticed, data from hairs from one head tend to become more similar when looking at the concentration of an element in hair (expressed as ppm) instead of looking at the absolute amount of an element (expressed as pg). This is pointed out by hairs Nos. 1, 3, 5 (Table 1). No positive explanation can be given for the low concentration in hair No. 2. Since the root-end of this hair shows the greatest discrepancy, we think it possible that either the root of this hair was lost during washing or that the hair was in the telogen (dormant) phase of the hair cycle. TABLE 1 RESULTS FOR WHOLE HAIRS 1
2
3
4
5
length (total), cm weight (total) vg weight per c d ug/cm
3.9 245 62.9
3.2 85 26.6
3.0 78 26.1
3.1 135 43.5
3.7 222 61.7
As (total), pg x 10-4 PPm
70.3 28.7
5.7 6.7
29.3 37.5
a)
62.6 28.2
Hair No. ;
a) sample No. 4 was lost after weighing. The total As-content was obtained by summing the results of the individual sections. TABLE 2 RESULTS FOR INDIVIDUAL HAIR-SECTIONS 1
Hair No. cm
a) b)
pprn
2 cm
pprn
4
3 cm
pprn
cm
pprn
the sections are tabulated with the root-end of the hair first. The first section includes the root. sample contaminated by unidentified activity.
Looking at Table 2, we notice a remarkable concentration of arsenic in the first 0.5 cm of hair (hair No. 1). This is consistent with the fact that scalp hair grows at a rate of 113 to 112 mm a day (Millar et al, 1961), (Montagna and Ellis, 1958) and that the hairs were plucked 5.8 days after the intake of the arsenic; this delay will result in a growth of 2 to 3 mm. All hair sections further away from the root show arsenic contents between 0.9 and 3.6 pprn except the tip end of hair No. 3, which shows 8.5 ppm. Since published distribution curves of arsenic in hair from healthy people show that 99% of all samples have a concentration of less than 4.5 pprn (Smith, 1963, Perkons and Jervis, 1965), the values found by us can be judged as lying within the normal range. Combining the facts that all excess arsenic is contained within the first 5 mm of hair and that the remainder of the hair shows a normal arsenic content of about 2 ppm, the arsenic content of the first 5 mm of hairs Nos. 2 , 3 and 4 can be calculated from the data in Table 3:TABLE 3 As-CONTENT OF FIRST 5 mm OF HAIR (INCLUDING ROOT) 210 ppm Hair No. 1 2 29 PPm 3 196 ppm
198 ppm
5
196
Neglecting the value of hair No. 2 (which has been discussed above), the mean value in the first 5 mm of hair (including the root) as observed 5.8 days after the intake of 8 g of As203 is approximately 200 ppm. Table 4 summarizes the four cases of acute poisoning where the amount of ingested arsenic is known and where analysis of head hair has been carried out subsequently. There are no indications in the literature on how the amount of ingested arsenic has been estimated in the first three cases. In the case reported by us, the patient stated to have ingested 8 g of As203 ; since he was apprenticed in a drug store and therefore accustomed to the handling and weighing of chemicals, we consider this statement as fairly accurate. As can be seen from Table 4, the arsenic content of hair increases approximately in proportion to the amount taken in. The data available a t present are certainly not numerous enough to propose a strict relation between intake and excretion, but they should be of value in fixing the order of magnitude of an unknown amount of ingested arsenic by measuring the arsenic content of head hair. TABLE 4 SUMMARY O F FOUR CASES O F ACUTE ARSENIC POISONING Arsenic time between content intake and of first 0.5 cm Amount of taking of (including As ingested taken as hair root) effect Literature 0.003 g 0.003 g 0.8 g 6 a)
g
As203 in tea As203 in tea As203
4 4
As203 in warm water
6.2 d
7
h h d
2.1 ppm 4.9 ppm a 20 ppm a)
not fatal not fatal not fatal
200 ppm
fatal
Smith, 1963 Smith, 1963 Smith and Lenihan, 1964 Smith, 1963 this work
value interpolated by us.
Literature GIBBONS, D., 1963, J. Forensic Sci. Soc., 4, 33. J. A., 1954, J . Chem. Phys., 22, 760. GREEN,M., and KAFALAS, JERVIS,R. E., 1967, Symposium on nuclear activation techniques in the life sciences, Amsterdam, 1967, paper SM-91/39. KERR,F. M., 1964, Thesis, University of Ottawa. C. A., 1961, J. Histochem. and MILLAR,M. S., VINCENT,N. R., and MAWSON, Cytochem. 9, 111. W., and ELLIS,R. A., 1958, The biology of Hair Growth, Academic MONTAGNA, Press, New York, 1958. PERKONS, A. K., and JERVIS,R. E., 1965, Intern. Conf. on modern trends in activation analysis, Texas A and M University, p. 295. SMITH,H., 1963, J. Forensic Sci. Soc., 4, 192. SMITH,H., and LENIHAN, J. M. H., 1964, Biological applications of activation analysis, in Methods of forensic science, vol. 3, Ed. by A. S. Curry, New York, 1964.
P. BARTI-IE U n i t of renal diseases, Universitatsklinik Base1 E. P. MARTIN I
Introduction I t is generally accepted that the intake of arsenic will manifest itself by an increased content of arsenic in the hair; thus the concentration and the distribution of arsenic along a hair can serve as proof for chronic or acute arsenic poisoning (Smith and Lenihan, 1964). However, information on the relation between ingested arsenic and arsenic excreted into the hair, which is of great significance to the forensic application of the phenomenon, is still scant. Two cases involving sublethal doses have been reported (Smith, 1963), and experiments with guinea pigs involving acute and sub-acute doses are mentioned by Jervis (Jervis, 1967). The present work reports on the findings in a case of acute, lethal poisoning with a known amount (6g) of arsenic. Since work of this kind involves arsenic concentration as low as 1 ppm and hair samples of 0.5 cm length (weighing approximately 3 x 10-5 g) an analytical method capable of measuring as little as 3 x 10-11 g of arsenic is of prime importance. Activation analysis does meet this requirement and is the method adopted by us. Its principles and applications to forensic investigations are covered by Gibbons (Gibbons, 1963). Case history
The patient was an 18-year-old male; he took 8 grams of arsenic dissolved in warm water in order to commit suicide. 60 minutes later onset of characteristic symptoms: heavy vomiting which increased in intensity, and severe muscle spasms especially of the lower limbs. 10 hours after the intake the patient was admitted a t the Emergency Unit, Dept. of Internal Medicine of the University Clinic a t Basle, where the following findings were made: Shock (blood pressure 75/20 mm Hg); heart rate 122/min; general dehydration, severe muscle spasms; subcutaneous emphysema on chin, neck and left side of chest. The emergency treatment consisted of infusions of plasma, sodium chloride, glucose, and plasma-expanders, and of injections of BAL as an antidote to the poison. One day later, after a transient reduction of urinary output, there was a slow recovery of the general condition. However, 80 hours after intake, acute hallucinosis made necessary a transfer to the Psychiatric Dept. of the University Clinic. The treatment was there continued with infusions and BAI,.
The next day the patient showed high fever and progressive loss of consciousness, whereupon he was transferred back to the Dept. of Internal Medicine. He was still reacting to pain and had continual convulsions of the whole body muscles. On his back appeared big blisters. Therapy was continued with BAL and Diazepam (ValiumR). 122 hours after the intake this respiration became irregular (Cheynes-Stokes) and changed finally to gasping. Death 158 hours after the intake was due to respiratory insufficiency and terminal blood aspiration. The main autopsy findings were: edema of the brain, degeneration of ganglion cells in the cerebrum and the cerebellum; bleeding ulcers of the gastric mucosa with terminal blood aspiration; congestion of the internal organs. 5 hairs were pulled from the right parietal side of the skull 150 hours after the intake, that is 8 hours before death.
Experimental Treatment of the samples prior to irradiation: One hair a t a time was weighed and then cut with a stainless steel scalpel into pieces of 0.5 to 1.6 cm length. These pieces were washed individually in acetone and water (for 5 minutes in each solvent), dried and slipped into envelopes of polyethylene for irradiation. All handling was done with platinum tipped tweezers. -
Irradiation: All samples from one hair were irradiated together with As-standards to an integrated flux of 1017 n/cm2. Radiochemical isolation of 76As: Work on the samples started one to two hours after the end of the irradiation. The hair sections were dissolved in a mixture of 1ml H2S04, 1 ml HN03 and a few drops of H202after addition of 30 mg As and 5 mg each of Cu and Sb. Upon completion of the dissolution the mixture was made 8 N in HC1 and As& precipitated with thioacetamid a t 0°C. The precipitate was centrifuged, washed and dissolved in 112 ml each of HzS04 and HzO2. The resulting solution was made 11 T\i in HC1 and As111 was extracted into benzene after addition of NaI and ascorbic acid (Green and Kafalas, 1954). The organic phase was washed and then back extracted with water. Finally, As-metal was precipitated with NaH2P02from 6 N HC1, collected on a disk of filter paper, washed, dried, weighed and mounted for counting. Recovery of As was usually about 70%. Measurement of the samples: Under the irradiation conditions chosen by us, most samples showed a rather weak activity, thus ruling out y-spectroscopy. The samples were, therefore, counted on a p-counter with anticoincidence-shielding. 3 x 10-11 g As resulted in 10 cpm. The background of the counter was 1.8 cpm. Samples were measured for four days, and the decay curves analysed by a least-square computer program. -411 samples except one decayed with the established half-life of 26.5 h, thus proving that they were free from contamination. Results and discussion Table 1 and 2 summarize the pertinent data of our samples and give the results of analyses for arsenic. The weight per cm of the hair analysed by us (Table 1) fluctuated between 26 and 63 pglcm, which was roughly consistent with the numbers given by Kerr (Kerr, 1964). Kerr, weighed 40 hairs from one head, finding a mean of 54 pglcrn and a range of 37 to 73 pg/cm. 195
As Kerr has noticed, data from hairs from one head tend to become more similar when looking at the concentration of an element in hair (expressed as ppm) instead of looking at the absolute amount of an element (expressed as pg). This is pointed out by hairs Nos. 1, 3, 5 (Table 1). No positive explanation can be given for the low concentration in hair No. 2. Since the root-end of this hair shows the greatest discrepancy, we think it possible that either the root of this hair was lost during washing or that the hair was in the telogen (dormant) phase of the hair cycle. TABLE 1 RESULTS FOR WHOLE HAIRS 1
2
3
4
5
length (total), cm weight (total) vg weight per c d ug/cm
3.9 245 62.9
3.2 85 26.6
3.0 78 26.1
3.1 135 43.5
3.7 222 61.7
As (total), pg x 10-4 PPm
70.3 28.7
5.7 6.7
29.3 37.5
a)
62.6 28.2
Hair No. ;
a) sample No. 4 was lost after weighing. The total As-content was obtained by summing the results of the individual sections. TABLE 2 RESULTS FOR INDIVIDUAL HAIR-SECTIONS 1
Hair No. cm
a) b)
pprn
2 cm
pprn
4
3 cm
pprn
cm
pprn
the sections are tabulated with the root-end of the hair first. The first section includes the root. sample contaminated by unidentified activity.
Looking at Table 2, we notice a remarkable concentration of arsenic in the first 0.5 cm of hair (hair No. 1). This is consistent with the fact that scalp hair grows at a rate of 113 to 112 mm a day (Millar et al, 1961), (Montagna and Ellis, 1958) and that the hairs were plucked 5.8 days after the intake of the arsenic; this delay will result in a growth of 2 to 3 mm. All hair sections further away from the root show arsenic contents between 0.9 and 3.6 pprn except the tip end of hair No. 3, which shows 8.5 ppm. Since published distribution curves of arsenic in hair from healthy people show that 99% of all samples have a concentration of less than 4.5 pprn (Smith, 1963, Perkons and Jervis, 1965), the values found by us can be judged as lying within the normal range. Combining the facts that all excess arsenic is contained within the first 5 mm of hair and that the remainder of the hair shows a normal arsenic content of about 2 ppm, the arsenic content of the first 5 mm of hairs Nos. 2 , 3 and 4 can be calculated from the data in Table 3:TABLE 3 As-CONTENT OF FIRST 5 mm OF HAIR (INCLUDING ROOT) 210 ppm Hair No. 1 2 29 PPm 3 196 ppm
198 ppm
5
196
Neglecting the value of hair No. 2 (which has been discussed above), the mean value in the first 5 mm of hair (including the root) as observed 5.8 days after the intake of 8 g of As203 is approximately 200 ppm. Table 4 summarizes the four cases of acute poisoning where the amount of ingested arsenic is known and where analysis of head hair has been carried out subsequently. There are no indications in the literature on how the amount of ingested arsenic has been estimated in the first three cases. In the case reported by us, the patient stated to have ingested 8 g of As203 ; since he was apprenticed in a drug store and therefore accustomed to the handling and weighing of chemicals, we consider this statement as fairly accurate. As can be seen from Table 4, the arsenic content of hair increases approximately in proportion to the amount taken in. The data available a t present are certainly not numerous enough to propose a strict relation between intake and excretion, but they should be of value in fixing the order of magnitude of an unknown amount of ingested arsenic by measuring the arsenic content of head hair. TABLE 4 SUMMARY O F FOUR CASES O F ACUTE ARSENIC POISONING Arsenic time between content intake and of first 0.5 cm Amount of taking of (including As ingested taken as hair root) effect Literature 0.003 g 0.003 g 0.8 g 6 a)
g
As203 in tea As203 in tea As203
4 4
As203 in warm water
6.2 d
7
h h d
2.1 ppm 4.9 ppm a 20 ppm a)
not fatal not fatal not fatal
200 ppm
fatal
Smith, 1963 Smith, 1963 Smith and Lenihan, 1964 Smith, 1963 this work
value interpolated by us.
Literature GIBBONS, D., 1963, J. Forensic Sci. Soc., 4, 33. J. A., 1954, J . Chem. Phys., 22, 760. GREEN,M., and KAFALAS, JERVIS,R. E., 1967, Symposium on nuclear activation techniques in the life sciences, Amsterdam, 1967, paper SM-91/39. KERR,F. M., 1964, Thesis, University of Ottawa. C. A., 1961, J. Histochem. and MILLAR,M. S., VINCENT,N. R., and MAWSON, Cytochem. 9, 111. W., and ELLIS,R. A., 1958, The biology of Hair Growth, Academic MONTAGNA, Press, New York, 1958. PERKONS, A. K., and JERVIS,R. E., 1965, Intern. Conf. on modern trends in activation analysis, Texas A and M University, p. 295. SMITH,H., 1963, J. Forensic Sci. Soc., 4, 192. SMITH,H., and LENIHAN, J. M. H., 1964, Biological applications of activation analysis, in Methods of forensic science, vol. 3, Ed. by A. S. Curry, New York, 1964.