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Abnormal tissue distribution of lead in amyotrophic lateral sclerosis
Journal of the Neurological Sciences, 1976, 29:259-265
259
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
A B N O R M A L TISSUE D I S T R I B U T I O N OF LEAD IN A M Y O T R O P H I C L A T E R A L SCLEROSIS
SEBASTIAN CONRAD1, LARS-OLOF RONNEV[ and OLOF VESTERBERG
Department of Clinical Neurology (SC), Karolinska Sjukhuset, Department of Anatomy (LOR), Karolinska Institutet, S-104 O1 Stockholm 60, and Chemical Division (OV), Occupational Health Department, The National Board of Occupational Safety and Health, S-10026 Stockhohn 34 (Sweden) (Received 31 January, 1976)
SUMMARY
The lead content of cerebrospinal fluid (CSF) was found to be significantly elevated in 12 patients with amyotrophic lateral sclerosis, when compared to 28 control subjects having non-degenerative neurological disorders. The difference could not be explained as being merely secondary to blood-CSF barrier damage. A hypothetical model of the pathogenesis of the disease is advanced and the results are discussed in relation to this model.
INTRODUCTION
There has been interest in studying the relationship between amyotrophic lateral sclerosis (ALS), and lead poisoning since the work of Wilson (1907), who described 4 cases of progressive amyotrophy accompanying lead poisoning. The question has gained in topicality through some recent work, and since knowledge on both the turnover of lead in the organism and the analytical methods available for determinations of lead in various tissues have improved in recent years, further work in the field seems likely to be of interest. In a series of ongoing studies, we are investigating the turnover of lead in patients with ALS. The present communication deals with the findings obtained to date on the lead content of cerebrospinal fluid (CSF) in persons with this disease which was found to be significantly raised when compared to controls. A full description of the turnover studies will be presented elsewhere. In 1968, Currier and Haerer reported that of a series of 31 patients suffering from ALS, 24 had previously been exposed to heavy metals. However, at postmortem analysis of the spinal cord in 2 cases, normal levels of lead were found. In a report by Campbell, Williams and Barltrop (1970) based upon 74 patients suffering from
260 ALS, the proportion of patients who had been exposed to lead was 3 times greater than in a control group, but the levels of lead in bone, as determined in biopsies of the iliac crest, were found to be normal. Treatment of a few cases with Ca-EDTA was reported to have given somewhat variable results. Recently, Petkau, Sawatzky, Hillier and Hoogstraten (1974) have found increased levels of lead in the spinal cord and in muscle obtained at postmortem from 6 patients suffering from the disease. Modern concepts of lead toxicology, work on exposure and uptake of lead in the human body and analytical methods for lead have recently been reviewed by Hernberg (1975). He pointed out that determinations of lead in biological material are still difficult to perform and are subject to large methodological errors, but reasonable accuracy can be achieved if the determinations are performed under strictly controlled conditions. Currently, the level of lead in whole blood is considered to reflect both recent exposure and the total burden of lead. Most of the body burden is deposited in bone, where it has a biological half-life of 10 years or more (see ICRP-commission 1959). Nearly all of the lead in blood is considered to be bound to the erythrocytes, and interestingly, the small fraction present in plasma has been considered independent of the total level of lead in blood (Rosen and Trinidad 1974). MATERIAL AND METHODS
Material Our material consisted of 12 patients in whom ALS was diagnosed on conventional clinical grounds, and 28 controls not having primary degenerative CNS disorders. Details on the localization and duration of symptoms and on the age of the patients in the ALS group are shown in Table 1. None of the ALS patients were found to have diabetes or malignancy. The clinical diagnosis and ages of the patients in the control group are shown in Table 2.
Sampling CSF was obtained by lumbar puncture and it was confirmed that the needles were not contaminated with lead. Two ml of CSF was collected in lead-free test tubes. In all of the ALS cases and in 19 of the control group, additional CSF was taken for paper electrophoresis, and in the case of the ALS patients, also for isoelectric focussing. In the ALS group, blood was also taken for determination of lead/whole blood.
Analyticalprocedures The electrophoresis and isoelectric focussing of CSF were performed according to techniques presented earlier (Ghrde and Kjetlin 1971 ; Kjellin and Stibler 1975). The lead concentrations were measured by atomic absorption in a Perkin Elmer Model 403, by using the Delves procedures (Delves 1970) in a modified version (Vesterberg and Wrangskog 1976). Microsampting cups type 303-0813 (PerkinElmer) were used, in which samples of liquor of 50 #1 were dried and ashed before introduction into the flame. The standard curve was prepared by adding known
Sex
M F M M F F M F F F M M
Initials
G.B. G.S. S.R. P.S. D.Z. E.S. R.N. S.J. S.E. H.B. E.L. H.B.
1928 1934 1932 1928 1912 1943 1906 1922 1900 1915 1925 1946
Year o f birth
1971 1971 1972 1971 1971 1973 1974 1974 1975 1975 1970 1970
Year o f onset
+ + 4- -,~ ~+ + + ++ 4- 4~ 44- 4~ 4+ 44- 44-4-4-
Peripheral paresis (4- 4-4-4-
Symptoms
4- 44 - 4 4-
-~ +
+ -~ + + +++ 4- 4-
Spasticity (4-4- 4- 4-)
4-4-4-
+ + + ++ + 4-
+ + ~
Bulbar symptoms (4- 4-4-4-)
<
< <
< <
20 12 11 16 10 10 15 10 10 12 10 10
Whole blood lead (/tg/100 ml) 2.55 1.86 2.46 2.22 1.60 2.72 1.56 2.82 3.52 2.42 5.30 4.85 m e a n : 2.84 S -- 1.19
C S F lead (/Jg/100ml)
54 55 74 49 53 73 102 38 41 35 63 76
Total protein (rag/100 ml)
CSF
38.6 29.5 38.9 28.0 27.0 16.0 57.6 ~ 25 24.3 20.5 38.2 48.0(1971)
Albumin (mg/100ml)
D E T A I L S O F P A T I E N T S IN T H E S E R I E S - - L E V E L S O F L E A D IN W H O L E B L O O D A N D CSF, T O T A L P R O T E I N A N D A L B U M 1 N I N C S F
TABLE 1
t,J
262 TABLE 2 CONTROL GROUP ALBUMIN IN CSF In rials
1.0. M.A. K.E. S.K. R.W. E.W. K.B. O.D. K.N. P.A. L.T. K.S. G.B. I.P. L.D. G.L. 15.I. I.K. E.S. E.J. S.J. M.S. A.B. L.B. L.L. U.R O.E. ~.L.
DETAILS OF CASES A N D OF LEAD. TOTAL PROTEIN AND
Sex
F F M M M M F F M M F M M F M M M M F F F M F F M F M M
Year of birth
Clinical diagnosis
CSF lead (itg/lO0ml)
CSF
1896 1923 1948 1904 1908 1905 1910 1895 1911 1952 1903 1907 1948 1921 1917 1945 1924 1943 1948 t932 1943 1912 1911 1948 1937 1926 1938 1922
brain infarct polyneuritis brain infarct brain infarct brain infarct brain infarct commotio brain infarct TIA normal peripheral paresis myetopathy MS? encephalitis spinal tumor encephalitis MS MS? peripheral paresis torticollis normal tremor tremor MS ataxia epilepsia MS ? diabetes L brain infarct
1.53 1.34 1.27 1.89 1.63 1.26 1.90 2.04 0.99 0.89 1.20 0.94 0.88 0.99 2.21 1.59 0.93 t.51 1.46 1.06 0.96 1.08 1.06 1.68 I. 19 1.09 1.07 2.61 mean : 1.41 S 0.49
6~ 98 44 82 44 49 68 66 80 38 47 58 72 198 134 86 70 49 37 49 48 37 49 58 49 41 56 56
albumin protein (regal00 (mg/100 mb mli total
2l. 1 30.0 30.7 41.0 101.0 75.8 55.0 38.2 31.8 20.2 29.5 27.6 20.7 29.2 30.0 29.9 _90.8 36.4 32.5
a m o u n t s o f l e a d in 0.01 M H N O a t o a p o o l o f l i q u o r . E v a l u a t i o n o f t h e a t o m i c a b s o r p t i o n signal was m a d e b y i n t e g r a t i o n in t h e i n s t r u m e n t . T h e c a l c u l a t i o n s w e r e m a d e in a C o m p u c o r p
M o d e l 445 b y l i n e a r r e g r e s s i o n analysis. T h e c o e f f i c i e n t o f
r e g r e s s i o n was o f t e n 0.99 ± 0.003 f o r s a m p l e s w i t h a d d e d s t a n d a r d . D e t e r m i n a t i o n s o f t e a d in w h o l e b l o o d w e r e p e r f o r m e d a c c o r d i n g t o t h e m e t h o d o f E i n a r s s o n a n d L i n d s t e d t (1969).
Processing of data T h e C S F c o n c e n t r a t i o n s o f l e a d in t h e A L S a n d c o n t r o l g r o u p s , r e s p e c t i v e l y , w e r e t a b u l a t e d (see T a b l e s 1 a n d 2) a n d t h e d i f f e r e n c e s b e t w e e n t h e g r o u p s w e r e t e s t e d b y t h e S t u d e n t t-test o n t h e 99.9 ~ level o f p r o b a b i l i t y . S i n c e a s l i g h t d a m a g e
263 CSE lead
6-
(#g/100 m0
4"
O
•
I
20
•
O0
OO
°o ~9o °Oo
lb
2'0
• ALS PATIENTS O CONTROLS
3b
~0
5'o
6'0
7'0
8b
9'0
~60 ,'0
CSF albumin (rag//100 rnt)
Fig. ]. Diagram showing CSF lead correlated to CSF albumin in ALS and control groups.
to the blood-CSF barrier to proteins is often found in ALS (Kjellin and Stibler 1976), the CSF lead levels were also correlated to the function of this barrier, in an attempt to exclude the possibility that elevated lead/CSF values were merely secondary to a defect in the blood-CSF barrier. Thus the lead levels were correlated to CSF[albumin], as calculated from electrophoresis, in a diagram (Fig. 1), and we tried to choose controls within the range from no ( < 35 mg albumin/100 ml CSF) to severe barrier damage. RESULTS As can be seen in Tables 1 and 2, the mean value of lead in the CSF in the group of ALS cases is significantly raised when compared to findings in the control group. Three out of the 12 ALS patients showed concentrations below 2.0 #g/100 ml, whereas 3 of the controls showed values above that limit. In Fig. 1, the values correlated with information on the blood-CSF barrier are presented. It can be seen that a few of the ALS patients show slightly raised CSF albumin values ( < 35 mg/100 ml), whereas in the control group there were samples with results ranging from none to severe barrier damage. The ALS patients having no or slight barrier damage showed higher figures for CSF/lead than the controls having similar values for CSF albumin, the borderline being located at about 1.6-1.8/~g lead/100 ml CSF. One of the control cases, showing a CSF lead above 2.0/zg/100 ml CSF and 1 of the ALS patients, having a value below this limit, both showed evidence of a considerable barrier damage. As can be seen in Table 1, all of the ALS patients had normal levels of whole blood lead ( < 40/~g/100 ml).
264 DISCUSSION AND CONCLUSIONS Against the background of the above-mentioned earlier suspected relationship between ALS and lead poisoning, and previous reports of generalized capillary damage occurring in ALS (Kniffer and Quick 1969; St6rtebecker, Nordstr6m, Pap de Pest6ny, Seeman and Bj6rkerud 1970) we have formulated a theory for the pathogenetic action of lead in the disease. Our further studies aim to test the relevance ot the theory, which consists of three premises, viz. : (1) Lead is pathogenetic in ALS; (2) Lead is abnormally available in nervous tissue in the disease, which may be due, at least partly, to generalized capillary damage; (3) Lead reaches the motor neurons from skeletal muscle. Lead is deposited in the muscles. It is conjugated, taken up by the motor end-plates and is transported to the motor neuron cell bodies by means of retrograde axoplasmic flow (Kristensson and Olsson 1971). Our studies follow 2 main pathways, one clinical, of which the present study is part, and one experimental, which is an extension of our earlier studies on cat motor neurons (see Conradi 1976; Ronnevi and Conradi 1974). The findings presented here are based upon a small series o f cases, but obviously demonstrate that lead is abnormally available to the nervous system in ALS. The mechanisms of entry of lead to CSF are not known, which may be related to the present lack of knowledge on the protein-binding of lead in plasma (see Hernberg 1975). Therefore, it cannot be decided whether the elevated CSF lead levels found here reflect capillary damage or some other mechanism. Determinations of lead in plasma in ALS, at present being studied, may elucidate the question. The lead might also be bound to other components in the blood than is normally the case. As mentioned, the levels of lead in whole blood were normal in the ALS cases, and there was no correlation between these values and those of lead in CSF. In view of the theory presented above, estimations of lead in muscle seem most pertinent. However, these estimations at present being performed, are technically complicated; nevertheless, the reported findings of elevated lead levels in muscle in ALS (Petkau et al. 1974) give some support to the theory. The observation of raised levels of lead in the CSF of ALS patients does not prove a pathogenetic effect of the metal, but its neurotoxic properties are well known (see Kehoe 1972). With that background, attempts to remove the lead with chelators are now being performed in an attempt to correlate clinical effects with lead determinations.
REFERENCES Campbell, A. M. (3., E. R. Williams and D, Badtrop (1970)Motor neuron disease and exposure to
2~5 Currier, R. D. and A. F. Haerer (1968) Amytrophic lateral sclerosis, Arch. environm, tteulth, J712-719. Delves, H. T. (1970) A micro-sampling method for the rapid determination of lead in blood by atomic-absorption spectrophotometry, Analyst, 95: 431-438. Einarsson, ~. and G. Lindstedt (1969) A non-extraction atomic absorption method for the determination of lead in blood, J. clin. Lab. hwest,. 23: 367-371. Ghrde, A. and K. G. Kjellin (1971) Diagnostic significance of cerebrospinal fluid examinations m myelopathy, Acta neurol, scand., 47: 555--568. Hernberg, S. (1975) Toxic metals and their compounds Lead. In: C. Zenz (Ed.), Occupational Medicine - - Principles and Practical Applications. Year Book Medical Publishers, Chicago, IU., pp. 715-770. International Commission on Radiological Protection (1959) Recommendations o f the ICRP .Report o f Committee H on Permissible Dose for International Radiation (ICRP Publication. No. 2), Pergamon Press, London, Kehoe, R. A. (1972~ Occupational lead poisoning, Part 2 (Chemical signs of the absorption of lead), J. occup. Med., 14: 390-396. Kjellin, K. G. and H. Stibler (1975) Cerebrospinal fluid protein patterns in spasmodic torticollis. Europ. Neurol., 13: 461-475. Kjellin, K. G. and H. Stibler (1976) Non-hypertrophic familial neuropathy associated with intention tremor A variety of Charcot-Marie-Tooth disease?. J. neuroL Set.. 27: 45-57. Kniffer, I. C. and D. T. Quick (1969) Skeletal muscle capillary abnormalities in amyotrophic lateral sclerosis. Neurology (Minneap. J. 19: 312-313. Kristensson, K. and Y. Olsson (1971) Uptake and retrograde axonal transport of peroxidase in hypoglossal neurones. Acta neuropath. (Bed.), 19: 1-9. Petkau. A,, A. Sawatzky, C. R. Hillier and J. Hoogstraten (1974) Lead content of neuromuscular tissue in amyotrophic lateral sclerosis Case report and other considerations, Brit. J. indust. Med., 31: 275-287. Ronnevi. L.-O. and S. Conradi [1974) Ultrastructural evidence for spontaneous elimination of" synaptic terminals on spinal motor neurons in the kitten. Brain Res., 80: 335-339. Rosen. J. F. and E. E. Trinidad (1974) Significance of plasma lead levels in normal and lead-intoxicated children. Environm. Health Persp., 7: 139-144. St6rtebecker, P.. G. Nordstr6m, M. Pap de Pest6ny, T. Seeman and S. Bj6rkerud (1970) Vascular and metabolic studies of amyotrophic lateral sclerosis, Part 1 (Angiopathy in biopsy specimens of peripheral arteries), Neurology (Minneap.), 20: 1157-1160, Wilson. S. A. K. (1907) The amyotrophy of chronic lead poisoning Amyotrophic lateral sclerosis of toxic origin, Rev. Neurol. Psychiat., 5: 441-455. Vesterberg, O. and K. Wrangskog (1976) Determination of lead in cerebrospinal fluid by atomic absorption using a sampling cup technique, In preparation.
259
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
A B N O R M A L TISSUE D I S T R I B U T I O N OF LEAD IN A M Y O T R O P H I C L A T E R A L SCLEROSIS
SEBASTIAN CONRAD1, LARS-OLOF RONNEV[ and OLOF VESTERBERG
Department of Clinical Neurology (SC), Karolinska Sjukhuset, Department of Anatomy (LOR), Karolinska Institutet, S-104 O1 Stockholm 60, and Chemical Division (OV), Occupational Health Department, The National Board of Occupational Safety and Health, S-10026 Stockhohn 34 (Sweden) (Received 31 January, 1976)
SUMMARY
The lead content of cerebrospinal fluid (CSF) was found to be significantly elevated in 12 patients with amyotrophic lateral sclerosis, when compared to 28 control subjects having non-degenerative neurological disorders. The difference could not be explained as being merely secondary to blood-CSF barrier damage. A hypothetical model of the pathogenesis of the disease is advanced and the results are discussed in relation to this model.
INTRODUCTION
There has been interest in studying the relationship between amyotrophic lateral sclerosis (ALS), and lead poisoning since the work of Wilson (1907), who described 4 cases of progressive amyotrophy accompanying lead poisoning. The question has gained in topicality through some recent work, and since knowledge on both the turnover of lead in the organism and the analytical methods available for determinations of lead in various tissues have improved in recent years, further work in the field seems likely to be of interest. In a series of ongoing studies, we are investigating the turnover of lead in patients with ALS. The present communication deals with the findings obtained to date on the lead content of cerebrospinal fluid (CSF) in persons with this disease which was found to be significantly raised when compared to controls. A full description of the turnover studies will be presented elsewhere. In 1968, Currier and Haerer reported that of a series of 31 patients suffering from ALS, 24 had previously been exposed to heavy metals. However, at postmortem analysis of the spinal cord in 2 cases, normal levels of lead were found. In a report by Campbell, Williams and Barltrop (1970) based upon 74 patients suffering from
260 ALS, the proportion of patients who had been exposed to lead was 3 times greater than in a control group, but the levels of lead in bone, as determined in biopsies of the iliac crest, were found to be normal. Treatment of a few cases with Ca-EDTA was reported to have given somewhat variable results. Recently, Petkau, Sawatzky, Hillier and Hoogstraten (1974) have found increased levels of lead in the spinal cord and in muscle obtained at postmortem from 6 patients suffering from the disease. Modern concepts of lead toxicology, work on exposure and uptake of lead in the human body and analytical methods for lead have recently been reviewed by Hernberg (1975). He pointed out that determinations of lead in biological material are still difficult to perform and are subject to large methodological errors, but reasonable accuracy can be achieved if the determinations are performed under strictly controlled conditions. Currently, the level of lead in whole blood is considered to reflect both recent exposure and the total burden of lead. Most of the body burden is deposited in bone, where it has a biological half-life of 10 years or more (see ICRP-commission 1959). Nearly all of the lead in blood is considered to be bound to the erythrocytes, and interestingly, the small fraction present in plasma has been considered independent of the total level of lead in blood (Rosen and Trinidad 1974). MATERIAL AND METHODS
Material Our material consisted of 12 patients in whom ALS was diagnosed on conventional clinical grounds, and 28 controls not having primary degenerative CNS disorders. Details on the localization and duration of symptoms and on the age of the patients in the ALS group are shown in Table 1. None of the ALS patients were found to have diabetes or malignancy. The clinical diagnosis and ages of the patients in the control group are shown in Table 2.
Sampling CSF was obtained by lumbar puncture and it was confirmed that the needles were not contaminated with lead. Two ml of CSF was collected in lead-free test tubes. In all of the ALS cases and in 19 of the control group, additional CSF was taken for paper electrophoresis, and in the case of the ALS patients, also for isoelectric focussing. In the ALS group, blood was also taken for determination of lead/whole blood.
Analyticalprocedures The electrophoresis and isoelectric focussing of CSF were performed according to techniques presented earlier (Ghrde and Kjetlin 1971 ; Kjellin and Stibler 1975). The lead concentrations were measured by atomic absorption in a Perkin Elmer Model 403, by using the Delves procedures (Delves 1970) in a modified version (Vesterberg and Wrangskog 1976). Microsampting cups type 303-0813 (PerkinElmer) were used, in which samples of liquor of 50 #1 were dried and ashed before introduction into the flame. The standard curve was prepared by adding known
Sex
M F M M F F M F F F M M
Initials
G.B. G.S. S.R. P.S. D.Z. E.S. R.N. S.J. S.E. H.B. E.L. H.B.
1928 1934 1932 1928 1912 1943 1906 1922 1900 1915 1925 1946
Year o f birth
1971 1971 1972 1971 1971 1973 1974 1974 1975 1975 1970 1970
Year o f onset
+ + 4- -,~ ~+ + + ++ 4- 4~ 44- 4~ 4+ 44- 44-4-4-
Peripheral paresis (4- 4-4-4-
Symptoms
4- 44 - 4 4-
-~ +
+ -~ + + +++ 4- 4-
Spasticity (4-4- 4- 4-)
4-4-4-
+ + + ++ + 4-
+ + ~
Bulbar symptoms (4- 4-4-4-)
<
< <
< <
20 12 11 16 10 10 15 10 10 12 10 10
Whole blood lead (/tg/100 ml) 2.55 1.86 2.46 2.22 1.60 2.72 1.56 2.82 3.52 2.42 5.30 4.85 m e a n : 2.84 S -- 1.19
C S F lead (/Jg/100ml)
54 55 74 49 53 73 102 38 41 35 63 76
Total protein (rag/100 ml)
CSF
38.6 29.5 38.9 28.0 27.0 16.0 57.6 ~ 25 24.3 20.5 38.2 48.0(1971)
Albumin (mg/100ml)
D E T A I L S O F P A T I E N T S IN T H E S E R I E S - - L E V E L S O F L E A D IN W H O L E B L O O D A N D CSF, T O T A L P R O T E I N A N D A L B U M 1 N I N C S F
TABLE 1
t,J
262 TABLE 2 CONTROL GROUP ALBUMIN IN CSF In rials
1.0. M.A. K.E. S.K. R.W. E.W. K.B. O.D. K.N. P.A. L.T. K.S. G.B. I.P. L.D. G.L. 15.I. I.K. E.S. E.J. S.J. M.S. A.B. L.B. L.L. U.R O.E. ~.L.
DETAILS OF CASES A N D OF LEAD. TOTAL PROTEIN AND
Sex
F F M M M M F F M M F M M F M M M M F F F M F F M F M M
Year of birth
Clinical diagnosis
CSF lead (itg/lO0ml)
CSF
1896 1923 1948 1904 1908 1905 1910 1895 1911 1952 1903 1907 1948 1921 1917 1945 1924 1943 1948 t932 1943 1912 1911 1948 1937 1926 1938 1922
brain infarct polyneuritis brain infarct brain infarct brain infarct brain infarct commotio brain infarct TIA normal peripheral paresis myetopathy MS? encephalitis spinal tumor encephalitis MS MS? peripheral paresis torticollis normal tremor tremor MS ataxia epilepsia MS ? diabetes L brain infarct
1.53 1.34 1.27 1.89 1.63 1.26 1.90 2.04 0.99 0.89 1.20 0.94 0.88 0.99 2.21 1.59 0.93 t.51 1.46 1.06 0.96 1.08 1.06 1.68 I. 19 1.09 1.07 2.61 mean : 1.41 S 0.49
6~ 98 44 82 44 49 68 66 80 38 47 58 72 198 134 86 70 49 37 49 48 37 49 58 49 41 56 56
albumin protein (regal00 (mg/100 mb mli total
2l. 1 30.0 30.7 41.0 101.0 75.8 55.0 38.2 31.8 20.2 29.5 27.6 20.7 29.2 30.0 29.9 _90.8 36.4 32.5
a m o u n t s o f l e a d in 0.01 M H N O a t o a p o o l o f l i q u o r . E v a l u a t i o n o f t h e a t o m i c a b s o r p t i o n signal was m a d e b y i n t e g r a t i o n in t h e i n s t r u m e n t . T h e c a l c u l a t i o n s w e r e m a d e in a C o m p u c o r p
M o d e l 445 b y l i n e a r r e g r e s s i o n analysis. T h e c o e f f i c i e n t o f
r e g r e s s i o n was o f t e n 0.99 ± 0.003 f o r s a m p l e s w i t h a d d e d s t a n d a r d . D e t e r m i n a t i o n s o f t e a d in w h o l e b l o o d w e r e p e r f o r m e d a c c o r d i n g t o t h e m e t h o d o f E i n a r s s o n a n d L i n d s t e d t (1969).
Processing of data T h e C S F c o n c e n t r a t i o n s o f l e a d in t h e A L S a n d c o n t r o l g r o u p s , r e s p e c t i v e l y , w e r e t a b u l a t e d (see T a b l e s 1 a n d 2) a n d t h e d i f f e r e n c e s b e t w e e n t h e g r o u p s w e r e t e s t e d b y t h e S t u d e n t t-test o n t h e 99.9 ~ level o f p r o b a b i l i t y . S i n c e a s l i g h t d a m a g e
263 CSE lead
6-
(#g/100 m0
4"
O
•
I
20
•
O0
OO
°o ~9o °Oo
lb
2'0
• ALS PATIENTS O CONTROLS
3b
~0
5'o
6'0
7'0
8b
9'0
~60 ,'0
CSF albumin (rag//100 rnt)
Fig. ]. Diagram showing CSF lead correlated to CSF albumin in ALS and control groups.
to the blood-CSF barrier to proteins is often found in ALS (Kjellin and Stibler 1976), the CSF lead levels were also correlated to the function of this barrier, in an attempt to exclude the possibility that elevated lead/CSF values were merely secondary to a defect in the blood-CSF barrier. Thus the lead levels were correlated to CSF[albumin], as calculated from electrophoresis, in a diagram (Fig. 1), and we tried to choose controls within the range from no ( < 35 mg albumin/100 ml CSF) to severe barrier damage. RESULTS As can be seen in Tables 1 and 2, the mean value of lead in the CSF in the group of ALS cases is significantly raised when compared to findings in the control group. Three out of the 12 ALS patients showed concentrations below 2.0 #g/100 ml, whereas 3 of the controls showed values above that limit. In Fig. 1, the values correlated with information on the blood-CSF barrier are presented. It can be seen that a few of the ALS patients show slightly raised CSF albumin values ( < 35 mg/100 ml), whereas in the control group there were samples with results ranging from none to severe barrier damage. The ALS patients having no or slight barrier damage showed higher figures for CSF/lead than the controls having similar values for CSF albumin, the borderline being located at about 1.6-1.8/~g lead/100 ml CSF. One of the control cases, showing a CSF lead above 2.0/zg/100 ml CSF and 1 of the ALS patients, having a value below this limit, both showed evidence of a considerable barrier damage. As can be seen in Table 1, all of the ALS patients had normal levels of whole blood lead ( < 40/~g/100 ml).
264 DISCUSSION AND CONCLUSIONS Against the background of the above-mentioned earlier suspected relationship between ALS and lead poisoning, and previous reports of generalized capillary damage occurring in ALS (Kniffer and Quick 1969; St6rtebecker, Nordstr6m, Pap de Pest6ny, Seeman and Bj6rkerud 1970) we have formulated a theory for the pathogenetic action of lead in the disease. Our further studies aim to test the relevance ot the theory, which consists of three premises, viz. : (1) Lead is pathogenetic in ALS; (2) Lead is abnormally available in nervous tissue in the disease, which may be due, at least partly, to generalized capillary damage; (3) Lead reaches the motor neurons from skeletal muscle. Lead is deposited in the muscles. It is conjugated, taken up by the motor end-plates and is transported to the motor neuron cell bodies by means of retrograde axoplasmic flow (Kristensson and Olsson 1971). Our studies follow 2 main pathways, one clinical, of which the present study is part, and one experimental, which is an extension of our earlier studies on cat motor neurons (see Conradi 1976; Ronnevi and Conradi 1974). The findings presented here are based upon a small series o f cases, but obviously demonstrate that lead is abnormally available to the nervous system in ALS. The mechanisms of entry of lead to CSF are not known, which may be related to the present lack of knowledge on the protein-binding of lead in plasma (see Hernberg 1975). Therefore, it cannot be decided whether the elevated CSF lead levels found here reflect capillary damage or some other mechanism. Determinations of lead in plasma in ALS, at present being studied, may elucidate the question. The lead might also be bound to other components in the blood than is normally the case. As mentioned, the levels of lead in whole blood were normal in the ALS cases, and there was no correlation between these values and those of lead in CSF. In view of the theory presented above, estimations of lead in muscle seem most pertinent. However, these estimations at present being performed, are technically complicated; nevertheless, the reported findings of elevated lead levels in muscle in ALS (Petkau et al. 1974) give some support to the theory. The observation of raised levels of lead in the CSF of ALS patients does not prove a pathogenetic effect of the metal, but its neurotoxic properties are well known (see Kehoe 1972). With that background, attempts to remove the lead with chelators are now being performed in an attempt to correlate clinical effects with lead determinations.
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