Increased Metallothionein in the Liver and Kidney of Patients With Amyotrophic Lateral Sclerosis Peter A. E. Sillevis Smitt, MD; Hugo van Beek; Aalbert-Jan Baars, PhD; Dirk Troost, MD; Elisabeth S. Louwerse, MD; Antonia C. M. Krops-Hermus; Frederik A. de Wolff, PhD; J. M. B. Vianney de Jong, MD

To evaluate the putative role of metals and trace elements in the pathogenesis of classic amyotrophic lateral sclerosis, we studied the metallothionein levels in liver and kidney samples obtained at autopsy from 24 patients with amyotrophic lateral sclerosis and 18 controls. To assay metallothioneins and copper, cadmium, and zinc bound to

comprise increased spinal cord lead,6 selenium,7 and man¬ ganese7,8 levels; increased cerebrospinal fluid lead levels9; and increased 24-hour urinary excretion of lead after edetic

spectrometry. Total cadmium, zinc, and copper concentrations were determined separately with the use of graphite furnace atomic absorption spectrometry with Zeeman background correction. The median liver metallothionein level was 60.3 mg/kg (range, 9 to 318 mg/kg) in the patients with amyotrophic lateral sclerosis and 12.6 mg/kg (range, 0 to 104.5 mg/kg) in the controls. In the kidney, median metallothionein levels were 126.9 mg/kg (range, 44 to 387 mg/kg) in the patients with amyotrophic lateral sclerosis and 64 mg/kg (range, 13.1 to 187 mg/kg) in the controls. Total zinc, cadmium, and copper concentrations, as measured by atomic absorption spectrometry, were not significantly different in patients vs controls. Our finding of elevated metallothionein levels in organs from patients with amyotrophic lateral sclerosis may indicate an increased ex-

are major metal-binding pro¬ include the detoxification functions proposed and storage of heavy metals.11 Metallothioneins are a group of nonenzymatic, low-molecular-weight (6500 d) proteins. Sulfur-containing cysteine represents about one third of the amino acids that make up MTs. Metallothio¬ neins bind with a decreasing order of avidity to copper, cadmium, and zinc ions. Transcription of MTs is induced by a wide variety of metal ions, hormones, and various pathophysiologic conditions associated with the acutephase response.11 Much less is known about the degrada¬ tion of MTs, which is thought to take place mainly in the

\s=b\

we used high-performance liquid chromatography directly coupled to flame atomic absorption

metallothioneins,

posure to

metals.

(Arch Neurol. 1992;49:721-724) has been hypothesized that classic amyotrophic lateral sclerosis (ALS) is caused by an environmental factor.1"3 The possibility of putative involvement of toxic metals and trace elements has the longest history and is based on ep¬ idemiologie considerations4 and an increasing number of case reports of ALS-like syndromes associated with longterm exposure to metals, particularly to lead5 and mer¬ trace elements have been studied exten¬ cury.5 Metals and with ALS. Reported metal disturbances sively in patients

It

Accepted

for publication January 30, 1992. Departments of Neurology (Drs Sillevis Smitt, Louwerse, and de Jong), Neuropathology (Dr Troost), and Occupational and Environmental Health (Dr de Wolff), Academic Medical Centre, Amsterdam, and the Department of Biochemistry and Toxicology, Central Veterinary Institute, Lelystad (Mr van Beek, Dr Baars, and Ms Krops-Hermus), the From the

Netherlands. Reprint requests to the Department of Neurology, Academic Medical Centre H-2-214, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands (Dr Sillevis Smitt).

acid (EDTA) treatment.10 However, most of these alter¬ ations could not be reproduced.5 Conflicting results, diffi¬ culties in obtaining uncontaminated samples, and lack of history of exposure in most patients with ALS have led to skepticism concerning the exposure to metal hypothesis.

Metallothioneins (MTs)

teins. Their

lysosomes.12 To establish a possible role of MTs in ALS, we have studied the hepatic and renal levels of MT, zinc, cadmium, and copper in liver and kidney samples obtained at autopsy from patients with classic ALS and controls. PATIENTS, SUBJECTS,

AND METHODS

At autopsy, 18 liver and 24 kidney samples were prospectively collected from 25 patients with classic ALS and 18 liver and 18 kidney samples were obtained from 19 sex- and age-matched controls. Seventeen patients with ALS had participated in an open clinical trial of N-acetyl-L-cysteine (NAC) in which high-dose NAC was administered subcutaneously daily. Eight patients had received NAC almost until the time of death, while the other nine had stopped taking NAC more than 3 months before death. In the eight patients, the median daily dose was 3.25 g (range, 1 to 5 g) for a median period of 13.5 months (range, 3 days to 26 months). The death-to-autopsy interval was less than 16 hours. No signif¬ icant relationship has been found to exist between the death-toautopsy interval and the concentrations of cadmium, zinc, copper, and MT.13 Samples were stored in aluminum containers at -80°C until assay. To simultaneously measure MT and MT-bound copper, cad¬ mium and zinc, we applied protein separation by highperformance liquid chromatography, with on-line combined di¬ ode array detection and flame atomic absorption spectrometry.14 Liver and kidney samples weighing 2 g were homogenized in

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Table 1.—Liver Metallothionein (MT), Zinc, MT-Zinc, Cadmium, MT-Cadmium, Copper, and MT-Copper Concentrations*

Group No.

Age,

ALS

Control

18

18

Sex, M/F

9/9 60.3(9-318)

MT

Zinc MT-zinc

Cadmium MT-cadmium

Copper MT-copper "Metals and MTs

62.6(37.8272.5) 3.3 (0.5-29.8) 2.6(0.4-9.1) 0.8(0-3.1) 4.5(1-14.4) 0.7(0.2-1.7) are

expressed

in

MT-Copper Concentrations*

Group .07

Age,

y

Sex, M/F .002

MT

Zinct

48.4(27.6-99.1) 0.8 (0-8.4) 1.8(0.1-6) 0.3(0-1.5) 4.3(1.4-10.6) 0.2(0-1.5)

milligrams

per

.06

MT-zinc

.004

Cadmiumt MT-cadmium

.9 .007 .95

.0007

kilogram of wet

weight. MT-zinc, MT-cadmium, and MT-copper denote the respective metal concentrations bound to MT. Values are expressed as median (reference range).

Control

ALS

No.

69(15-90) 9/9 12.6(0-104)

66(41-75)

y

Table 2.—Kidney Metallothionein (MT), Zinc, MT-Zinc, Cadmium, MT-Cadmium, Copper, and

Copperf MT-copper *Metals and MTs

24

18

66(41-77) 12/12 126.9(44-387) 26.8(15.9-89) 1.9(0.5-15) 21.9(1.8-49) 7.6(1.3-26.5) 1.8 (0.5-7.5) 0.8(0.3-1.7)

69(15-90) 9/9 64(13.1-187) 24.9 (9.1-48.3) 0.6(0.1-4.8) 14.9(1.3-48.4) 3.6(0.7-13.1) 1.9(0-9.5) 0.4 (0-0.8)

are

expressed

in

milligrams

per

.17

.003 .4 .009

.16 .003

.9 ' ^

frV^y^jOf*»

_^ -»^ ~—« I

J.^-

Zinc

Copper

J 30 ng/mL A.

fi

li

I.M' llnWllh^^flliÎi'iiÎii^M"1·-"^^'-·· T"r"TTTT""""rweipy K'HllW*1"1 W|Wf

J20 ng/mL Copper

J30 ng/mL

I

10

20

30

40

10

20

30

40

Fig 1.—Chromatograms of metallothionein and protein-bound zinc, cadmium, and copper in liver (left) and kidney (right) specimens from a patient with amyotrophic lateral sclerosis. Metallothioneins elute between 23 and 27 minutes.

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between grouped data were assessed with the Mann-Whitney U test. A two-tailed level of P.7). Kidney MT concentration was 155 mg/kg (range, 46 to 264 mg/kg) and 115 mg/kg (range, 44 to 387 mg/kg) in NAC-treated and non-NAC-treated patients with ALS,

respectively (P>.6).

COMMENT of elevated MT levels in liver and kidney finding specimens from patients with ALS may indicate either in¬ creased synthesis or decreased breakdown of these pro¬ teins. Ions of zinc, cadmium, and copper are potent inducers of MT synthesis.11 The normal tissue levels of cadmium and copper (Tables 1 and 2) indicate that MTs have not been induced by a local excess of these metals. Liver zinc levels in patients with ALS are very close to being signif¬ icantly higher than those in controls (P=.06). It is possible that a small difference in (long-term) exposure to zinc has resulted in increased liver and kidney MT concentrations. A number of other metals may be responsible for the in¬ creased MT levels in patients with ALS: mercury, manga¬ nese, and lead have been implicated in the etiology of ALS5 and are able to induce MT11 as well. Aluminum, much dis¬ cussed in cases of ALS in the western Pacific region, has not been studied well in tissues from patients with classic ALS.1 According to one report, aluminum can induce and bind to MT.16 However, this finding was not confirmed by other reports,17 and aluminum is not generally regarded as an inductor of MT.11 Metallothionein levels could have increased due to overproduction of defective proteins. Although this issue was not addressed in our study, the normal percentage of

Our

(MT) in liver and kidney specimens from patients with amyotrophic lateral sclerosis and controls. Horizon¬ tal bars indicate the medians.

Fig 2.—Levels of metallothionein

10 mmol/L of ammoniumdihydrogenphosphate (pH 8.2), heated for 90 minutes at 100 000g for 2 minutes at 100°C, and and 4°C. The resulting liver and kidney supernatants were filtered through 0.8-µ and 0.45-µ polymer filters (Millipore, EttenLeur, the Netherlands), respectively. Appropriate amounts (0.2 to 1 mL) were injected into a reversed-phase Polymer Laboratories Reversed Phase-S high-performance liquid chromatography col¬ umn (PLRP-S, Polymer Laboratories, Church Stretton, United Kingdom) and eluted with a gradient of 10 mmol/L of ammoni¬ umdihydrogenphosphate (pH 8.2) and acetonitrile. Protein con¬ centration was measured by diode array detection at 245 nm, and zinc, cadmium, and copper concentrations were measured by flame atomic absorption spectrometry at 213.9, 228.8, and 324.8

centrifugea

nm,

respectively.

Total cadmium, zinc, and copper concentrations in liver and kidney samples were determined separately with the use graph¬ ite furnace atomic absorption spectrometry with Zeeman back¬ ground correction.15 Group data are given as median and ranges. Differences

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MT bound to zinc, cadmium, and copper in patients with ALS defied this notion. Synthesis of MTs, which are rich in cysteine, could hypothetically be induced by NAC, which had been taken by eight patients with ALS until the time of death. No signif¬ icant differences in MT levels were found between patients with ALS who did and did not receive NAC treatment. Many other factors are known to induce MT synthesis in animal models and cultured cells.11 Because much less is known about MT induction in humans, it is difficult to discern potentially confounding factors between patients with ALS and controls. It is possible that low-grade differ¬ ences in MT-inducing agents (known or unknown) have existed for a prolonged period before death. Wasting and weight loss are prominent features in most patients with terminal ALS. Starvation is known to induce MT synthesis in rats18; however, the patients in the study of Lui et al13 on Alzheimer's disease retained normal liver MT levels de¬ spite severe weight loss. Sulfoxidation is impeded in ALS.3 It is unknown whether the breakdown of MTs, which are rich in the sulfur-containing cysteine, requires sulfoxidation. De¬ creased sulfoxidation does not seem to lead necessarily to an increase in MT, as in Alzheimer's disease hepatic MT

levels

are

normal13

despite disturbed sulfoxidation.19 References

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1989;119:1062-1070.

13. Lui E, Fisman M, Wong C, Diaz F. Metals and the liver in Alzheimer's disease: an investigation of hepatic zinc, copper, cadmium, and metallothionein. J Am Geriatr Soc. 1990;38:633-639. 14. van Beek H, Baars AJ. Direct coupling of HPLC to flame AAS to isolate and quantitate metalloproteins. Atomic Spectroscopy. 1990;11:70\x=req-\

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ground correction. Talanta. 1987;34:580-582. 16. Jefferey EH, Jansen HT, Dellinger JA. In vivo interactions of aluminum with hepatic cytochrome P-450 and metallothionein. Fundam Appl Toxicol. 1987;8:541-548.

17. Van der Voet GB, Brandsma AE, Heijink E, de Wolff FA. Accumulation of aluminium in rat liver: association with constituents of the cytosol. Pharmacol Toxicol. In press. 18. Bremner I, Davies NT. The induction of metallothionein in rat liver by zinc injection and restriction of food intake. Biochem J. 1975; 149: 733-738. 19. Steventon GB, Heafield MTE, Sturman S, Waring RH, Williams AC. Xenobiotic metabolism in Alzheimer's disease. Neurology. 1990;40:1095\x=req-\ 1098.

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