Manganese, Copper, and Zinc in Cerebrospinal Fluid ...

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Frank and M. Samter, eds., Little Brown, Boston, pp. 1081–1101 ... P. Dore-Duffy, F. Catalanotto, J. O. Donaldson, et al., Zinc in multiple sclerosis, Ann. Neurol.

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Manganese, Copper, and Zinc in Cerebrospinal Fluid from Patients with Multiple Sclerosis TORUN M. MELØ,1 CECILIE LARSEN,1 LINDA R. WHITE,2,3 JAN AASLY,3 TORILL E. SJØBAKK,1 TROND P. FLATEN,1 URSULA SONNEWALD,2 AND TORE SYVERSEN*,2 Departments of 1Chemistry and 2Clinical Neurosciences, Norwegian University of Science and Technology, N-7491 Trondheim, Norway; and 3Department of Neurology, Regional and University Hospital, N-7006 Trondheim, Norway Received July 8, 2002; Accepted August 20, 2002

ABSTRACT The concentrations of manganese, copper, and zinc were measured in cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) and patients with no known neurological disease (control group). Manganese and copper levels were determined by two different analytical methods: atomic absorption spectrometry (AAS) and high-resolution inductively coupled plasma–mass spectrometry (HR-ICP-MS), whereas zinc levels were determined by HR-ICP-MS only. Manganese levels (mean±SEM) were significantly decreased in the CSF of MS patients (1.07±0.13 µg/L, ICP-MS; 1.08±0.11 µg/L, AAS) compared to the levels in the control group (1.78±0.26 µg/L, ICP-MS; 1.51±0.17 µg/L, AAS). Copper levels were significantly elevated in the CSF of MS patients (10.90±1.11 µg/L; ICP-MS, 11.53±0.83 µg/L, AAS) compared to the levels in the control group (8.67±0.49 µg/L, ICP-MS; 9.10±0.62 µg/L, AAS). There were no significant differences between the CSF zinc levels of MS and control patients. The physiological basis for the differences in manganese and copper concentrations between MS patients and controls is unknown, but could be related to alterations in the manganese-containing enzyme glutamine synthetase and the copper-containing enzyme cytochrome oxidase, respectively. Index Entries: multiple sclerosis; atomic absorption spectrometry; HR-ICP-MS; copper; manganese; zinc.

*Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research


Vol. 91, 2003


Melø et al.

INTRODUCTION Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS). Why demyelination develops is not known. Studies have shown that MS is an immunologically mediated disease with a genetic predisposition (1–3). The onset, however, is probably provoked by multiple unknown factors. These factors may be environmental, virus mediated, and/or arise from metabolic changes resulting from excessive production of nitric oxide (NO). Active nitrogen species are overproduced in inflammatory brain lesions in MS (4,5). NO has been shown to mediate the death of oligodendrocytes, the myelin-producing cells that are primary targets of damage in MS (4,6–8). The concentrations of metals in the cerebrospinal fluid (CSF) are lower and held at more constant levels compared with those in blood, because of the carefully controlled metal permeability of the blood–brain barrier (BBB). As there is no barrier separating the brain from the CSF, the composition of the CSF will reflect metabolic processes in the brain more closely than will that of the blood. However, CSF obtained through lumbar puncture reflects processes occurring in the spinal cord as well as in the brain, and this should be taken into consideration when examining its composition. Abnormalities of transition metal metabolism are increasingly implicated in the pathogenesis of neurodegenerative disorders (9–11). Limited work has been done on the possible roles of metals and trace elements in MS. Therefore, examining the levels of manganese (Mn), copper (Cu), and zinc (Zn) may provide some insight into the underlying metabolic changes that are related to the disease process.

MATERIALS AND METHODS The CSF samples were among those taken for diagnostic assessment of patients with suspected neurological diseases at the University Hospital of Trondheim. There were 18 patients diagnosed with MS. Of these, 15 patients had the relapsing–remitting form of MS, all but one in the active phase. The remaining three patients had the primary chronic form of the disease. Diagnosis was made according to the Poser criteria (12). Evidence of demyelination was confirmed by magnetic resonance imaging in all patients for whom electrophoresis confirmed oligoclonal bands in the CSF. The MS group was compared with age- and-sex-matched patients with no known neurological disease or deficit (controls, n = 19). The cerebrospinal fluid was obtained by lumbar puncture performed in the lateral recumbent position with a stainless-steel needle (0.7×88 mm, Spinocan, articlenr.04507908; B. Braun). The CSF ran through polyethylene tubing (Optidynamic, Althin) into an ice-cooled polystyrene tube. The samples were centrifuged to remove cells (3000g for 10 min at 4°C), and the supernatant was transferred to a new polystyrene tube. The samples were stored at –80°C. Biological Trace Element Research

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Mg, Cu, and Zn in Multiple Sclerosis


The concentrations of Mn and Cu were determined by atomic absorption spectrometry (AAS; Perkin-Elmer, model 5100PC) equipped with a HGA600 graphite furnace, Zeeman background corrector, and AS60 autosampler. Because of the limited amounts of CSF available, Zn could not be determined by AAS. For analysis, 200-µL aliquots of the CSF samples were transferred into new, acid-washed (with 6 M HNO3) polyethylene tubes. The samples were diluted with ultrapure water, and HNO3 (Merck, p.a.) was added to a final concentration of 0.03 M. Blind samples (tests and blanks) were prepared by the same procedure, using ultrapure water instead of CSF samples for the blanks. All plasticware was presoaked in 6 M HNO3 overnight to remove surface metal contamination, and then thoroughly rinsed with ultrapure water. The wavelengths used were 279.5 nm for Mn and 324.8 nm for Cu. Pyrocoated tubes were used throughout, and, in addition, an L’Vov platform was employed for the measurement of Mn. Calibration curves were prepared from standard stock solutions (BDH Chemicals Ltd., Poole, UK). The inductively coupled plasma–mass spectrometric (ICP-MS) analysis of Mn, Cu, and Zn was performed using a high-resolution ICP-MS (Thermo [Finnigan] model Element) with seven scans per sample. The radio-frequency power was 1150 W. The sample was introduced using a Cetac autosampler (AS×500) with a peristaltic pump (pump speed at 1 mL/min). A concentric Meinhard nebulizer connected to a Scott spray chamber was used. All of the elements were analyzed at medium resolution. For analysis, 140–200 µL of CSF, depending on the available amount of sample, was transferred into new polyethylene tubes (Merck Eurolab AS). The samples were diluted in ultrapure water to a total volume of 2.90 mL before the addition of 100 µL concentrated HNO3 (Merck, Suprapure). The instrument was calibrated using a 0.5M HNO3 solution containing Cu, Zn, and Mn at appropriate concentrations. To check for possible drift in the instruments, a standard solution with known elemental concentrations were analyzed for every 10 samples. In the ICP-MS analysis, two aliquots of a certified reference material (Seronorm animal reference/control serum; Nycomed, Oslo) were also analyzed. The samples were analysed in random order, and the analyst did not know the identification of the samples. Serum and CSF albumin were determined by the routine procedure at the University Hospital. The albumin ratio (QA = albumin in CSF/albumin in serum) was used as a measure of the disturbance of the BBB (13). Statistical analysis was carried out using the Mann–Whitney U-test (14), and p

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