Article pp.112-115 du Vol.23 n°1 (2003)

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112-115 Sci. Aliments 23(1), 2003 O. Robert et al.

FOCUS : JSMTV

Use of proton nuclear magnetic resonance (

¹

H-NMR) for brain comparison of healthy

and scrapie sheep

O. Robert

¹

, M.C. Aoust de Rouveze

¹

, V. Gilard

¹

,

M. Malet-Martino

¹

, R. Martino

¹

, V. Gayrard

²

, N. Picard-Hagen

²

INTRODUCTION

Scrapie is a transmissible spongiform encephalopathy, which affects the central nervous system of sheep and goats. It is characterised by neurodegenerative lesions that systematically occur in the cerebral trunk region (pons or obex). Our aim was to compare with ¹H NMR the metabolic profiles of two brain regions (cerebellar hemisphere and obex) in healthy sheep and in sheep suffe- ring from natural scrapie.

EXPERIMENTAL PART

Four healthy and six scrapie animals of Manech redhead race were used for these experiments. Samples of brain tissue were about 55 mg on average. They were removed immediately after death in the obex part (14 and 18 samples for healthy and scrapie animals, respectively) and in the cingular gyrus (CG) located in the cerebral hemispheres (12 and 19 samples for healthy and scrapie sheep, respectively). The samples were frozen at –80°C in the 30 minutes after death.

Perchloric acid extracts placed in a sealed tube were analysed with ¹H NMR at 400 MHz on a Bruker ARX 400 spectrometer.

1. Groupe de RMN Biomédicale, Laboratoire des IMRCP (UMR CNRS 5623), Université Paul Sabatier, 118 route de Narbonne, 31062 TOULOUSE Cedex.

2. Laboratoire de Physiopathologie et Toxicologie Expérimentales (UMR INRA 181), École Nationale Vétéri- naire de Toulouse, 23, chemin des Capelles, 31076 Toulouse Cedex.

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Use of proton nuclear magnetic resonance (1H-NMR) for brain comparison of healthy and scrapie sheep 113

RESULTS AND DISCUSSION

Using 1D and 2D NMR techniques, signals were identified, and attributed to metabolites as follows: lactate, alanine, γ-aminobutyric acid (GABA), acetate, N- acetylaspartate (NAA), N-acetylaspartylglutamate (NAAG), glutamate (Glu), glutamine (Gln), succinate, aspartate, créatine, choline (Cho), glycerophosphocholine (GPC), phosphocholine (PC), inositol and scylloinositol (Figure). NAAG found in the obex extract was not detected in the CG extract.

FIGURE

High-resolution ¹H NMR spectrum of a PCA extract from a scrapie brain (obex part).

(I: spectral region from 1.8 to 3.1 ppm; II: spectral region from 3.0 to 4.5 ppm)

Table reports the concentrations of metabolites (in µmoles/g of wet weight tissue) according to the pathological status and the nature of the cerebral sam- ple (obex, CG). In healthy sheep, the concentrations of lactate, alanine and GPC + PC were higher in obex than in CG. In scrapie brains, the concentrations of NAA, GABA, Glu and Gln were lower in obex than in CG, whereas those of GPC + PC and choline were more elevated in obex. The concentrations of NAA and NAAG in obex were lower in scrapie than in healthy animals, when the disease increased the GABA concentrations. In the CG part, the concentrations of alanine, NAA, GABA, Glu, Gln, and GPC + PC were higher in scrapie brains.

Creatine

GABA Aspartate GABA

NAA+NAAG NAA+NAAG + aspartate

Succinate Gln Glu

Glu+Gln NAAG

NAA

Acetate GABA

ppm

3.0 2.8 2.6 2.4

3.8 4.0

4.2

4.4 3.6 3.4 3.2

2.2 2.0

NAA

Lactate Inositol

Creatine

Glu+Gln

Inositol+Impurities

Scylloinositol Inositol

GPC Creatine

PC Cho I

II

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114 Sci. Aliments 23(1), 2003 O. Robert et al.

TABLE

Concentrations of metabolites (lactate, alanine, N-acetylaspartate (NAA), N-acetylaspartylglutamate (NAAG), γ-aminobutyric acid (GABA), glutamate, glutamine, creatine, choline, glycerophosphocholine + phosphocholine (GPC + PC)

and inositol (in µmoles/g of wet weight tissue) measured by ¹H NMR in perchloric acid extracts of obex and cingular gyrus regions

from four healthy and six scrapie sheep.

To our knowledge, the literature only reports ¹H NMR studies of brain extracts of experimental murine models of scrapie (1,2). It is surprising to see that differences in the metabolic profiles between healthy and scrapie sheep are more numerous in CG than in obex where the neurodegenerative lesions are located. The increase of NAA in the CG of scrapie sheep was not observed in the murine model of scrapie (1,2). The increased inositol concentration reported in a murine model of scrapie (2) was not observed in our study. However, the authors quantify this metabolite by integration of the signal at 3.55 ppm, an area strongly “contaminated” by signals of other metabolites (Figure), whereas we quantified this metabolite by integration of its isolated resonance at 3.30 ppm.

The increase of GABA in the CG of scrapie animals is in agreement with the results of Lu et al. (3), who suggested a modification of the GABA distribution in the brain of hamster experimentally infected by scrapie.

Obex Cingular Gyrus

Metabolites Healthy Scrapie Healthy Scrapie

Lactate 14 ± 5 11 ± 2 9.2 ± 3.2 11 ± 3

Alanine 0.48 ± 0.18 0.40 ± 0.08 0.32 ± 0.15 0.42 ± 0.14

NAA 2.8 ± 1.1 1.8 ± 0.4 3.2 ± 1.2 3.9 ± 0.8

NAAG 1.0 ± 0.5 0.59 ± 0.36

GABA 0.72 ± 0.23 0.88 ± 0.28 0.61 ± 0.27 1.2 ± 0.3

Glutamate (Glu) 3.1 ± 1.0 2.6 ± 0.3 3.8 ± 1.5 5.9 ± 1.2 Glutamine (Gln) 1.5 ± 0.4 1.6 ± 0.4 1.6 ± 0.5 2.1 ± 0.5

Creatine 4.8 ± 1.6 4.9 ± 1.2 3.9 ± 1.6 5.0 ± 1.3

Choline (Cho) 0.18 ± 0.09 0.15 ± 0.05 0.15 ± 0.06 0.11 ± 0.05

GPC+PC 1.5 ± 0.5 1.8 ± 0.5 1.0 ± 0.4 1.5 ± 0.3

Inositol 7.5 ± 2.8 8.3 ± 2.0 6.4 ± 2.7 8.6 ± 2.2

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Use of proton nuclear magnetic resonance (1H-NMR) for brain comparison of healthy and scrapie sheep 115

CONCLUSION

In conclusion, this ¹H NMR study demonstrates a modification of the metabolic profiles in sheep with clinical symptoms of scrapie. This study could be extended to brain samples of animals in the incubation phase of scrapie to see if the metabolic modifications precede the clinical phase. Moreover, other samples like muscles or cerebrospinal fluid could be studied, with the advan- tage that their analysis does not need the animal sacrifice and limits the risk of contamination.

REFERENCES

1. BELL J.D., COX I.J., WILLIAMS S.C.R., BELTON P.S., MCCONNELL I., HOPE J., 1991. J. Gen. Virol., 72, 2419-2423.

2. CHUNG Y.L., WILLIAMS A., CHONG A.,

JAMES H., WILLIAMS S.C.R., BELL J.D., 1996. NMR in Biomedicine, 9, 359-363.

3. LU P., STURMAN J.A., BOLTON D.C., 1995. Brain Res., 681, 235-241.