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Calcium content of mice brain lipids during myelination
Neuroscience Letters, 3 (1976) 311--314
311
©Elsevier/North-Holland Biomedical Press
CALCIUM CONTENT OF MICE BRAIN LIPIDS DURING MYELINATION
SERGE POLLET, GERARD CHAIX and NICOLE BAUMANN Laboratoire de Neurochimie, INSERM U.134, H6pital de la Salpdtri~re, 75634 Paris Cedex 13 (France)
(Received September 30th, 1976) (Accepted October 8th, 1976)
SUMMARY Calcium levels have been determined in brain of C57B1 mice and in the Quaking dysmyelinating m u t a n t during the myelination period. In control mice, calcium c o n t e n t per gram brain is maximum at 20 days, at the acme of myelination, and decreases thereafter to reach a plateau at 30 days. This variation does n o t occur in the m u t a n t where the level is 50% lower than maxim u m in controls and stable at 15 days after birth. In control mice, the variation may be related to the level or to the qualitative variation of some glycolipids and proteolipids as it disappears in lipid extract after Folch wash.
Calcium takes part in the constitution of membranes and in their physicochemical and pharmacological properties [ 2,3,5] but its localizations and modes of action remain unclear. As we have been working on a dysmyelinating mutant, the Quaking mouse in which there is an important decrease in the brain c o n t e n t of myelin sphingolipids [1], a m e t h o d for the determination of calcium in brain lipid extracts was devised and a study of calcium level as a function of age was undertaken during myelination (this developmental process occurs in brain between the 10th and the 50th day with a m a x i m u m at 18 days). Each experiment was performed on 15--30 male C57B1 mice of the same age and homogeneous in weight. The Quaking mutation is recessive and not detectable before 12 days after birth; therefore the study was only done after this date in the mutant. Glassware was washed carefully with acid (concentrated HNO3 diluted twice) and abundantly rinsed with distilled and quartz bidistilled water. Lipid extract was performed on lyophilized brains and extracted twice in the cold, each time for 30 min, with chloroform-methanol 2:1 (V/V), 1 ml for 20 mg and in a minimal volume of 5 ml. Gangliosides and proteolipids were separated from total lipid extract by a Folch wash [4], with 0.2 vol.
312
bidistilled water and also with 0.15 vol. chloroform-methanol-water 3: 48:47 (V/V/V). Lipid extracts, either washed or non-washed, were kept in chloroform-methanol 2:1 (V/V) at a concentration ranging from 2 to 5 mg/ml. Before calcium determination, a mineralization of a dry lipid extract is performed during 1 h 15 rain at 500°C. Thereafter, 0.5 ml of concentrated HNO3 diluted 10-fold are added and the mineralized extract is heated at 80°C for 20 min,to obtain nitrates. The excess of acid is eliminated by evaporation to dryness. After mineralization, 1 ml of a solution of lanthanum chloride is added. This solution is made up from 11.7 g lanthanum oxide, 50 ml concentrated HC1 and completed to 200 ml with bidistilled water; it is diluted 100-fold before usage. Calcium determination is carried out with a Varian-Techtron atomic absorption spectrophotometer, equipped with a 6 mA calcium hollow cathode lamp. A mixture of air (11 liter/min) - acetylene (1 liter/min) is necessary for the flame. The resonance wave-length for calcium is 422.7 nm. The standard solution is made of Ca(NO3 )2 in bidistilled water and the standard curve is constituted of samples of 0.5, 1, 2, 3, 4 and 5 t~g/nl which have been previously mineralized under the same conditions as the lipid extracts. So as to control a possible influence of the lipid matrix, the determinations are made with a constant a m o u n t of lipid extract to which are added increasing quantities of calcium standards (standard additions method). On Fig. 1 are indicated the levels of calcium in lipid extracts before and after Folch wash. In non-mutant mice there is a maximum at 20 days, period
30
- - b
10
N
~QK
o = t O
4
8
10 12 15
18
21
25
30
....o N *OK
Adul't
Age in d a y s (post n a t a l )
Fig. 1. C a l c i u m level in m i c e brain lipid e x t r a c t s as a f u n c t i o n o f age. E], t o t a l lipid e x t r a c t s in c o n t r o l s ; ©, t o t a l lipid e x t r a c t s in c o n t r o l s a f t e r F o l c h w a s h ; A, t o t a l lipid e x t r a c t s in Q u a k i n g ; o, t o t a l lipid e x t r a c t s in Q u a k i n g a f t e r F o l c h wash. E a c h e x p e r i m e n t was perform e d a m i n i m u m o f t h r e e t i m e s . T h e r e is a v a r i a t i o n o f 15% in c a l c i u m values o b t a i n e d .
313
of active myelination, followed by a decrease, and a plateau occurs at 30 days. Variation is more important before than after Folch wash. In the washed extract, there is little modification after 10 days. Fig. 1 shows also the values obtained in the mutant. Calcium c o n t e n t is lower (50% lower compared to m a x i m u m in controls) and there is little variation in lipid extracts, either washed or non-washed. The level is practically constant after 15 days. Calcium detected in total lipid extracts may be related to lipids or to proteolipids. Free calcium in water, coming from the extracellular space and solubilized by the mixture of chloroform-methanol, could be another possibility. In Fig. 2 is indicated the variation of water per gram brain as a function of age. No relation can be established between water and calcium contents, as the level of water is constant when there is maximal variation for calcium, in both normal and Quaking mice; on the other hand, partitioning of 4s CaC12 between lower and upper phases after Folch wash indicates an equal quantity in the two phases; if the difference found was related to free calcium taken up during lipid extraction, a similar type of curve would be obtained before and after Folch wash and this is n o t the case.
c 900 .Q
©
A
•
8O(3
700
0
zl
8 1012 15 18 2'1 2'5 Age in days (postnatal)
L
3'0
~
-
~N •QK
-
Adult
Fig. 2. Water c o n t e n t in controls ( A ) and Quaking ( • ) mice.
As the variation in the a m o u n t of calcium as a function of age occurs only before Folch wash, it may be related to proteolipids or to those glycolipids which are eliminated by this procedure. Further work is necessalw to see which of these constituents this variation is related to and why it disappears in the mutant. Nevertheless, this work shows for the first time variation in lipid calcium content related to brain maturation.
ACKNOWLEDGEMENTS
This work was supported by I.N.S.E.R.M. and D.R.M.E.
314
REFERENCES 1 Baumann, N.A., Jacque, C., Pollet, S. and Harpin, M.L., Fatty acid and lipid composition of the brain of a myelin deficient mutant, the Quaking mouse, Europ. J. Biochem., 4 (1968) 340--344. 2 Brink, F., The role of Ca ions in neural processes, Pharmacol. Rev., 6 (1954) 243--285. 3 Deamer, D.W. and Cornwell, D.G., Calcium action on fatty acid and phospholipid monolayers and its relation to cell membrane, Biochem. biophys. Acta (Amst.), 116 (1966) 555--562. 4 Folch, J., Ascoli, I., Lees, M., Meath, J.A. and Lebaron, N., Preparation of lipid extracts from brain tissue, J. biol. Chem., 191 (1951) 833--841 . 5 Shah, D.O. and Schulman, J.H., Interactions of Ca ++ with lecithin and sphingomyelin monolayers, Lipids, 2 (1967) 21.
311
©Elsevier/North-Holland Biomedical Press
CALCIUM CONTENT OF MICE BRAIN LIPIDS DURING MYELINATION
SERGE POLLET, GERARD CHAIX and NICOLE BAUMANN Laboratoire de Neurochimie, INSERM U.134, H6pital de la Salpdtri~re, 75634 Paris Cedex 13 (France)
(Received September 30th, 1976) (Accepted October 8th, 1976)
SUMMARY Calcium levels have been determined in brain of C57B1 mice and in the Quaking dysmyelinating m u t a n t during the myelination period. In control mice, calcium c o n t e n t per gram brain is maximum at 20 days, at the acme of myelination, and decreases thereafter to reach a plateau at 30 days. This variation does n o t occur in the m u t a n t where the level is 50% lower than maxim u m in controls and stable at 15 days after birth. In control mice, the variation may be related to the level or to the qualitative variation of some glycolipids and proteolipids as it disappears in lipid extract after Folch wash.
Calcium takes part in the constitution of membranes and in their physicochemical and pharmacological properties [ 2,3,5] but its localizations and modes of action remain unclear. As we have been working on a dysmyelinating mutant, the Quaking mouse in which there is an important decrease in the brain c o n t e n t of myelin sphingolipids [1], a m e t h o d for the determination of calcium in brain lipid extracts was devised and a study of calcium level as a function of age was undertaken during myelination (this developmental process occurs in brain between the 10th and the 50th day with a m a x i m u m at 18 days). Each experiment was performed on 15--30 male C57B1 mice of the same age and homogeneous in weight. The Quaking mutation is recessive and not detectable before 12 days after birth; therefore the study was only done after this date in the mutant. Glassware was washed carefully with acid (concentrated HNO3 diluted twice) and abundantly rinsed with distilled and quartz bidistilled water. Lipid extract was performed on lyophilized brains and extracted twice in the cold, each time for 30 min, with chloroform-methanol 2:1 (V/V), 1 ml for 20 mg and in a minimal volume of 5 ml. Gangliosides and proteolipids were separated from total lipid extract by a Folch wash [4], with 0.2 vol.
312
bidistilled water and also with 0.15 vol. chloroform-methanol-water 3: 48:47 (V/V/V). Lipid extracts, either washed or non-washed, were kept in chloroform-methanol 2:1 (V/V) at a concentration ranging from 2 to 5 mg/ml. Before calcium determination, a mineralization of a dry lipid extract is performed during 1 h 15 rain at 500°C. Thereafter, 0.5 ml of concentrated HNO3 diluted 10-fold are added and the mineralized extract is heated at 80°C for 20 min,to obtain nitrates. The excess of acid is eliminated by evaporation to dryness. After mineralization, 1 ml of a solution of lanthanum chloride is added. This solution is made up from 11.7 g lanthanum oxide, 50 ml concentrated HC1 and completed to 200 ml with bidistilled water; it is diluted 100-fold before usage. Calcium determination is carried out with a Varian-Techtron atomic absorption spectrophotometer, equipped with a 6 mA calcium hollow cathode lamp. A mixture of air (11 liter/min) - acetylene (1 liter/min) is necessary for the flame. The resonance wave-length for calcium is 422.7 nm. The standard solution is made of Ca(NO3 )2 in bidistilled water and the standard curve is constituted of samples of 0.5, 1, 2, 3, 4 and 5 t~g/nl which have been previously mineralized under the same conditions as the lipid extracts. So as to control a possible influence of the lipid matrix, the determinations are made with a constant a m o u n t of lipid extract to which are added increasing quantities of calcium standards (standard additions method). On Fig. 1 are indicated the levels of calcium in lipid extracts before and after Folch wash. In non-mutant mice there is a maximum at 20 days, period
30
- - b
10
N
~QK
o = t O
4
8
10 12 15
18
21
25
30
....o N *OK
Adul't
Age in d a y s (post n a t a l )
Fig. 1. C a l c i u m level in m i c e brain lipid e x t r a c t s as a f u n c t i o n o f age. E], t o t a l lipid e x t r a c t s in c o n t r o l s ; ©, t o t a l lipid e x t r a c t s in c o n t r o l s a f t e r F o l c h w a s h ; A, t o t a l lipid e x t r a c t s in Q u a k i n g ; o, t o t a l lipid e x t r a c t s in Q u a k i n g a f t e r F o l c h wash. E a c h e x p e r i m e n t was perform e d a m i n i m u m o f t h r e e t i m e s . T h e r e is a v a r i a t i o n o f 15% in c a l c i u m values o b t a i n e d .
313
of active myelination, followed by a decrease, and a plateau occurs at 30 days. Variation is more important before than after Folch wash. In the washed extract, there is little modification after 10 days. Fig. 1 shows also the values obtained in the mutant. Calcium c o n t e n t is lower (50% lower compared to m a x i m u m in controls) and there is little variation in lipid extracts, either washed or non-washed. The level is practically constant after 15 days. Calcium detected in total lipid extracts may be related to lipids or to proteolipids. Free calcium in water, coming from the extracellular space and solubilized by the mixture of chloroform-methanol, could be another possibility. In Fig. 2 is indicated the variation of water per gram brain as a function of age. No relation can be established between water and calcium contents, as the level of water is constant when there is maximal variation for calcium, in both normal and Quaking mice; on the other hand, partitioning of 4s CaC12 between lower and upper phases after Folch wash indicates an equal quantity in the two phases; if the difference found was related to free calcium taken up during lipid extraction, a similar type of curve would be obtained before and after Folch wash and this is n o t the case.
c 900 .Q
©
A
•
8O(3
700
0
zl
8 1012 15 18 2'1 2'5 Age in days (postnatal)
L
3'0
~
-
~N •QK
-
Adult
Fig. 2. Water c o n t e n t in controls ( A ) and Quaking ( • ) mice.
As the variation in the a m o u n t of calcium as a function of age occurs only before Folch wash, it may be related to proteolipids or to those glycolipids which are eliminated by this procedure. Further work is necessalw to see which of these constituents this variation is related to and why it disappears in the mutant. Nevertheless, this work shows for the first time variation in lipid calcium content related to brain maturation.
ACKNOWLEDGEMENTS
This work was supported by I.N.S.E.R.M. and D.R.M.E.
314
REFERENCES 1 Baumann, N.A., Jacque, C., Pollet, S. and Harpin, M.L., Fatty acid and lipid composition of the brain of a myelin deficient mutant, the Quaking mouse, Europ. J. Biochem., 4 (1968) 340--344. 2 Brink, F., The role of Ca ions in neural processes, Pharmacol. Rev., 6 (1954) 243--285. 3 Deamer, D.W. and Cornwell, D.G., Calcium action on fatty acid and phospholipid monolayers and its relation to cell membrane, Biochem. biophys. Acta (Amst.), 116 (1966) 555--562. 4 Folch, J., Ascoli, I., Lees, M., Meath, J.A. and Lebaron, N., Preparation of lipid extracts from brain tissue, J. biol. Chem., 191 (1951) 833--841 . 5 Shah, D.O. and Schulman, J.H., Interactions of Ca ++ with lecithin and sphingomyelin monolayers, Lipids, 2 (1967) 21.