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VoI. 1, No. 1, pp. 59-64,1983
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Pergamon PressLtd. 0 1983ISDN
PHOSPHOGLYCERIDES AND THEIR ACYL GROUP COMPOSITION IN MYELIN AND MICROSOMES OF RAT SPINAL CORD DURING DEVELOPMENT GRACE Y. SUN,*? BYRON N. DESOUSA,$ VICKI DANOPOULOS” and LLOYD A. HORROCKSS
*Sinclair Comparative Medicine Research Farm and Biochemistry Department, University of Missouri, Columbia, MO 65201 and SDepartment of Physiological Chemistry, The Ohio State University, Columbus, OH, U.S.A. (Received 29 September 1982; accepted 21 January 1983)
Abstract -The phosphoglycerides and their acyl group composition in myelin and microsomes isolated from rat spinal cords were examined at various stages of the postnatal developmental period. In the immature cord, diacyl-GPC is the major phosphoglyceride in both microsomal and myelin fractions. The proportion of this phosphoglyceride decreased steadily with age during development and the decrease was marked by an increase in diacyl-GPE in microsomes and ethanolamine plasmalogen in both fractions. Marked changes in acyl group composition were observed in the microsomes during development of spinal cord, whereas acyl groups of myelin showed less extensive changes A considerable amount of monoenoic fatty acid is already present in the myelin of 7-day-old cord. Acyl groups of diacyl-GPC in the microsomal fraction indicated a decrease in 16:0,16: 1,20:4(n-6) and an increase in 18:0,X3: 1 and 20: 1 with respect to development. On the other hand, microsomal diacyl-GPE and alkenylacyl-GPE indicated a decrease in 16:0,18:0 and 20:4(n-6), but an increase in 18: 1 and 20: 1. An exception is the proportion of 22:6(n-3) which increased in diacyl-GPE and decreased in alkenylacyl-GPE during development. The lack of obvious acyl group changes in myelin phosphoglycerides after 7 days of age is in agreement with the results of deSousa & Horrocks (Devl Neurosci. 1,114120,1979) that myelination in spinal cord took on an early course and reached the adult composition sooner than the cerebrum. Key words: Myelin, Microsomes, Spinal cord, Phospholipid composition, Development, Acyl group composition.
Plasmalogens,
The spinal cord is evolved from cells in the neural tube which is part of the CNS. Previous studies from deSousa & Horrocks’,2 showed that during CNS dcvelopm~nt, the spinal cord exhibits characteristics of maturity at an earlier date than the cerebrum. The spinal cord is more susceptible to trauma than other parts of the brain7 although this may or may not be directly related to its early maturation characteristics. As compared to the cerebrum, the spinal cord seems to be enriched in myelin lipids such as cerebrosides and ethanolamine plasmalogens.’ Nevertheless, a detailed analysis of the phosphoglycerides and their acyl group profiles has not been achieved with the subcellular fractions. This study was made to examine the phospholipids and their acyl group profiles of the myelin and microsomes isolated from rat spinal cord at different ages during postnatal development. EXPERIMENTAL
PROCEDURES
Spragu~Dawley rats (Madison, WI) were selected at ages 7,9,17,25,30 and 40 days for the experiment. Within 1 min after decapitation without anesthesia, the spinal cords were ejected in a cold room according to the procedure described by deSousa & Horrocks.’ The tissues were stored at -20°C until required for further analysis. After weighing, thawed spinal cords were suspended in 20 vol. of 0.85 M sucrose with 50 mM Tris-HCl (pH 7.4) and homogenized in a Potter-Elvejhem tissue homogenizer with a motor-driven Teflon pestle. The nervous tissue homogenates (from pooled samples of 6-g spinal cords) were subjected to differential and sucrose gradient centrifugation for isolation of microsomal and myelin fractions. l3
Abbrev~tio~: GPC, glycero-3-phosph~holines; serines; GPI, gly~ro-3-phosphoinositols. t To whom correspondence should be addressed.
GPE, glycero-3-phosphoethanolamines;
59
GPS, gly~ro-3-phospho-
G. Y. Sun er al.
60
The myelin and microsomal-enriched pellets were suspended in water, and 5 vol. of chloroform methanol 2: 1 (v/v) were added for extraction of the lipids. Portions of the lipid extracts were applied to thin-layer plates prepared with silica gel G (Brinkman Instruments, Inc., Westbury, NY). Phospholipids were separated by a previously described two-dimensional TLC procedure” except that the second dimension solvent system was changed to chloroform : methanol : acetone: acetic acid:O. 1 M ammonium acetate (120: 50: 60:2.5: 15, by vol.). After solvent development, lipid spots were visualized by spraying the thin-layer plates with 0.1% 2’7’-dichlorofluorescein. The lipid spots were scraped into test tubes for phosphorus assay3 or for conversion to fatty acid methyl esters by alkaline methanolysis. 8 Analysis of the fatty acid methyl esters was performed on a Hewlett-Packard 583OA gas-liquid chromatograph equipped with dual flame ionization detectors and automatic peak area integration device. Two 6 ft x % in. stainless steel columns packed with 10% EGSS-X coated on Gas Chrom-P (Applied Science Laboratories, State College, PA) were used for analysis and conditions for chromatography of the methyl esters were the same as described previously. ‘I A known amount of heptadecanoic acid methyl ester (C17:O) was added to each sample for quantitative determination. Repeated analysis of the same sample showed variation of < 5% of the peak area. RESULTS Prior to this study, a comparison of the phospholipid composition of rat spinal cord and cerebrum was made.’ The proportion of ethanolamine plasmalogens was higher in spinal cord than in cerebrum. Results from this study show substantial differences in phospholipid composition between the microsomal and myelin-enriched fractions of rat spinal cord (Table 1). At all ages examined, the proportion of ethanolamine plasmalogen in myelin was higher than that in the microsomal fraction. Diacyl-GPC, diacyl-GPE and alkenylacyl-GPE comprised the major phospholipid constituents of myelin and microsomes, but only trace amounts of diacyl-GPI were detected. During postnatal development, there was a decrease in the proportion of diacyl-GPC and an increase in alkenylacyl-GPE and sphingomyelin in both myelin and microsomal fractions. The increase in diacyl-GPE and diacyl-GPS was found mainly with the microsomal fraction. The acyl groups of diacyl-GPC from spinal cord microsomes were enriched in 16:0, 18:0 and 18: 1. Marked changes in the acyl group composition of this phosphoglyceride were observed during development. As shown in Table 2, the spinal cord microsomes from 7 to 40 days of age indicated a decrease in 16:0 (from 56.5 to 34.1%) and an increase in 18:O (from 7.7 to 14.4%) and 18: 1 (from 26.3 to 39.3%). Small amounts of 20: 1,20:4 and 22:6 were also present in the microsomal diacyl-GPC, and the proportion of 20: 1 increased from 0.4 to 4.7% during the same period. A small decrease in 20:4 and increase in 22:6 was also observed in this phosphoglyceride. The acyl groups of diacyl-GPE in microsomes were comprised mainly of 18:0, 18: 1 and 22:6 with smaller proportions of 20: 1 and 20:4. With the exception of 18:0, which indicated a decrease with age, acyl group changes in diacyl-GPE were similar to diacyl-GPC. The alkenylacyl-GPE in adult cord microsomes were comprised mainly of 18: 1,20: 1 and 22:6. During the developmental period, the increase in monoenoic acyl groups was most striking in alkenylacyl-GPE. A distinct difference in Table I. Percent distribution of phosphohpids in the microsomal and myelin fractions from rat spinal cord during maturation Phospholjpids Diacyl-GPE Alkenylacyl-GPE Diacyl-GPC Spningomyelin Diacvl-GPS Diacyl-GPI Others
5
7
9
Age (days) 11 13
17
30
40
Microsomes 10.4 13.7 21.3 17.4 16.5 19.8 25.4 25.X 7.9 6.9 7.9 9.1 13.0 7.8 11.3 17.4 60.5 59.6 51.7 57.5 48.9 44.3 37.5 33.5 6.0 4.3 4.8 4.8 6.7 8.4 11.3 9.7 8.0 9.7 8.1 8.7 10.1 13.3 9.7 12.1 2.2 tr tr tr tr ;2 5.8 6.2 2.5 ZS :4 2.6 1.5
3
7
Y
Age (days) 13 20
25
30
411
Myeiin 14.5 14.3 15.4 16.3 14.8 16.4 14.3 16.1 17.3 19.1 20.4 23.2 21.6 25.1 30.2 28.1 52.8 47.2 45.5 43.2 45.0 39.2 34.5 31.7 3.1 7.0 6.3 6.Y 7.7 9.8 X.6 Y.6 7.2 7.0 6.7 6.4 6.5 7.3 x.4 7.8 2.5, 3.0 2.3 2.5 1.9 1.9 1.6 2.2 2.2 2.4 3.4 1.5 2.5 2.4 2.4 4.5
Lipids were extracted from microsomal and myelin fractions and were separated by reactional two-dimensional TLC as described in the text. individual lipid spots were quantitated by phosphorus assay.’ tr = Only trace amounts were detected.
Spinal cord fatty acids during development
61
Table 2. Acyl group composition of phosphoglycerides in microsomes of rat spinal cords during postnatal development 40
37.2 14.4 37.3 2.1 0.7 3.4
34.1
-2.7 1.7
z6 0.6 2.6
9.1 28.1 29.1 0.6 1.1 6.0 0.4 7.3 2.5 15.9
6.6 29.3 30.5 1.0 1.3 6.7 0.4 7.5 2.0 0.3 14.5
Alkenylacyl-GPE 5.3 3.2 7.0 3.7 1.5 6.9 32.3 44.9 20.9 0.8 0.6 1.9 2.4 8.6 17.2 1.6 1.6 1.4 13.4 7.6 21.1 8.1 7.2 7.9 1.0 0.8 23.4 12.3 34.7
3.4 1.8 47.3 0.8 2.7 20.2 1.2 6.2 6.0 0.9 9.6
7
9
16:O 16:l 18:O 18:l 18:2 20:o 2O:l 20:3 (n-6) 20:4(n-6) 22:4(n-6) 22:6(n-3)
56.5 4.0 7.7 26.3 1.0
54.4 3.7 7.7 25.4 1.2 0.7
16:O
17.4 35.1 22.8 12.7 2.3 0.8 8.9
18:O 18:l 18:2 20:o 2O:l 20:3(n-6) 20:4(n-6) 22:4(n-6) 22:5 (n-6) 22:6(n-3) 16:O 18:0 18:l 18:2 20:o 2O:l 20:3 (n-6) 20:4(n-6) 22:4(n-6) 22:5 (n-6) 22:6(n-3)
0.4 3.2 0.9
10.0 6.7 20.3 2.2 19.1 9.6 2.3 29.8
Age Ways)
30
Fatty acids
17
Diacyl-GPC 44.3 12.5 33.6 1.7 0.5 2.2 2.9 3.9 0.5 0.3 1.7 1.1
13.3 37.7 19.6 0.8 0.2 0.6 0.2 13.2 3.2 1.0 9.2
Diacyl-GPE 7.9 31.2 26.9 1.1 0.6 2.5 0.5 10.7 2.8 0.4 15.3
13.4 39.3 1.9 0.8 4.7
Results are expressed as weight percent. Lipid extracts from microsomes were separated by reactional two-dimensional TLC as described in the text. The acyl groups in the phosphoglycerides were converted to methyl esters, and fatty acid methyl esters were analyzed by GLC.
developmental profile is observed with regard to the proportion of 22:6(n-3) of diacyl-GPE and alkenylacyl-GPE. The proportion of 22:6(n-3) in diacyl-GPE increased with age, but that in alkenylacyl-GPE decreased. Data (Table 2) show that the major phosphoglycerides from early spinal cord microsomes were almost devoid of 20: 1, but the proportion of this fatty acid increased almost lo-fold beween 9 and 40 days. Similarly, the proportion of 18: 1 in alkenylacyl-GPE increased from 20.9 to 47.3% during the same period. The diacyl-GPC from myelin had a lower propo~ion of 16:0 and a higher proportion of 18: 1 as compared to that in the microsomal fraction (Tables 2 and 3). In general, the acyl groups of diacylGPE and alkenylacyl-GPE in the myelin fraction indicated a higher proportion of 18: 1 and a lower proportion of 22:6 as compared to those in the microsomal fraction. In contrast to the marked changes shown during development in the microsomal fraction, relatively small changes in acyl group composition were observed in the phosphoglycerides from myelin. Considerable amounts of 20: 1 were already present in the myelin phosphoglycerides from 7-day-old spinal cords, although only trace amounts of this acyl group were present in the microsomes of a corresponding age group (Tables 2 and 3). In the myelin, the proportion of 22:6(n-3) in both diacyl-GPE and alkenylacylGPE decreased during the early developmental period. Data also show that a considerable amount of 22:6(n-3) (19.7%) was present in alkenylacyl-GPE in the 7-day-old myelin preparation; how-
G. Y. Sun et al.
62
Table 3. Acyl group composition of phosphoglycerides in myelin of rat spinal cords during postnatal development
Age (days) Fatty acids
7
17
16:O 18:O 18:l 18:2 20:o 2O:l 20:3 (n-6) 20:4(n-6) 22:4(n-6) 22:6(n-3)
29.7 17.2 41.2 2.8 1.0 3.3 0.5 2.8 0.4 0.9
26.6 17.5 43.2 3.1 1.1 3.4 0.4 3.2 0.7 0.9
16:0 18:O 18: 1 18:2 20:o 2O:l 20:3 (n-6) 20:4(n-6) 22:4(n-6) 22:6(n-3)
4.2 31.9 40.1
10.1 26.6 36.7 0.6 1.0 5.4 0.9 8.7 2.4 7.6
16:O 18:0 18:l 18:2 20:o 2O:l 20:3 (n-6) 20:4(n-6) 22:4(n-6) 22:6(n-3)
2.2
25
30
40
24.7 17.7 43.6 2.8 1.1 4.3 0.3 3.1 0.8 0.9
25.1 16.9 4.5.2 3.3 0.8 4.6 0.2 2.5 0.6 0.8
7.x 24.3 42.0 1.3 1.3 10.0 0.7 6.2 1.6 4.7
6.0 22.9 38.7 0.9 1.3 10.5 0.7 8.3 2.3 x.2
3.2
3.6 1.7 54.7 0.7 1.5 18.3 I.5 5.x 6.0 5.7
Diacyl-GPC
3.2 8.6 12.1
Diacyl-GPE 6.4 24.1 38.3 1.3 1.1 9.3 1.1 8.4 2.3 7.4
Alkenylacyl-GPE 3.4 1.h 50.3 0.7 2.1 16.H 1.7 8.1 6.6 8.0
37.0 3.5 15.3 1.9 10.3 10.1 19.7
Results are expressed tion.
as weight percent.
I .3 S2.8 0.7 2.0 16.7 1.9 7.4 6.6 7.3
See Table 2 for descrip-
Table 4. Acyl group composition of diacyl-GPS and diacyl-GPI in microsomes and myelin from rat spinal cords during postnatal development Age (days) Fatty acids
7
Microsomes 17
30
17
Myelin 30
40
1.7 38.0 41.8 1 .O 4.9 1 .o 5.3 2.2 4.2
Myelin 1.2 42.2 39.0 1.0 4.6 1.1 5.X 1.8 3.4
0.7 40.2 39.1 1.1 6.8 0.9 5.4 1.9 4.0
Diacyl-GPS 16:0 18:0 18:l 20:o 2O:l 20:3(n-6) 20:4(n-6) 22:4(n-6) 22:6(n-3)
6.5 47.7 25.5
16:0 18:0 18:l 20:4(n-6) 22:6(n-3)
6.0 50.2 10.1 31.0 2.6
0.3 5.1 2.8 11.1
Microsomes 2.3 39.6 33.8 1.1 3.5 0.8 4.7 3.5 9.6
6.6 47.6 12.8 29.4 3.6
2.1 38.8 39.7 0.9 4.8 0.7 3.7 1.8 7.6 Diacyl-GPI 8.6 47.8 12.1 27.6 3.9
9.5 42.9 14.9 28.9 2.6
Spinal cord fatty acids during development
63
ever, the proportion of this fatty acid decreased more than two-fold between 7 and 25 days with a concomitant rise in 18: 1. The acyl group composition of diacyl-GPS in microsomes and myelin was also examined (Table 4). In both membrane fractions, 18:0 and 18:l comprised nearly 80% of the acyl groups of this phosphoglyceride. In the microsomal fraction, an increase in 18: 1 and 20: 1 and a decrease in 16:0, 18:0 and 22:6 was observed between 7 and 17 days of age. Again, relatively few changes with age were observed with the myelin phospholipid. The acyl groups of diacyl-GPI in microsomes and myelin were enriched in 18:0,18: 1 and 20:4 (Table 4). Except for a small decrease in the proportion of 20:4, the acyl group composition of diacyl-GPI in the cord microsomes remained fairly constant with age.
DISCUSSION As shown in a previous study, * the phospholipids in rat spinal cord are more enriched in alkenylacyl-GPE than those in the cerebrum. Since a high content of ethanolamine plasmalogen is also a characteristic feature of myelin, this data suggests that the spinal cord contains a higher proportion of myelin than the cerebrum. Nevertheless, a comparison of the phospholipid profile of purified myelin from adult rat brain and spinal cord revealed little difference. l3 The phospholipids of rat spinal cord myelin seem to follow a similar developments profile as compared to the brain.’ Results here revealed that both subcellular fractions from spinal cord indicated changes in phospholipid com~sition during development. These changes were correlated to an increase in both diacyl-GPE and alkenylacyl-GPE and a decrease in diacyl-GPC. Although a similar trend of lipid changes was reported for the brain microsomes, these changes were comparatively less extensive than those shown in the spinal cord.5 Both similarities and differences are found in the acyl group profiles of phosphoglycerides from spinal cord subcellular fractions and those from brain. 9~11For example, there is a lower proportion of 16:0 and a higher proportion of 18: 1 in the diacyl-GPC from spinal cord microsomes as compared to that from brain. The spinal cord microsomes also showed higher 18: 1 and 20: 1 and lower 20:4 and 22:6 in alkenylacyl-GPE and diacyl-GPE. Microsomes from CNS tissues are known to be heterogeneous, comprising of membranes from neuronal and glial origin. Microsomes from both brain and spinal cord showed similar acyl group changes during development. These changes include a decrease in 16: 0 and an increase in 18: 1 and 20: 1 in diacyl-GPC, and a dramatic increase in 20: 1 in alkenylacyl-GPE, The abrupt increase in 20: 1 suggests that phosphoglyce~de species with this fatty acid are synthesized in brain and spinal cord postnataly. It is interesting to note the high proportion of 22:6(n-3) in alkenylacyl-GPE in microsomes during the early postnatal period and the rapid decrease during development. The results further suggest differences in the rate of synthesis and metabolism of specific phosphoglyceride species during the developmental period. Myelin isolated from spinal cord also showed differences and similarities as compared to that from cerebrum. The acyl composition of diacyl-GPC from myelin of adult rat spinal cord has a lower proportion of 16:0 and higher 18: 1 than that in brain’i (Tables 2 and 3). Similarly, the proportions of 18: 1 and 20: 1 in alkenylacyl-GPE and diacyl-GPE were also higher in the cord myelin than in brain. The brain myelin, however, showed a more dramatic postnatal change in acyl group profile than the spinal cord myelin. ‘* The lack of acyl group change in myelin isolated from the spinal cord is probably due to its earlier maturation schedule. The monoenoic acid 2O:l is especially enriched in myelin and is concentrated in the ethanolamine plasmalogens. The fact that myelin isolated from 7-day-old spinal cord has already attained a high proportion of 20: 1 suggests that maturation of spinal cord myelin is commencing at an earlier time than brain myelin. The lack of accumulation of this fatty acid in the microsomal phosphoglycerides during the early developmental period seems to indicate the presence of a rapid transfer mechanism for phosphoglyceride from microsomes to myelin. It is generally known that myelin synthesis is a continuous process throughout life, although the rate of synthesis decreased markedly after the developmental period. 4~10,12However, whether this developmental profile is the same for spinal cord myelin remains to be studied.
64
G. Y. Sun et al,
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