SHORT COMMUNICATION Stearate desaturase' in rat brain and liver (Received 28 January 1976. Accepted 6 April 1976)
THE FORMATION of oleic acid from stearic acid is known to take place in the liver, especially in microsomes (PAULSR U D ct ~ 7 \ . , 1970). It has also been known for a long time that Ct8 monoenoic components are major constituents of the glycerophospholipids of brain which account for about 25-50",; of the total fatty acids there (KLENKer a[,, 1953; DEBUCH,1956). However, until recently, the question of whether o r not brain tissue itself can introduce double bonds into saturated fatty acid chains had not been examined to any extent. COOK& SPENCE(1973) recently demonstrated oleic acid formation by homogenates of weanling rat brains. We confirmed their results with brain tissue slices but we could not detect desaturase activity with brain microsomes (SENG& DEBUCH,1975). EXPERIMENTAL PROCEDURES These were essentially as given in SENG& DEBUCH (1975). Wistar rats of either sex (Hannover), 4, 7, 14 and 90 days' old were decapitated. Tissue pieces of 0.3g (chopped at 0.4 m m perpendicularly, with the tissue chopper of MCILWAIN& BUDDLE(1953)) were suspended in a final VOI of 3 ml containing (in mmol/l.): [l-'"C]stearic acid 0.533 (1.2 x lo6 d.p.m./pmol); glucose, 20; MgCI2, 2.5; KCI, 4.6; NaCI, 120; NalHPO,, 4.4; Tris-HCI. 28.2 (pH 7.4); bovine serum albumin, 0.14;,. All incubations were gassed for the first 30 s with O 2 then stoppered. After 15 rnin at 37'C the material was extracted by the procedure of BLlGH & DYER(1959). slightly modified in that homogenization with the solvents was performed ultrasonically (3' 70 W). Transesterification of the total lipid extract and radio GLC analysis of fatty acid methyl esters were performed as described before (SENG& DEBUCH,1975). Zero time incubations and enzyme blanks showed that the substrate [I-'4C]stearate (Amersham Buchler) contained 0.36 _+ 0.079!/, (n = 15) of the radioactivity in a GLC peak appearing at the retention time of oleate. This impurity was subtracted from all percentage conversion rates (18:O 1 8 : l ) prior to calculation of molar rates (Fig. 1). RESULTS AND DISCUSSION Our previously published data (SENG& DEBUCH. 1975) showed that a low desaturating capacity in 7-day-old brain is reduced with age and development, whereas in liver a much higher level of activity develops with age. Incubations of 30 min gave higher oleic acid formation rates than e.g. 12Omin incubations, The aims of the present experiments were therefore (1) to investigate even younger rat brains (4-day-old), (2) to incubate only for 15 min. (3) to incubate in the presence of albumin and (4) to exploit the
pmole5 oleale gfw.
min
-.-
- -,-
AOOO
Liver
brain
,I
I I
I
4
7
age (log days)
14
90 days
FIG. 1. Stearic acid dehydrogenase = (desaturase) measured in slices of rat brain and liver (X s) (pmol/g fresh tis.sue/min). Number of incubations and significance of data are given in Table 1. sensitivity of the radio gaschromatographic equipment by using substrate of higher specific radioactivity. Figure I shows that, under the conditions described here, the rate of desaturation expressed as pmol oleate formed per g fresh weight/min was higher than found before (SENG & DEBUCH,1975). However, the desaturating capacity of liver increases very considerably with age whereas that of brain tissue diminishes to values near to zero in the adult. Statistical evaluation (paired r-tests, see Table 1) showed significant differences ( P < 0.05) between blanks and brain percentage conversion rates at ages 4 and 7 days, between blanks and liver values from 7 to 90 days. When brain desaturation rates were compared, significant differences were found between 4 days and 90 days; liver values dif-
TABLE1. EVALUATION OF PAIRED I-TESTS (CALCULATED FROM PERCENTAGE CONVERSION RATES) A. Comparison of blanks (n Age in days 4
Brains incubated n P< Livers incubated n
Pi
=
15) with tissue values 7 14 90
5 0.001
3 0.01
n.s.
3 n.s.
2 n.s.
4 0.01
2 0.05
0.001
5
3
B. Comparison of activities in the same tissue at different ages 'Ompared 4-7 ages in days 4-90 7-14 7-90
Brains P < 'More correctly named: stearoyl-CoA: (acceptor)-dehyd- ~i~~~~ p < rogenase. EC 1.3.'?.? 825
n.s. n.s.
(0.10) 0.02 n s . 0.02
n.s. n.s.
n.s. 0.01
n.s. 0.02
826
Short communication
fered significantly between 90 days and 14. 7. and 4 days respectively. According to these and o w earlier data we conclude that the low capacity of brain to carry out desaturation is due primarily to a low level of the desdturase itself. Activation of fatty acids was found in adult brain microsomes. in the same order of magnitude as in adult 1975). According to liver microsomes (SEX & DEBUCH. & REHA (1975) and a very recent study by PLZLARRAT in contrast to COOK & S P t N c t (1973) the low brain microsoma1 desaturase can be measured using other techniques.
Ph!.sio/qisc/ir C/icni;t, der Unirersitar, D 5 Kolri 41, Josepli-Srel_rilurl,I-Str.52 Medical Reseurch Cenrre. Polish Acadeniy qf Sciences. W m u w 36. 3 Dworkowa Srrecr. Polllntl
insririct fiir
REFERENCES
BLIGHE. G. & DYERW . J. (1959) Con. J . Biochern. Physiol. 37, 91 1-917. COOKH. W. & SPENCE M. W . (1973) J. biol. Chem. 248, 1793-1 796. DEBUCH H. (1956) Z.pk!~.sio/.Chent. 304, 109-137. KLENKE., DEBUCH H. & D ~ i l hH. (1953) Z . phjsiol. Cheni. 292, 241-250. MCILWAIN J. & BUDDLE H . L. (1953) Biochem. J . 53, 4 12420. P. N.Smc PAULSRUD J. R.. STEWART S. E., GRAFFG. & HOLMAN H. DEBUCH R. T. (1970) Lipids 5, 61 1-616. PULLARKAT R . K. & REHA H . (1975) J . Neurockem. 25, J . STROSZNAJDER607-610 SENGP. N. & DEBUCH H. (1975) Z.physiol. C h e n ~ .356, 1043-1053.
THE FORMATION of oleic acid from stearic acid is known to take place in the liver, especially in microsomes (PAULSR U D ct ~ 7 \ . , 1970). It has also been known for a long time that Ct8 monoenoic components are major constituents of the glycerophospholipids of brain which account for about 25-50",; of the total fatty acids there (KLENKer a[,, 1953; DEBUCH,1956). However, until recently, the question of whether o r not brain tissue itself can introduce double bonds into saturated fatty acid chains had not been examined to any extent. COOK& SPENCE(1973) recently demonstrated oleic acid formation by homogenates of weanling rat brains. We confirmed their results with brain tissue slices but we could not detect desaturase activity with brain microsomes (SENG& DEBUCH,1975). EXPERIMENTAL PROCEDURES These were essentially as given in SENG& DEBUCH (1975). Wistar rats of either sex (Hannover), 4, 7, 14 and 90 days' old were decapitated. Tissue pieces of 0.3g (chopped at 0.4 m m perpendicularly, with the tissue chopper of MCILWAIN& BUDDLE(1953)) were suspended in a final VOI of 3 ml containing (in mmol/l.): [l-'"C]stearic acid 0.533 (1.2 x lo6 d.p.m./pmol); glucose, 20; MgCI2, 2.5; KCI, 4.6; NaCI, 120; NalHPO,, 4.4; Tris-HCI. 28.2 (pH 7.4); bovine serum albumin, 0.14;,. All incubations were gassed for the first 30 s with O 2 then stoppered. After 15 rnin at 37'C the material was extracted by the procedure of BLlGH & DYER(1959). slightly modified in that homogenization with the solvents was performed ultrasonically (3' 70 W). Transesterification of the total lipid extract and radio GLC analysis of fatty acid methyl esters were performed as described before (SENG& DEBUCH,1975). Zero time incubations and enzyme blanks showed that the substrate [I-'4C]stearate (Amersham Buchler) contained 0.36 _+ 0.079!/, (n = 15) of the radioactivity in a GLC peak appearing at the retention time of oleate. This impurity was subtracted from all percentage conversion rates (18:O 1 8 : l ) prior to calculation of molar rates (Fig. 1). RESULTS AND DISCUSSION Our previously published data (SENG& DEBUCH. 1975) showed that a low desaturating capacity in 7-day-old brain is reduced with age and development, whereas in liver a much higher level of activity develops with age. Incubations of 30 min gave higher oleic acid formation rates than e.g. 12Omin incubations, The aims of the present experiments were therefore (1) to investigate even younger rat brains (4-day-old), (2) to incubate only for 15 min. (3) to incubate in the presence of albumin and (4) to exploit the
pmole5 oleale gfw.
min
-.-
- -,-
AOOO
Liver
brain
,I
I I
I
4
7
age (log days)
14
90 days
FIG. 1. Stearic acid dehydrogenase = (desaturase) measured in slices of rat brain and liver (X s) (pmol/g fresh tis.sue/min). Number of incubations and significance of data are given in Table 1. sensitivity of the radio gaschromatographic equipment by using substrate of higher specific radioactivity. Figure I shows that, under the conditions described here, the rate of desaturation expressed as pmol oleate formed per g fresh weight/min was higher than found before (SENG & DEBUCH,1975). However, the desaturating capacity of liver increases very considerably with age whereas that of brain tissue diminishes to values near to zero in the adult. Statistical evaluation (paired r-tests, see Table 1) showed significant differences ( P < 0.05) between blanks and brain percentage conversion rates at ages 4 and 7 days, between blanks and liver values from 7 to 90 days. When brain desaturation rates were compared, significant differences were found between 4 days and 90 days; liver values dif-
TABLE1. EVALUATION OF PAIRED I-TESTS (CALCULATED FROM PERCENTAGE CONVERSION RATES) A. Comparison of blanks (n Age in days 4
Brains incubated n P< Livers incubated n
Pi
=
15) with tissue values 7 14 90
5 0.001
3 0.01
n.s.
3 n.s.
2 n.s.
4 0.01
2 0.05
0.001
5
3
B. Comparison of activities in the same tissue at different ages 'Ompared 4-7 ages in days 4-90 7-14 7-90
Brains P < 'More correctly named: stearoyl-CoA: (acceptor)-dehyd- ~i~~~~ p < rogenase. EC 1.3.'?.? 825
n.s. n.s.
(0.10) 0.02 n s . 0.02
n.s. n.s.
n.s. 0.01
n.s. 0.02
826
Short communication
fered significantly between 90 days and 14. 7. and 4 days respectively. According to these and o w earlier data we conclude that the low capacity of brain to carry out desaturation is due primarily to a low level of the desdturase itself. Activation of fatty acids was found in adult brain microsomes. in the same order of magnitude as in adult 1975). According to liver microsomes (SEX & DEBUCH. & REHA (1975) and a very recent study by PLZLARRAT in contrast to COOK & S P t N c t (1973) the low brain microsoma1 desaturase can be measured using other techniques.
Ph!.sio/qisc/ir C/icni;t, der Unirersitar, D 5 Kolri 41, Josepli-Srel_rilurl,I-Str.52 Medical Reseurch Cenrre. Polish Acadeniy qf Sciences. W m u w 36. 3 Dworkowa Srrecr. Polllntl
insririct fiir
REFERENCES
BLIGHE. G. & DYERW . J. (1959) Con. J . Biochern. Physiol. 37, 91 1-917. COOKH. W. & SPENCE M. W . (1973) J. biol. Chem. 248, 1793-1 796. DEBUCH H. (1956) Z.pk!~.sio/.Chent. 304, 109-137. KLENKE., DEBUCH H. & D ~ i l hH. (1953) Z . phjsiol. Cheni. 292, 241-250. MCILWAIN J. & BUDDLE H . L. (1953) Biochem. J . 53, 4 12420. P. N.Smc PAULSRUD J. R.. STEWART S. E., GRAFFG. & HOLMAN H. DEBUCH R. T. (1970) Lipids 5, 61 1-616. PULLARKAT R . K. & REHA H . (1975) J . Neurockem. 25, J . STROSZNAJDER607-610 SENGP. N. & DEBUCH H. (1975) Z.physiol. C h e n ~ .356, 1043-1053.
