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Activity levels of cholesterol ester metabolizing enzymes in brain in multiple sclerosis: Correlation with cholesterol ester concentrations
EXPERIMENTAL
NEUROLOGY
68, 601-604 (1980)
RESEARCH
NOTE
Activity Levels of Cholesterol Ester Metabolizing Enzymes in Brain in Multiple Sclerosis: Correlation with Cholesterol Ester Concentrations
University
of California, Langley Porter Sonoma State Hospital, Received
Institute, Eldridge, November
Brain-Behavior California
Research 95431
Center
at
5, 1979
We determined the concentrations of free and esterified cholesterol, and the activities of cholesterol esterifying and cholesterol ester hydrolysing enzymes, in brain tissue from patients with multiple sclerosis and from individuals with no known neurological disease. Our results showed an increase in cholesterol ester concentrations in multiple sclerotic brain accompanied by a significant reduction in the activity of the ester hydrolases. There was also a slight reduction in the activity of the esterifying enzyme. These results suggest that the elevated concentrations of cholesterol esters in multiple sclerotic brain are not due to increased synthesis but are rather due to a reduction in the ester hydrolase activity.
An increase in the concentrations of cholesterol esters, although variable, occurs in both demyelinated and histologically normal regions of brain (l-4, 6, 13, 14) from patients with multiple sclerosis (MS). The presence of cholesterol esterifying enzyme and cholesterol ester hydrolase(s) in brain (5, 10) suggests that brain cholesterol esters are of endogenous origin, and that the increase in cholesterol ester concentrations in MS brain might be due to a change in the activity of the ester metabolizing enzyme (s). We measured the activities of these enzymes in brain tissue from individuals with and without MS and correlated the Abbreviation: MS-multiple sclerosis. 1 The authors are grateful to Dr. W. W. Tourtellotte, Director, Human Neurospecimen Bank, Los Angeles, CA, for providing us tissue samples. This investigation was supported by National Institutes of Health grants NSl1670 and NS14938. 601 0014-4886/80/060601-04$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved
602
SHAH
AND
JOHNSON
changes in enzyme activity with alterations in cholesterol ester concentrations to determine whether the ester concentrations in MS brain increase because of an increase in esterifying activity or a decrease in hydrolase activity. Cerebral white matter from four patients with MS (plaque and periplaque regions) and from eight individuals with no known neurological diseases used in the present study were supplied by Dr. W. W. Tourtellotte (Director, Human Neurospecimen Bank, Los Angeles, California). Autolysis periods for brain tissue samples were 7 to 30 h and the patients’ ages were 23 to 68 years. The periplaque regions corresponded to a normal appearing area 0.6 cm around the plaque. ]C’~C]cholesterol and ]1J4C]oleic acid with sp. act. 50 mCi/mmol and cholesterolll-‘“Cloleate sp. act. 50 to 60 mCi/mmol were purchased from New England Nuclear Corporation, Boston, Massachusetts, and checked for radio purity prior to use. Unlabeled cholesterol, oleic acid, and cholesterol oleate were the products of Applied Sciences Laboratories, State College, Pennsylvania. For cholesterol ester hydrolases in white matter, incubation mixtures contained ]4J4C]cholesterol oleate (100,000 cpm) plus 0.5 pmol unlabeled cholesterol oleate, 4 ~1 Triton X-100, and either 0.8 ml 0.15 M citrate phosphate buffer (PH 4.5) or 0.8 ml 0.1 M sodium phosphate buffer (PH 6.5). The mixtures were sonicated 30 s, then 0.2 ml (1 mg protein) 800-g supernatant was added and the mixtures incubated 3 h at 37°C with shaking. The reaction was terminated and the lipids were then extrated from the reaction mixtures. Cholesterol and oleic acid were separated from cholesterol oleate and the radioactivity assayed by procedures described elsewhere (10). For measurement of cholesterol esterifying activity, incubation mixtures containing [4-14C]cholesterol (120,000 cpm) plus 0.6 pmol unlabeled cholesterol and 1.2 pmol unlabeled oleic acid in 50 ~1 acetone and 0.8 ml 0.15 M citrate phosphate buffer were sonicated. The reaction was initiated by adding 0.2 ml (1 mg protein) of 800-gsupernatant from white matter. The mixtures were incubated 3 h at 37°C with shaking. The lipids were free and esterified cholesterol were extracted following incubation, separated, and the radioactivity was assayed as before (10). For determining the concentrations of free and esterified cholesterol, the lipids were extracted from a portion of 10% homogenate from white matter and free and esterified cholesterol were separated by column chromatography on silica gel G. The amounts of free cholesterol were estimated as reported earlier (lo), and the amounts of esterified cholesterol were estimated by a more sensitive method described recently by Heider and Boyett (8). Results presented in Table 1 show that the amounts of free cholesterol in white matter from normal individuals and from MS patients did not differ
CHOLESTEROL
ESTER METABOLISM TABLE
603
IN MS BRAIN
I
Cholesterol Ester Concentrations and the Activities of Cholesterol Ester Metabolizing Enzymes in Human Brain White Matter” Cholesterol Cholaterol
e\ter
(nmoUmg
hydrolyced
ester5
formed
Estelified
(nmollmg
protein/h)
Free
profemlhl pH
MS
(plaque)
MS
~periplaquel
(41
(41 Normal
” The on
each
(81
numbers tissue.
’ S~gmficantly
PH65
pH
5 5
concentration
(wkl
0.13’
0.63
+ 0.X’
0.242
t
o.ll*
25.01
c
9.63
t
? 29’
1.57
? 0.57’
0.316
t
0.19
XX?
5 2.43
15.5
t
4.06
2.64
?
0.481
0.19
26.15
f
parentheses
represent
concentrationa
different
from
of normal
the free
and
number esteritied
i
of
tissue
ample?
cholesterol
are
4.26
KS9
I 88
3.50
II.08
analyzed. expressed
PUCKitage estenfied
Iwk)
r
I.19
sterol
concentration
I.57
m The
4.5
~rerol
Duphcate ai
mgig
2
1.30’
2
1.04’
23.31
6.42
? 2.99*
2
0.04
0.35
2
analya~r wet
weight
was of
white
2 6.E’
0.18
carned
out
matter.
P < 0.025.
significantly. However, both periplaque and plaque regions from MS brains showed a significant increase in the amounts of esterified cholesterol. The highest proportion (23%) of esterified cholesterol was found in the plaque region. The cholesterol esterifying activity in the white matter (plaque region) from patients with MS was significantly lower than the activity in white matter from normal individuals. The reduction of esterifying activity in MS white matter (periplaque region) was however not significant. The hydrolase (s) activities, on the other hand, were significantly lower in both plaque and periplaque regions of MS brains. The reduction in the periplaque region was about 40%, whereas the reduction in the plaque region was about 85%. These data on reduction in hydrolase activities are similar to the reduction in acid lipase activity observed in patients with MS by Hirsch et al. (9). Results of our study show that, as reported earlier by other investigators (l-4, 6. 13, 14), there is a significant increase in the amounts of esterified cholesterol in white matter (normal appearing periplaque and plaque regions) from MS patients. Because cholesterol esters in brain are synthesized in situ, one might expect the increase in cholesterol ester concentrations in MS brain to be a result of increased synthesis. Our data show, however, that the cholesterol esterifying enzyme activity in white matter from MS brain is actually less than in white matter from the normal brain. It is therefore unlikely that the elevated cholesterol ester concentrations in MS brain are caused by changes in the activity of the esterifying enzyme. The esterifying enzyme in brain utilizes free fatty acids (5, lo), and, in the absence of added fatty acid, cholesterol esterification is stimulated by phospholipase A, (11). An increase in free fatty acid concentrations (7) and in phospholipase A, activity (15) in MS brain has been reported. Thus, in spite of the reduced activity of esterifying enzyme in MS
604
SHAH AND JOHNSON
brain, the extent of esterification actually taking place in viva may be high. This is consistent with the suggestion (12) that steryl esters serve as carrier and storage points for free fatty acids that would otherwise be toxic. There is a greater likelihood, however, that the accumulation of cholesterol esters in MS brain is due to reduction in hydrolase(s) activity for the following reasons. The increase in esterified cholesterol is maximal in the plaque region, and is accompanied by an almost total lack of hydrolase(s) activity. In periplaque regions, on the other hand, the increase in cholesterol ester concentration is small as is the reduction in hydrolase activity. REFERENCES 1. ALLING, C., M. VANIER, AND L. SVENNERHOLM. 1971. Lipid alterations in apparently normal white matter in multiple sclerosis. Bruin Res. 35: 325-336. 2. CUMINGS, J. N. 1953. The cerebral lipids in disseminated sclerosis and in amaurotic family idiocy. Brain 76: 55 l-562. 3. CUMINGS, J. N. 1955. Lipid chemistry of the brain in demyelinating diseases. Bruin 78: 554-563. 4. DAVISON, A. Neurochem.
N.,
AND
M.
WAJDA.
1962. Cerebral lipids in multiple
sclerosis. J.
9: 427-432.
5. ETO, Y., AND K. SUZUKI. 1971. Cholesterol ester metabolism in brain: properties and subcellular distribution of cholesterol esterifying enzymes and cholesterol ester hydrolases in adult rat brain. Biochim. Biophys. Acta 239: 293-311. 6. GERSTL, B., M. J. KAHNKE, J. K. SMITH, M. G. TAVASTJERNA, AND R. B. HAYMAN. 1961. Brain lipids in multiple sclerosis and other diseases. Brain 84: 310-319. 7. GERSTL, B., M. G TAVASTJERNA, L. F. ENG, AND J. K. SMITH. 1972. Sphingolipids and their precursors in human brain (normal and MS). Z. Neural. 202: 104- 120. 8. HEIDER, J. G., AND R. B. BOYETT. 1978. The picomole determination of free and total cholesterol in cells in culture. J. Lipid Res. 19: 114- 118. 9. HIRSCH, H. E., P. DUQUETTE, AND M. E. PARKS. 1976. The quantitative histochemistry of multiple sclerosis plaques; acid proteinase and other acid hydrolases. J. Nelrrothem. 26: 505-512. 10. JOHNSON, R. C., AND S. N. SHAH. 1978. Cholesterol ester metabolizing enzymes in human brain: properties, subcellular distribution and relative levels in various diseases conditions. J. Neurochem. 31: 895-902. 11. RAMSEY, R. B., AND A. N. DAVISON. 1974. Effect of phospholipase A upon brain cholesterol ester formation. Lipids 9: 440-442. 12. RAMSEY, R. B., AND A. N. DAVISON. 1974. Steryl esters and their relationship to normal and diseased human central nervous system. J. Lipid Res. IS: 249-255. 13. WENDER, M., Z. ADAMEZEWSKA, AND A. WAJGT. 1973. Cerebral lipids in a case with clinically silent isolated plaque of the multiple sclerosis type. Eur. Neurol. 9: 21-29. 14. WENDER, M., H. FILIPEK-WENDER, AND B. STANISLAWSKA. 1973. Cholesteryl esters in apparently normal white matter in multiple sclerosis. Eur. Neurol. 10: 340-348. 15. WOELK, H., AND K. PEILER-ICHIKAWA. 1974. Zur Aktivitat der Phospholipase A2 gegenuber verschiedenen I-alk-I’-enyl-2-acyl-und-1-alkyl-2-acyl verbindungen wahrend der Multiplen Sklerose. J. Neurol. 207: 319-326.
NEUROLOGY
68, 601-604 (1980)
RESEARCH
NOTE
Activity Levels of Cholesterol Ester Metabolizing Enzymes in Brain in Multiple Sclerosis: Correlation with Cholesterol Ester Concentrations
University
of California, Langley Porter Sonoma State Hospital, Received
Institute, Eldridge, November
Brain-Behavior California
Research 95431
Center
at
5, 1979
We determined the concentrations of free and esterified cholesterol, and the activities of cholesterol esterifying and cholesterol ester hydrolysing enzymes, in brain tissue from patients with multiple sclerosis and from individuals with no known neurological disease. Our results showed an increase in cholesterol ester concentrations in multiple sclerotic brain accompanied by a significant reduction in the activity of the ester hydrolases. There was also a slight reduction in the activity of the esterifying enzyme. These results suggest that the elevated concentrations of cholesterol esters in multiple sclerotic brain are not due to increased synthesis but are rather due to a reduction in the ester hydrolase activity.
An increase in the concentrations of cholesterol esters, although variable, occurs in both demyelinated and histologically normal regions of brain (l-4, 6, 13, 14) from patients with multiple sclerosis (MS). The presence of cholesterol esterifying enzyme and cholesterol ester hydrolase(s) in brain (5, 10) suggests that brain cholesterol esters are of endogenous origin, and that the increase in cholesterol ester concentrations in MS brain might be due to a change in the activity of the ester metabolizing enzyme (s). We measured the activities of these enzymes in brain tissue from individuals with and without MS and correlated the Abbreviation: MS-multiple sclerosis. 1 The authors are grateful to Dr. W. W. Tourtellotte, Director, Human Neurospecimen Bank, Los Angeles, CA, for providing us tissue samples. This investigation was supported by National Institutes of Health grants NSl1670 and NS14938. 601 0014-4886/80/060601-04$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved
602
SHAH
AND
JOHNSON
changes in enzyme activity with alterations in cholesterol ester concentrations to determine whether the ester concentrations in MS brain increase because of an increase in esterifying activity or a decrease in hydrolase activity. Cerebral white matter from four patients with MS (plaque and periplaque regions) and from eight individuals with no known neurological diseases used in the present study were supplied by Dr. W. W. Tourtellotte (Director, Human Neurospecimen Bank, Los Angeles, California). Autolysis periods for brain tissue samples were 7 to 30 h and the patients’ ages were 23 to 68 years. The periplaque regions corresponded to a normal appearing area 0.6 cm around the plaque. ]C’~C]cholesterol and ]1J4C]oleic acid with sp. act. 50 mCi/mmol and cholesterolll-‘“Cloleate sp. act. 50 to 60 mCi/mmol were purchased from New England Nuclear Corporation, Boston, Massachusetts, and checked for radio purity prior to use. Unlabeled cholesterol, oleic acid, and cholesterol oleate were the products of Applied Sciences Laboratories, State College, Pennsylvania. For cholesterol ester hydrolases in white matter, incubation mixtures contained ]4J4C]cholesterol oleate (100,000 cpm) plus 0.5 pmol unlabeled cholesterol oleate, 4 ~1 Triton X-100, and either 0.8 ml 0.15 M citrate phosphate buffer (PH 4.5) or 0.8 ml 0.1 M sodium phosphate buffer (PH 6.5). The mixtures were sonicated 30 s, then 0.2 ml (1 mg protein) 800-g supernatant was added and the mixtures incubated 3 h at 37°C with shaking. The reaction was terminated and the lipids were then extrated from the reaction mixtures. Cholesterol and oleic acid were separated from cholesterol oleate and the radioactivity assayed by procedures described elsewhere (10). For measurement of cholesterol esterifying activity, incubation mixtures containing [4-14C]cholesterol (120,000 cpm) plus 0.6 pmol unlabeled cholesterol and 1.2 pmol unlabeled oleic acid in 50 ~1 acetone and 0.8 ml 0.15 M citrate phosphate buffer were sonicated. The reaction was initiated by adding 0.2 ml (1 mg protein) of 800-gsupernatant from white matter. The mixtures were incubated 3 h at 37°C with shaking. The lipids were free and esterified cholesterol were extracted following incubation, separated, and the radioactivity was assayed as before (10). For determining the concentrations of free and esterified cholesterol, the lipids were extracted from a portion of 10% homogenate from white matter and free and esterified cholesterol were separated by column chromatography on silica gel G. The amounts of free cholesterol were estimated as reported earlier (lo), and the amounts of esterified cholesterol were estimated by a more sensitive method described recently by Heider and Boyett (8). Results presented in Table 1 show that the amounts of free cholesterol in white matter from normal individuals and from MS patients did not differ
CHOLESTEROL
ESTER METABOLISM TABLE
603
IN MS BRAIN
I
Cholesterol Ester Concentrations and the Activities of Cholesterol Ester Metabolizing Enzymes in Human Brain White Matter” Cholesterol Cholaterol
e\ter
(nmoUmg
hydrolyced
ester5
formed
Estelified
(nmollmg
protein/h)
Free
profemlhl pH
MS
(plaque)
MS
~periplaquel
(41
(41 Normal
” The on
each
(81
numbers tissue.
’ S~gmficantly
PH65
pH
5 5
concentration
(wkl
0.13’
0.63
+ 0.X’
0.242
t
o.ll*
25.01
c
9.63
t
? 29’
1.57
? 0.57’
0.316
t
0.19
XX?
5 2.43
15.5
t
4.06
2.64
?
0.481
0.19
26.15
f
parentheses
represent
concentrationa
different
from
of normal
the free
and
number esteritied
i
of
tissue
ample?
cholesterol
are
4.26
KS9
I 88
3.50
II.08
analyzed. expressed
PUCKitage estenfied
Iwk)
r
I.19
sterol
concentration
I.57
m The
4.5
~rerol
Duphcate ai
mgig
2
1.30’
2
1.04’
23.31
6.42
? 2.99*
2
0.04
0.35
2
analya~r wet
weight
was of
white
2 6.E’
0.18
carned
out
matter.
P < 0.025.
significantly. However, both periplaque and plaque regions from MS brains showed a significant increase in the amounts of esterified cholesterol. The highest proportion (23%) of esterified cholesterol was found in the plaque region. The cholesterol esterifying activity in the white matter (plaque region) from patients with MS was significantly lower than the activity in white matter from normal individuals. The reduction of esterifying activity in MS white matter (periplaque region) was however not significant. The hydrolase (s) activities, on the other hand, were significantly lower in both plaque and periplaque regions of MS brains. The reduction in the periplaque region was about 40%, whereas the reduction in the plaque region was about 85%. These data on reduction in hydrolase activities are similar to the reduction in acid lipase activity observed in patients with MS by Hirsch et al. (9). Results of our study show that, as reported earlier by other investigators (l-4, 6. 13, 14), there is a significant increase in the amounts of esterified cholesterol in white matter (normal appearing periplaque and plaque regions) from MS patients. Because cholesterol esters in brain are synthesized in situ, one might expect the increase in cholesterol ester concentrations in MS brain to be a result of increased synthesis. Our data show, however, that the cholesterol esterifying enzyme activity in white matter from MS brain is actually less than in white matter from the normal brain. It is therefore unlikely that the elevated cholesterol ester concentrations in MS brain are caused by changes in the activity of the esterifying enzyme. The esterifying enzyme in brain utilizes free fatty acids (5, lo), and, in the absence of added fatty acid, cholesterol esterification is stimulated by phospholipase A, (11). An increase in free fatty acid concentrations (7) and in phospholipase A, activity (15) in MS brain has been reported. Thus, in spite of the reduced activity of esterifying enzyme in MS
604
SHAH AND JOHNSON
brain, the extent of esterification actually taking place in viva may be high. This is consistent with the suggestion (12) that steryl esters serve as carrier and storage points for free fatty acids that would otherwise be toxic. There is a greater likelihood, however, that the accumulation of cholesterol esters in MS brain is due to reduction in hydrolase(s) activity for the following reasons. The increase in esterified cholesterol is maximal in the plaque region, and is accompanied by an almost total lack of hydrolase(s) activity. In periplaque regions, on the other hand, the increase in cholesterol ester concentration is small as is the reduction in hydrolase activity. REFERENCES 1. ALLING, C., M. VANIER, AND L. SVENNERHOLM. 1971. Lipid alterations in apparently normal white matter in multiple sclerosis. Bruin Res. 35: 325-336. 2. CUMINGS, J. N. 1953. The cerebral lipids in disseminated sclerosis and in amaurotic family idiocy. Brain 76: 55 l-562. 3. CUMINGS, J. N. 1955. Lipid chemistry of the brain in demyelinating diseases. Bruin 78: 554-563. 4. DAVISON, A. Neurochem.
N.,
AND
M.
WAJDA.
1962. Cerebral lipids in multiple
sclerosis. J.
9: 427-432.
5. ETO, Y., AND K. SUZUKI. 1971. Cholesterol ester metabolism in brain: properties and subcellular distribution of cholesterol esterifying enzymes and cholesterol ester hydrolases in adult rat brain. Biochim. Biophys. Acta 239: 293-311. 6. GERSTL, B., M. J. KAHNKE, J. K. SMITH, M. G. TAVASTJERNA, AND R. B. HAYMAN. 1961. Brain lipids in multiple sclerosis and other diseases. Brain 84: 310-319. 7. GERSTL, B., M. G TAVASTJERNA, L. F. ENG, AND J. K. SMITH. 1972. Sphingolipids and their precursors in human brain (normal and MS). Z. Neural. 202: 104- 120. 8. HEIDER, J. G., AND R. B. BOYETT. 1978. The picomole determination of free and total cholesterol in cells in culture. J. Lipid Res. 19: 114- 118. 9. HIRSCH, H. E., P. DUQUETTE, AND M. E. PARKS. 1976. The quantitative histochemistry of multiple sclerosis plaques; acid proteinase and other acid hydrolases. J. Nelrrothem. 26: 505-512. 10. JOHNSON, R. C., AND S. N. SHAH. 1978. Cholesterol ester metabolizing enzymes in human brain: properties, subcellular distribution and relative levels in various diseases conditions. J. Neurochem. 31: 895-902. 11. RAMSEY, R. B., AND A. N. DAVISON. 1974. Effect of phospholipase A upon brain cholesterol ester formation. Lipids 9: 440-442. 12. RAMSEY, R. B., AND A. N. DAVISON. 1974. Steryl esters and their relationship to normal and diseased human central nervous system. J. Lipid Res. IS: 249-255. 13. WENDER, M., Z. ADAMEZEWSKA, AND A. WAJGT. 1973. Cerebral lipids in a case with clinically silent isolated plaque of the multiple sclerosis type. Eur. Neurol. 9: 21-29. 14. WENDER, M., H. FILIPEK-WENDER, AND B. STANISLAWSKA. 1973. Cholesteryl esters in apparently normal white matter in multiple sclerosis. Eur. Neurol. 10: 340-348. 15. WOELK, H., AND K. PEILER-ICHIKAWA. 1974. Zur Aktivitat der Phospholipase A2 gegenuber verschiedenen I-alk-I’-enyl-2-acyl-und-1-alkyl-2-acyl verbindungen wahrend der Multiplen Sklerose. J. Neurol. 207: 319-326.