Neurochemical Research, Vol. I0, No. 5, 1985, pp. 677-689
METABOLISM OF I N T R A C E R E B R A L L Y INJECTED M E V A L O N A T E IN BRAIN OF S U C K L I N G A N D Y O U N G A D U L T RATS RONALD
C.
JOHNSON
AND SHANTILAL
N.
SHAH*
University of California, San Francisco Department of Psychiat~ Brain-Behavior Research Center Sonoma Developmental Center EIdridge, California
Accepted January 5, 1985
We have investigated the in vivo metabolism via sterol and nonsterol pathways of intracerebrally injected mevalonate (MVA) in brains from suckling (10-day-old) and young adult (60-day-old) rats. Results of our study indicated that increasing the amounts of MVA injected increased MVA incorporation into all the lipid fractions examined. The incorporation of MVA into nonsaponiable lipids (NSF) and digitonin precipitable sterols (DPS) was similar in brains from adult and suckling rats. In brain tissue from both suckling and young adult rats the synthesis of dolichot from MVA varied with the amounts of MVA injected. Significant amounts of MVA were recovered in phosphorylated and free polyprenols (farnesol and geraniol) in brain tissue from rats of both ages. Also in both groups of animals, the amounts of MVA incorporated in phosphorylated and free farnesol were higher than the amounts recovered in either, phosphorylated or free geraniol. The amounts of MVA incorporated into the prenoic/fatty acid fraction by brain tissue from both suckling and young adult rats were less than 1% of the total MVA incorporated (nonsaponifiable and saponifiable lipids). Incorporation of MVA into the prenoic/fatty acid fraction by brain tissue was higher in suckling than in young adult rats. These data indicate that the brain tissue from suckling and young adult rats do not differ in their capacity to metabolize MVA into squalene and sterols and that in brain, metabolism of MVA by a shunt pathway is minimal. This suggests that in vivo regulation of cholesterol synthesis during brain development must occur at a step(s) in the sterol synthetic pathway prior to mevalonate, and that metabolism of mevalonate by shunt pathway did not play a role in the developmental regulation of brain sterol synthesis. The data also suggest that in both * Address all correspondence to: Shantilal N. Shah, Brain-Behavior Research Center, Sonoma Developmental Center, Eldridge, CA 95431, (707) 938-4701.
677 0364-3190/85/0500-0677504.50/0 @ 1985 Plenum Publishing Corporation
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JOHNSON AND SHAH
groups of animals the synthesis of squalene by synthetase may in part control brain sterol synthesis and the synthesis of dolichol is regulated by MVA concentration in the tissue.
INTRODUCTION It has been reported that brains of 9-day-old rats utilize the carbon skeleton of mevalonate (MVA) to a significant extent (7-33%) for biosynthesis of compounds other than sterols via a shunt pathway (3). It has not as yet been determined whether the metabolism of mevalonate by a shunt pathway occurs in brains of adult animals or whether metabolism of mevalonate by this pathway is related to regulation of sterol synthesis in brain during development. Although the synthesis of squalene and sterols from mevalonate by brain tissue from adult rats has been studied (6, 10, 12) the capacity of the brain tissue from suckling and young adult rats to metabolize MVA in vivo by sterol pathways has not been compared. The purpose of the present study is to compare the in vivo metabolism of mevalonate by sterol and nonsterol (shunt) pathways in brains of young adult and suckling rats.
EXPERIMENTAL PROCEDURE R,S-(2-~4C)-mevalonic acid (MVA) dibenzylethylenediamine salt (DBED), specific activity 50.1 mCi/mmol (New England Nuclear Corp., Boston, Massachusetts) was diluted with varying amounts of unlabeled MVA DBED salt (Calbiochem, San Diego, California) and converted to the sodium salt before use. Cholesterol, lanosterol, farnesol, dolichol, geraniol and squalene were purchased from Sigma Chemical Co., St. Louis, Missouri. Pre-coated silica gel G plates were purchased from Analtech, Inc., Newark, Delaware. Suckling (10-day-old) and young adult (60-day-old) rats were used for in vivo studies. To inject the suckling animals, an incision was made through the skin and the injection was delivered into the brain at a depth of 0.5 cm. Young adult animals were subjected to light ether anaesthesia and an incision was made through the skin. A small hole was made through the skull above the right hemisphere, and the injection was delivered into the brain while the animal was still under anaesthesia. Animals were injected with 50 ~1 of a saline solution containing lfxCi of [2-~4C] MVA plus either 0.2, 0.5, 1.0 or 2 fxmoles, of unlabeled MVA, and sacrificed after 0.5, 2.0 and 4.0 hrs. The brains were removed, weighed and minced. One-half of the tissue mince was homogenized with 3 vol. of 0.9% NaCl. Radioactivity was measured in an aliquot (1%) of the homogenate to determine the amounts of MVA recovered in brain. To the remaining homogen~ite a 50 txl of benzene solution containing 50 ~xg each of farnesol, geraniol, dolichol, squalene and lanosterol were added as carrier and the mixture was saponified by adding 15% KOH in 50% ethanol and heating the mixture for 60 min at 80~ Nonsaponifiable lipids and saponifiable lipids (phosphorylated polyprenols and prenoic/ fatty acid fractions) were isolated and extracted essentially as described by Christophe and Popjak (2). The nonsaponifiable lipids (NSF) were extracted from the saponified mixture
METABOLISM OF MEVALONATE
679
with petroleum ether. An aliquot (1%) was counted to determine the total radioactivity in NSF. A portion of NSF (50%) was used to precipitate sterols as digitonide and assay the radioactivity in digitonin precipitable sterols. Lipids in the remaining portion of the NSF, were separated by thin-layer chromatography on silica ge! G using dichloromethane-ethyl acetate, 97:3, as the developing solvent (17). Individual spots on the TLC plates were identified by the use of authentic compounds, scraped and transferred into scintillation vials for determination of radioactivity. For estimation of phosphorylated polyprenols formed, the aqueous phase after removal of NSF was acidified to pH 2 and allowed to stand overnight to hydrolyse the phosphorylated polyprenols. A 50 ixl benzene solution containing 50 txg each of farnesol, geraniol and dolichol were again added. The mixture was adjusted to pH 12 and free polyprenols were extracted with petroleum ether (2). A portion of the extract was used for determination of radioactivity in total phosphorylated polyprenols. The individual polyprenols were separated by TLC using the remainder of the extract. The alkaline aqueous phase remaining after extraction of the polyprenols was again acidified to pH 2. A 50 p~l of benzene solution containing 50 fxg each of stearic and oleic acid were added and the fatty/prenoic acids were extracted with petroleum ether (2). The radioactivity was assayed using a small portion of the latter extract and fatty/prenoic acids after methylation were separated by thin-layer chromatography on silver nitrate-impregnated silica gel G plates (3). The incorporation of MVA into various fractions is expressed as nmoles per brain and was calculated as follows: cpm recovered x nmoles of MVA administered/cpm administered.
RESULTS Approximately 40-50 and 30-40 percent of the injected mevalonate is present in brain tissue 30 mins after the administration of mevalonate to suckling and young adult rats, respectively, (Table I). In suckling rats the amounts of mevalonate recovered in brain tissue decreased with time only when the amount of MVA administered was 0.2 p~mol. In adult rats, on the other hand, the amounts of mevalonate recovered in brain declined with time at all (except 2 ~xmol) concentrations of MVA administered. Low MVA recovery values at 30 min after injection as compared to those for 2 and 4 hrs after injection may reflect variability in the amount of substrate retained. Relatively lower recovery of injected MVA in brain of young adult rats as compared to that of suckling animals could be also due to variable retention or to rapid clearance of MVA from brain of adult rats. The incorporation in vivo of MVA into total nonsaponifiable lipids and digitonin precipitable sterols is shown in Figure 1. In brain tissue from both suckling and young adult rats an increase in the amount of MVA injected results in increased incorporation of MVA into total nonsaponifiable lipids (Figure 1A). In spite of the relatively lower recovery of injected MVA in brains from adult rats the total amounts of MVA incorporated into NSF (nmoles/brain) in brain tissue from suckling and young adult rats was not significantly different. The incorporation of MVA into
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TABLE I RECOVERY OF MEVALONATE ( M V A ) IN BRAIN TISSUE AFTER INTRACEREBRAL (IC) INJECTION
MVA Recovered as % of Injected Time After Administration MVA injected 0xmol)
0.5 hr
2.0 hr
4.0 hr
I. 10-day-old-rats 0.2 0.5 1.0 2,0
52.3 41.1 40.4 41.8
_+ _+ _+ _+
2.3 5.0 1.8 3.5
37.6 45.0 49.2 41.8
_+ _+ _+ _+
1.2 5.9 7.6 1.5
28.7 40.8 45.5 39.8
+ 0.3 _+ 3.6 _+ 2.3 _+ 0.7
II. 60-day-old rats 0.2 0.5 1.0 2.0
39.5 26.5 31.3 31.6
_+ _+ _+ _+
6.0 5.1 1.3 1.0
26.1 21.6 22.4 39.5
_+ _+ _+
3.7 3.6 1.2 3.1
13.5 17.9 21.2 37.5
_+ _+ _+ +_
1.3 2.3 2.1 1.2
Suckling (10-day-old) pups and y o u n g adult (60-day-old) male rats were injected intracerebrally with 1 ixCi of [2-~4C]mevalonate plus varying amounts of unlabeled mevalonate as indicated above. The animals were sacrificed at indicated times after injection. Brain tissue was removed, weighed and h o m g e n i z e d as described in the text. Total radioactivity recovered in brain tissue was determined by measuring the radioactivity in small aliquot (1%) of brain h o m o g e n a t e and multiplying it by the dilution factor. The percent MVA recovered was calculated as follows:
cpm recovered in brain cpm injected
x 100.
E a c h value in the table represents the average - SD for three animals.
DPS also increased with an increase in the amount of MVA injected (Figure 1B). The optimal incorporation into DPS in adults is lower than that in suckling rat brain when the amount of MVA injected was 0.2 and 0.5 ixmol. The reverse was the case when the amount of MVA injected was l and 2 txmol. The amount of MVA recovered in both the phosphorylated polyprenols (Figure 2A) and the prenoic/fatty acid fraction (Figure 2B) increased with increase in the amount of MVA injected. The amount of MVA recovered in the phosphorylated polyprenols fraction was higher in the adult rat brain than in the brain of suckling rats when 2 txmol of MVA was injected. In the case of the prenoic/fatty acid fraction, however, the reverse was true. When 1 or 2 ixmol of MVA was injected the amounts recovered in the fractions from the brains of suckling rats were at all time intervals higher than the amounts recovered in the fractions from adult brain. In
681
METABOLISM OF MEVALONATE
60
10 DAY
200[ I
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--g
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DAY
A
0
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O E E
0 E
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2
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1~
60 DAY
B
o~
~m S~
25 ~
~
1
2
3
4
1
TIME
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3
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(hrs)
FIG. 1. Incorporation of intracerebrally injected mevalonate into total nonsaponifiable lipids (A) and digitoniu precipitable sterols (B). Suckling (10-day-old) pups and young adult (60day-old) rats wre injected (1 Ixc of [2-14C] MVA) with varying amounts ( O - - O : 0.2 Ixmol; e--e: 0.5 p~mole; ~ - - [ ] : 1.0 p.mole; and R - - i : 2.0 jxmoles) of unlabeled MVA and sacrificed at the indicated time after injection, as described in the text and in Table I. The details of procedure for isolating and assaying the radioactivity recovered in nonsaponifiable lipids and digitonin precipitable sterols is described in the text. Each point in the figure represents average and range for three animals.
682
JOHNSON AND SHAH
10
•
DAY
60
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E
E
~ 0
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0
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x
1
2
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(hrs)
FIG. 2. Incorporation of intracerebrally injected mevalonate ( Q - - O : 0.2 ~mole; O - - O : 0.5 ixmole; C ] - - D : 1.0 Ixmol and I - - I : 2.0 ixmol) into phosphorylated polyprenols (A) and prenoic/fatty acid (B) fraction. The details for isolating phosphorylated polyprenols and prenoic/fatty acid fraction are described in the text. Each point in the figure represents the average and range for three animals.
683
METABOLISM OF MEVALONATE
TABLE II TYPICAL DISTRIBUTION OF M V A IN NONSAPON1FIABLE LIPIDS, PttOSPHORYLATED PRENOLS, AND PRENOIC/FATTY ACID FRACTIONS OF BRAIN Mevalonate incorporated (nmol/brain) 10-day-old rats Lipid Components in
2 hrs
4 hrs
80 12.0 10.6 13.1 30.4 1.9 5.2 3.4
Nonsaponifiable Fraction 142 201 83 128 31.0 45.0 1.1 2.0 28.8 52.7 10.9 38.1 21.4 29.2 19.6 33.2 20.6 15.0 34.8 24.6 3.8 5.3 1.3 3.9 9.2 12.8 6.1 11.3 5.7 12.8 2.0 5.7
148 0.9 56.4 39.8 21.6 4.2 12.7 6.0
Total Geraniol-pp Farnesol-pp Dolichol-pp
9.9 0.7 6.0 2.9
Saponifiable Fraction Phosphorylated Polyprenols 15.8 14.3 10.6 19.6 2.1 3.0 1.2 2.6 7.3 6.1 6.1 10.6 5.7 4.1 2.5 4.7
24.8 3.2 12.3 4.8
Total Unidentified (origin) Prenoic Acid Fatty acids
0.9 0.4 0.1 0.3
Total Unidentified (origin)
Cholesterol (C27 sterols) Lanosterol (C29 sterols) Squalene Geraniol Farnesol Dolichol
0.5 hr
2 hrs
60-day-old rats
1.5 0.5 0.3 0.5
4 hrs
0.5 hr
Fatty/prenoic 1.7 0.5 0.3 0.6
Acids 0.8 0.2 0.2 0.3
0.8 0.2 0.2 0.4
1.0 0.2 0.2 0.4
The data are for a single animal injected with 1 ixc of [2-14C]MVA plus 2 txmol of unlabeled MVA and sacrificed at indicated times after injection. The individual components of NSF, phosphorylated polyprenols and prenoic/fatty acid fractions were separated by thin layer chromatography as described in the text. Each lipid component was identified on the basis of its ability to co-chromatograph with authentic standards.
both groups of animals the total amounts of MVA recovered in the fatty/ prenoic acid fraction, were less than 2 nmol/brain (i.e., less than 1% of the total MVA incorporated into NSF). It is recognized that a variable amount of sterol fraction is precipitated with digitonin hence the values for the recovery of label in DPS do not reflect the true measure of sterol synthesis (15). Also, to obtain data for the incorporation of MVA into squalene, C27 sterols and dolichol, a portion of the NSF was separated by thin-layer chromatography. The data of a representative experiment presented in Table II indicate that the
684
JOHNSON AND SHAH
~
" lo oar
~ ~ 4~
A
i i
2
3
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1
TIME
2
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(hrs)
10 DAY
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o 5
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TIME lo
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(hrs)
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g
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1
2
3
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FIG. 3. Incorporation of intracerebrally injected M V A (O - - O: 0.2 ~Lmol; 9 - - 9 0.5 p,mol; E l - - [ ] : 1.0 i~mol; and m - - B : 2.0 i~mol) in squalene (A); C27 sterols (B); and dolichol (C), Each point represents the average and range for three animals.
METABOLISM OF MEVALONATE
685
incorporation of MVA in cholesterol (C27sterols) and lanosterol (C29sterols) increases with time while incorporation into squalene decreases. These changes occur in brain tissue from rats of both age groups. It may be noted, however, that in the NSF fraction from brain tissue of suckling rats significant radioactivity is recovered in an unidentified compound(s) which remain at the origin. This compound(s) is (are) not present in the NSF fraction from brain of young adult rats. Separation of the phosphorylated polyprenol fraction by thin-layer chromatography presented in Table II showed that in all cases the amounts of MVA recovered in phosphorylated farnesol is higher than that recovered in phosphorylated geraniol and phosphorylated dolichol. The radioactivity in these three compounds accounted for 90% of the total radioactivity in the fraction. The amount of MVA recovered in phosphorylated farnesol and geraniol was higher with the adult than with the suckling rats. The amount of MVA recovered in phosphorylated dolichol, however, did not differ significantly in the two groups of animals. Separation of prenoic/fatty acid fraction by thin-layer chromatography indicated that the radioactivity in the fatty acid spot (F) corresponding to authentic stearic and oleic acid accounted approximately 35% of the total radioactivity. The total amount of MVA recovered into the fatty acid fraction was approximately 0.5 nmol or less per brain (Table II). The amounts of MVA incorporated into fatty acids by adult rat brain were somewhat lower than the amounts of MVA incorporated by suckling rat brain. The data on incorporation of MVA into squalene and Czv sterols as a function of time and the amounts of MVA injected are shown in Figures 3A and 3B, respectively. The incorporation into both squalene and C27 sterols increases with an increase in the amount of MVA injected, and the amounts incorporated in both fractions (nmol/brain) by brain tissue were higher in the adult than in the suckling rats. The incorporation into squalene reached maximum within 30 min and declined thereafter with time (Figure 3A), with a corresponding increase in the incorporation into C27 sterols (Figure 3B). The incorporation of MVA into dolichol increased with increase in the amounts of MVA injected (Figure 3C). The total amount of MVA incorporated into dolichol (nmol/brain) is, however, small (less than 10 nmol representing less than 5% of total MVA recovered in NSF). The incorporation of MVA into dolichol by brain tissue from adult and suckling rats did not differ significantly. DISCUSSION Results of this study indicate that (1) the in vivo capacity of young adult rat brain to synthesize squalene and sterols from mevalonate is equal to
686
JOHNSON ANDSHAH
or greater than the capacity of the immature brain; (2) the in vivo synthesis of dolichol from mevalonate by brain tissues from suckling and young adult rats did not differ significantly; (3) the incorporation of mevalonate into fatty acid was quite low (less than 1% of the total MVA incorporated into NSF) in brain tissue from rats of both ages; (4) the amounts of MVA recovered into phosphorylated polyprenols in brain tissue from young adult rats were slightly higher with the adult than with the immature rats. Although in vivo synthesis of squalene and cholesterol from mevalonate by brain tissue from adult rats has been reported earlier (6, 10, 12), the optimal capacity of the brain from suckling and young adult rats to synthesize squalene and cholesterol from mevalonate has neither been examined nor compared. In view of the finding in this and other laboratories (9, 11) that the in vitro activity of pyrophospho-mevalonate decarboxylase is lower in brain from adult rats, one would expect adult rats to have a lesser capacity to synthesize cholesterol from mevalonate. Contrary to this expectation, however, we find that in vivo synthesis of squalene and cholesterol in brain tissue from young adult rats is at least equal to that in immature rats. This discrepancy may reflect the differences between the metabolism of mevalonate by intact cell and cell-free preparations. Variations in rates of substrate oxidation by different brain preparations (dissociated brain cells and brain homogenates) have, in fact, been reported (16), and differences between in vivo and in vitro metabolism of MVA by sterol and nonsterol pathways have been observed in brain as well as in other tissues (3, 8, 14). Since the rate of accumulation of cholesterol in adult rat brain is much lower than in brain from suckling rats and since the turnover of cholesterol in brain tissue is quite low in both groups of animals, the actual synthesis of cholesterol in the adult brain must be quite small as compared to the synthesis in immature brain. Our data show that the brain tissue from adult rats is able to synthesize cholesterol from mevalonate to the same extent as or to a greater extent than the brain tissue from suckling rats. The developmental regulation of sterol synthesis in brain must therefore occur at a step prior to mevalonate formation in the metabolic pathway for sterol synthesis. Since the HMG CoA reductase step is considered to be a primary regulatory reaction for cholesterol synthesis in most tissues, the regulation in vivo of cholesterol synthesis in brain might also occur at this step. A somewhat higher recovery of MVA into phosphorylated polyprenols (farnesol-pp and geraniol-pp) as compared to free polyprenols in brain tissue from young adult rats indicates that polyprenol phosphate phosphatase activity may be reduced in brains of these rats, and that the accumulation of the polyprenol phosphates by way of feedback inhibition
METABOLISM OF MEVALONATE
687
may regulate the synthesis of MVA. Also, the incorporation of MVA into phosphorylated farnesol was higher than that in phosphorylated geraniol. This may occur as a result of a reduction in the synthesis of squalene from farnesol pyrophosphate. In the present study the incorporation of intracerebrally injected MVA into acidic lipids (fatty/prenoic acid) from brain was less than 1% of total MVA incorporated into nonsaponifiable lipids. This amount is much smaller than the incorporation into brain tissue of intramuscularly injected MVA (7-33%) reported by Edmond and Popjak (3). The reasons for this difference are not clear at present. It is likely that the difference may be related to the mode of MVA administration. Relatively poor recovery of intraperitoneally injected MVA in brain tissue of newborn 15-day-old rats has been reported by Ramsey et al. (13). These investigators also found that intracerebrally administered MVA gave the best yield of labeled cholesterol. This suggests that only intracerebrally injected precursors or (substrate) can provide data for the measure of true capacity of the brain to metabolize the particular substrate. If this is true then our data of less than 1% incorporation of MVA into fatty acids indicate that the metabolism of mevalonate by a nonsterol (shunt) pathway is negligible in brain tissue from both suckling and young adult rats. It is also likely that this small amount of incorporated label could have occurred by a carbon dioxide fixation reaction and not via the shunt pathway. The labeled carbon dioxide in this case could result from the oxidative demethylation of (C29 sterols) lanosterol to C27 sterols. It is unlikely, therefore, that the metabolism of MVA by a shunt pathway plays a role in regulating sterol synthesis in brain. Age-dependent changes in the in vitro synthesis of dolichol from acetate in mouse brain have been reported (4). Our data, however, indicate that synthesis of dolichol from mevalonate in brain tissue of suckling and young adult rats did not differ. Developmental change in the dolichol synthesis in brain in vivo must also, therefore, occur at a step prior to mevalonate. In summary, the results of our study indicate that, unlike the findings on in vitro MVA metabolism (9, 11, 13), the in vivo metabolism of mevalonate in brain of suckling and young adult rats did not differ. The possibility remains, however, that different regions of brain from suckling and young adult rats may vary in their capacity to synthesize cholesterol from mevalonate. These data also indicate that the developmental regulation of sterol synthesis and the synthesis of nonsterol components such as dolichol in brain must occur at a step or steps in the metabolic pathway prior to mevalonate.
688
JOHNSON A N D S H A H
ACKNOWLEDGMENT This investigation was supported by research grant NS11670 from National Institutes of Health.
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11. RAMACHANDRAN,C. K., and SHAH, S. N. 1977. Studies on mevalonate kinase and decarboxylase in developing rat brain. J. Neurochem. 28:751-757. 12. RAMSEY, R. B., JONES, J. P., and NICHOLAS, H. J. 1971. The biosynthesis of cholesterol and other sterols by brain tissue: Distribution in subcellular fractions as a function of time after intracerebral injection of (2-14C) mevalonic acid. J. Neurochem. 18:14851493. 13. RAMSEY, R. B., JONES, J. P., NAQUI, S. H. M., and NICHOLAS, H. J. 1971. The biosynthesis of cholesterol and other sterols by brain tissue: I1. A comparison of in vitro and in vivo methods. Lipids 6:225-232. 14. RHIGHETTI,M., WILEY, M., MURRILL,P., and SIPERSTEIN,M. 1976. The in vitro metabolism of mevalonate by sterol and nonsterol pathways. J. Biol. Chem. 251:27162721.
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15. TABACIK,C., ALLAN,S., and DE-PAULET,A. C. 1983. Digitonin-precipitable sterols as a measure of cholesterol biosynthesis: Contradictory results. Lipids 18:641-649. 16. TILDON,J. T., MERRILL,S., and ROEDER, L. M. 1983. Differential substrate oxidation by dissociated brain cells and homogenate during development. Biochem. J. 216:21-25. 17. WONG, T., and LENNARZ, W. 1982. Biosynthesis of dolichol and cholesterol during embryonic development of the chicken. Biochim. Biophys. Acta 710:32-38.