Jowiml oJ Nei,i.ucheiiiisli-~. 1975. Vol. 25. pp. 659-665. Pergamon Press. Prmtcd in Great Britain.
THE DISPOSITION OF [24-3H]CEREBROSTEROL IN DEVELOPING RAT BRAIN* Y . Y . La. and L. L. SMITH Division of Biochemistry, Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77550, U S A . (Received 25 February 1975. Accepted 30 April 1975) Abstract-The uptake into subcellular fractions of developing rat brain in vivo of intracerebrally injected [4-'4C]cholesterol, [24-3H]cerebrosterol, and [24-3H]24-epicerebrosterol was measured for periods up to 30 days following administration. [4-'4C]cholesterol was accumulated rapidly in nuclei, nerve endings, and microsomes, more slowly in myelin and mitochondria. [24-3H]cerebrosterol was accumulated rapidly in myelin, nerve endings, and microsomes, more slowly in nuclei and mitochondria. The uptake of [24-3H]24-epicerebrosterol was essentially the same as that of [24-3H]cercbrosterol. Ratios of radioactivities of [24-3H]cerebrosterd and [4-'4C]cholesterol accentuated the early accumulation of [24-3H]cerebrosterol in myelin, nerve endings, and microsomes, and declining 3H:'4C ratios disclosed the rapid elimination of [24-3H]cerebrosterol and [24-3H]24-epicerebrosterol relative to [4-L4C]cholesterol in nerve endings and microsomes. The data suggest that the removal of [24-3H]cerebrosterol from brain results from an enzymic metabolism of the sterol, therefore that cerebrosterol exists in brain in a dynamic state of biosynthesis and catabolism.
WE IJAVE demonstrated previously the presence of Tex: (SD) strain, 18-days old, weighing 35-50 g, purchased cerebrosterol (24-hydroxycholesterol, (24S)-cholest-5- from the Tcxas Inbred Mice Co., Houston, Texas were en-3/3,24-diol)and its biosynthesis from cholesterol maintained on Purina lab chow fed ad lib. Intracerebral injection of 15 p1 of an ethanol solution (cholest-5-en-3/3-01)in crude microsomal fractions of containing 24 pg of [4-'4C]cholesterol (10" d.p.m.) and bovine (DHARet al., 1973) and rat (LIN & SMITH, 1974~)brain. In efforts to discover what role, if any, 6 pg of [24-3H]cerebrosterol (10' d p m . ) or containing 24 pg of [4-'4C]cholesterol (10" d.p.m.) and 6 pg of [24that cerebrosterol plays in brain function we sought 3H]24-epicerebrosterol (lo6 d.p.m.) was achieved using a to determine whether cerebrosterol is metabolized by No. 26 stainless steel hypodermic needle as described clscbrain tissue. Preliminary experiments with rat brain where (NICHOLAS & THOMAS, 1959~).Although this trcathomogenates in vitvo using [24-3H]cerebrosterol (LIN ment caused an immediate nervous reaction in the animals, & SMITH,1974b) as substrate did not provide encour- none became ill, incapacitated, or otherwise affected by agement, but in vivo experiments reported herein the injections and none dicd from the injection or during using [24-3H]cerebrosterol and [24-3H]24-epicere- the course of the experiments. No covering or sealing of brosterol ([24-3H] (24R)-cholest-5-ene-3B,24-diol)the wound was attempted. A preliminary experiment inprovide evidence that a selective metabolism of cere- volved intracerebral injection of [24-3H]cerebrosterol brosterol occurs among subcellular fractions of intact (2.3 x 10" d.p.m.) alone, without co-administration of [4' 4C]cholesteroL developing rat brain. Animals were killed in pairs immediately and after 0 5 , EXPERIMENTAL [4-'4C]cholestcrol (60 mCi/m-mol, 98% pure) purchased from New England Nuclear Co., Boston, Mass. and [243H]cerebrosterol and [24-3H]24-epicerebrosterol prepared in our laboratory (LIN & SMITH,19746) were purified prior to use by additional thin-layer chromatography using benzene-ethyl acetate, ethyl acetate-hexane, and chloroformacetonc solvent mixtures previously described (Lm & SMITH,1974a, 19746). Immature malc outbred albino rats
* Paper XXXV of the series "Sterol Metabolism". For Paper XXXIV, see SMITHL. L. 62 KULIGM. J. (1975) Cancer Biochem. Bioplzys. 1, 79-84, Financial support of these studics by the U S . Public Health Service (research grant NS-08 106) is gratefully acknowledged. 655.t
I , 5, 24 and 72h in one experiment, after 1. 7 and 30 days in another experiment. Animals injcctcd with 1243H]cerebrosterol alone were killed after 1, 12 and 52 days. Excised whole brains were rinsed with 0.9% saline, minced, and then homogenized at 4°C in 0.32 M-sucrose solution madc with 0.015 u-phosphate buffer (PH 7.4), as previously ; & DAVISON, 1968). described (LIN& SMITH,1 9 7 4 ~CUZNER Subcellular fractionation of the homogenate by centrifugation and by discontinuous gradient centrifugation using our modifications of the methods of Cuzner & Davison & DAVISON,1968) yielded nuclei, myelin, nerve (CUZNER cndings, mitochondria, microsomes and cytosol fractions, all operationally defined (LIN & SMITH.1 9 7 4 ~ ;CUZNER & DAVISON,1968). Each subcellular fraction was extracted with chloroform-methanol (2: 1, v/v) three times, and the cxtracts were
660
Y. Y. LIN and L. L. SMITH
washed twice with an equal volume of chloroform-methanol-0.1 M-tripotassium citrate (3:48:47, v/v/v). The organic layer was separated, dried over anhydrous sodium sulfate, and evaporated under vacuum. The total lipid residue was dissolved in chloroform-methanol (2: 1, v/v), and half of the solution was used for radioactivity assay in the total lipids fraction. To the remaining half 100 pg of inactive cerebrosterol was added as carrier. The total lipids were chromatographed on a 0.25 mm thick 20 x 20 cm chromatoplate of Silica Gel HF,,, (E. Merck GmbH., Darmstadt) using chloroform-acetone (21 :1, v/v) in double ascending development. Resolved zones containing sterols of interest were excised from the chromatoplate, and ',C and 3H activities were measured on the material added to 10 ml of toluene-absolute ethanol (100:3, v/v) containing 0 5 % PPO and 0.025% POPOP using a Packard Model 2420 Automatic Tri-Carb scintillation counter. Control experiments established that this simplified chromatographic procedure afforded good resolution of the sterols of interest from other brain lipids, yielding recovered [24-3H]cerebrosterol containing no [4-'4C]cholesterol and recovered [4-'4C]cholesterol containing no [24-3H]cerebrosterol.Over 90% of the radioactivity associated with the total lipids fraction was accounted for by the radioactive sterols. Material eluted from regions of the chromatoplate immediately ahead of [4-'4C]cholesterol, behind [24-3H]cerebrosterol, or lying between the two radioactive sterol zones was essentially devoid of measurable radioactivity.
I n uitro incubations of whole brain homogenate prepared in 032 ~-sucrose-0~015 M-phosphate buffer (pH 7.4) as described and of slices of brain in 0.32 M sucrose-4.015 M-phosphate buffer (pH 7.4) were conducted at 37°C following addition of lo6 d.p.m. each of [24-3H]cerebrosterol and [4-'4C]cholesterol. After 30 min the brain slices were homogenized at 4"C, and both homogenates were fractionated by centrifugation as described for the in uiuo experiments.
RESULTS
The changes in the amount of radioactivity associated with cholesterol, cerebrosterol, and 24epicerebrosterol recovered from subcellular fractions of developing rat brain at short times following intracerebral administration of [4-14C]cholesterol and the [243H]24-hydroxysterols are displayed in Fig. 1. The levels of radioactivity are plotted as per cent of the total amounts recovered from each subcellular fraction at the given time interval in order to accentuate the early features. Plots of percent of radioactivity administered rather than of total radioactivity recovered gave very similar curves which led to identical conclusions as drawn from the data presentations of Fig. 1. The total amount of radioactivity recovered from all subcellular fractions during the first 24 hr following sterol administration was fairly constant, D
FIG. 1. Levels (percent of radioactivity found in all subcellular fractions) of [4-14C]cholesterol, [243H]cerebrosterol, and [24-3H]24-epicerebrosterol in subcellular fractions of developing rat brain following intracerebral injection of lo6 d.p.m. each of [4-14C]cholesterol and [24-3H]cerebrosterol or [24'H]24-epicerebrosterol. A, nuclei; B, myelin; C, nerve endings; D, mitochondria; E, microsomes; F, cytosol.
Cercbrosterol disposition in rat brain TABLE1. LOUG-TERM RETENTION OF
Subcellular
fraction Nuclei Myelin Nerve endings Mitochondria Microsomes Cytosol Totals
661
RADIOACTIVITY IN SLBCELLULAR FRACTIOM OF RAT BRAIN
Radioactivity retained, % of administered amount [24-3H] [4-'4C] [24-3H] [24-'H] cerebrosterol* cholesterolt cerebrosterolt 24-epicerebrosterolt 0.75 day 12 days 52 days 1 day 7 days 30 days 1 day 7 days 30 days 1 day 7 days 30 days 1.2 0.06 7.6 0.29 45 0.26 1.6 0.04 0.48
THE DISPOSITION OF [24-3H]CEREBROSTEROL IN DEVELOPING RAT BRAIN* Y . Y . La. and L. L. SMITH Division of Biochemistry, Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77550, U S A . (Received 25 February 1975. Accepted 30 April 1975) Abstract-The uptake into subcellular fractions of developing rat brain in vivo of intracerebrally injected [4-'4C]cholesterol, [24-3H]cerebrosterol, and [24-3H]24-epicerebrosterol was measured for periods up to 30 days following administration. [4-'4C]cholesterol was accumulated rapidly in nuclei, nerve endings, and microsomes, more slowly in myelin and mitochondria. [24-3H]cerebrosterol was accumulated rapidly in myelin, nerve endings, and microsomes, more slowly in nuclei and mitochondria. The uptake of [24-3H]24-epicerebrosterol was essentially the same as that of [24-3H]cercbrosterol. Ratios of radioactivities of [24-3H]cerebrosterd and [4-'4C]cholesterol accentuated the early accumulation of [24-3H]cerebrosterol in myelin, nerve endings, and microsomes, and declining 3H:'4C ratios disclosed the rapid elimination of [24-3H]cerebrosterol and [24-3H]24-epicerebrosterol relative to [4-L4C]cholesterol in nerve endings and microsomes. The data suggest that the removal of [24-3H]cerebrosterol from brain results from an enzymic metabolism of the sterol, therefore that cerebrosterol exists in brain in a dynamic state of biosynthesis and catabolism.
WE IJAVE demonstrated previously the presence of Tex: (SD) strain, 18-days old, weighing 35-50 g, purchased cerebrosterol (24-hydroxycholesterol, (24S)-cholest-5- from the Tcxas Inbred Mice Co., Houston, Texas were en-3/3,24-diol)and its biosynthesis from cholesterol maintained on Purina lab chow fed ad lib. Intracerebral injection of 15 p1 of an ethanol solution (cholest-5-en-3/3-01)in crude microsomal fractions of containing 24 pg of [4-'4C]cholesterol (10" d.p.m.) and bovine (DHARet al., 1973) and rat (LIN & SMITH, 1974~)brain. In efforts to discover what role, if any, 6 pg of [24-3H]cerebrosterol (10' d p m . ) or containing 24 pg of [4-'4C]cholesterol (10" d.p.m.) and 6 pg of [24that cerebrosterol plays in brain function we sought 3H]24-epicerebrosterol (lo6 d.p.m.) was achieved using a to determine whether cerebrosterol is metabolized by No. 26 stainless steel hypodermic needle as described clscbrain tissue. Preliminary experiments with rat brain where (NICHOLAS & THOMAS, 1959~).Although this trcathomogenates in vitvo using [24-3H]cerebrosterol (LIN ment caused an immediate nervous reaction in the animals, & SMITH,1974b) as substrate did not provide encour- none became ill, incapacitated, or otherwise affected by agement, but in vivo experiments reported herein the injections and none dicd from the injection or during using [24-3H]cerebrosterol and [24-3H]24-epicere- the course of the experiments. No covering or sealing of brosterol ([24-3H] (24R)-cholest-5-ene-3B,24-diol)the wound was attempted. A preliminary experiment inprovide evidence that a selective metabolism of cere- volved intracerebral injection of [24-3H]cerebrosterol brosterol occurs among subcellular fractions of intact (2.3 x 10" d.p.m.) alone, without co-administration of [4' 4C]cholesteroL developing rat brain. Animals were killed in pairs immediately and after 0 5 , EXPERIMENTAL [4-'4C]cholestcrol (60 mCi/m-mol, 98% pure) purchased from New England Nuclear Co., Boston, Mass. and [243H]cerebrosterol and [24-3H]24-epicerebrosterol prepared in our laboratory (LIN & SMITH,19746) were purified prior to use by additional thin-layer chromatography using benzene-ethyl acetate, ethyl acetate-hexane, and chloroformacetonc solvent mixtures previously described (Lm & SMITH,1974a, 19746). Immature malc outbred albino rats
* Paper XXXV of the series "Sterol Metabolism". For Paper XXXIV, see SMITHL. L. 62 KULIGM. J. (1975) Cancer Biochem. Bioplzys. 1, 79-84, Financial support of these studics by the U S . Public Health Service (research grant NS-08 106) is gratefully acknowledged. 655.t
I , 5, 24 and 72h in one experiment, after 1. 7 and 30 days in another experiment. Animals injcctcd with 1243H]cerebrosterol alone were killed after 1, 12 and 52 days. Excised whole brains were rinsed with 0.9% saline, minced, and then homogenized at 4°C in 0.32 M-sucrose solution madc with 0.015 u-phosphate buffer (PH 7.4), as previously ; & DAVISON, 1968). described (LIN& SMITH,1 9 7 4 ~CUZNER Subcellular fractionation of the homogenate by centrifugation and by discontinuous gradient centrifugation using our modifications of the methods of Cuzner & Davison & DAVISON,1968) yielded nuclei, myelin, nerve (CUZNER cndings, mitochondria, microsomes and cytosol fractions, all operationally defined (LIN & SMITH.1 9 7 4 ~ ;CUZNER & DAVISON,1968). Each subcellular fraction was extracted with chloroform-methanol (2: 1, v/v) three times, and the cxtracts were
660
Y. Y. LIN and L. L. SMITH
washed twice with an equal volume of chloroform-methanol-0.1 M-tripotassium citrate (3:48:47, v/v/v). The organic layer was separated, dried over anhydrous sodium sulfate, and evaporated under vacuum. The total lipid residue was dissolved in chloroform-methanol (2: 1, v/v), and half of the solution was used for radioactivity assay in the total lipids fraction. To the remaining half 100 pg of inactive cerebrosterol was added as carrier. The total lipids were chromatographed on a 0.25 mm thick 20 x 20 cm chromatoplate of Silica Gel HF,,, (E. Merck GmbH., Darmstadt) using chloroform-acetone (21 :1, v/v) in double ascending development. Resolved zones containing sterols of interest were excised from the chromatoplate, and ',C and 3H activities were measured on the material added to 10 ml of toluene-absolute ethanol (100:3, v/v) containing 0 5 % PPO and 0.025% POPOP using a Packard Model 2420 Automatic Tri-Carb scintillation counter. Control experiments established that this simplified chromatographic procedure afforded good resolution of the sterols of interest from other brain lipids, yielding recovered [24-3H]cerebrosterol containing no [4-'4C]cholesterol and recovered [4-'4C]cholesterol containing no [24-3H]cerebrosterol.Over 90% of the radioactivity associated with the total lipids fraction was accounted for by the radioactive sterols. Material eluted from regions of the chromatoplate immediately ahead of [4-'4C]cholesterol, behind [24-3H]cerebrosterol, or lying between the two radioactive sterol zones was essentially devoid of measurable radioactivity.
I n uitro incubations of whole brain homogenate prepared in 032 ~-sucrose-0~015 M-phosphate buffer (pH 7.4) as described and of slices of brain in 0.32 M sucrose-4.015 M-phosphate buffer (pH 7.4) were conducted at 37°C following addition of lo6 d.p.m. each of [24-3H]cerebrosterol and [4-'4C]cholesterol. After 30 min the brain slices were homogenized at 4"C, and both homogenates were fractionated by centrifugation as described for the in uiuo experiments.
RESULTS
The changes in the amount of radioactivity associated with cholesterol, cerebrosterol, and 24epicerebrosterol recovered from subcellular fractions of developing rat brain at short times following intracerebral administration of [4-14C]cholesterol and the [243H]24-hydroxysterols are displayed in Fig. 1. The levels of radioactivity are plotted as per cent of the total amounts recovered from each subcellular fraction at the given time interval in order to accentuate the early features. Plots of percent of radioactivity administered rather than of total radioactivity recovered gave very similar curves which led to identical conclusions as drawn from the data presentations of Fig. 1. The total amount of radioactivity recovered from all subcellular fractions during the first 24 hr following sterol administration was fairly constant, D
FIG. 1. Levels (percent of radioactivity found in all subcellular fractions) of [4-14C]cholesterol, [243H]cerebrosterol, and [24-3H]24-epicerebrosterol in subcellular fractions of developing rat brain following intracerebral injection of lo6 d.p.m. each of [4-14C]cholesterol and [24-3H]cerebrosterol or [24'H]24-epicerebrosterol. A, nuclei; B, myelin; C, nerve endings; D, mitochondria; E, microsomes; F, cytosol.
Cercbrosterol disposition in rat brain TABLE1. LOUG-TERM RETENTION OF
Subcellular
fraction Nuclei Myelin Nerve endings Mitochondria Microsomes Cytosol Totals
661
RADIOACTIVITY IN SLBCELLULAR FRACTIOM OF RAT BRAIN
Radioactivity retained, % of administered amount [24-3H] [4-'4C] [24-3H] [24-'H] cerebrosterol* cholesterolt cerebrosterolt 24-epicerebrosterolt 0.75 day 12 days 52 days 1 day 7 days 30 days 1 day 7 days 30 days 1 day 7 days 30 days 1.2 0.06 7.6 0.29 45 0.26 1.6 0.04 0.48
