Brain Cholesterol. XII. The Incorporation of 1-14C-Acetate into Baboon Sterol JON J. KABARA, Department of Chemistry, University of Detroit, Detroit, Michigan, and N. T. WERTHESSEN, Institute of Health Science, Brown University, Providence, Rhode Island ABSTRACT
frozen and stored in an ice chest. Free and esterified sterols (after hydrolysis) were isolated, measured, and assayed by the method previously reported (7). Radiochemical purity of the isolated free cholesterol was established (7,8). The resulting data are reported in terms o f specific activity, ( D / M / m g ) and activity p e r gram tissue ( D / M / g tissue). Because of uncertainties in the sterol ester measurements, concentration of ester in the individual tissues~ is not reported at this time.
The incorporation of l-z4C-Acetate into tissue cholesterol of the baboon was measured. Using this indicator gray matter of the cerebrum indicated greater metabolic activity than did white matter. Other tissues besides neural tissue were examined. The peak of radioactivity occurred between 3 and 4 hr. The highest incorporation of radioactivity was measured in the adrenal gland. Liver, spleen and kidney values were of intermediate order.
RESULTS INTRODUCTION
ONTRARY TO EARLIER CONCLUSIONS of others (1), our investigations in the mouse (2-6) indicated that active lipid metabolism continues in the brain during adult life. Because of these preliminary conclusions, and possible relationship to cerebral function, further investigations on brain cholesterol were made. The following is a continuation of the study of cholesterol biosynthesis in primates. The present investigation brings us closer to understanding similar processes in man by reporting on acetate incorporation into brain tissue cholesterol of the baboon.
C
EXPERIMENTAL
Five mate African baboons weighing between 21 and 25 kg., representing ages approximately 4-6 years, were used for these studies. Live animals were imported from Kenya, East Africa, and acclimated for 3 to 4 months before use. Diet, during captivity, consisted of Purina Monkey Chow supplemented with fresh fruit and whole corn. Anesthetized animals were used in these experiments. Both Nembutol (5 m g / k g ) and phencyclidine (Sernylan) (2 m g / k g ) were used. The unconscious animals were injected intravenously with 100 ~ g / k g of 1-~4C-Acetate and then killed at five time intervals: 171, 184, 188, 248, and 294 min. F o r analysis, samples of cerebrum white, cerebrum gray, liver, spleen, kidney and adrenals were surgically removed, then washed,
Values for cholesterol specific activity ( D / M / m g ) , after 1-14C-Acetate injection, are affected by many factors; of those that can b e readily ascertained, one is the dilution of specific activity by sterol concentration in each individual organ. To measure the effect of sterol concentration, radioactivity was calculated on a gram tissue basis ( D / M / g tissue). Results of both calculations are presented in Table I. The specific activity of free cholesterol was lowest in cerebrum (white and gray) and highest in the adrenal gland. Liver, spleen, and kidney values were of intermediate order. D / M / g tissue values from these same tissues indicated a similar relative order of incorporation. The two large divisions of brain matter, white and gray, vary in biosynthetic capacity as measured by 1-14C-Acetate incorporation. By this measure, gray matter of the cerebrum seemed metabolically more active than did the white matter. These differences were evident in comparisons of both free specific activity and activity per gram tissue values. It should be noted that the specific activity values for ester cholesterol are higher than for free cholesterol. This is true for all tissues under investigation. Because the hydrolysis of the esterified fraction was thought not to be quantitative in these particular experiments, only ester-specific activity values are reported at this time. Blood values were not available for these studies.
494
495
I N C O R P O R A T I O N OF 1-14C-ACETATE I N T O B A B O O N STEROL TABLE I I n c o r p o r a t i o n o f 1-14C-Acetate into Tissue Cholesterol of the B a b o o n Ester D ) I~-) mg
m g / g Tissue
Free D/M/mg
16.7 17.2 22.0 13.7 18.5
27 75 26 66 40
450 1285 570 905 740
356 350 429 264 157
11.7 12.7 13.7 8.75 I3.5
103 97 55 308 93
1200 1230 755 2695 1250
2600 1000 1900 2238 1615
Liver
1.35 1.04 0.99 1.18 1.34
458 2148 5604 1450 4291
618 2234 5565 1711 5750
15733 28944 31257 76519 22116
Spleen
1.13 1.48 1.07 1.73 1.08
912 3677 707 5551 203
1031 5442 756 9603 219
11326 8675 6855 13465 7326
Kidney
2.00 1.27 2.08 2.06 2.10
101 268 121 994 115
202 340 252 2048 242
55238 22128 50850 15446 23075
Adrenals
1.84 1.68 2,24 2.27 2.29
4713 8746 3346 5065 2429
8672 14693 7495 11498 5562
19743 12333 11486 12297 7713
Organ Cerebrum White 2:51 h r 3:04 h r 3:08 h r 4:08 h r 4:54 h r ~Jray
D / M / g Tissue
DISCUSSION
More knowledge of the basic biochemical mechanism taking place in neural tissue is needed before we can understand the mechanism of brain function. Studies dealing with the neurochemistry of higher primates, especially man, are extremely scarce. As an approach to this problem, we have investigated the biosynthesis of cerebral cholesterol in the baboon. There are comparatively few recorded studies of the biosynthesis of cholesterol in this primate. Savage et al. (9) reported on the biosynthesis of cholesterol from 2-14C-Sodium pyruvate in normal and diabetic baboons. These workers reported only total radioactivity in various organs at a given time after isotope administration. Kritchevsky et al. (10,11) studied the biosynthesis of cholesterol with labeled mevalonic acid as the precursor. The peak specific activity of the serum-free cholesterol was attained between 4 and 10 hr; peak specific activity of serum cholesterol ester was reached
between 9 and 26 hr. No data on cerebral biosynthesis were presented. This paper represents our initial research studies on brain colesterol in the baboon. Certain weaknesses of our approach ale evident: absence of blood values and the nonquantitative recovery of esterified cholesterol. Despite these shortcomings, the data proved of value as a basis for future studies. Because of the difficulty in obtaining large primates and their concurrent cost, only 5 animals were used for this experiment; data from these animals did not indicate peak time of labeling. It was expected that in such a small series, individual biological variations might be large. In collecting the data, it was noted that the animal killed at 4:08 hr exhibited variance from the other 4 baboons. This was true for all tissue values of this animal. If we exclude this animal from our consideration, a better and probably accurate picture seems to emerge. The exclusion of the single aberrant animal indicates that the peak of tissue labeling took place between 3 and 4 hr. Of the tissues examined, biosynthetic capacity as measured by 1-14C-acetate incorporation, showed the adrenal gland to be most active, followed by liver, spleen, and kidney. Brain cholesterol metabolism was tow but significant in the adult baboon. This agreed with previous findings in mice (2-6) using 1-14C-Acetate. A better indicator of brain synthetic capabilities in regard to sterol synthesis would be U-14Cglucose (5). Of particular interest and confirming our past results in mice (6) was the high specific activity of cholesterol ester in contrast to free sterol. These ester values were interpreted to mean that equilibration of newly synthesized free cholesterol with total organ cholesterol were slower than the esterification reaction. This may be interpreted as evidence for the existence of at least two free sterol pools. Of additional interest was the difference in metabolism between cerebral gray and white tissue. In this baboon experiment with 1-14CAcetate as the precursor, two facts emerge about brain cholesterol metabolism. First, higher activity was measured for cholesterol extracted from gray cerebrum than white cerebrum matter. Second, in contrast to gray cerebrum matter and other tissues, the ester activity of cholesterol assayed from white cerebrum matter was higher on a D / M / g tissue than D / M / m g basis. This is the result of the greater amount (mass) of ester found in white material as compared to gray. LIPIDS,
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JON J. KABARA AND N. T. WERTHESSEN
I n a g r e e m e n t w i t h o u r o w n in v i v o findings, w e r e t h e r e s u l t s r e p o r t e d b y D a v i s o n a n d cow o r k e r s ( 1 2 ) in r a b b i t s . U t i l i z i n g l a b e l e d c h o lesterol, t h e y f o u n d t h e t u r n o v e r o f s t e r o l t o b e g r e a t e r in g r a y c e r e b r u m t h a n in w h i t e . I n contrast, Korey (13) found that using labeled m e v a l o n a t e o r g l u c o s e in v i t r o , w h i t e c e r e b r u m m a t t e r ( r a t ) w a s m o r e a c t i v e in lipid s y n t h e s i s than the gray of cerebral cortex. As measured by present methodology, the p r o b l e m of relative metabolic activity b e t w e e n g r a y a n d w h i t e c e r e b r u m m a t t e r is still to b e solved, Final conclusions concerning brain cholesterol metabolism must wait until more i n - d e p t h e x p e r i m e n t s a r e m a d e in a v a r i e t y o f species and with different p r e c u r s o r s u n d e r cont r o l l e d c o n d i t i o n s . M o r e definitive e x p e r i m e n t s r e g a r d i n g r e g i o n a l c h o l e s t e r o l b i o s y n t h e s i s in t h e brain of b a b o o n s are presently being conducted in this l a b o r a t o r y b y t h e s i m u l t a n e o u s u s e o f 2 - ~ H - A c e t a t e , 1-14C-Acetate, o r u - r 4 C - g l u c o s e . ACKNOWLEDGMENT
Supported (in part) by Public Health Service Research Grant No. NB-02235 from the National Institute of Neurological Diseases and Blindness and Public Health Service Research Grant No. HD-02191 from the National Institute of Child Health.
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