Cholesterol Ester Hydrolase Activity Human Cerebrospinal Fluid N.
Center, University of Catifornie, Langley Znstitste, So~aonm State Hospital, Eldridge, California 95431
This study demonstrates for the first time the presence of cholesterol ester hydrolase (EC 22.214.171.124) in human cerebrospinal fluid. The pH optimum of cholesterol ester hydrolase in that fluid is approximately 6.0. The activity of the enzyme is optimal when the substrate is introduced either as an acetone solution or as a suspension in Triton X-100. Cholesterol ester hydrolase activity was not detected in human serum. It is suggested that the cholesterol ester hydrolase in cerebrospinal fluid is derived from brain and that the assay of this enzyme in cerebrospinal fluid may be of diagnostic values in various demyelinating diseases.
INTRODUCTION The concentration of cholesterol esters in brain is high just prior to the onset of active myelination, but declines during myelination and thereafter (1, 4, 12, 21). N ormal adult brain contains only minute amounts of cholestero1 esters (2, 3, 6, 21), however, there is an increase in esterified cholesterol in brain in various pathological demyelinating conditions (5, 19, 20, 22). The occurrence of the cholesterol ester-synthesizing enzyme and three different cholesterol ester hydrolases in rat brain was reported recently (7, 8). A n increase in the concentration of one of the three hydrolases in brain, that has a pH optimum of 7.2, coincides with the period of rapid myelination (9). The activity of this enzyme is low in the brains of myelin-deficient mutant mice (10) and in degenerating hen sciatic nerve (14). Moreover, the enzyme is present in purified myelin (8). Abbreviation : CSF-cerebrospinal fluid. 1 This investigation was supported by National NS-11670 and HD-01823. 68 0014-4886/78/0581-0068$02.00/O Copyright 0 1978 by Academic Press.
These facts suggest that cholesterol esters, and the cholesterol ester-metabolizing enzyme, namely, cholesterol ester hydrolase, may play a role in the process of myelination and demyelination. Cerebrospinal fluid (CSF) that bathes the brain is a convenient material for assessing homeostasis of the brain tissue. Human CSF contains cholesterol, and approximately 50% of this cholesterol is in esterified form (15). According to recent reports the concentration of esterified cholesterol is decreased in the CSF from patients with phenylketonuria (16) and increased in the CSF from patients with multiple sclerosis (17). This suggests a possible correlation between the concentrations of esterified cholesterol in CSF and myelin abnormality. Aside from the report of Illingworth and Glover (13) that lecithin cholesterol acyltransferase is present in human CSF, there is a paucity of information on cholesterol ester-metabolizing enzymes in human CSF. The present communication reports for the first time the presence of cholesterol ester hydrolase activity in human CSF. Because cholesterol ester hydrolase was not detected in the human serum the enzyme in CSF most likely comes from the brain. MATERIALS
Cholesterol [‘“C] oleate was purchased from New England Nuclear Corp., Boston, Massachusetts. Unlabeled cholesterol oleate was obtained from Applied Science Laboratories. Human CSF samples were obtained from local hospitals and kept frozen at -80°C until used. After thawing, the CSF samples were pooled and centrifuged 10 min at 100 g to remove cellular material. Standard assay mixture contained 100,000 cpm cholesterol [14C]oleate (sp act 800 cpm/nmol). The substrate suspended in 0.2 ml 1% Triton X-100 in methanol was first added to the tubes. After evaporating the methanol, 0.8 ml 0.15 M citrate phosphate buffer (pH 6.0) and 0.2 ml CSF were added. The tubes containing the reaction mixture were flushed with nitrogen, capped with rubber stoppers, and incubated 3 h at 37°C with gentle shaking. Incubations carried out without CSF served as controls. The reaction was terminated by adding 5 ml 2 : 1 (v/v) chloroform : methanol mixture. Cholesterol, oleic acid, and cholesterol oleate, 100 pg each, were added as carriers and the lipids were extracted essentially as described by Folch-Pi et al. (11). Free fatty acids (oleic acid) and free cholesterol were separated from cholesterol oleate by thin-layer chromatography. A mixture of hexane, diethyl ether, and acetic acid (80 : 20 : 1, v / v / v ) was used as a developing solvent. Spots corresponding to cholesterol oleate and oleic acids were scraped from the plates and transferred to scintillation vials for assay of radioactivity. The amount (nanomoles) of cholesterol ester hydrolyzed was calculated from the difference between radioactivity recovered in oleic acid
FIG. 1. Effect of pH of incubation mixture on cholesterol ester hydrolase activity of human CSF. The incubation mixture contained 100,000 cpm cholesterol [“Cloleate (sp act 800 cpmjnmol), 2 ~1 Triton X-100, 0.2 ml CSF (pooled sample), and 0.8 ml either 0.15 M citrate phosphate buffer (for pH range 4.5 to 6.0) or 0.15 M potassium phosphate buffer (for pH range 6.5 to 7.5). Other details of experimental procedures are described in the text. Each point represents the average of duplicate determinations with variation less than 5%.
in experimental and control samples and the specific activity strate used. RESULTS
of the sub-
Because brain tissue is known to contain three different cholesterol hydrolases, each with a different $H optimum, we first determined the effect of varying pH on cholesterol ester hydrolase activity of human CSF.
FIG. 2. Effect of incubation time and substramte concentration on cholesterol ester hydrolase activity of human CSF. A-The incubation mixture contained 100,000 cpm cholesterol [14C]oleate (sp act 800 cpm/nmol), 2~1 Triton X-100, 0.2 ml CSF (pooled sample), and 0.8 ml 0.15 M citrate phosphate buffer (pH 6.0). Incubations were carried out for various time intervals. B-The incubation mixture contained various amounts of cholesterol [Yloleate, 2 ~1 Triton X-100, 0.2 CSF, and 0.8 ml 0.15 M citrate phosphate buffer (PH 6.0). The incubations were carried out for 3 h. Each ‘point is the average of closely agreeing (variation less than 5%) duplicate determinations.
of the Amounts Enzyme
CSF CSF CSF CSF Boiled Serum
on Cholesterol - .-____
Cholesterol ester hydrolyzed (1111101/3 h)
(0.1 ml) (0.2 ml) (0.4 ml) (0.6 ml) CSF (0.2 ml) (0.1 ml)
0.160 0.343 0.747 0.969 0.023 0.026
” The incubation conditions were the same as described amount of CSF added to the incubation mixture was varied. duplicate determinations with variation less than 5(;1,.
in Fig. 1 except that Each value is an average
Results in Fig. 1 show that cholesterol ester hydrolase activity in a pooled sample of human CSF is optimal approximately at pH 6.0. Two other pooled samples of CSF when tested showed that the hydrolase activity varied from sample to sample, but the activity was optimal at pH 6.0. Results in Fig. 2 show that the reaction rate is linear to 4 h and is optimal at substrate concentrations of 10 to 15 nmol. The effects of various solubilizing agents and the amount of CSF on cholesterol esterase activity were then examined. Results in Table 1 show that increasing the amount of CSF increased the cholesterol esterase activity and that cholesterol ester hydrolase activity was lost by boiling. The results also show that the cholesterol esterase activity is not present in human serum. The cholesterol TABLE Effect
None Acetone (0.05 ml) Sodium taurocholate Triton X-100 (0.27,) Tween 20 (0.2’55) Sodium deoxycholate
Cholesterol ester hydrolyzed (nmol/0.2 ml CSF/3
0.012 0.160 0.123 0.143 0.099 0.075
a Cholesterol [KJoleate used as substrate was suspended in the respective detergents; other incubation conditions are described in the text. Pooled CSF (0.2 ml) was used as the enzyme source. Each value represents the average of duplicate determinations with variation less than 5y0.
esterase activity of CSF was maximal when substrate was either introduced as acetone solution or suspended in Triton X-100 and the activity of the enzyme was about half-maximal in the presence of either bile acids (sodium taurocholate or sodium deoxycholate) or Tween 20 (Table 2). The components of CSF originate from central nervous system and serum. The data presented here clearly demonstrate that cholesterol ester hydrolase is present in human CSF. As the activity of this enzyme was not detected in human serum the enzyme in CSF is most likely derived from brain. The concentration of cholesterol ester in brain is increased in various demyelinating conditions (5, 19, 20, 22). In the case of multiple sclerosis this increase was found to be reflected in CSF (17). It was shown that in other cholesterol ester storage diseases a severe deficiency of acid cholesterol ester hydrolase occurs in other tissues (18). It is likely, therefore, that the increase in brain cholesterol ester concentrations is associated with a cerebral deficiency in the hydrolase activity, and that this later deficiency may in turn be reflected in reduced hydrolase activities in CSF. It would thus be of interest to determine whether or not a change in the activity of this enzyme in CSF can be correlated with myelin abnormalities in the central nervous system. Such a correlation may prove to be of diagnostic value in demyelinating diseases. The report of Illingworth and Glover that cholesterol esterase activity is absent from human CSF ( 13) is inconsistent with our present finding. The failure of those investigators to detect the cholesterol esterase activity in CSF may be attributed to the following reasons. First, the pH of their incubation medium was 7.2 ; our results (Fig. 1) show that the cholesterol esterase of human CSF is almost inactive at this PH. Second, sodium deoxycholate was used as the detergent in their study ;‘according to our data, the activity of the hydrolase in CSF is very low in the presence of sodium deoxycholate. Finally, it would be difficult to detect cholesterol ester hydrolase activity with the low specific activity of the substrate used in their study. REFERENCES C. W. M., AND A. N. DAVISON. 1959. The occurrence of esterified cholesterol in the developing nervous system. .I. Neurockem. 4 : 282-289. 2. ALLING, C., AND L. SVENNERHOLM. 1969.Concentrationand fatty acid composition of cholesterylestersof normalhumanbrain.J. Neurochem. 16: 751-759. 3. CLARENBERG, R., I. L. CHAIKOFF, AND M. D. MORRIS. 1963.Incorporation of injected cholesterolinto the myelinatingbrain of the 17-day-oldrabbit. J. Neurothem. 10: 135-143. 4. DAVISON, A. N. 1965.Brain sterolmetabolism. A&V. Lipid Res. 3: 171-196. 5. DAVISON, A. N., AND W. WAJDA. 1962.Analysis of lipids from fresh and preservedadult humanbrain. Biochem. J. 82 : 113-117. 6. Era, Y., AND K. SUZUKI. 1972.Cholesterolestersin developingrat brain: Concentrationandfat,@acid composition. J. Neurochem. 19: 109-115. 1. ADAMS,
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