Comparison of the inhibition by methylmercury and triethyllead of galactolipid accumulation in rat brain

ENVIRONMENTAL

RESEARCH

Comparison Triethyllead

23, 282-291 (1980)

of the Inhibition by Methylmercury and of Galactolipid Accumulation in Rat Brain I. K. GRUNDT*

AND N. M. NEsxovrct

*Laboratory of Clinical Biochemistry, University of Bergen, School of Medicine, N-5016 Haukeland Sykehus, Bergen, Norway, and Kentre National de la Research Scientifique, Centre de Neurochimie, II, Rue Humann. 67085 Strasbourg, France Received December 27, 1979 Methylmercury chloride (MeHg) and triethyllead chloride (Et,Pb) interfere with the myelin deposition in young rats. In order to establish how myelin synthesis and development are affected by these neurotoxins, we have investigated the synthesis of myelin galactolipids using two different systems: (1) slices of cerebellum from 2-week-old rats, (2) enzyme preparations from rat brain. MeHg (50 PM) or Et,Pb (5 PM) gave maximum inhibition of the uptake of ?SO, by sulfatides (63%). The [3H]serine uptake by cerebrosides with normal unsubstituted fatty acids (NFA) was inhibited up to 45% at 70 and 7 FM of MeHg and Et,Pb, respectively, after incubation of the slices for 16 hr in an Eagle basal medium at 37°C. At the same concentrations the inhibition of [3H]serine uptake in cerebrosides with cu-hydroxy fatty acids (HFA) was about 35%. MeHg (So-100 PM) inhibited UDPgalactose:ceramide galactosyltransferase (CGalT) and phosphoadenosine-5’-phosphosulfate:cerebroside sulfotransferase (CST) activities up to 50%, while the inhibition with Et,Pb was about 30% at 2.5 mM for the enzymes.

INTRODUCTION

Previous studies have demonstrated that the environmental poisons, methylmercury (MeHg) and triethyllead (Et,Pb), hamper the cerebral myelinating processes in young rat (Grundt et al., 1980; Konat and Clausen, 1974, 1976; Konat et al., 1976). As sulfatides and cerebrosides are characteristic myelin components, the biosynthesis of these glycolipids in brain tissue has frequently been used as a measure of myelination and demyelination (Silberberg et al., 1972; Fry et al., 1974; Hayes and Jungalwala, 1976). Cerebrosides with nonsubstituted fatty acids (NFA cerebrosides) or with a-hydroxy fatty acids (HFA cerebrosides) are wellestablished markers of myelin and their content in the brain increases with increasing myelin deposition. We previously found in MeHg-intoxicated rats that the level of cr-hydroxy fatty acids in the myelin cerebrosides was lower than that in normal rats (Grundt, 1980). On the other hand the accumulation of 3H-labeled NFA cerebrosides was more inhibited than the accumulation of labeled HFA cerebrosides, when [3H]serine was added to the nerve cell culture grown in the presence of Et,Pb (Grundt et al., submitted for publication). Also in these experiments with Et,Pb the sulfatide synthesis was more affected by the poison than the cerebroside synthesis (Grundt et al., submitted for publication). In studies of ’ Abbreviations used: MeHg, methylmercury chloride; Et,Pb, Triethyllead chloride; NFA cerebrosides, cerebrosides with nonsubstituted fatty acids; HFA cerebrosides, cerebrosides with (Yhydroxy fatty acids; CGalT, UDPgalactose:ceramide galactosyltransferase (EC 2.4.1.45); CST, phosphoadenosine-5’-phosphosulfate:cerebroside sulfotransferase (EC 2.8.2.11). 282 0013-9351/80/060282-10$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

MeHg

AND

EtaPb,

EFFECTS

ON

BRAIN

LIPIDS

283

sulfatide labeling from [35S]sulfate, using cerebellum slices of rat, we observed that Et,Pb was more potent as an inhibitor of the sulfatide biosynthesis than MeHg (Grundt et al., 1974). We have now compared the effects of MeHg and Et,Pb on the biosynthesis of sulfatides and cerebrosides in developing rat brain, using two different systems. Using cerebellum slices of young rat, we compared the effects of MeHg and Et,Pb on [35S]sulfate incorporation into sulfatides, and on the uptake of [3H]serine into cerebrosides. To get more information about possible sites of Et,Pb and MeHg action, this study was extended to the investigation of their effects on the two enzymes involved in the biosynthesis of glycolipids, i.e., UDPgalactose:ceramide galactosyltransferase (EC 2.4.1.45, CGalT) and phosphoadenosine-5’-phosphosulfate:cerebroside sulfotransferase (EC 2.8.2.11, CST). MATERIALS

AND

METHODS

Triethyllead chloride was kindly supplied as a gift from the Associated Octel Company Ltd., London. Methylmercury chloride was from K & K Laboratories. L-r3-3H]serine, 17 Ci/mmole, and [35S]Na.$0,, 5 Ci/mg S, were purchased from The Radiochemical Centre, Amersham, England. The sources of UDP [14q galactose, HFA cerebroside; of [35S]PAPS and sulfatide, have been given before (Neskovic et al., 1974; Sarlieve et al., 1974). Unless otherwise stated, the chemicals were of analytical grade from Merck, Darmstadt, West Germany. Incubation Procedure for Tissue Slices A system of tissue slices, previously described (Grundt et al., 1974), was used to study the incorporation of radioactive precursors into galactolipids. Cerebellum (about 0.1 g) from lCday-old rats of the strain BD IX, was cut into 6 slices. The slices were incubated with 0.1 mCi of a radioactive precursor in 4 ml of a nutrition medium for 16 hr at 37°C. The radioactive precursor in the study of sulfatide labeling was [35S]Na,S04; in the study of cerebroside labeling [3H]serine was the precursor. The nutrition medium was Eagle basal medium (MEM, Gibco Bio-Cult, Scotland), supplemented with 20% fetal calf serum, (Gibco Bio-Cult), and the incubations were performed in Falcon tissue culture flasks with 25 cm2 growth area. The effect of MeHg and Et,Pb on the labeling of cerebrosides and sulfatides was studied after addition of different concentrations of MeHgCl up to 70 PM and of Et,PbCl up to 7 pM to the incubation medium, immediately before incubation. At the end of the incubation period the samples were transferred into centrifuge tubes in an ice bath and the medium was removed by centrifugation (4°C 2440g in a Beckman Model J-21 centrifuge). The slices were then washed three times with 10 ml of 0.9% ice-cold NaCl and centrifuged as above. Separation

of Lipids

and Radioactivity

Determination

The cerebellum slices were dispersed in 0.3 ml of water by shaking in a Vortex mixer. The lipid extract was then prepared in chloroform:methanol (2: l), filtered to remove the precipitated proteins, and washed to eliminate the water-soluble contaminants (Folch-Pi et al., 1957). When [35S]sulfate was the precursor, the radioactivity of the total lipid extract was counted. When [3H]serine was incorporated into the slices, NFA cerebrosides and HFA cerebrosides were first

284

GRUNDT

AND

NESKOVIC

separated from the lipid extract. The lipid extract was subjected to a mild hydrolysis according to Dittmer and Wells (1969). NFA cerebrosides and HFA cerebrosides were separated on precoated silica gel plates (Kieselgel 60, Merck, Darmstadt, West Germany) in a system described by Morel1 and Radin. (1969) The plates were developed twice in chloroform:methanol:water (144:25:2.8) and dried for 15 min at 60°C between the runs. NFA cerebrosides and HFA cerebrosides in a natural mixture from calf brain white matter were used as reference lipids. The lipids were visualized by spraying the plates with 2% iodine in petroleum ether (60-80°C). The iodine was allowed to evaporate and the spots of NFA cerebrosides and HFA cerebrosides were scraped from the plates into the scintillation vials. Ten milliliters of Stint-Hei 3 (Koch-Light Laboratories Ltd., England) were added to samples of dried lipids (for sulfatides) or silica gel (for cerebrosides). Counting was done in the H-3 channel, and for 35S in the C-14 channel of a Packard Tri-Carb liquid scintillation counter, Model 2409, with external correction. Estimation

of Hg and Pb in Cerebellum

Slices

Mercury was determined in the protein precipitate obtained after extraction with chloroform:methanol(2: 1). The proteins were dissolved in a mixture of equal volumes of 45% NaOH, 1% cystein, and 0.9% NaCl, 30 min at 6O”C, and Hg was measured by atomic absorption as described earlier (Magos and Clarkson, 1972). Lead was measured from the dispersion prepared from the cerebellum slices as described above. The samples were lyophilized, dissolved in HNO,, and Pb was determined by atomic absorption in a Perkin- Elmer 372 instrument. Protein was measured according to Lowry et al. (1951). Effect of MeHg

and EtJ’b

on Enzyme Activities

The activities of CGalT and CST were determined as previously described (Neskovic et al., 1974; Sarlieve et al., 1974). As the enzyme source a 10% (w/v) whole brain homogenate in 50 mM K-phosphate, 50% (v/v) glycerol (pH 7.6) was used. In some experiments the partially purified CGalT (fraction V, Neskovic et al., 1976) was used. The composition of the incubation mixtures is given in the legend to corresponding figures. To study the effect of MeHg and Et,Pb on the enzyme activities the procedure was as follows. The enzyme preparation was added to the assay mixture from which the radioactive precursor was omitted, an appropriate amount of the poison was added as solution in water, and the mixture was incubated for 15 min at 0°C. The enzymatic reaction was then commenced by adding the radioactive precursor and incubation proceeded for 15 min at 27°C for CGalT activity or 30 min at 36°C for CST activity. The labeled lipids were extracted and radioactivity was determined (Neskovic et al., 1974; Sarlieve et al., 1974). RESULTS

Both MeHg and Et,Pb had an inhibitory effect on the incorporation of radioactive precursors into the different glycolipids, but the poisons were active at different concentrations. Et3Pb gave a similar effect as MeHg at about 10 times lower

MeHg

AND

Et,Pb,

EFFECTS

ON

BRAIN

LIPIDS

285

concentration (Table 1). When compared with control groups, both poisons inhibited the incorporation of [35S]sulfate about 60-65% within a concentration range from 12.5 to 70 PM or 1.25 to 7 PM of MeHg or Et,Pb, respectively. The incorporation of [3H]serine into cerebrosides was gradually decreasing with increasing concentrations up to 70 PM MeHg or 7 PM Et,Pb. The labeling of NFA cerebrosides was decreased to about 45% of control values and that of HFA cerebrosides to about 35%. In the control samples less [3H]serine was incorporated in the NFA cerebrosides than in HFA cerebrosides. These initial observations were extended by a study of the Hg and Pb uptake into the cerebellum slices. Table 2 shows the amount of Hg and Pb in the cerebellum slices after incubation of the slices in a nutrient medium, which contained MeHgCl or Et,PbCl. The measurements were made after incubation with two different concentrations of the alkylmetal compound. All the Hg added as MeHgCl to the nutrient medium was recovered in the cerebellum slices, whereas Pb was equilibrated between medium and tissue, and about 50% of Pb in the medium was taken up by the sliced cerebellum. The effect of MeHg and Et,Pb on the enzymatic synthesis of HFA cerebrosides and sulfatides, was studied by adding different concentrations of the poisons to the assay mixtures of CGalT and CST and incubating for 15 min at 0°C before the enzymatic reaction started. Preliminary experiments have shown that after 15 min maximum effects of both poisons were obtained. The effect of increasing concentrations of MeHg on CGalT activity is presented in Fig. 1. When the whole brain homogenate was used as the enzyme source CGalT activity was virtually unchanged up to about 50 PM MeHg, then dropped to approximately 50% of the control value of 100 PM MeHg. At higher concentrations of MeHg the enzyme activity decreased more slowly and attained about 17% of the original activity at 250 PM MeHg. When partially purified CGalT was used, a rapid loss of the enzyme activity occurred so that no lag phase could be observed, and the inhibition attained about 88% at 50 pM MeHg. However, if a heat-inactivated brain homogenate was added to the purified enzyme so that total protein content of the incubation mixture was the same as that in experiments with brain homogenate, the observed decrease of the enzyme activity fitted well with the inhibition curve for brain homogenate. The inhibitory effect of Et,Pb on CGalT activity was observed at much higher concentrations (Fig. 1). CGalT activity of brain homogenate was almost unchanged up to about 1 mM Et,Pb, then decreased gradually to approximately 45% of the original value at 5 mM Et,Pb. The CGalT activity in the purified enzyme preparation was apparently more sensitive to the inhibitory action of Et,Pb; a 20% inhibition was observed at 0.1 mM Et,Pb and about 50% inhibition resulted in the presence of 1 mM Et,Pb. In the presence of the heat-inactivated protein, the values were close to those obtained for brain homogenate. Compared to CGalT, the effect of MeHg on the CST activity in a brain homogenate was more pronounced and almost 70% inhibition was obtained at 50 PM MeHg (Fig. 2). Et,Pb produced an inhibitory effect on CST at much higher concentrations than MeHg. However, compared to CGalT, the inhibition of CST was

2288 k 550(9) 1444 2 299(6) 497 2 92(6)

Control

3105 2 439(9) 1303 2 239(3) 450 -t 141(2)

12.5 /.LM 2542 k 664(15) 970 f 128(3) 339 2 90(2)

MeHgCl 25 /LM 1112 k 382(4) 861 2 191(2) 328 k 15(2)

50 /..bM

1.25 /AM 3052 2 23(2) 1194 ” 363(2) 432 2 63(3)

incorporated

1350 k 89(2) 962 f 259(3) 428 + 63(3)

Et,PbCl 2.5 /.LM

VITRO

1131 f 22(2) 671 ? 7(2) 285 2 68(3)

5 PM

INTO NFA- ANDHFA-CEREBROSIDESOFRATCEREBELLUMIN

Note. Results are given as cpm incorporated per cerebellum. Values are means + SD for the number of experiments given in brackets. Cerebellum from 14-day-old rat (0.1 g) was cut into slices and incubated for 16 hr at 37°C in 4 ml of Eagle’s basal medium supplemented with 20% fetal calf serum, with 0.1 mCi of [YS]sulfate or [3H]serine, and with MeHgCl or Et,PbCI at different concentrations. The inhibition of the uptake of radioactivity into sulfatides and NFA cerebrosides at 50 pM MeHgCl and at 5 pM Et,PbCl was significant (P < 0.05); the inhibition of the radioactive uptake into HFA cerebrosides was not significant.

Sulfatides NFA cerebrosides HFA cerebrosides

Glycolipid

1

Radioactivity

TABLE INHIB~TIONOFTHEUPTAKEOF[~~S]SULFATEINTOSULFATIDESANDOF~H]SERINE

e: E

mz

; > 2

z

0

M&g

UPTAKE

BY

CEREBELLUM

AND

SLICES

Et,Pb, EFFECTS

OF

Added to medium (/a)

ON

BRAIN

TABLE 2 MeHgCl AND Et,PbCl

FROM

THE

Uptake from medium 649

Hg

NUTRIENT

MEDIUM

Percentage uptake

10.3 k 2.8 20.9 k 9.9

10 20

287

LIPIDS

103 109

Pb 0.47 t 0.045 2.25 k 0.22

1.04 4.14

42.3 54.3

Note. 0.1 g sliced cerebellum was incubated 16 hr 37°C with 4 ml of Eagle’s basal medium supplemented with 20% fetal calf serum, together with MeHgCl or Et,PbCl at different concentrations. The concentration of Hg and Pb in the slices was determined after the incubation. The amount of Hg and Pb taken up from the medium is given as means &cerebellum 2 SD.

observed at lower concentrations of Et,Pb, and at 1 mM Et,Pb the inhibition was almost 50%. To compare the effects of MeHg and Et,Pb on glycolipid biosynthesis in the two systems, the MeHg and Et,Pb concentrations, which produced similar inhibitory

0100

500

50

MM

1000

FM

Hg

Pb

250

100

2500

5000

FIG. I. Inhibition by MeHg and Et,Pb of CGalT. The incubation mixture contained (in 0.25 ml final volume); a-hydroxy fatty acid ceramide, 0.1 pmole; lecithin, 0.077 pmole; Triton X-100, 0.5 mg; UDP [‘“Clgalactose, 0.01 pmole (64,000 cpm); MgCl,, 1 pmole; Tris-HCI buffer (pH 8.0), 10 pmole; enzyme preparation. The incubation time was 15 min at 27°C. (Neskovic et al.. 1974). Enzyme preparations were preincubated with varying concentrations of MeHg and Et,Pb as described under Material and Methods. The following enzyme preparations were used: (1) purified enzyme (Fraction V, Neskovic et al. 1976), 8 pg of protein (0); (2) as in (1) plus heat-inactivated brain homogenate, 200 pg protein (0); (3) brain homogenate, 200 fig protein (x). For other details see Material and Methods.

288

GRUNDT

I, 01

AND NESKOVIC

, 05

lmMPb2

25

5

FIG. 2. Inhibition by MeHg and Et,Pb of CST activity in a 10% homogenate of rat brain. The incubation mixture contained (in 0.5 ml final volume) [YSJPAP, lOO,OOO- 120,000 cpm; cerebroside, 0.12 pmole; Brij 96, 4 mg; MgC12, 20 pmole; ATP, 5 pmole; imidazole buffer (pH 7.0), 50 pmole; enzyme preparation. The incubation time was 30 min at47”C. (Sarlitve et al., 1973). Enzyme preparation (brain homogenate, 200 pg of protein) was treated with MeHg and Et,Pb as described in the legend to Fig. 1.

effects on HFA enzyme assays similar effects magnitude. On

cerebroside and sulfatide biosynthesis in cerebellum slices and in are listed in Table 3. The concentrations of MeHg which produced in brain slices and in enzyme assay were of the same order of the contrary, the concentrations of Et,Pb, which produced compaTABLE

3

COMPARJ~~N OF MeHg AND Et,Pb CONCENTRATIONS WHICH PRODUCE SIMILAR INHIBITORY EFFECTS ON CEREBROSIDE AND SULFATIDE BIOSYNTHESIS IN EXPERIMENTS WITH CEREBELLUM SLICES, AND IN ASSAYS OF ENZYME ACTIVITIES IN TOTAL HOMOGENATE OF RAT BRAIN

Inhibitor/protein (nmol/mg)

Inhibition

(%)

Glycolipid formed (enzyme)

Inhibitor

Cerebellum slices

Enzyme assay

Cerebellum slices

Enzyme assay

HFA cerebrosides (CGalT)

MeHg Et,Pb

13.0 0.87

94 3125

32 37

30 35

Sulfatides (CST)

MeHg EtsPb

18.9 0.87

72 3125

63 62

50 50

Note. Results from Table 1 and Fig. 1 and 2, describing effects from MeHg and Et,Pb on the synthesis of HFA cerebrosides and sulfatides in experiments with tissue slices (Table 1) and in enzyme assays (Figs. 1 and 2), are for a comparison recalculated to give concentrations of MeHg and Et,Pb in nmoUmg protein in the assay medium, which give similar inhibitory effect.

MeHg

AND

Et,Pb,

EFFECTS

ON

BRAIN

LIPIDS

289

rable effects on sulfatide (50-60% inhibition) and on cerebroside biosynthesis (30-32% inhibition) in the two systems studied, were several thousand times lower in experiments with cerebellum slices. DISCUSSION

In agreement with earlier studies of sulfatide metabolism in brain tissue exposed to toxic metals (Grundt er al., 1974), the present work shows that MeHg and Et,Pb markedly reduce the rate of myelin glycolipid accumulation in rat brain. So far little is known about the neurotoxic mechanism of these poisons and the biochemical processes underlying the myelination defect they produce. It is therefore difficult to propose specific mechanisms by which Et,Pb and MeHg can affect glycolipid biosynthesis. In view of the results obtained in the present and from previous studies, different possibilities can be envisaged. The decrease of the cerebroside and sulfatide accumulation in the exposed brain slices may result from a more general effect of the poison on the protein biosynthesis as described for MeHg (Yoshino er al., 1966; Syversen, 1977). The reduction of the energyproducing metabolic processes has also been proposed as the main factor in the neurotoxic action of Et,Pb (Aldridge et al., 1962). Et,Pb has been shown to interfere with protein synthesis secondary to the suppression of cellular energyproducing processes (Konat et al., 1978), and the synthesis of myelin proteins was more severely affected than other brain proteins (Konat et al., 1978). Evidence for a selective interference with the deposition of myelin components was given from observations of decreased levels of sulfatides in myelin preparations isolated from brains of developing animals treated with Et,Pb (Konat and Clausen, 1974; 1976) and of HFA cerebrosides in myelin from MeHg poisoned rats (Grundt et al., 1980). The study of MeHg and Et,Pb effects on the enzyme activities, although not conclusive, provide complementary data indicating that different mechanisms may be involved in the actions of MeHg and Et,Pb. There is a great quantitative difference between the Et,Pb effects in studies with tissue slices and the enzyme preparations. The effect of Et,Pb on the galacto- and sulfotransferase activities was relatively small; conversely, the results of experiments with brain slices indicate that one or several components in the biosynthetic chain of galactolipids are particularly sensitive to the toxic action of Et,Pb. This action may be related to the above-mentioned inhibition by Et,Pb of energy-producing processes in brain. On the other hand, the effects of MeHg on cerebellum slices and in enzyme assays were quantitatively comparable. It is possible, therefore, that effects of MeHg on the cerebroside and sulfatide formation result, at least in part, from a direct action of the poison on the transferase system involved in the biosynthesis of these lipids. However, it should be pointed out that the comparison of the Et,Pb and MeHg effects in terms of their tissue contents may be misleading since local effective concentrations of each poison may vary due to different physicochemical properties and hence different distributions between the hydrophilic and the hydrophobic phases of cellular membranes.

290

GRUNDT

AND NESKOVIC

ACKNOWLEDGMENTS The authors express their gratitude to Dr. 0. D. Laerum Institute of Pathology, University of Bergen, Norway, for kind support, to T. L. M. Syversen, Laboratory of Pharmacology, University of Trondheim, Norway, for helping us with determinations of Hg; to J. P. Rambaek, Institute for Atomic Energy, Kjeller, Norway, for performing the analyses of Pb. This study was supported in parts by a grant from Fondation Simone et Cino de1 Duca, Paris, France.

REFERENCES Aldridge, W. N., Cremer, J. E., and Threlfall, C. J. (1962). Trialkylleads and oxidative phosphorylation: A study of the action of alkylleads upon rat liver mitochondria and rat brain cortex slices. Biochem. Pharmacol. 11, 835-946. Dittmer, J. C., and Wells, M. A. (1969). Quantitative and qualitative analysis of lipids and lipid components. Selective hydrolysis procedures. In “Methods in Enzymology” (J. M. Lowestein, Ed.), Vol. 14, pp. 518-519. Academic Press, New York/London. Folch-Pi, J., Lees, M., and Sloane-Stanley, G. H. (1957). A simple method for the isolation and purification of total lipid from animal tissues. J. Biol. Chem. 226, 497-509. Fry, J. M., Lehrer, G. M., and Bornstein, M. B. (1974). Sulfatide synthesis: Late inhibition in developing cultures of mouse spinal cord. J. Neurochem. 22, 459-460. Grundt, I. K., Offner, H., Konat, G., and Clausen, J. (1974). The effect of methylmercury chloride and triethyllead chloride on sulfate incorporation into sulfatides of rat cerebellum slices during myelination. Environ. Physiol. Biochem. 4, 166- 171. Grundt, I. K., Stensland, E., and Syversen, T. L. M. (1980). Changes in fatty acid composition of myelin cerebrosides after treatment of developing rat with methylmercury chloride and diethylmercury. J. Lipid Res. 21, 162-168. Grundt, I. K., Ammitzboll, T., and Clausen, J. (1980). Inhibition by triethyllead of the sulfatide and cerebroside accumulation in cultured brain cells. Submitted for publication. Hayes, L. W., and Jungalwala, F. B. (1976). Synthesis and turnover of cerebrosides and phosphatidylserine of myelin and microsomal fractions of adult and developing rat brain. Biochem. J. 160, 195-204. Konat, G., and Clausen, J. (1974). The effect of long-term administration of triethyllead on the developing rat brain. Environ. Physiol. Biochem. 4, 236-242. Konat, G., and Clausen, J. (1976). Triethyllead-induced hypomyelination in the developing rat forebrain. Exp. Neurol. 50, 124-133. Konat, G., Offner, H., and Clausen, J. (1976). Triethyllead restrained myelin deposition and protein synthesis in the developing rat forebrain. Exp. Neural. 52, 58-65. Konat, G., Offner, H., and Clausen, J. (1978). Effect of triethyllead on protein synthesis in rat forebrain. Exp. Neural. 59, 162-167. Konat, G., Offner, H., and Clausen, J. (1979). The effect of triethyllead on total and myelin protein synthesis in rat forebrain slices. J. Neurochem. 32, 187- 1%. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Magos, L., and Clarkson, T. W. (1972). Atomic absorption determination of total, inorganic and organic mercury in blood. J. Assoc. Official Anal. Chemists 55, 966971. More& P., and Radin, N. S. (1969). Synthesis of cerebroside by brain from uridine diphosphate galactose and ceramide containing hydroxy fatty acid. Biochemistry 8, 506-512. Neskovic, N. M., Sarlieve, L. L., and Mandel, P. (1974). Purification and properties of UDPgalactose:ceramide galactosyltransferase from rat brain microsomes. Biochim. Biophys. Acta. 334, 309-315. Neskovic, N. M., Sarheve, L. L., and Mandel, P. (1976). Brain UDPgahmtose:ceramide galactosyltransferase. Purification of a catalytically active protein obtained after proteolytic digestion. Biochim. Biophys. Acra 429, 342-351. Sarlibve, L. L., Neskovic, N. M., Rebel, G., and Mandel, P. (1974). PAPS-cerebroside sulphotransferase activity in developing brain of neurological mutant mouse (MSD). Exp. Brain Res. 19, 158-165.

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ON BRAIN LIPIDS

291

Silberberg, D., Benjamins, J., Herschkowitz, N., and McKhann, G. M. (1972). Incorporation of radioactive sulphate into sulphatide during myelination in cultures of rat cerebellum. J. Neurochem. 19, ll- 18. Syversen, T. L. M. (1977). Effects of methylmercury on in vivo protein synthesis in isolated cerebral and cerebellar neurons. Neuropathol. Appl. Neurobiol. 3, 225-23. Yoshino, Y., Mozai, T., and Nakao, K. (1966). Biochemical change in the brain in rats poisoned with an alkyl mercury compound with special reference to the inhibition of protein synthesis in brain cortex slices. J. Neurochem. 13, 1223- 1230.