Vitamin B12 and the lipids of the central nervous system

Clin. Biochem. 2, 1-11 (1968)

V I T A M I N B12 AND T H E L I P I D S OF T H E C E N T R A L NERVOUS

SYSTEM*

DAVID ALAN T U R N E R x AND WILLIAM H. CEVALLOS2

Research Institute of the Hospital for Sick Children, Toronlo, Canada, Biochemistry Research Division, Department of Medicine, Sinai Hospital of Baltimore, Inc. and Johns Hopkins School of Medicine, Baltimore, l]Iaryland (Received April 30, 1968)

SUMMARY

1. A vitamin B12 deficient diet containing antibiotic to inhibit intestinal flora and endogenous B12 was fed to female Wistar rats for four weeks. The rats were then bred and the newborn were divided into two groups one of which continued to receive the B~2 deficient antibiotic supplemented regimen. The second group was fed the identical diet plus vitamin B12 (Merck Sharp and Dohme). This group served as a control to the experimental group with which they were paired. 2. Attempts to produce a B~ deficiency with neurological complications such as subacute combined degeneration of the spinal cord, have not been successful in rats. It was postulated that maternal dietary deficiency of B 12would sufficiently deprive the fetus during the period of maximum growth and nutritional demand, to result in initial nerve damage such that a lesion resembling the histology observed in pernicious anaemia in man would result if the newborn were continued on the B~ deficient diet in the postnatal period. 3. BI~ concentration of brain, spinal cord, sciatic nerve, plasma, spleen, liver, and kidney was measured at four, eight, and twelve months of age. Lipid analyses were confined to brain, spinal cord, and sciatic nerve tissue. 4. B12 concentration was markedly diminished in all tissues of the deficient animals, compared to the controls, and weight gain was greatly reduced. 5. At four months of age a marked decrease in the cephalin and lecithin fractions of the brain and spinal cord was observed in the B12 deficient animals. No additional fall occurred at eight and twelve months of age. The triglycerlde concentration of sciatic nerve was markedly decreased during the entire study. 6. Gas chromatography analysis of the total lipid extracts from these tissues showed the fatty acids were not significantly different between the test and control groups in any period. *This project was supported by USPHS Grant No. E-3529. ~Present address to which reprint requests and correspondence should be sent, Doctor David Alan Turner, The Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto 2, Ontario. 2Present address, Department of Biochemistry and Physiology, Division of Research, Lankenau Hospital, Philadelphia, Pennsylvania.

2

TURNER

& CEVALLOS

7. Histological examination failed to show any change in the myelin. 8. The administration of inorganic phosphorus (p3~.) eight dab's prior to terminating each experimental period indicated decreased incorporation of pa2 into all of the phospholipids. 9. Vitamin B~2 deficiency in rats from the fetal stage for one year failed to produce any clinical symptoms in the rat resembling those observed in B,2 deficiency in man. Significant changes in the phospholipid fractions were not coincident with measurable clinical symptoms suggesting that the biochemical lesion must be extensive to produce symptoms and must precede the clinical findings by a lengthy period of time.

N U M B E R O F F A C T O R S C O N T R O L L I N G L I P I D M E T A B O L I S M and the morphological function of lipids in the brain and spinal cord remain obscure. Sub-acute combined degeneration of the spinal cord and cerebral malfunction are known complications of pernicious anaemia in man which respond in specific fashion to a single nutritional compound, vitamin Bw.. The purpose of the present investigation was to study the role of vitamin Bx~ (cyanocobalamin) in the metabolism of the neutral lipids and phospholipids of brain, spinal cord, and peripheral (sciatic) nerve. Lower animals on a vitamin Bx~. deficient diet have not been found to develop the clinical picture of pernicious anaemia, as this disease is observed in man. Biochemical changes in nerve tissue may, however, precede clinical symptoms by long periods of time; therefore the investigation reported here was designed to study the composition and changes in nerve lipid in a successive generation of rats maintained on either a vitamin BI2 deficient or a vitamin B 1 o_ supplemented regimen.

A

~'IETHODS

An attempt to overcome the great difficulty in producing a clinical vitamin B12 deficiency in rats was made by placing pregnant rats on the deficient diet and continuing this diet with the offspring. Rats of the Wistar strain were fed a soybean diet deficient in vitamin Bm during pregnancy and lactation, and their progeny selected for the test group were continued on the same diet. The newborn rats were divided into two groups after weaning, and placed in individual cages with wide mesh screen bottoms under comparable environmental conditions. The wide mesh floors prevented collection and ingestion of faecal material, which might have contained vitamin B12 in spite of antibiotic sterilization of the intestine. During the entire period of the experiment, one group was offered a vitamin B12 deficient diet consisting of 60% soybean protein, 32~o sucrose, 4% salt mixture "Hegsted" (1), 4o-/ocorn oil, 0.1~o succinyl sulphathiazole and a vitamin supplement containing all known essential vitamins (2), except vitamin B12. The other group which served as a control, was offered the same basal ration supplemented with vitamin B12 (50 mcg/kilogram of diet). All rats were fed ad libitum and weighed regularly in order to record their growth response. The

Bl2 A N D CNS LIPIDS

3

control (normal) and the experimental groups were further subdivided into three groups and were sacrificed at four months (Group 1), eight months (Group 2), and twelve months (Group 3) of age. At these times, the animals were sacrificed under light ether anaesthesia, blood was drawn from the abdominal aorta, and the tissues to be examined were quickly removed, weighed, and placed in iced solvents (3-~). In order to study the lipid composition of brain, spinal cord, and peripheral nerve, the tissues were ground and extracted with chloroform-methanol 2:1 in an Elvehjenl-Potter homogenizer. The extract was washed with normal saline, the chloroform layer was filtered, dried over anhydrous sodium sulphate, evaporated under vacuum, and the lipids were redissolved in hexane and placed on 18-g colunms of activated silicic acid for fractionation. The activation of the silicic acid was carried out according to the method of Homing (6). Table 2 shows the concentrations of the elution solvents and the procedure used to fractionate the lipids of the nerve tissues. The precautions recommended by Rouser (7) were used throughout to minimize oxidative degradation of the lipids. In order to determine the degree of purity and for identification, the lipids eluted from the column were subjected to silicic acid impregnated paper chromatography, according to the method of Marinetti (8), and thin-layer chromatography according to the method of Malins and Mangold (9). The neutral lipid fractions were analyzed for cholesterol by the method of Hanel and Dam (10), glycerol by the method of Van Handel and Zilversmit (11), and ester bonds by the method of Snyder and Stephens (12). Phospholipids were analyzed by the method of Bartlett (18). Following quantitative analysis, the total lipids were transinethylated in sulphuric acid and methanol under nitrogen at 65 ° for four hours. The methyl esters of the fatty acids were extracted with hexane, washed with water, dried, and stored under nitrogen. Gas liquid phase chromatography (GLC) was performed at 180 ° and 24 p.s.i, inlet pressure using a U-shaped 4 inm I.D. 6-ft glass column containing 120-140 mesh siliconized chromosorb-W coated under vacuum with 15% diethylene glycol succinate (DEGS). The identity of the methyl esters was established by comparing the retention time with those of pure fatty acid standards and the fatty acid methyl esters from menhaden oil of known fatty acid composition and retention times (lg). The areas under the peaks were estimated by triangulation. The phospholipids in the plasma and red blood cell membranes of patients with pernicious anaemia have been reported to differ from the normal (15, 16). In this study, the metabolism of nerve phospholipid in the vitamin Bto deficient rats was followed using radioactive phosphorus (p32) as a tracer. Radioactive phosphorus (p32) was administered intraperitoneally as sodium monohydrogen phosphate 30- dissolved in physiological saline solution (E. R. Squibb and Sons, New York). Each rat received one microcurie p3~. per gram of body weight. The animals were killed eight days after the administration of the labelled phosphate compound. The feeding schedule was not interrupted during the course of the tracer experiments. The tissues were removed and prepared by the same techniques used in the nonradioactive studies. There was one exception ;

4

TURNER & CEVALLOS

i.e., during the lipid extraction, the c o n t a m i n a t i n g acid-soluble phosphorus was removed by three successive washings of the chloroform-methanol extract with 0.1 N H C I , as described by Dawson (17). T h e silicone acid columns were monitored by measuring the phosphorus and radioactivity content of aliquots from each eluted fraction. T h e radioactivity was measured in an a u t o m a t i c T r i - C a r b liquid scintillation counter (18, 19). RESULTS T h e results shown in T a b l e 1 indicate t h a t the v i t a m i n B ~ deficient animals gained weight more slowly t h a n the controls; their average weight was approxim a t e l y 22ialo less after twelve m o n t h s of t r e a t m e n t . In all the tissues analyzed, TABLE 1 TISSUE CONCENTRATIONS OF VITAMIN

Group No. 1

Body weight 4-S.E. gm Plasma4-S.E. m~/ml Liver 4-S.E. m/~/ml Kidney 4-S.E. m/~/m[ Brain 4-S.E. m/x/ml Spleen 4-S.E. m/,/ml

B,~.

Group No. 2

Group No. 3

Normal

B~., Deficient

Normal

B~ Deficient

Normal

B~.~Deficient

3444-17.7

2474-14.0

4374-21.2

2394-9.5

4194-23.7

1554-16.0

2274-9.2

584-5.0

5794-30.0

754-8.2

166-4-8.6

204-3.0

4624-18.2

104-1.8

4954-28.0

174-3.0

6054-67.8

484-2.8

20344-186.4

274-4.0

15344-106.0

674-11.4

544-3,1

74-0.5

784-3.7

34-0.4

774-2.8

44-0.4

704-3,0

124-1.1

694-1.6

184-1.2

884-3.6

124-0.9

467-4-29.0 3194-8.7

Group No. 1:14 rats, sacrificed at 4 months. Group No. 2:19 rats, sacrificed at 8 months. Group No. 3:15 rats, sacrificed at 12 months. the concentration of v i t a m i n B12 was less in the experimental groups t h a n in their respective controls. For example, at four m o n t h s of age (Group 1) the level in the liver, kidney, and brain was less than one-eighth of t h a t found in the controls ; in the plasma a b o u t one-third, and in the spleen a b o u t one-sixth. T h e concentrations of v i t a m i n BI2 in the eight- (Group 2) and t w e l v e - m o n t h (Group 3) periods were significantly lower than the v i t a m i n B12 levels of the v i t a m i n B12 s u p p l e m e n t e d group. A t eight months (Group 2), the v i t a m i n B~2 level in the plasma and the spleen of the test animals was a p p r o x i m a t e l y 2 5 % of the normal value; the liver 2~o, the kidneys 1%, and the brain 4 % . A t twelve months (Group 3), the plasma and the spleen of the v i t a m i n BI.~ deficient animals had v i t a m i n B12 values which were a p p r o x i m a t e l y 14~o, liver 3.4%, kidney 4.3%, and brain 5 % of the values found in the controls. T h e findings showed t h a t the experimental animals were m a r k e d l y v i t a m i n B~. deficient t h r o u g h o u t the entire period of study. T h e results of the q u a n t i t a t i v e analyses are expressed as mg of lipid per g r a m

B,~ AND CNS LIPIDS

5

of tissue. In order to have adequate quantities of lipid for analysis, it was necessary to pool the tissues of all rats in each individual group prior to homogenization and extraction. Unfortunately, such t r e a t m e n t precludes statistical analysis of the results but specific trends are a p p a r e n t from the pooled averages. T h e results shown in Table 2 indicate that, after four months of dietary vitamin Bt2 deficiency, the total phospholipid content of the brain and spinal cord was significantly lower than in the vitamin B~2 supplemented animals. T h e observed decrease can be a t t r i b u t e d to the fall in the concentrations of the cephalin and lecithin. Additional quantitative change in these fractions were not a p p a r e n t after eight or twelve months of vitamin Bao deficiency. TABLE 2 QUANTITATIVE ANALYSIS OF R A T N E R V E TISSUE LIPIDS M G . LIPID PER GM. FREStl TISSUE GROUP 1

Brain Lipid Fraction Total Phospholipids Total Cholesterol Total Glycerides Triglycerides Diglyeerides M onoglycerides 6% Methanol-Chloroform Fraction Cephalin Lysocephalin Lecithin Sphingomyelin Lysolecithin

Spinal Cord

Sciatic Nerve

Norm.

Exp.

Norm.

Exp.

Norm.

Exp.

44.6 29.0 17.8 5.1 7.0 5.7

32.1 30.7 18. O 6.2 4.8 6.9

89.2 50.0 37.0 20.4 7.8 8.8

72.2 59.9 31.4 13.3 9.1 8.9

33.6 25.0 50.0 47.4 1.3 .7

;36.4 23.9 33.3 30.7 1.0 1.5

1.7 21.2 4.1 15.9 1.5 .2

1.1 15.4 3.1 11.1 1.1 .3

4.6 41.4 10.6 27.4 4.4 .8

3.2 29.4 10.9 22.9 4.9 .8

.7 14.4 1.4 5.7 10.7 .0

.5 12.2 1.3 9.5 12.3 .4

A marked decrease in the total glyceride content of sciatic nerve in tile fourmonth experimental group was observed and this decrease could be a t t r i b u t e d to a decrease in the triglyceride concentration. After eight months of vitamin B12 deficiency (Table 3) the experimental group was found to have less than half as much triglyceride content in the sciatic nerve as the control animals. Table 4 shows the results from rats fed the vitamin BI~ deficient diet for twelve months. There were no marked changes in the concentration of the lipids of brain, spinal cord, and sciatic nerve, except for the sciatic nerve triglyceride concentration which was less than half the value found in the control sciatic nerve. No consistent changes in the phospholipid concentration were observed with vitamin B12 deficiency on the basis of the experiments in which the incorporation of radioactive phosphorus (ps2) into nerve tissue phospholipids was studied. T h e results shown in Tables O, 6, and 7 suggest less ps2 incorporation by most of the phospholipids in the brain, spinal cord, and sciatic nerve of the vitamin B~2 deficient animals at eight and twelve months of age, but these results were not conclusive. Because of the design of the study, it was not possible to draw a n y conclusions regarding relative rates of uptake or turnover.

6

T U R N E R & CEVALLOS TABLE 3

QUANTITATIVE ANALYSIS OF RAT NERVE TISSUE LIPIDS MG. LIPID PER GM. FRESH TISSUE GROUP 2 Brain Lipid Fraction Total Phospholipids Total Cholesterol Total Glycerides Triglycerides Diglycerides M onoglycerides 6% Methanol-Chloroform Fraction Cephalin Lysocephalin Lecithin Sp h i ngom .,,'elin Lysolecithin

Spinal Cord

Sciatic Nerve

Norm.

Exp.

Norm.

Exp.

Norm.

Exp.

47.8 24.6 18.8 9.1 3.2 6.4

40.4 23.5 20.4 7.1 5.3 8.0

57.6 44.6 33.2 21.9 3.0 8.3

60.0 43.0 30.0 17.4 3.9 8.7

36.9 21.5 43.1 40.7 .8 1.6

37.2 23.2 19.4 18.2 .4 .7

2.3 15.3 4.0 24.4 1.4 .4

2.1 13.1 9.1 14.1 1.5 .5

3.1 18.1 16.8 16.8 2.0 .8

2.7 18.6 18.6 17.1 2.2 .8

1.2 16.4 4.6 9.4 5.1 .0

1.9 17.4 2.6 9.1 6.0 .2

TABLE 4 QUANTITATIVE ANALYSIS OF RAT NERVE TISSUE LIPIDS MG. LIPID PER GM. FRESH TISSUE GROUP 3 Brain Lipid Fraction Total Phospholipids Total Cholesterol Total GI.vcerides Triglycerides Diglycerides M onoglycerides 6% Methanol-Chloroform Fraction Cephalin l.ysocephalin Lecithin Sphingomyelin Lysolecithin

Spinal Cord

Sciatic Nerve

Norm.

Exp.

Norm.

Exp.

Norm.

Exp.

40.5 29.3 17.8 6.9 7.5 :3.4

:37.8 28.2 18.8 4.9 8.3 5.6

59.9 40.1 :34.7 23.9 5.0 5.8

58.7 35.9 :34.3 22.5 5.6 6.1

21.3 23.8 72.0 71.3 .3 .4

28.1 24.3 32.7 32.0 .3 .3

1.7 15.3 6.9 13.8 2.3 .4

1.4 15.4 5.4 1:3.I 2. O .4

2.5 17.2 18.8 17.5 3.0 .9

3.5 15.8 18.1 17.6 2.7 1.0

1.7 7.5 .3 5.2 6.4 .0

1.4 8.6 1.0 8.2 8.7 .2

TABLE 5 THE UPTAKE OF p32 BY RAT NERVE TISSUE PHOSPHATIDES C.P.M./MG. PItOSPHOLIPID GIiOUP 1 Brain Lipid Fraction 6% MethanoI-Chlorofornl Fraction Cephalin L ysocepha lin I,eci th in Sphingom)'elin Lysolecithin

Spinal Cord

Sciatic Nerve

Norm.

Exp.

Norm.

Exp.

Norm.

Exp.

;3526 3097 348.5 4241 1424 3255

4690 4f188 4651 5467 1936 3307

2707 1546 812 2111 367 755

2276 1540 590 1933 822 1857

2671 1905 1498 1462 '2582 --

2120 1518 12;36 89 1695 757

Bl2 A N D CNS L I P I D S TABLE 6 THE UPTAKE OF pa~ BY RAT NERVE TISSUE PHOSPHATIDES C.P.M./MG. PHOSPHOLIPID GROUP 2 Brain Lipid Fraction 6% Methanol-Chloroform Fraction Cephalin Lysocephalin Lecithin Sphingomyelin Lysolecithin

Spinal Cord

Sciatic Nerve

Norm.

Exp.

Norm.

Exp.

Norm.

Exp.

7908 5404 4410 4691 3021 5482

6089 3262 2176 3488 1723 3002

7314 2431 1423 335l 1335 2056

3559 1249 787 1878 729 1436

3508 1656 1564 2745 1146 --

1977 999 1100 1617 564 769

TABLE 7 THE UPTAKE OF p32 nv RAT NERVE TISSUE PHOSPHATIDES C.P.M./iVIG. PI-IOSPHOLIPID GROUP 3 Brain Lipid Fraction 6% Methanol-Chloroform Fraction Cephalin Lysocephalin Lecithin Sphingomyelin Lysolecithin

Spinal Cord

Sciatic Nerve

Norm.

Exp.

Norm.

Exp.

Norm.

Exp.

6745 5591 3746 6917 2434 3163

5222 4017 2843 5377 2009 3200

8323 1935 1441 3445 1324 2039

6054 2392 1314 3295 1244 2940

3877 1603 1953 1139 2664 --

4330 1719 845 1096 2408 1625

TABLE 8 TOTAL LIPID FATTY ACIDS (% COMPOSITION)* GROUP 1 Brain

Spinal Cord

Sciatic Nerve

F a t t y Acid

Norm.

Exp.

Norm.

Exp.

Norm.

14 15 16 16:1 17 18 18:1 18:2 20 18:3 22 20:4 24 22:5 22:6

2.1 18.3 0.4 0.8 17.1 21.6 0.9 0.6 4.0 9.2 8.8 -5.4 9.2

3.0 21.3 -0.8 19.7 31.1 1.1 1.1 4.0 0.8 10.6 -6.0 --

0.3 4.2 15.7 0.4 0.6 16.4 32.1 1.2 1.7 10.6 1.9 5.1 2.2 5.0 1.7

4.7 12.2 0.3 0.8 12.5 33.9 1.7 1.7 10.0 -6.3 4.4 7.:3 3.5

2.6 1.2 18.5 3.8 0.4 5.2 38.5 21.3 0.5 1.6 1.1 1.9 2.0 ---

*By gas liquid phase chromatography analysis.

Exp, .8 1.8 18.0 2.0 0.4 6.6 35.4 18.8 1.0 0.9 2.0 3.0 2.6 2.1 --

8

TURNER Tables

the

8, 9, a n d

brain,

deficient tween

spinal

10 show the fatty cord,

and

diet for periods

the experimental

& CEVALLOS acid composition

sciatic

nerve

of rats

of up to twelve and

the control

months. values

of the total lipids found

maintained

on a vitamin

No significant

were

differences

found.

TABLE 9 TOTAL LIPID FATTY ACIDS ( % COMPOSITION)* GROUP 2 Brain

Spinal C o r d

Sciatic N e r v e

Fatty Acid

Norm.

Exp.

Norm.

Exp.

Norm.

Exp.

14 15 16 16:1 17 18 18:1 18:2 20 21 22 20:2 20:3 20:4 24 24:1 22:5

0.1 2.4 23.8 0.4 3.0 23.6 23.9 0.8 1.5 2.7 3.0 . . 4.4 2.0 7.5 --

0.2 1.9 21.7 0.1 1.8 30.9 19.4 0.7 0.4 3.3 1.1 . . 5.1 -7.4 --

0.1 3.3 12.1 0.1 0.1 14.5 21.7 0.9 1.6 11.3 1.4

0.2 5.2 16.8 0.3 0.9 21.9 27.3 1.3 1.6 8.1 2.0

0.6 0.5 19.4 0.8 0.8 9.6 16.1 7.6 1.9 ---

3.3 19.8 . 6.6

3.0 -. 6.0

1.0 0.4 20.1 -2.1 4.6 17.2 9.8 0.9 --32.0 1.9 -3.0 11.6

20.0

. .

. . .

5.2 1.9 6.4

.

* B y g a s liquid p h a s e c h r o m a t o g r a p h y a n a l y s i s

TABLE

10

TOTAL LIPID FATTY ACIDS ( % COMPOSITION)* GROUP 3 Brain Fatty Acid

Norm.

14 15 16 16:1 17 18 18:1 18:2 20 18:4 20:1 20:3 20:4 20:5 22:5 22:6

0.2 3.3 23.6 -0.2 19.6 20.9 0.5 0.8 -3.9 3.9 7.2 ----

Spinal C o r d Exp. 0.2 0.6 20.2 -1.7 18.6 22.5 0.9 0.2 0.8 3.3 0.2 7.6 0.4 1.7 --

Sciatic N e r v e

Norm.

Exp.

Norm.

Exp.

0.4 3.0 15.2 0.3 -13.4 37.8 -1.4 9.8 . 1.7 4.8 --1.6

0.3 2.2 15.0 0.4 0.3 15.6 38.1 0.2 1.5 9.7 . 2.0 4.3 --1.7

0.9 0.1 29.7 2.9 0.2 6.4 21.1 13.4 0.4 --

1.3 0.4 25.4 0.8 0.4 6.3 25.8 10.2 0.9 0.4

1.7 0.8 2.3 10.9 --

2.4 1.1 0.4 13.7 --

* B y g a s liquid p h a s e c h r o m a t o g r a p h y analysis.

.

.

in B12 be-

Bl2 AND CNS LIPIDS TABLE 11 SILICIC ACID COLUMNCHROMATOGRAPHY: SOLVENT SYSTEMAND ELUTION PATTERNOF FOLCH EXTRACTOF NERVE TISSUE Lipid Moiety Eluted Solvent System 1% 4% 8% 15% 30% 100% 2% 6% 15%

Ether-hexane Ether-hexane Ether-hexane Ether-hexane Ether-hexane Ether Methanol-chloroform Methanol-chloroform Methanol-chloroform

25% 30% 40% 70% 100%

Methanol-chloroform Methanol-chloroform Methanol-chloroform Methanol-chloroform Methanol

Neutral Lipids

Phospholipids

Cholesterol ester Triglyceride Free Fatty Acids Cholesterol Diglyceride M onoglyceride Phosphatidic acid Cerebroside Cephalins (serine and ethanolamine) Lysocephalin Phosphoinositol Lecithin Sphingomyelin Lysolecithin

DISCUSSION

Neurological symptoms of B12 deficiency which appear in about 900/0 of untreated pernicious anaemia patients include subacute combined degeneration of the posterior and lateral columns of the spinal cord, peripheral neuritis, cerebellar and cerebral disturbances. Usually, in patients the sensory systems are affected first, and motor tracts commonly become involved later in the course of the disease. If the deficiency continues untreated in man, the neurological changes are irreversible. Although vitamin B12 arrests or alleviates the early neurological complications of pernicious anaemia, very little is known about the biochemical function of vitamin B12 in the metabolism of the central nervous system (20, 21, 22). Lipid studies in pernicious anaemia patients were done thirty years ago, but these were concerned only with the plasma and the red blood cells. Williams and his co-workers (18), observed that remission of anaemia was accompanied by increases in the total plasma phosphate, lecithin, and sphingomyelin, and a decrease in the cephalin concentration. Kirk (16) observed that the feeding of liver to pernicious anaemia patients caused a marked rise in the plasma sphingomyelin level, and in the cerebrosides of the red blood cells. The present investigation was an attempt to correlate the neurological changes produced by vitamin B12 deficiency with changes in the lipid distribution in central and peripheral nerve tissue. The albino rat was selected for study because, like man, it is omnivorous, but we were aware of the difficulty in producing overt neurological symptoms of vitamin B12 deficiency in these animals. Nevertheless, it was felt that biochemical changes in nerve tissue, which presumably precede clinical signs and symptoms, might be detectable, especially if the vitamin B12 deficiency was initiated in the foetal stage when the growth rate and nutritional demand are at a maximum.

10

TURNER & CEVALLOS

The results of this investigation indicate that rats maintained for up to twelve months on a vitamin B~2 deficient diet do not follow a normal growth curve of weight gain. Significantly lower levels of vitamin Ba,. in the plasma, liver, kidney, brain, and spleen when compared with the controls, were attained. A vitamin Ba2 depletion had thus been satisfactorily produced and maintained. Unfortunately, the deficient state appears to have been insufficiently severe to produce neurological s y m p t o m s similar to those which appear in man after prolonged vitamin B~_ deprivation. Histological examination of nerve and brain from the experimental animals also failed to reveal any changes in the myelin of these tissues. Significant changes in the lipid concentration of peripheral nerve were observed with a marked decrease in the triglyceride concentration which was a p p a r e n t in the fourth month and became more marked after eight and twelve months of vitamin B~. deprivation. Quantitative phospholipid levels did not change significantly during this period, however, it is possible that the rate of turnover was reduced. T h e total incorporation of p~2 into the phospholipids of the rat brain, spinal cord, and sciatic nerve was found to be a very slow process, less than 0.1% of the injected dose. This was in agreement with the findings of Fries (18) and Changus (19) indicating perhaps that the phospholipid content of a tissue m a y be independent of its phospholipid or phosphorus turnover.

ACKNOWLEDGMENT T h e authors wish to t h a n k Doctor Elmer Alpert and the Merck Sharp and Dohme C o m p a n y , West Point, Pennsylvania, for their generous supply of all the vitamin Bt2 used in this investigation; and to express their gratitude to Miss Aurora Alcaraz and Miss B e t t y Padilla for their able assistance in carrying out m a n y of the numerous analyses involved in this work.

1. 2. 8. 4. 5. 6. 7. 8.

REFERENCES HEGSTED,D. M., MILLS, R. C., ELVEHJE,Xl,C. A. & HART,E.B. Choline in the nutrition of chicks. J. Biol. Chem. 138, 459-466 (1941). LEA'ZAN,E. A. & C~ow, B.F. Effect of Vitamin B~2 deficiency on cholesterol metabolism. J. Nutrition 78, 109-114 (1962). SKEGGS,H. R., NEPPLE, H. M., VALENTIK,K. A., HUFF, J. W. & WInG,T, L.D. Observations on the use of lactobacillus leichmannii 4797 in the microbiological assay of vitamin B~. J. Biol. Chem. 184, 211-221 (19.50). YAMAMOTO,R. S., OKUDA,K. & CHOW,B.F. Effect of carbon tetrachloride injury on plasma and liver vitamin B~2 levels. Proc. Soc. Exp. Biol. and Med. 98,497-500 (1957). GAFFNEV,G. W., HOROmCK,W., OKUDA,K., MELEE, P., CHOW,B. F. & SHOCK,N. Vitamin Bl2 serum concentrations in 528 healthy human subjects of ages 12-94. J. Gerontol. 12, 32-38 (1957). HORNING, M. G., V~IILLIAMS,E. A. & HORNING, E. C. Separation of tissue cholesterol esters and triglycerides by silicic acid chromatography. J. Lipid Research 1,482-48.5 (1960). ROUSER,G., O'BRIEN,J. & HELLER, D. The separation of phosphatidyl ethanolamine and phosphatidyl serine by column chromatography. J.A.O.C.S. 38, 14-19 (1961). MARmETTbG. V., ERBLAND,J. & KOCHEN,J. Quantitative chromatography of phosphatides. Federation Proc. 16, 837-844 (1957).

By., A N D CNS L I P I D S

11

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