Incorporation of (1-14C) palmitate into lipid fractions of developing rat brain in response to maternal feed restriction

NUTRITION RESEARCH, Vol. 9, pp. 1033-1040, 1989 0271-5317/89 $3.00 + .00 Printed in the USA. Copyright (c) 1989 Pergamon Press plc. All rights reserved.

INCORPORATION OF D E V E L O P I N G

OF RAT

_k(i_14C)P A L M I T A T E B R A I N IN R E S P O N S E RESTRICTIO N

I N T O LIPID F R A C T I O N S TO M A T E R N A L FEED

P a d m i n i Shantaram M.Sc., Vijaya R. Karandikar P. Srinivasa Rao* Ph.D.

M.Sc.

National Institute of Nutrition, Indian Council of Medical Research J a m a i O s m a n i a PO, Hyderabad - 500 007, India

ABSTRACT Studies were undertaken to ascertain relative utilization of intracerebrally injected labelled palmitate, in14normal and feed restricted rats, at early stages of development. IC palmitate incorporation into total brain lipids was low at selected time intervals in undernourished progeny. Distribution of the radioactivity from labelled palmitate a m o n g s t lipid fractions in general, indicated a predominant incorporation of the label into polar phospholipid fractions, c o m p a r e d to neutral lipids. However, the radioactivity in the cholesterol and free fatty acid fractions in brains of undernourished pups was relatively higher, with concomitant decreases in total phospholipid fraction. Distribution of radioactivity amongst phospholipid fractions indicated an increase in the case of phosphatidyl choline as against phosphatidyl ethanola mine in feed restricted animals, with distinct difference in 7 day old animals. The decreased incorporation of labelled precursor into total lipids, and higher accretion of radioactivity in free fatty acid fractions probably reflects an impaired capacity of feed restricted animals to utilise precursor fatty acid towards synthesis of specific lipid constituents of the brain. KEY

WORDS:

1-14C

Palmitate incorporation, brain lipids, undernutrition. INTRODUCTION

Several reports exist documenting the fact that, mild to moderate nutritional stress, i m p o s e d on the developing brain causes lasting and irreversible deficits (both functional, and structural) in its final form (1,2). Of several compositional changes that occur during the growth spurt period of the brain, those associated with the unique process of myelinogenesis have attracted greater attention (3,4). Myelin membrane being predominantly lipid in composition, alterations in the accretion and synthesis of lipids from fatty acid precursors, at early stages of d e v e l o p m e n t (5,6), might contribute to hypomyelination, which is k n o w n to occur in response to nutritional insufficiency. Palmitate, which is the major product of the fatty acid synthesizing system in the brain in situ, undergoes transformation through chain elongation and desaturation reactions, prior to incorporation ~i~to m e m brahe lipids. The relative utilization of intra-cerebrally injected (I -'~ C ) palmitate towards synthesis of lipids in the brain, at different stages of its

To

whom

correspondence

should be sent

1033

1034

P. SHANTARAMet al.

d e v e l o p m e n t was therefore studied. The present report includes data on the uptake of IC palmitate by developing rat brain, and its incorporation into total lipids and phospholipid fractions of control and nutritionally deprived animals. MATERIALS

AND

METHODS

(1-14C) Palmitate (57 m ci/m mole) was obtained from the Radiochemical Center A mersham. Radiopurity, as assessed by TLC, was greater than 9 8 % . Lipid standards were purchased from Sigma Chemical, St. Louis, M O. Solvents were of analytical grade. Animals and treatment Undernutrition was i m p o s e d by feed restriction. Ten day pregnant Wistar rats fed a standard laboratory rat chow (which provided 21 per cent protein, and was adequate with respect to all essential nutrients including fat, vitamins and minerals) (Table I) ad libitum represented the control group. Those receiving restricted a m o u n t s of feed '(50% of that c o n s u m e d by control animals) were considered as experimental. 8 pups were retained with each mother. They were administered labelled palmitate intracerebrally, at 7, 14 and 21 days of age. TABLE Composition

I

of stock diet

Constituents

Carbohydrates Protein Fat Salt Mixture Vitamin and choline chloride with starch M oistur e

Weight (percentage)

52.6 21.3 12.0 4.8 mixture 0.2 9.1

A standard laboratory stock diet with the above composition was used in this study. This was prepared from wheat flour (15%), roasted bengal gram flour (57%), groundnut flour (10%), skim reed milk p o w d e r (5%) and casein (4%). Salt and vitamin mixture were added separately. In addition, vanitine is added to provide 7000 IU of vitamin A and 300 IU of vitamin D per kg diet. 3 For intracerebral injections, a portion of (I-14C) pal mitate in benzene (containing 50 uci) was taken to dryness under N~, and an equimolar a m o u n t of O.01N KOH was added. This was followed by 0.6~ ml of 1 2 % albumin (in 0.9% Nacl). The mixture was sonicated for 30 sec. Aliquots of 10 ul of fatty acid-albumin c o m p l e x (eqivalent to 0.588 uci of (I-14C) palmitic acid) were intracerebrally injected using a Hamilton microsyringe, (I0 ul capacity) at a midline point b e t w e e n the eyes and ears, just under the skull (I m m deep). The microsyringe used was equipped with a needle guard, to prevent penetration deeper than I m m. Pups which bled at the point of injection were not considered for analysis. After allowing a 2 hour period for incorporation, these animals were sacrificed by decapitation, the whole brains r e m o v e d and im mediately i m m e r s e d in 10 ml of cold chloroform:methanol (2:1 v/v) solvent.

PALMITATE UPTAKE BY BRAIN LIPIDS Extraction

and separation

1035

of lipids

Brain tissues were homogenized with 40 volumes of chloroform:methanol, (2:1 v/v) and total lipids were extracted by the m e t h o d of Folch et al (7). Total lipid extracts were suspended in a k n o w n v o l u m e of benzene, aliquots of b e n z e n e solution of lipids were used for determination of total radioactivity and separation of lipid classes (neutral lipids, cholesterol, triglycerides and free fatty acids) by thin layer chromatography. Lipid extracts of brain tissues from animals not administered isotope, were used for separation of free fatty acids by T L C (solvent system :petroleum ether:ether:acetic acid 80:20:1, by volu m e) for deter ruination of palmitate pools by gas liquid c h r o m a t o g r a p h y . For separation of phospholipids, total lipid extracts were spotted on silica gel H plates and developed in the solvent system described a b o v e to r e m o v e neutral lipids. Phospholipids which r e m a i n e d at the origin were eluted with a solvent mixture described by Skipski et al 1964 (8). These eluents were concentrated, dried and dissolved in suitable v o l u m e of benzene. An aliquot of this was used for determination of radioactivity of total phospholipids. The remaining portion was subjected to separation into phospholipid fractions by T L C , using a solvent system co m prising chloroform : m ethanol:acetic acid: w ater (I 00:60:1 6:8 by volume). Spots visua13zed by brief exposure to iodine, were scraped off, eluted, as described for total phospholipids (8), and concentrated eluents transferred to counting vials. After evaporation to dryness under N 2, a toluene based scintillation fluid w a s added, and the radioactivity w a s d e t e r m z n e d in a Tricarb scint~ffi~t~on counter. Counting efficiency w a s 85 per cent as d e t e r m i n e d by an internal (IC) toluene standard. RESULTS

AND

DISCUSSION

The primary objective of these investigations was to e x a m i n e the relative utilization of labelled palmitate precursor by developing brains subjected to nutritional stress. Palmitate has been chosen for these e x p e r i m e n t s because of its p r e d o m i n a n t occurrence in situ, and, its ready transformation to higher homologues through reactions involving chain elongation and desaturation (6) prior to incorporation into brain lipids. Table 2 depicts data on body weights, brain weights, total lipids, palmitate pools, (as per cent of free fatty acid) and, the extent of incorporation of labelled palmitate into total lipids of the brain, as function of age and as affected by nutritional stress. A progressive increase in body and brain weights with e concomitant increase in total lipid content was noticeable with age. Feed restriction brought about a generalized decrease in these p a r a m e t e r s at each age (although significant c h a n g e s were observed in body weights at all ages, brain weight was affected only .~t 21 days). Palmitate pools in brain were not altered by nutritional stress. (I -j C) Palmitate incorporation into brain lipids was found to be r e d u c e d at all ages, in undernourished progeny. Significant differences in the specific activity, in t e r m s of total lipids ( D P M / m g total lipid) were evident at days 14 and 21. A b o u t 11.3 per cent of the injected label entered the total lipid fraction at day 7 in control pups, as against 6.8% in the case of undernourished ones (P 0.001). The a m o u n t of radioactivity associated with the total lipids increased to 27 and 25 per cent at 14 and 21 days respectively, in control animals, as against 2 5 % and 2 0 % in feed restricted ones (a figure of ~ 0 % has however b e e n reported in e x p e r i m e n t s e m p l o y i n g older animals (9)). With regard to the neutral lipid fraction (Table 3), both cholesterol end free fatty acids in

1036

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1039

14 and 21 day undernourished animals s h o w e d a higher proportion of radioactivity from palmitate, c o m p a r e d to control pups. This followed concomitant decreases in the incorporation of the label into total phospholipid fractions (Table 4). We have observed an increased -oxidation of palmitate in the case of undernourished rats (data not reported here). Preferential catabolism of available palmitate (as an energy source), to acetate, and subsequent ut~3isation of acetate for cholesterol synthesis, could probably explain increased radioactivity in cholesterol fractions of undernourished animals. (I _14C) palmitate is channelized maximally towards phospholipid biosynthesis, and largely influences the continuous process involving turnover of c eilular membranes (10). The results of these experiments suggest a predominant incorporation of the label into phospholipids of the brain during early periods of development. Experimental animals, when c o m p a r e d to controls s h o w e d significant decreases in the incorporation of precursor label into total phospholipids at 7 and 21 days (Table 4). Most of the radioactivity of phospholipids is accreted in phosphatidyl choline at the 14 and 21 day stages. Experimental animals incorporated relatively lesser radioactivity irrespective of the nature of phospholipid fractions. Significant diminutions were observed, in radioactivity with phosphatidyl ethanolamine (at 7 and 21 d) and phosphatidyl serine (at 21 d) (these phospholipid fractions are relatively richer in the long chain polyunsaturated fatty acids). The decreased incorporation of labelled precursor into phospholipids in general, and the above phospholipid fractions in particular, probably reflects an impaired capacity of undernourished animals to uti3ize palmitate precursor towards specific phospholipid synthesis. These results are in line with earlier in vitro observations (6) wherein microsomal system for chain elongation, desaturation and incorporation of labelled palmitate into brain phospholipids were s h o w n to be influenced by early nutritional stress. ACKNOWLEDGEMENTS The authors thank Or. B . S . Narasinga Rao, Director, National Institute of Nutrition, Hyderabad, for his keen interest in the work and helpful suggestions. Technical assistance of Mrs. Padmini Prakash is gratefully acknowledged. REFERENCES I.

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1040

P. SHANTARAM et a l .

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m e m branes.

Science

N.Y.

1 986;

1 53: