Dietary lipids influence the activity of

D5-desaturase and phospholipid fatty acids

in rat enterocyte microsomal membranes

M. Keelan, M.T. Clandinin, and A.B.R. Thomson

Abstract: Previous studies have shown that isocaloric modifications in the type of lipids in the diet alter the nutrient uptake

and lipid composition of the intestinal brush border membrane. In this study adult rats were fed for 2 weeks isocaloric

semisynthetic diets with triglycerides enriched with either saturated (S) or polyunsaturated (P) fatty acids. Enterocyte

microsomal membranes (EMMs) were isolated from along the jejunal villus. The activity of ∆5-desaturase was higher in the

upper than in the lower portions of the villus, and was greater in P than in S. The activity of ∆9- and ∆6-desaturases did not

vary along the villus or with changes in dietary S or P. The two predominant EMM phospholipids were phosphatidylcholine

and phosphatidylethanolamine, and these did not vary along the villus or with changes in diet. The major EMM fatty acids in

phosphatidylcholine and phosphatidylethanolamine were 16:0, 18:0, 18:2ω6, and 20:4ω6; none of the individual fatty acids

varied along the villus or with diet, although minor changes in fatty acid classes were observed. Thus, alterations in dietary

lipids modify the activity of ∆5-desaturase in the EMMs collected from the upper portion of the villus, but this does not result

in the expected changes in fatty acids in the EMM phospholipids.

Key words: adaptation, crypt–villus axis, desaturases, polyunsaturated fatty acid diet, saturated fatty acid diet.

Résumé: Des études antérieures ont montré que des modifications isocaloriques au niveau du type de lipides de la diète

affectent la capture des nutriments et la composition des lipides de la membrane de bordure en brosse intestinale. Dans la

présente étude, des rats adultes ont été soumis à des diètes semi-synthétiques isocaloriques, composées de triglycérides

enrichis d’acides gras polyinsaturés (P) ou saturés (S). Les membranes microsomales des entérocytes (MME) ont été isolées

de la villosité jéjunale. L’activité de la ∆5-désaturase a été plus importante dans la partie supérieure que dans la partie

inférieure de la villosité, et elle a été plus forte avec les acides gras P qu’avec les acides gras S. L’activité de la ∆9-désaturase

et de la ∆6-désaturase n’a pas varié le long de la villosité ni avec les variations de P ou S alimentaires. Les deux plus

importants phospholipides des MME ont été la phosphatidylcholine (PC) et la phosphatidyléthanolamine (PE), qui n’ont pas

varié le long de la villosité ni avec les variations de la diète. Les principaux acides gras de la PC et de la PE des MME ont été

les suivants : 16:0, 18:0, 18:2ω6 et 20:4ω6; aucun acide gras n’a varié le long de la villosité ni avec le type de diète, bien que

des variations mineures des classes d’acides gras aient été observées. Ainsi, des variations au niveau des lipides alimentaires

modifient l’activité de la ∆5-désaturase dans la MME prélevée dans la partie supérieure de la villosité, mais elles n’induisent

pas les variations d’acides gras attendues dans les phospholipides des MME.

Mots clés : adaptation, axe crypte–villosité, désaturases, diète composée d’acides gras polyinsaturés, diète composée d’acides

gras saturés.

[Traduit par la Rédaction]

Introduction

Isocaloric variations in the type of lipids in the diet modify thetransport function of the intestine (Thomson et al. 1986). Inchow-fed animals there are only modest variations along theintestine in the composition of the phospholipids in the brushborder membrane (BBM) (Dudeja et al. 1990; Meddings et al.1990). When mixtures of enterocytes are collected from along

the villus, this functional adaptation in response to changes indietary lipids is associated with variations in the lipid compo-sition of the BBM and of the enterocyte microsomal membrane(EMM), and it is also associated with alterations in the activityof EMM phospholipid and fatty acid metabolic enzymes (Garget al. 1988a, 1988b, 1988c; Keelan et al. 1994). This study wasundertaken to determine the effect of feeding isocaloric semi-synthetic diets with triglycerides enriched with saturated fattyacids (S) versus polyunsaturated fatty acids (P) on cholesterol,phospholipids, their fatty acyl constituents, and desaturase ac-tivities in EMM obtained from enterocytes isolated from alongthe various depths or heights of the villus of the jejunum.

Methods and materials

Animals and dietsThe guiding principles in the care and use of laboratory animals,approved by the Canadian Council on Animal Care, were strictly

Received May 3, 1996.

M. Keelan,1 M.T. Clandinin, and A.B.R. Thomson. Nutritionand Metabolism Research Group, Department of Medicine(Division of Gastroenterology), and Department of FoodSciences and Nutrition, Edmonton, AB T6G 2C2, Canada.

1 Author for corresponence at 519 Robert Newton ResearchBuilding, University of Alberta, Edmonton, AB T6G 2C2,Canada.

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observed in the conduction of this study. The animal rooms were keptat 21°C, in a 12 h of light : 12 h of dark cycle. Male Wistar ratsweighing 300–350 g were fed one of two isocaloric semisyntheticdiets for 2 weeks: a saturated fatty acid diet (S) or a polyunsaturatedfatty acid diet (P). Both diets were nutritionally adequate for all essen-tial nutrients and contained 20% fat; S was enriched in palmitic andstearic acid, while P was enriched in ω6 polyunsaturated fatty acids(Table 1) (Thomson et al. 1986).

Enterocyte fractionations from along the villusAnimals were sacrificed by intrahepatic injection of Euthanyl®

(800 mg sodium pentobarbital/kg body weight). A 60-cm segment ofproximal small intestine beginning at the ligament of Treitz was re-moved from each animal. The small intestines of eight animals wereused for the preparation of each fractionation. The following stepswere carried out at 4°C. Each intestinal segment was rinsed withice-cold 0.9% NaCl, cut into four 10-cm segments, and everted ontopegs. The pegs were placed directly into a Plexiglas pegboard cylin-der containing preoxygenated 300 mM D-mannitol, 10 mM Tris-HClin 2.5 mM EDTA buffer, pH 7.4. Fraction I was from the upper por-tion of the villus, whereas fraction IV was from the lower portion ofthe villus. The validation of this fractionation technique has beenreported (Keelan et al. 1995). The cylinder was then placed onto amodified shaker (IKA-VIBREX-VXR® horizontal shaker, TerochemScientific, Edmonton, Alta.), set at 1400 rpm to release enterocytes bymechanical vibration. Enterocyte fractions I, II, III, and IV were col-lected after shaking the tissue for a total of 5, 25, 45, and 65 min,respectively. The cylinder was filled with fresh buffer after collectingeach fraction. Each fraction was then centrifuged for 15 min at 300 × g(JA-14 rotor, Beckman J2-21 high speed centrifuge) to pellet theenterocytes. The supernatants (S1) were discarded and the pellets (P1)were resuspended in 250 mM sucrose, 100 mM KH2PO4, 1 mMEDTA, in 1 mM glutathione buffer, pH 7.2. The resuspended pelletswere homogenized with a Polytron® homogenizer at setting 8 for30 s on ice. Aliquots were taken for protein, and stored at –80°C untilthe time of assay for protein (Lowry et al. 1951), alkaline phosphatase(Bowers et al. 1976), sucrase (Dahlqvist 1964), thymidine kinase(Bresnick and Karjala 1964; Salser and Ballis 1973), NADPH cyto-chrome c reductase (Sotocasa et al. 1967), and DNA (Ma et al. 1992).

Preparation of enterocyte microsomal membranesThe enterocyte microsomal membranes (EMMs) were prepared ac-cording to a modification of the method of Lindskog and co-workers(Lindeskog et al. 1986). Each fraction homogenate was centrifugedfor 15 min at 2500 × g (JA-20 rotor). The pellet (P2) was discarded,and the supernatant (S2) was centrifuged for 15 min at 18 000 × g.The pellet (P3) was discarded, and the supernatant (S3) was centri-fuged for 20 min at 44 000 × g. The pellet (P4) was discarded, and thesupernatant (S4) was centrifuged for 70 min at 90 000 × g (type 30rotor, Beckman L5-65 ultracentrifuge). The supernatant (S5) was dis-carded, and the pellet (P5) was resuspended in 250 mM sucrose,150 mM KC1, in 40 mM Tris buffer, pH 7.4, and was homogenizedwith the Polytron® at setting 8 for 20 s on ice. Aliquots were takenfor protein, contaminating brush border membrane marker enzymes(sucrase and alkaline phosphatase), for immediate lipid extraction,and for assessment of EMM desaturase enzyme activities. NADPHcytochrome c reductase activity was determined to assess the purifi-cation of the microsomal preparations, which were enriched at least10–15 fold over homogenates (Sotocasa et al. 1967).

Lipid extraction and lipid analysesLipids were extracted according to a modification of published meth-ods (Bowyer and King 1977; Folch et al. 1957). Aliquots of the chlo-roform layer were taken for the quantitation of total phospholipid(Sunderman and Sunderman 1960) and total cholesterol (Allain et al.1974; Morin 1976), phospholipid composition (Touchstone et al.

1979; Vitiello and Zanetta 1978), as well as for the qualitative analy-ses of phospholipid fatty acid composition (Keelan et al. 1990a).

Desaturase enzyme activitiesThe measurements of ∆9-, ∆6-, and ∆5-desaturase activities were car-ried out according to a minor modification of a published method(Garg et al. 1986, 1988b). Each 1.0-mL reaction mixture contained5 mM ATP, 0.1 mM coenzyme A, 1.25 mM NADH, 0.5 mM niaci-namide, 2.25 mM glutathione, and 5 mM MgCl2, dissolved in62.5 mM KH2PO4 and 62.5 mM NaF buffer, pH 7.4. A volume of0.050 mL containing 200 nmol of cold substrate with 1% labelledsubstrate (∆9-desaturase: 0.1 µCi 1-[14C]16:0/assay (1 Ci = 37 GBq);∆6-desaturase: 0.1 µCi 1-[14C]18:2ω6/assay; ∆5-desaturase: 0.05 µCi1-[14C]20:3ω6/assay) was solubilized with fatty acid free bovine se-rum albumin (fraction V, Sigma, St. Louis, Mo.; 10 mg BSA/mLphosphate buffer) and was added to the reaction mixture, and pre-incubated for 5 min at 37°C. The reaction was started by the additionof EMM (2 mg protein), and the mixture was incubated in a shakingwater bath for exactly 15 min at 37°C. The reaction was terminatedby the addition of chloroform–methanol (2:1) containing 0.5% butyl-ated hydroxytoluene. Lipids were double extracted with chloroform–methanol (2:1), and the chloroform layer was dried under nitrogen at50°C. Samples were reconstituted in hexane, and fatty acids wereconverted to methyl esters by heating for 1 h at 100°C with 14%(w/w) boron trifluoride in methanol. Deionized water was added tothe cooled samples, and the upper hexane layer was transferred to testtubes and dried under nitrogen at 50°C. Samples were stored at –80°Cuntil AgNO3 plates were prepared. Fatty acid methyl esters were sepa-rated based on the degree of their unsaturation, using thin layer chro-matography plates impregnated with 10% (w/w) AgNO3 in silicagel G. Plates were developed for 35 min in hexane – diethyl ether –glacial acetic acid (94:4:2), followed by 25 min in hexane – diethylether – glacial acetic acid (90:10:2). Fatty acid methyl esters weresprayed with 0.15 ANSA (8-anilino-1-naphthalene-sulfonic acid),and were visualized under ultraviolet light. Spots were scraped intoscintillation vials, Scientiverse II (Fisher Scientific Limited,Edmonton, Alta.) was added, and the samples were counted in a liquidscintillation counter (Beckman LS 5801, Beckman Instruments, PaloAlto, Calif.) with a counting efficiency of more than 90%. Desaturaseactivity was expressed as picomoles of desaturated product formedper minute per milligram of EMM protein (pmol⋅mg–1 protein⋅min–1).

Expression of results of statistical analysesThe data are means ± SEM of 3 or 4 preparations, each comprisingpooled results from 8 rats. Two-way ANOVA was used to comparethe results of fractions I–IV. A p value of 0.05 or less was accepted as

Fatty acid P S

14:0 0.30 3.52

14:1ω9 0.01 0.21

15:0 0.04 0.55

16:0 11.24 25.04

16:1ω9 0.15 2.02

17:0 0.11 1.32

18:0 3.73 19.18

18:1ω9 21.80 28.73

18:1ω7 1.46 3.08

18:2ω6 50.09 8.29

18:3ω3 5.98 0.72

18:4ω3 1.01 0.29

19:0 0.00 0.09

20:0 0.27 0.20

20:1ω9 0.19 0.06

Table 1.Fatty acid composition of diets.

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representing a statistically significant difference between the meanvalues.

Results

Enterocyte microsomal membrane cholesterol andphospholipids

There were no differences along the villus (fractions I, II, III,or IV), or between rats fed saturated (S) or polunsaturated (P)diets, in enterocyte microsomal membrane (EMM) total cho-lesterol, total phospholipids, or individual phospholipids

(Table 2). The ratio of phospholipid to cholesterol, and ofphosphatidylcholine, PC, to phosphatidylethanolamine, PE(PC/PE), did not vary along the villus in rats fed S or P.

Enterocyte microsomal membrane phospholipid fattyacyl constituents

The fatty acyl constituents were determined in the two pre-dominant EMM phospholipids, PC and PE (Tables 3 and 4).The major EMM fatty acids in PC and PE were 16:0, 18:0,18:2ω6, and 20:4ω6. None of the individual fatty acids in PCor PE in EMM varied along the length of the villus. There

Saturated fatty acid diet (S) Polyunsaturated fatty acid diet (P)

I II III IV I II III IV

Total cholesterol

nmol/mg protein 74±19 58±20 53±21 73±28 59± 12 68±21 61±24 65±18

nmol/mg DNA 191±84 102±49 89±22 79±20 451±427 165±89 117±55 140±16

Total phospholipids

nmol/mg protein 360± 21 379± 54 246± 54 320±47 220± 63 427± 94 349±107 344± 97

nmol/mg DNA 661±193 563±272 388±130 422±56 1219±732 822±402 581±289 663±210

PL/Chol 5.8±1.4 7.7±2.7 4.5±0.6 5.8±1.2 3.2±1.0 5.3±0.2 6.8±1.9 6.1±1.3

Phospholipid composition

nmol/mg protein

PC 174±12 186±27 129±17 152±21 132±29 241±53 212±50 162±52

SM 31± 6 34± 6 25± 6 16± 2 9± 2 19± 6 16± 3 22±13

PE 108±11 98±12 82±14 113±22 65±18 120±32 88±19 80±26

PI 31± 9 33± 2 26± 5 39± 8 14± 6 26± 9 23± 6 26±13

PC/PE 1.7±0.3 1.6±0.2 1.6±0.3 1.2±0.3 2.1±0.2 2.1±0.2 2.7±0.8 2.1±0.1

% of total phospholipids

PC 49.5±3.0 51.1±4.1 48.6±2.9 46.0±3.5 56.2±1.5 56.6±1.2 8.6±1.8 55.3±1.4

SM 8.9±1.7 9.8±2.1 8.7±1.2 8.7±0.4 4.0±0.8 4.3±0.8 4.9±0.8 6.6±1.9

PE 30.6±2.6 27.7±3.2 30.0±3.0 30.9±4.3 26.9±1.8 26.9±1.8 24.9±2.9 27.1±1.0

PI 8.5±2.1 9.4±0.6 9.9±1.4 9.9±2.0 5.8±1.8 6.8±1.8 6.9±1.7 7.6±2.8

Note: Values are means±SEM. No statistically significant differences were detected. Chol, total cholesterol; PE, phosphatidylethanolamine; PC,

phosphatidylcholine; PI, phosphatidylinositol; PL, total phospholipids; SM, sphingomyelin. I, II, III, and IV, fractions I, II, III, and IV, respectively.

Table 2.Effect of dietary lipid unsaturation on enterocyte microsomal membrane lipids in enterocytes isolated from along the jejunal villus.

Saturated fatty acid diet (S) Polyunsaturated fatty acid diet (P)

I II III IV I II III IV

16:0 8.3±0.8 9.0±2.0 11.6±1.3 12.6±2.2 10.2±2.0 7.6±1.3 9.6±0.9 8.6±1.1

18:0 32.3±2.5 24.7±5.7 31.1±1.2 31.4±1.8 29.5±2.0 31.2±1.1 29.3±1.5 29.0±1.3

18:1ω9 6.9±1.0 6.0±1.6 6.6±0.7 7.7±0.4 5.0±0.5 4.9±0.3 5.2±0.2 5.5±0.5

18:1ω7 1.0±0.4 1.4±0.5 1.4±0.2 0.7±0.2 1.1±0.3 1.6±0.1 1.8±0.3 1.7±0.3*

18:2ω6 27.9±1.7 25.0±5.8 36.0±1.1 33.9±0.5 33.0±3.2 38.5±0.5 38.2±2.3 36.9±2.0

18:3ω3 1.2±1.0 0.1±0.0 nd 0.5±0.4 0.1±0.1 0.4±0.4 0.2±0.2 0.2±0.1

20:0 1.5±0.8 1.4±0.9 1.3±0.8 1.0±0.5 0.2±0.1 0.2±0.1 0.2±0.1 0.8±0.4

20:4ω6 10.1±0.5 7.1±1.7 21.3±10.9 9.5±0.9 12.2±2.0 11.0±1.7 9.6±0.5 10.1±0.5

20:5ω3 nd 0.9±0.5 0.5±0.5 nd 0.6±0.6 0.1±0.1 0.2±0.2 0.7±0.4

22:6ω3 2.7±2.0 1.0±0.3 2.0±1.4 0.9±0.5 1.2±0.7 0.3±0.2 0.6±0.4 1.0±0.5

Total ω9 8.4±1.0 7.4±1.9 6.7±0.7 8.2±0.5 8.4±1.3 6.9±0.9 6.4±0.6 7.9±1.5

Total ω6 40.9±1.5 35.1±7.7 60.3±13.2† 44.7±1.5 47.0±1.7 50.8±1.2 49.6±1.5 48.5±1.8

Total ω3 4.7±2.4 2.0±0.5 3.3±2.2 1.4±0.9 2.0±1.4 0.8±0.4 1.1±0.7 2.0±0.9

Unsaturation index 140±13 111±25 191±63 126±4 142±13 138±3 135±3 140±6

Note: Values are means±SEM. nd, not detected. I, II, III, and IV, fractions I, II, III, and IV, respectively.

*p < 0.05, P vs. S.

†p < 0.05, vs. fraction I.

Table 3.Effect of dietary fat unsaturation on microsomal membrane phosphatidylcholine (PC) fatty acid composition in enterocytes isolated

from along the jejunal villus.

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were minor changes in the classes of fatty acids: total ω6 fattyacids were higher in PC of III than of I of rats fed S, and theunsaturation index of IV of PC of rats fed P was greater thanin S (Table 3), while total ω3 fatty acids were increased in PEof II of rats fed P compared with those fed S, or compared withI of rats fed P (Table 4).

Microsomal membrane desaturasesIn the jejunum of rats fed S or P, the activities of EMM ∆9- and∆6-desaturases (pmol⋅mg–1 protein⋅min–1) were similar alongthe villus (Table 5). In contrast, in both diet groups higherlevels (p < 0.05) of ∆5-desaturase were present in I comparedwith II–IV. The EMM activities of ∆9- and ∆6-desaturase werenot affected by diet, whereas ∆5-desaturase was higher in I (butnot in the other fractions) of P compared with S.

Discussion

The nutrient transport function of the BBM varies along the

length of the villus, with most uptake occurring from the upperportion of the villus (Fingerote and Thomson 1993). This vari-ation in absorption is associated with different types andamounts of phospholipids along the villus, with little change inthe amount of cholesterol (Meddings et al. 1990). The findingin this study of a similar content of cholesterol in EMM alongthe villus was, therefore, not surprising. However, because ofthe variations in BBM phospholipids along the villus, we hadexpected different amounts and (or) proportions of phos-pholipids in EMM from the upper (I) compared with the lowervillus fractions (II, III, and IV). This was not observed(Table 2), and raises the possibility that there may be some stepbeyond the microsomes that modifies the phospholipid contentof the EMM vesicles, which are subsequently inserted into theBBM. Phosphatidylethanolamine methyl transferase (PEMT)activity has been reported in the BBM (Garg et al. 1988a;Dudeja and Brasitus 1987), and it is possible that this or otherphospholipid metabolic enzymes may be responsible for thistailoring of BBM phospholipids.

Saturated fatty acid diet (S) Polyunsaturated fatty acid diet (P)

I II III IV I II III IV

16:0 9.3±5.4 8.2±3.8 4.2±0.4 8.4±4.3 3.3±1.3 3.7±1.1 6.9±5.2 4.6±2.4

18:0 39.2±1.5 39.9±2.0 39.6±1.0 35.1±5.9 29.0±2.1 33.4±3.0 26.7±10.7 32.8±4.7

18:1ω9 8.4±0.8 8.0±1.1 6.9±0.5 9.1±1.1 6.5±0.6 10.6±0.7 10.6±3.0 8.8 ±1.2

18:1ω7 0.8±0.3 0.7±0.3 0.9±0.1 1.0±0.5 1.0±0.1 0.4±0.4 1.3±0.2 1.0±0.3

18:2ω6 16.2±0.8 18.4±1.8 19.5±1.1 20.6±2.6 16.8±2.4 19.3±4.5 32.1±15.1 26.0±5.4

18:3ω3 0.1±0.1 0.5±0.3 0.1±0.1 0.1±0.1 nd 1.3±1.3 0.1±0.1 nd

20:0 0.2±0.1 1.0±0.7 0.1±0.1 0.2±0.1 0.5±0.3 1.7±1.5 0.5±0.1 0.6±0.2

20:4ω6 16.0±4.6 16.0±3.2 19.1±1.8 17.6±4.1 13.6±0.1 14.4±2.6 11.9±6.8 13.9±2.6

20:5ω3 nd 0.6±0.4 0.4±0.2 nd 0.2±0.2 1.2±1.2 0.4±0.2 1.0±0.5

22:6ω3 2.4±1.3 1.7±0.4 2.4±0.2 2.0±0.4 2.0±0.1 2.3±0.7 1.8±1.5 1.5±0.7

Total ω9 9.0±0.7 8.4±1.1 8.0±0.9 9.5±1.4 20.2±6.6 11.7±0.8 12.2±2.0 11.6±2.3

Total ω6 34.1±5.1 36.2±3.4 41.6±2.0 44.0±1.3 32.9±2.3 38.1±1.4 46.6±6.8 41.5±2.9

Total ω3 3.4±1.7 3.1±0.4 3.0±0.3 2.4±0.3 3.1±0.9 9.9±0.9*† 2.3±1.5‡ 3.4±1.0‡

Unsaturation index 132±26 132±14 154±5 142±10 162±12 158±17 149±11 145±9

Note: Values are means±SEM. nd, not detected. I, II, III, and IV, fractions I, II, III, and IV, respectively.

*p < 0.05, P vs. S.

†p < 0.05, vs. fraction I.

‡p < 0.05, vs. fraction II.

Table 4.Effect of dietary fat unsaturation on microsomal membrane phosphatidylethanolamine (PE) fatty acid composition in enterocytes

isolated from along the jejunal villus.

Saturated fatty acid diet (S) Polyunsaturated fatty acid diet (P)

I II III IV I II III IV

Protein (mg/fraction) 3.6±0.6 8.2±1.8 5.8±0.7 5.0±1.0 3.9±0.9 5.9±0.8 7.3±0.9 6.5±0.9

∆9-Desaturase

(pmol⋅mg–1 protein⋅min–1) 661±156 269±70 292±47 275±81 784±221 403±158 208±52 282±58

∆6-Desaturase

(pmol⋅mg–1 protein⋅min–1) 2040±585 583±118 1150±247 789±154 1314±718 1013±314 767±187 362±56

∆5-Desaturase

(pmol⋅mg–1 protein⋅min–1) 1889±769 796±118† 862±265† 887±270† 3872±384* 951±302† 238±84† 311±158†

Note: Values are means±SEM. I, II, III, and IV, fractions I, II, III, and IV, respectively.

*p < 0.05, P vs S.

†p < 0.05, vs. fraction I.

Table 5.Effect of dietary lipid unsaturation on enterocyte microsomal membrane desaturase activities in enterocytes isolated from along the

jejunal villus.

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Different classes of BBM phospholipids tend to be enrichedwith different fatty acyl constituents (Keelan et al. 1990a,1990b). While it is known that BBM phospholipids vary alongthe length of the villus (Meddings et al. 1990), it has not yetbeen reported whether there are associated alterations in thephospholipid fatty acids. With the absence of alterations inEMM phospholipid classes along the villus (Table 2), it wasnot expected that there would be changes in the EMM phos-pholipid fatty acyl constituents. Indeed, this was the case: noneof the individual fatty acids in PC or PE varied along the villus,although there were minor differences between the classes offatty acids (Table 4).

The microsomal membrane desaturases modify the fattyacyl constituents of phospholipids (Garg et al. 1986, 1988a,1988b, 1988c). Because there was higher activity of EMM∆5-desaturase in the upper compared with the lower portion ofthe villus (I compared with II–IV; Table 5), it had been ex-pected that there would be greater EMM PC or PE content of20:4 and 20:5, and less 18:2 and 18:3. This was not observed(Tables 3 and 4). We speculate that the ∆5-desaturase alteredfatty acids in the EMM that did not remain in the membrane,but may have been used for lipoprotein synthesis.

The enterocyte is unique in its capacity to transport fat inthe form of chylomicrons. The origin of phospholipids se-creted with chylomicrons is primarily luminal phosphatidyl-choline, which must be absorbed as lysophosphatidylcholinefollowing its hydrolysis by phospholipases, and then reacy-lated to phosphatidylcholine by lysophosphatidylcholine acyl-transferase in the presence of acylCoA within the enterocyte.This pool of phosphatidylcholine is specifically directed tolipoprotein synthesis (Mansbach 1972, 1973, 1975, 1977).Chylomicron triacylglycerol fatty acid composition reflectsthe fatty acid composition of the diet, unlike the fatty acidcomposition of chylomicron phospholipids and cholesteryl es-ters (Feldman et al. 1983a, 1983b).

Isocaloric changes in the lipids in the diet result in altera-tions in the BBM uptake of sugars and lipids (Thomson et al.1986). Feeding S compared with P had no effect on EMMphospholipids (Table 2), or on the fatty acyl constituents in PCand PE (Tables 3 and 4). However, the general properties ofthe fatty acid composition were influenced by diet: for exam-ple, when feeding P compared with S, there were more totalpolyunsaturates and a higher unsaturation index in PE(Table 4); feeding P was associated with an increase in ω3fatty acids in PE in the lower portion of the villus. Thus, vari-ations in the fat content of the diet result in minor alterationsin the classes of the fatty acids in the phospholipids of EMM,and these changes vary between phospholipids and betweenenterocyte location along the villus. This raises the possibility,therefore, that the control of membrane phospholipid fattyacids may be different in the enterocytes in the upper comparedwith the lower portions of the villus.

In summary, (i) isocaloric changes in the fatty acids in thediet resulted in minor alterations in the classes of fatty acylconstituents of the phospholipids in the enterocyte microsomalmembranes; (ii) these changes depended upon the location ofthe enterocyte along the villus; and (iii) alterations in dietarylipids modified the activity of ∆5-desaturase in the EMM col-lected from the upper portion of the villus, but this increasedactivity of ∆5-desaturase did not result in the expected changesin fatty acids in the EMM phospholipids.

Acknowledgments

The authors are grateful for the financial support of the Medi-cal Research Council of Canada and the Natural Sciences andEngineering Research Council of Canada. The excellent tech-nical assistance of Elizabeth Wierzbicki, Kim Doring, andTony Wierzbicki is warmly acknowledged. The authors alsothank Chandra Messier for word-processing assistance.

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