340
Biochimica et Biophysica Acta. 1086(1991) 340-348 © 1991ElsevierScience PublishersB.V. All rightsreserved0005-2760/91/$03.50
Changes in fatty acid composition during cell differentiation in the small intestine of suckling piglets J-M. A l e s s a n d r i , P h . G u e s n e t , T . S . A r f i a n d G . D u r a n d Laboratoir; de Nutrition el S~curit~ Alimentaire, Institat National de la Recherche Agronomique, INRA.CRI, Jouy.en-Josas (France)
(Received 2 October 1990) (Revised manuscriptreceived3 July 1991)
Key words: Fatty acid composition;Cell differentiation;Mucosa;Brush-bordermembrane;(Pig smallintestine) Alterations of phospholipid fatty acid composition in the renewing intestine were studied in the infant pipet. Newborn piglets were fed from birth to 2 weeks of age a concentrated cow's milk which defined a standard supply of dietary fatty acids. Phospholipids were isolated from the whole nmeosa, isolated intestinal cells and purified brush border membranes. Intestinal cells were isolated accordhtg to their position along the crypt-villus axis and cell phospholipids were extracted at each step of differentiation. Changes in fatty acid composition of cell phospholipids were related to these of lactase activity in th~ corresponding cell homogenates. In cell phospholipids, the relative content of linoleic and linolenie acids '-ucreased about 2-fold from crypt base to villus tip. Substantial contents of alkenylacyl glycerophospholipiO~ ~plasmalogens) were found in crypt cell phospholipids and in purified brush border membrane phosphatidylethanolamine (11 and 14% of alkenyl groups by weight of total fatty acids, respectively). The proportion of alkenylacyl glycerophospholipids decreased as cells ascended the villus column and became more differentiated. The results show that fatty acid compositional changes in differentiating cell phospholipids occurred in the immature intestine (before weaning) and suggest that these alterat|ons might be related to the ~ppearance of specific functions.
Introduction Morphologic and metabolic changes occur in the small intestine of mammalian neonates during the suckling period, leading to the development of mature mucosa properties Ill. It has been shown that cell differentiation processes, which account for the normal regeneration of intestinal epithelium throughout life, do not mimic those taking place during postnatal maturation [2]. Changes in mucosal lipid composition were shown to occur throughout ,:ell n'.igration along the crypt-villus axis on the one baad [3-6], and from newborn to adulthood on the other [7-9]. In the rat small
Abi~reviations:DHA. docosahexaenoicacid; DMA. dimethylacetals; ECL. equivalent chain length; PC. phospha~idylcholine; PE, phosphatidylethanolamine; P1. phosphatidylinositol; PS, phosphatidylserine;SM, sphingomyeline. Correspondence: J-M. Alessandri, Laboratoire de Nutrition et Sdeurit6 Alimentaire,lnstitut National de la Recherche Agronomique, INRA-CRJ.Jouy-en-Josas,78352 Cedex. France.
intestine, phospholipid, cholesterol and glycosphingolipid contents increased along crypt to villus path. Changes in the distribution of phospholipid polar head groups and alterations of glycosphingolipid molecular species occurred in brush border membranes during both postnatal maturation and crypt-villus differentiation (reviewed in Ref. 10). During the suckling period, the immature digestive tract is adapted to milk digestion and macromolecular transport. Neonatal pecularities are lost at weaning with dietary transition and development of selective permeability. It ~:a.~, been suggested that modifications in lipid composition - and therefore in fluidity - of intestinal apical membranes may account for variations in mucosal barrier to microorganisms and antigens during the postnatal period [9,11]. Differences in lipid composition and fluidity of microvillus membranes in villus and crypt enterocytes could also have functional significance [12], leading to the concept that a decrease in plasma membrane fluidity during both cell differentiation and postnatal development might be a general feature of the developing intestine [5,13]. Besides, it
342 Complete scouring of the mncosa needs up to 16 sequential incubations. Cell fractions harvested from the simultaneous incubation of two half-segments were pooled, and each pool was subsequently treated as one unique cell fiaction. Isolated cells were washed by centrifugation and pellets were homogenized in 5 ml of a preservation-glycerol buffer composed of 137 mM NaCl, 8.16 mM Na2HPO 4, 3.22 mM KCI, 1.47 mM KH~PO4, 5 mM EGTA, 6.81 M glycerol (pH 7.4). Homogenization was made by repeated injections of cell suspensions through the needle of a 5 ml-glass syringe. Cell homogenates were stored at -80°(2 until analyses. Assaying samples were rapidly thawed, diluted and homogenized in distilled water for protein [22] and enzymatic assays.
Light microscopy Histological analyses were undertaken on samples of isolated cells and on st.etions of resin-embedded intestinal tissue. An aliquot of pelleted cells was taken off immediately after the fourth and seventh extractions. Cells were stained with toluidin blue and mounted on glass slides for light microscopy observation (Nomarski interference contrast). In order to check the efficiency of scouring, pieces of the remnant intestinal tissue were fixed immediately after the final extraction with glutaraldehyde 2% in 0.1 M sodium cacodylate buffer for 2 h and then rinsed overnight in the same buffer. After dehydratation in graded ethanol, pieces were embedded in Epon 812 according to routine techniques. Epon sections were cut at a thickness of 0.5-1 /~m, mounted on glass slides and directly stained with toluidin blue for light microscopy observation.
Measurement of enzyme activities Disaccharidases and alkaline phosphatase were routinely used as markers of intestinal cell differentiation [21]. Lactase was measured using lactose as previously described [23]. M e a s u r e m e n t of alkaline pnitrophenylphosphatase activity [24] was adapted with minor modifications. Briefly, cell fractions were 1020-fold diluted and 100 p,I of the suspension were incubated for 20 rain at 37°C with 1 ml of a reaction buffer composed of 60 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (pH 9.5), 11 mM MgCI 2, 0.3 mM CaCI 2, 0.11 mM ZnCI~ ~,~ld 5 mM pnitrophenylphosphate. The reaction was stopped by 2 ml of 1.125 N Na0H and the suspension was cleared by centrifugation. Absorbance of the supernatant was measured at 400 nm and released p-nitrophenol was estimated by comparison with standards.
Purification of brush border membranes Frozen pieces of jejunum (20 to 30 g) were rapidly thawed at 37°C and washed with saline to remove
glycerol. Pieces were drained and immersed into 60 ml of an HMBA-buffer [25] composed of 10 mM (Hepes), 7 mM n-butylamine, 0.02% LiN 3 and 300 mM sorbitol, adjusted to pH 7.4 with maleic acid. The MgCI 2 precipitation method [26] was used for the purification of brush border membrane vesicles. Pelleted vesicles were finally suspended in 3 - 4 ml of the preservation-glycerol buffer and stored at -80°C. Purification steps were checked using lactase activity as specific marker c[ brush border membrane. The specific activity of this enzyme was enriched 12-15-fold in the final suspension of brush border membrane vesicles. 1 g of fresh tissue yielded 0.5 to 0.8 mg of brush border proteins.
Lipid analysis The fatty acids of cow's milk were extrac:ed and esterified according to the method of Wolff and Fabien [27]. Milk fatty acid propyl esters were identified using a Varian 3600 gas chromatograph equipped with a DB WAX capillary column (25 m x 0.32 mm I.D.) and coupled with a Spectra Physics 4270 integrator. The injector was heated from 30 to 150°C and the detector was set to 230°C. The oven temperature was programmed from 40 to 60°C at a heating rate of 2°C/rain and from 60 to 230°C at a heating rate of 5°C/rain. The main fatty acids of the concentrated cow's milk were short- and medium-chain fatty acids (13 to 14% by weight of total fatty acids), oleie (26-28%), palmitic (25-27%), stearic (11-12%) and myristie (10-10.5%) acids. Milk supplied essential fatty acids as linoleic (1.5%) and a-linolenic (0.7%) acids. Isolated cells and brush border membrane phospholipids were purified according to the methods previously described [19,20] except for the chloroform to methanol ratio which was 3:1 (v/v) instead of 2:1. The presence of glycerol in the samples made this modificatiov necessary. The bottom phase was dried under nitrogen and the dry matter was resuspended in 1 ml of 0.15 M NaCl before being extracted .vith chloroform/methanol 2:1 (v/v) to remove traces of glycerol. Other subsequent conditions remain unchanged [6]. The phospholipid classes of brush border membranes were separated by high-performance liquid chromatography [28]. Separation was achieved by a Zorbax Sil column (250 m m × 4.6 m m I.D) at a flow rate of 1.5 m l / m i n . Eluted material was detected at 206 nm. The chromatographic system was composed of two mobile phases:solvent A, n-hexane/2-propanol (3 : 2, v/v) and solvent B, n-hexane/2-propanol/water (6:4:0.55, v/v). Solvent A flew in the column at "he beginning of the elution. Then, a 50 to 100% linear gradient of solvent B was set between 14 and 30 min. All phospholipid classes were separated within 45 min. -* Peaks were identified using commercial phospholipid standards purchased from Sigma (St. Louis, MO,
343 U.S.A.) and quantitative estimation of each class was done using the Bartlett procedure [29]. Transmethylation of phospholipids [30] was performed at 70"C in a medium composed of methanol/ HCI/2,2-dimethoxypropane (I 0:1 : 0.4, v/v) and 0.05% (w/v) 2,6-di-t-bul~,l-p-cresol (BHT). lnt.ubation times were 4 h for total phospholipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), 4.5 h for phosphatidylinositoi (PI) and 5 h for phosphatidylserine (PS) and sphingomyeline (SM), respectively. Fatty acid methyl esters were identified using a Packard Model 427 gas chromatograph 427 equipped with a flamme ionization detector and a CP WAX 52 CB bonded fused-silica capillary column (50 m x 0.2 mm I.D.) purchased from Chrompack International B.Y. (Middelburg, The Netherlands). Hydrogen was the carrier gas, with a flow rate of 1-2 ml/min. Oven temperature was programmed from 140 to 2200C at a heating rate of 4*C/rain. Fatty acids were identified by comparison of equivalent chain lengths (ECL) with those of authentic fatty acid methyl esters. Dimethylacetals (DMA) derived from transmethylation of alkenyl-glycerophospholipids were identified by comparison of their ECL with those of standard DMA. The latter were obtained by transmethylation of plasmalogen-rich ethanolamine glycerophosphulipids purchased from Merck (Darmstadt, Germany). This compound was purified from bovine brain and contained about 60% of plasmalogeos. All fatty acid compositions were expressed as percent (by weigh0 of total fatty acids.
Treatment of data Lactase activity. Each cell fraction harvested from one piglet was set on the crypt-villus axis between 0 and 100% of isolated cells [21]. The 100% of isolated cells corresponds to the sum of the whole fractions expressed as proteins. Laetase activity was computed relative to the highest value reached by one cell fraction of each series. In that way, distribution profiles of this en~jme could be drawn along the crypt-villus axis as a percentage of the corresponding maximum value [6]. This treatment allowed us to superimpose the typical profiles of six individual piglets and thus define a common profile representative of the group. A common curve was automatically fitted from fourth-order polynomial regression which took into account the whole data. As shown in Fig. 3, lactase activity appeared for a suitable differentiation-marker of epithelial cells harvested from milk fed piglets. Fatty acids. The crypt-villus evolution of the cell fatty acid composition was plotted relative to the level of differentiation, the latter being given by the lactase specific activity of the corresponding cell homogenate. The curves were automatically fitted from fourth-order
"ttl
*Tt
Fig. I. The light microscOl~of mid villus cells (A) and lowervillus cells (B). get from fourthand seventh incubation~ respectively.The brush b~rder membraneis indicated by an arrowou cells loose from the mass.Scalebar is equal to 5 p.m.
polynomial regression which cumulated the raw data from six piglets. Each computer compiled fit was characterized by the coefficient of nonlinear regression, r. Results
Histological observations The nature of isolated cells was evidenced by light microscopy (Fig. i). Cells harvested from the fourth and seventh incubations (mid villus and lower villas, lcspectivcly) were found to differ in the thickness of brush border: the more mature the ceils, the thicker was the brush border (Fig. I). The extraction procedure led to complete scouring of the mucosa, as shown in Fig. 2. By comparison with untreated tissue, it can be seen that epithelial cells were entirely removed and that crypt areas became empty at the term of extraction.
344
Fig. 2. Semithin sections of Epon-embedded intestinal tissue ( X 200): treatment was made immediately after thawing (A) and at tl extraction procedure (B). Arrows indicate crypt sites (C) and Lamina propria (L).
Enzyme acticities Mean values of alkaline phosphatase activity increased from 10-20 nmol m i n - i m g - i in crypt cells to
°°1 ..
"k
150-230 nmol rain - t mg - t in upper villus not shown). This range o f activity was simih previously reported from adult rat intestine wise, lactase activity regularly increased fro mature cells, maximum activity (150 to 175 n m g - l) being achieved in upper villus cells (F profile depicted in Fig. 3 warranted the use activity as a pre-weanling differentiation small intestine epithelial cells.
Phospholipid fairy acid composition of the wh,
40
eu
I R:o91
°
ol
11K
. . . . . . . . . 0
20
40
Villus tip ~
60
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T o t a l proteins e x t r a c t e d (el.) Fig. 3, Evolution of lactase relative specific activity along the cryptvillus axis in neonatal piglet. Each point was ;.he mean value of a triplicated assay and represented one cell fraction from one piglet. [~.'tt:l #~htalnotl frnm ~iv nlaJolc ~ r o © . . . . . ; . . . . . . . A TK. . . . . . . . . . . . . . .
In scraped mucosa total phospholipids, acids were 16:0, 18:0, 1 6 : l ( n - 7), 1 8 : l ( n - 6), 20:4(n - 6), 18:3(n - 3), 20:5(n - 3), : and 22:6(n - 3) (Table 11). The precursors c 3), namely 18:3(n - 3), 20:5(n - 3) and 22:50 found to bc unusually high in the intestin milk-fed piglet. Consequently, docosahexa (DHA) represented only 20% of total (n ~eids (see Table II), whereas it accounted fi in the intestinal brush border membranes piglet [32]. This unusual distribution within family was also found in isolated cells and brush border membranes (see below).
345 versely, the sum of total DMA regularly decreased from crypt stem cells to upper villus cells (Fig. 4B). The crypt-villus decrease of total DMP content was not associated with significant changes in main alkenyl
II). T h e main alkenyl groups detected in whole m u c o s a phospholipids were 18:0, 16:0, 18:1 a n d 18:2. Minor groups such as D M A 16:1 were not detected at this step of the analysis.
Phospholipid fatty acid composition in isolated intestinal cells Phospholipid fatty acid composition varied according to cell p~,~'tion along the crypt-villus axis (Fig. 4). T h e most m a r k e d evolutions c o n c e r n e d essential fatty acids ( 1 8 : 2 ( n - 6) a n d 1 8 : 3 ( n - 3)) on the one hand, a n d total D M A on the o t h e r hand. Linolenic acid c o n t e n t was f o u n d to be as high in isolated cells as in t h e whole mucosa. Essential fatty acid contents increased about 2-fold from undifferentiated crypt cells to m a t u r e cells. T h e relative accretion o f 1 8 : 2 ( n - 6) a n d 18:3(n - 3) in cell phospholipids coincide well with t h e crypt-villus increase o f lactase specific activity in the corresponding cell h o m o g e n a t e s (Fig. 4A). Con-
group distribution (data not shown). T h i s m e a n s that, whatever the level of cell differentiation, the distribution of main D M A in cell phospholipids was apparently similar to those of the whole mucos~. (reported in Table ll). It is noteworthy, however, that crypt s t e m cells contained m u c h m o r e total D M A in their phospholipids (11% by weight of total methylesters) t h a n did u p p e r villus cells (3%) or the whole m u c o s a (6%). For this reason, low levels of D M A 16:1 could be occasionally detected in crypt s t e m cell phospholipids w h e r e a s it could not be detected in t h e o t h e r samples. Lastly, arachidonic acid content a n d 20:4(n - 6 ) to 1 8 : 2 ( n - 6) ratio in isolated cell phospholipids (Fig. 4C) were m u c h lower t h a n those found in rat epithelial cells [6]. Moreover, 2 0 : 4 ( n - 6) to 1 8 : ~ n - 6) ratio
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25 50 75 100 125 150 175 Lactose nmol. rain. -1. mg -1 Crypt oree L V i l l u s tip
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Loct,',se(nrnol m i n ' l rng-'l ) Crypt Qreo m Villus tip
Fig. 4. Changes in fatty acid composition in relation wilh cell differentiation step, from cnjpt-area to villus tip, as defined by lactase specific activity. Each single cell fraction was plotted as a function of its fatty acid content to its lactase activity. The data obtained from six piglets were superimposed and the curves were fitted from fourlh-order polynomial regression. (A) |8:2(n - 6) (cross symbols) and |8:3(n - 3) (b|ack squares) contents in total phospholipids. (B) Total DMA content in transmethylated phospholipids (the composition of DMA is given in Table !I). (C) 20:4(n - 6 ) to 18:2(n - 6 ) ratio computed from the individual values. Coefficients of polynomial regression were equal to 0.89, 0.53, 0.86 and 0.74 for 18:2,18:3, DMA and 20:4/18:2, respectively.
346 FABLE II Fatty acid compr.~ition in scraped mucosa total phospholipids in suckling piglets Mean values from six piglets (+S.D.). SFA, saturated fatty acids: MUFA, monounsaturatedfatty acids % (w/w) of total fatty acids 16:0 12.2 18:0 19.8 SFA 33.5
±S.D. 1.3 1.8 2.5
16:ln -7 18:1n -9 ,XMUFA
1.4 28.3 33.6
0.2 1.7 2.6
20:3n -9
l.l
0.09
18:2n -6 20:3n -6 20:4n --6 v n- 6
11.1 0.9 4.0 16.9
1.6 0.1 0.8 2.3
18:3n -3 20:5n - 3 22:5n - 3 22:6n -- 3 Vn - 3
2.3 1.7 1.8 1.4 7.2
0.4 0.2 0.2 0.2 0.7
DIvlA16:0 DMA 18:0 DMA 18:1 DMA 18:2 ,~ DMA
1.5 3.3 0.8 0.3 5.9
0.2 0.3 0.1 0.06 1.1
n - 6/n - 3 20:4/18:2 20:3/20:4
2.3 0.4 0.3
0.4 0.07 0.03
dropped from the crypt stem cells (0.6-0.8) to the differentiating cells of the villus base (0.2-0.4) whereas it remained about constant in the rat [6]. Phospholipid fatty acid composition of brush border membranes The former analysis .of.phospholipid fatty acids raised the question o f the unusual 18:3(n- 3) levels found in the whole mficosa and isolated cells. Furthermore, results reported in Table I! did not allow us to set up the actual DMA distribution in membrane phospholipids. Brush border membranes were purified in order (i) to prove the involvement of linolenic acid as membrane component and (ii) to appraise the distribution of alkenyl groups in a plasmalogen-rich class of purified membrane phospholipids, i.e. phosphatidylethanolamine (PE). Phospholipid class fatty acid compositions are reported in Table Ill. Linolenic acid was undoubtelly present in brush border membranes of suckling piglets as it accounted for more than 1% by weight of total methylesters in the two main phospholipid classes, PE and PC. Total DMA accounted for 14 and 1% by weight of methylesters in brush border membrane PE and PC, respectively. As previously sug-
gested (Table II), the major alkenyl groups found in the brush border PE were 18:0 and 16:0, which ac. counted for 52% and 29% of total identified DMA, respectively (Table Ill). Other alkenyl groups were DMA 18:1 (11% of total DMA), DMA 18:2 (4%) and DMA 16:1 (3%).
Discussion Until now, the Weiser's method was routinely used to isolate intestinal cells from rodents. The present study based on histological observations and biochemical analyses shows that it quite applies to piglet intestine. The low 20:4(n - 6) to 18:2(n - 6) ratio and 22:6(n - 3) content in cell or membrane pbospholipids (Fig. 4C, Tables II and l i d might signify that infant piglet intestine is not mature enough to convert essential tatty acids into long chain derivatives (desaturase activities) a n d / o r to incorporate these long chain fatty acids into mucosal phospholipids (acyltransferase activities). Desaturase activities were previously evidenced in ~eonatal piglet liver and brain [33] but information as to the ability of the intestine to desaturate fatty acids is lacking. The intestinal mucosa of the adult rat has been shown to desaturate both palmitic and linoleic acid [34]. These desaturase activities (A9 and A6) were recently detected in dog duodenal microsomes [35]. However, desaturase activities have not been identified in the neonate intestine and it is not known whether intestinal long chain polyunsaturated fatty acids originate from liver synthesis or are synthesized locally (or both). Changes in fatty acid composition along the cryptvillus axis were described only in two rat studies [5,6]. Comparison of Fig. 4A with results obtained in the rat [6] suggests that linolei~ acid profiles in piglet and rat intestines share a common pattern (provided that animals received sufficient amounts of essential fatty acids). In both cases, the crypt to villus path was characterized by an increasing incorporation of linoleic acid into cell phospholipids. Contrary to rat and weaned piglet, membrane phospholipids of suckling piglets contained rather high amounts of 18:3(n- 3), whose phospholipid incorporation also increased during cell migration. In agreement with these data, Reynier et al. [36] have recently shown that 18:2(n - 6) and 18:3(n 3) contents were much higher in phospholipids (PE and PC) of differentiated HT29 cells than in their undifferentiated counterparts. Moreover, they reported that arachidonic acid content in PE increased 2-fold with enterocytic differentiation of HT29 cells. This data is compatible with the crypt-villus evolution of 20:4(n - 6) we have previously profiled in the rat [6]. As suggested by Reyuier et al., the decreased contents of ( n - 6) and (n - 3 ) fatty acids in undifferentiated
347 cells b y c o m p a r i s o n w i t h m o r e m a t u r e cells m i g h t b e related to an increased bioconversion of these precurs o r s in e i c o s a n o i d s . T h e r e l a t i v e l y h i g h c o n t e n t o f i i n o l e n i c a c i d f o u n d in mucosa! phospholipids of suckling piglets could result in p a r t f r o n , t h e low ( n - 6) t o ( n - 3) r a t i o o f c o w ' s m i l k lipids ( r e l a t : v e e x c e s s o f ( n - 3)). N o w , l i n o l e n i c acid preferentially enters the pathways of bioconvers i o n o r / / - o x i d a t i o n [37] a n d is c o n s e q u e n t l y a p o o r source for membrane accretion. An impairment of the o x i d a t i v e p a t h w a y s in i n f a n t p i g l e t [38], c o n n e c t e d w i t h a relative excess of dietary (n - 3) by comparison with (n -6) fatty acids, could explained the appearance of l i n o l e n i c a c i d in m e m b r a n e p h o s p h o l i p i d s o f t h e i m m a ture intestine. O f p a r t i c u l a r i n t e r e s t is t h e p r o f i l e o f p l a s m a l o g e n derived DMA. Although intestinal mucosa phospho-
lipids a r e k n o w n t o be r e l a t i v e l y rich in alkyl- a n d a l k e n y l g l y c e r o p h o s p h o l i p i d s [39], a l t e r a t i o n s o f e t h e r lipids r e l a t e d t o p r o c e s s e s o f d i f f e r e n t i a t i o n a n d m a t u r a t i o n in t h e d e v e l o p i n g i n t e s t i n e h a v e n e v e r b e e n considered. Anyway, DMA (issued from transmethylation of total alkenylacyl-glycerophospholipids) are generally e x c l u d e d f r o m c u r r e n t m e m b r a n e lipid r e p o r t s . Taking into account this class of compound, an unexpected result was that tot& DMA significantly dec r e a s e d a s cells a s c e n d e d t h e villus c o l u m n a n d b e c a m e more differentiated. To some extent, decreasing content of total DMA compensated for increasing content of 18:2(n6 ) ( c o m p a r e F i g . 4 A a n d B). It m u s t b e n o t e d t h a t R e y n i e r e t al. [36] f o u n d s o m e w h a t o p p o s i t e results: n o r m a l d i f f e r e n t i a t e d h u m a n c o l o n i c cells c o n t a i n e d 2-fold m o r e p l a s m a l o g e n s in t h e i r g l y c e r o p h o s p h a t i d y l e t h a n o l a m i e : s ~han H ' I 2 9 cells, t h e l a t t e r b e -
TABLE Ill
Fatty acid composition in HPLC-isolatedphospholipid classesfrom purified brush border membranes Mean values from six piglets ( + S.D.)
Phosphdipid class (% w / w of total)
PE
S.D.
PC
S.D.
PS
S,D.
S.D.
SM
S.D.
35.4
3.5
37.3
4.6
I I. I
I. I
PI 4.0
0.7
I 1.9
1.8
Fatty acids (% w / w of total) 16:0 18:0 ,~ SFA
I0.0 16.5 29.3
0.6 0.6 1.0
21.4 18.2 46.3
2.8 1.3 2.3
8.6 29.8 44,3
1.1 1,4 2.8
12.1 27.1 43.7
].i 1.2 1.7
33.b 1!.8 59.8
8.1 1.7 7.7
16:1 n - 7 18:1 n - 9
!.3 22.0 1.9 26.8
0.4 0.6 0.5 0.8
0.6 29.0 !.6 34.7
0.1 0.9 0.2 0.8
0.7 23.9 1.5 28.5
0.1 t.6 0.3 1.6
0.7 25.0 !.5 29.7
0.1 i.! 0.1 0.7
0.7 16.3 0.9 26.0
0.2
18:1 n - 7 ,~ MUFA
20:3 n - 9
1.4
0.3
0.3
1.0
0.1
2.4
0.5
-
-
18:2 n - 6 20:9 n - 6 20:4 n - 6 22:4 n - 6 22:5 n - 6 .~ n - 6
9.8 0.7 6.7 1.2 0.4 19.4
2.0 0. i 0.4 0.2 0.1 1.9
6.4 0.5 2.0 0.3 0.3 I 1.6
0.2 0. I 0.2 0.2 0.9
8.1 1.2 4.3 0.7 0.6 15.4
1.2 0.8 0.1 0.4 0.8
4.5 1.4 8.9 0.5 0.2 15.7
0.2 0.2 0.6 0.1 0.1 0.9
3.5 0.2 0.9 0.3 5.5
0.8 0.1 0.8
18:3 n - 3 20:5 n - 3 22:5 n - 3 22:6 n - 3 Z n- 3
1.3 1.7 2.8 2.1 8.5
0.4 0.2 0.6 0.5
1.0 0.2 0.6 3.0 5.4
0.3 0.3
I.O 0.8 1.8 2.9 6.6
0.1 0. l 0.8 1.0
0.8 1.2 I. 1 1.6 5.2
O.l 0.4 0.1 0.3 0.6
0.4 0.2 0.3 2.2 3.1
0.2 0.3
n - 6/n - 3 20:4/18:2 20:3/20:4
2.3 0.7 0.2
0.3 d.2
2. I 0.3 0.2
0.1 -
2.3 0.5 0.2
0.3 0.2 -
3.0 2,0 0.3
0.3 0.1 -
1.8 0.3
0.3 0.1 -
14.0
1.2
1.1
0.2
1.7
0.8
0.9
0.2
0.8
0.3
29.2 3.4 52.4 11.0 3.8
0.7 0.9 0.9 0.7 0.2
,~ DMA Percent of total: DMA 16:0 DMA 16:1 DMA 18:0 DMA 18:1 DMA 18:2
-
3.1
348 ing differentiated o r not. T h e biological significance o f such evolutions is at p r e s e n t u n k n o w n . O t h e r p h o s p h o lipids of the alkyl variety w e r e not e x a m i n e d in the p r e s e n t study. Indeed, specific analyses are necessary to evaluate the respective roles o f e t h e r lipids in cell differentiation. It has bee~ p o s t u l a t e d t h a t plasmalog e n s m a y play a role in ion t r a n s p o r t system, in the control of w a t e r m o v e m e n t across p l a s m a m e m b r a n e s a n d in the p r o t e c t i o n o f biological m e m b r a n e s f r o m oxidative d a m a g e s [40-42]. P l a s m a l o g e n s m a y also function as a specific reservoir o f t h r o m b o x a n e a n d p r o s t a g l a n d i n p r e c u r s o r s [40]. T h u s , t h e putative role of e t h e r lipids in intestinal cell differentiation a n d the significance o f their evolution a l o n g the crypt-villus axis a r e o p e n to conjecture. C h a n g e s in fatty acid c o m p o s i t i o n d u r i n g intestinal cell differentiation m i g h t b e r e l a t e d to the a p p e a r a n c e of specific functions. T h e a p p a r e n t relation b e t w e e n increasing 1 8 : 2 ( n - 6) c o n t e n t a n d lactase specific activity d u r i n g differentiation provides evidence t h a t lipid compositional c h a n g e s c o u l d be involved in t h e n o r m a l process o f intestinal cell differentiation. By c o m p a r i s o n with previous studies, it c a n b e c o n c l u d e d t h a t this process o c c u r r e d in the d e v e l o p i n g intestine o f the n e w b o r n as well as in the m a t u r e intestine o f t h e a d u l t . Acknowledgments T h e m a n a g e m e n t o f the axenic piglet unit by Y. Duval-Iflah is gratefully a c k n o w l e d g e d . W e also t h a n k V. D u c r u e t , J. Th~venoux, A. L i n a r d a n d S. D e l p a l for technical assistance. T.S. Arfi w a s the recipient o f a g r a n t f r o m the F o n d a t i o n Fran~aise p o u r la Nutrition. P a r t of this w o r k was s u p p o r t e d by g r a n t s f r o m t h e ' A g r o b i o ' I N R A - p r o g r a m (projet Nutrition, S~curit~ Alimentaire). References 1 Moog, F. (1979) J. Anim. Sci. 49, 239-249. 2 Bouhours, D. and Bouhours, J.F. (1981) Biochem. Biophys. Res. Commun. 99, 1384-1389. 3 Bouhours, J.F. and Glickman, R.M. (1976) Biochim. Biophys. Acta 441,123-133. 4 Breimer, M.E., Hansson, G.C., Karlsson, K.A. and Leffler, H. (1981) Exp. Cell Res. 135, 1-13. 5 Brasitus, T.A. and Dudeja, P.K. (1985) Arch. Biochem. Biophys. 240, 483-488. 6 Alessandri, J.-M., Arfi, T.S., Thevenoux, J. and I..~ger, C.L. (1990) J. Pediatr. Gastroenterol. Nutr., 10, 504-515. 7 Pang, K-Y., Bresson. J.L. and Walker, W.A. (1983) Biochim. Biophys. Acta 727, 201-208. 8 Schwarz, S.M., Hostetler, B., Ling, S., Mone, M. and Watkins, J.B. (1985) Am. J. Physiol., 248, G200-G207.
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