Purified Brush Border Membrane Research Articles

Production, digestibility and allergenicity of hemp (Cannabis sativa L.) protein isolates

Hemp (Cannabis sativa L.), traditionally cultivated for industrial use and harvested for fibers and seeds, has raised much interest as a sustainable crop in the last years. Recently, hemp seeds and derived oil have started to be used in a variety of food products. Hemp-based food products are considered less allergenic than those from other edible seeds, although this statement has never been experimentally verified. In this study high purity grade hemp flour (HF) and hemp protein isolate (HPI) were obtained through a fast and cheap process starting from defatted hemp cakes, a residue of hempseed oil extraction. HPI resulted enriched at nearly 86% protein, mainly constituted by the storage protein edestin (accounting for 70% total protein). In vitro protein digestibility was determined using a static model of gastrointestinal digestion (GID), which included a final step with purified brush border membrane (BBM) enzyme preparations. HF and HPI showed a high degree of digestibility. The survival of potential bioactive and/or allergenic peptide sequences in digests was investigated by peptidomic analysis. Only a limited number of sequences survived GID. Among them, fragments from 12 seed proteins. These fragments were precursors of sequences with potential bioactive peptides, which might justify the bioactivity of HPI hydrolysates, reported in previous studies. More importantly, all known hemp allergens, including the major thaumatin-like protein and LTP, were entirely eliminated by the HPI production process, neither fragments of the proteins were present after GID. These data support the use of HPI as an ingredient for hypoallergenic foods.

Evaluation of Schizochytrium meal in microdiets of Pacific white shrimp ( Litopenaeus vannamei ) larvae

A feeding trial was conducted to assess the effects of dietary Schizochytrium meal supplementation on survival, growth performance, activities of digestive enzymes and fatty acid composition in Pacific white shrimp (Litopenaeus vannamei) larvae (initial body weight 4.21 ± 0.10 mg). Four isonitrogenous and isolipidic diets were formulated to contain graded levels of Schizochytrium meal: 0% (S0, the control diet), 2% (S2), 4% (S4) and 6% dry matter (S6). Results showed that there was no significant difference in survival of shrimps among dietary treatments (P > 0.05). Shrimps fed diets with 2% and 4% microalgae meal had significantly higher specific growth rate (SGR) than that of shrimps fed diets with 0% and 6% microalgae meal, and no significant differences were observed between shrimps fed diets with 2% and 4% microalgae meal (P > 0.05). Activity of trypsin in the pancreatic and intestinal segments, and activity of amylase in the pancreatic segments were not significantly affected by dietary microalgae meal levels (P > 0.05). Specific activities of both alkaline phosphatase and leucine-aminopeptidase in intestine and purified brush border membrane of intestine were significantly higher in shrimps fed diet with 2% microalgae meal (P < 0.05). There were no significant differences in C18:2n-6, n-3 fatty acids, n-6fatty acids, PUFA and n-3/n-6 in muscle samples among dietary treatments. C16:1n-7, C18:1n-9, MUFA, C18:3n-3 and C20:5n-3 decreased, however, C20:4n-6 increased in the muscle as dietary microalgae meal level increased. In conclusion, 4% Schizochytrium meal in microdiets of shrimps can improve growth performance and may be a valuable additive in the microdiets of shrimps.

Mechanisms Of Fructose Absorption

Mechanisms Of Fructose Absorption

Sodium/glucose cotransporter-1, sweet receptor, and disaccharidase expression in the intestine of the domestic dog and cat: two species of different dietary habit

The domestic cat (Felis catus), a carnivore, naturally eats a very low carbohydrate diet. In contrast, the dog (Canis familiaris), a carno-omnivore, has a varied diet. This study was performed to determine the expression of the intestinal brush border membrane sodium/glucose cotransporter, SGLT1, sweet receptor, T1R2/T1R3, and disaccharidases in these species adapted to contrasting diets. The expression (this includes function) of SGLT1, sucrase, maltase and lactase were determined using purified brush border membrane vesicles and by quantitative immunohistochemistry of fixed tissues. The pattern of expression of subunits of the sweet receptor T1R2 and T1R3 was assessed using fluorescent immunohistochemistry. In proximal, middle, and distal small intestine, SGLT1 function in dogs was 1.9- to 2.3-fold higher than in cats (P = 0.037, P = 0.0011, P = 0.027, respectively), and SGLT1 protein abundance followed an identical pattern. Both cats and dogs express T1R3 in a subset of intestinal epithelial cells, and dogs, but not cats, express T1R2. In proximal and middle regions, there were 3.1- and 1.6-fold higher lactase (P = 0.006 and P = 0.019), 4.4- and 2.9-fold higher sucrase (both P < 0.0001), and 4.6- and 3.1-fold higher maltase activity (P = 0.0026 and P = 0.0005), respectively, in the intestine of dogs compared with cats. Dogs have a potential higher capacity to digest and absorb carbohydrates than cats. Cats may suffer from carbohydrate malabsorption following ingestion of high-carbohydrate meals. However, dogs have a digestive ability to cope with diets containing significant levels of carbohydrate.

Quantification of myo‐inositol cotransporter expression in control and streptozotocin‐treated rats.

Myo‐inositol (MI) uptakes in cells is essentially performed by two Na+‐coupled transport systems; SMIT1, a basolateral system inducible through hypertonicity and SMIT2, an apical system responsible for MI uptake in the kidney and intestine, amongst other tissues. In diabetes, urinary MI excretion is increased by 1 order of magnitude through a non‐established mechanism. The purpose of this study is thus to investigate the influence of an acute diabetic condition (5‐day streptozotocintreatment) on the expression of both SMIT mRNA's in rat brain, liver, muscle, kidney and intestine through quantitative‐RT‐PCR (qRT‐PCR). Also, SMIT2 activity was determined by measuring MI transport in purified brush border membranes (BBMv) from kidney and intestine. SMIT1 transcripts are mainly found in the kidney where it doubles in diabetic rats. Other tissues show more modest mRNA levels and were not altered in diabetes. SMIT2 transcripts were found in all tested tissues in varying levels but were not modified in diabetes. MI uptakes performed on kidney and intestine BBMv confirmed the qRT‐PCR results showing no variations of transport in STZ rats due to SMIT2. We thus conclude that the increase in MI urinary excretion is not due to a reduction in the apical expression of MI cotransporter SMIT2.

In vitro translation of RNA to lactase during postnatal development of rat intestine

mRNA levels encoding lactase were detected by Northern blot analysis using two different probes in developing rat intestine. Probe I and probe II corresponding to second half of prolactase gene showed a 6.8 kb mRNA transcript in 7, 14, 21 and 30 day old rat intestine. There was no change in quantity of lactase mRNA detected using probe II, but hybridization with probe I showed a progressive decrease in mRNA transcript encoding lactase with age. At day 7 and 14 of postnatal development, the lactase mRNA was quite high, but it reduced upon weaning. The in vitro translation products of RNA detected by Western blot analysis using brush border lactase antibodies showed several isoforms of lactase antigen with molecular weight ranging from 100-220 kDa. Analysed at days 7 and 30 of postnatal development, lactase isoforms of molecular weight 130 kDa and 220 kDa were similar to those found in purified brush border membranes. The translation of RNA to 220 kDa lactase protein was high in 7 and 14 day old pups, but it was markedly reduced in 30 day old animals. These findings support the contention that translation of mRNA to lactase is impaired in weaned animals, which may also be responsible for the maturational decline in lactase activity in adult rat intestine.

Activities of selected digestive enzymes during larval development of large yellow croaker ( Pseudosciaena crocea)

Large yellow croaker is an important marine fish species for aquaculture in China. Larval rearing of this species is still based on live prey feeding. The aim of this study was to describe the onset and development of the main digestive enzymes during larval development of this species, in order to obtain essential data for the formulation of a compound diet adapted to larvae. Enzymatic assays were conducted from day 1 post hatching to day 40 in larvae fed the following live prey sequence: rotifers, Artemia nauplii, copepods, and then frozen copepods. Assays were done on whole larvae until day 19, then on dissected larvae and on purified brush border membranes of intestine. Pancreatic enzymes, amylase, and trypsin, showed high activities from early stages, even before mouth opening. Mechanisms of pancreatic secretion were achieved from day 19. Enzymes of brush border membranes, alkaline phosphatase, and aminopeptidase N, increased abruptly between day 23 and 25, showing maturation of the intestinal digestive process. These data, together with data describing mouth enlargement during development, suggest that large yellow croaker larvae could be fed successfully with a compound diet from early stages, at least from day 23.

Na+ gradient-dependent transport of hypoxanthine by calf intestinal brush border membrane vesicles.

The properties of hypoxanthine transport were investigated in purified brush border membrane vesicles isolated from calf proximal and distal jejunum. Hypoxanthine uptake in the vesicles was stimulated by a transmembrane Na(+) gradient and an inside negative potential resulting in a transient accumulation of intravesicular hypoxanthine, especially in the proximal jejunum. Na(+)-dependent hypoxanthine uptake at this site seemed to occur by two saturable transport systems, a high affinity (K(m)=0.33 micromol/l) and a low affinity (K(m)=165 micromol/l) transporter. Guanine, hypoxanthine, thymine and uracil inhibited intravesicular hypoxanthine uptake, whereas adenine and the nucleosides inosine and thymidine were without effect. These findings represent the first demonstration of active Na(+) gradient-dependent nucleobase transport in intestinal brush border membrane vesicles.

Immunolocalization of chitin synthase in the tobacco hornworm.

To start investigation of chitin synthesis and peritrophic membrane formation in the midgut of Manduca sexta, we have cloned a cDNA fragment encoding chitin synthase. Northern blots with a corresponding RNA probe revealed a single transcript of 4.7 kb, which was most prominent in poly(A) RNA isolated from the anterior and median midgut as well as from tracheal cells. In situ hybridization showed that the amount of chitin synthase transcripts in the cytoplasm of columnar cells decreased from the anterior to the posterior midgut. Moreover, in the anterior midgut they were localized in the apical region of columnar cells. Southern blots suggested more than one gene locus for chitin synthase in the Manducagenome. To analyze the distribution of chitin synthases on the protein level, we expressed a polymerase chain reaction (PCR) fragment of 119 amino acids in Escherichia coli and generated polyclonal antibodies to the purified recombinant protein. In immunoblots of crude extracts derived from the anterior midgut as well as from partially purified brush border membranes of columnar cells the affinity-purified anti-chitin synthase antiserum labeled a single protein with an apparent molecular mass of 150-200 kDa. Immunohistochemistry showed intense labeling in midgut brush border membranes. Immunofluorescence was restricted to the apical ends of microvilli. Apical membranes of salivary glands and tracheal cells were labeled as well, but not those of Malpighian tubules. This is the first time that chitin synthase expression has been visualized in insect tissues on the level of proteins.

Cloning and complete sequence characterization of two gypsy moth aminopeptidase-N cDNAs, including the receptor for Bacillus thuringiensis Cry1Ac toxin

The complete cDNAs corresponding to two distinct gypsy moth ( Lymantria dispar) larval gut aminopeptidases, APN1 and λAPN2, were cloned and sequenced. The 3.4 kilobasepair cDNA of APN1 which encodes a 1017 amino acid prepro-protein corresponds to the previously-identified gypsy moth APN (APN-1) that specifically binds the Cry1Ac δ-endotoxin of Bacillus thuringiensis. Analysis of the primary structure of APN1 revealed a cluster of five potential N-linked glycosylation sites near the N-terminus and a C-terminal sequence characteristic of a putative glycosylphosphatidyl-inositol (GPI) anchor signal sequence. The cDNA of APN1 encodes the N-terminal peptide sequence and nine internal sequences obtained from the purified brush border membrane vesicle Cry1Ac receptor by protein sequencing. The λAPN2 cDNA encodes a shorter protein with 51% similarity to APN1 that also appears to have a GPI anchor signal sequence. Expression of the APN1 cDNA in a baculovirus vector was confirmed by immunoblotting.

Sodium-Dependent Amino Acid Transport Is Preserved in Lyophilized Reconstituted Apical Membranes from Intestinal Epithelium

We demonstrate for the first time that functional electrogenic Na+-dependent amino acid transport is preserved for extended periods when purified brush border membranes prepared in hypotonic media are lyophilized and then rehydrated in buffer containing mannitol, NaSCN, and/or KSCN/valinomycin. Reconstituted lyophilized apical membranes from small intestine formed morphologically, physiologically, and thermodynamically normal vesicles which transportedl-alanine via system B into an osmotically active space energized by secondary active transport, as measured under equilibrium and nonequilibrium conditions. The lyophilized membranes are readily prepared and stored, thereby providing a means to pool large quantities of formed vesicles that are useful in examining cloned and reconstituted native amino acid transporter polypeptides.

Algal addition in sea bass ( Dicentrarchus labrax) larvae rearing: effect on digestive enzymes

Two groups of Dicentrarchus labrax were reared from day 5 to day 20 post-hatching with live prey in running seawater with or without algae. The concentration of algae Isochryisis galbana was maintained between 20 000 to 40 000 cells ml −1. On day 20, each group was divided into two subgroups, one still receiving live prey, the other fed a compound diet until day 32. On day 32, larvae fed live prey in the presence of algae exhibited an 18% higher survival than larvae reared without algae; in group fed compound diet, the algae supply induced a 26% survival enhancement. At day 16, it was observed a 40% weight improvement in the group reared in the presence of algae compared to the group reared without algae. At day 32, a slight but significant positive effect of algae on larvae weight was still noted in the group fed compound diet. From day 8 to day 16, the presence of algae in the rearing water resulted in an increase in trypsin activity whereas amylase and chymotrypsin were not affected. At day 26, alkaline phosphatase and maltase assayed in purified brush border membranes of intestine were significantly higher in larvae reared in seawater with algae than in those reared in clear water. This result suggests that the presence of algae facilitates the onset of hydrolytic functions of cell membranes. The significant improvement in survival of larvae reared in the presence of algae was related to the early development of brush border membranes. It is concluded that the algae acts by triggering digestive enzyme production, at both the pancreatic and intestinal level.

Streptozotocin diabetes and the expression of GLUT1 at the brush border and basolateral membranes of intestinal enterocytes

Changes in membrane expression of sodium-dependent glucose transporter (SGLT1) and glucose transporter isoform (GLUT2) protein have been implicated in the increased intestinal glucose transport in streptozotocin-diabetes. The possible involvement of GLUT1 in the transport response, however, has not previously been studied. Using confocal microscopy on tissue sections and Western blotting of purified brush border membrane (BBM) and basolateral membrane (BLM), we have examined enterocyte expression of GLUT1 in untreated and in 1 and 21 day streptozotocin diabetic rats. In control enterocytes, GLUT1 was absent at the BBM and detected at low levels at the BLM. Diabetes resulted in a 4- to 5-fold increased expression of GLUT1 at the BLM and the protein could also be readily detected at the BBM. Insulin treatment of diabetic rats increased GLUT1 level at the BBM but was without effect on expression of the protein at the BLM.

The renal brush border membrane sodium/sulfate cotransporter functions in situ as a homotetramer

The functional molecular size of the renal Na + SO 4 2− cotransporter was analysed with the radiation inactivation and fragmentation method. Purified brush border membrane vesicles preserved in a cryoprotective medium were exposed to γ-radiations. Initial rates of SO 4 2− influx into these vesicles were estimated with membranes irradiated with 0, 4 and 8 Mrad. In each case, SO 4 2− uptake by irradiated membranes was significantly reduced but remained linear during the first 5 sec of incubation. To avoid artifacts arising from a decrease in the driving force caused by modifications in membrane permeability, this incubation period was chosen to measure the effect of irradiation on the SO 4 2− transport activity. Increasing irradiation doses resulted in a monoexponential decrease in transport activity allowing the molecular size to be estimated at 238 ± 6 kDa (SD, n = 3). Recently, a cDNA for the Na + /SO 4 2− cotransporter was cloned and expressed in Xenopus laevis oocytes (Markovich D. et al. (1993) Proc. Natl Acad. Sci. U.S.A. 90, 8073–8077). The deduced amino acid sequence of this cotransporter predicts a molecular weight of 66 kDa. We suggest that the in situ activity of the renal brush border membrane Na + SO 4 2− cotransporter requires the presence of four intact and identical subunits arranged as a homotetramer.

IMMUNOLOCALIZATION OF AN ANTIGEN ASSOCIATED WITH THE INVERTEBRATE ELECTROGENIC 2Na+/1H+ ANTIPORTER

Epithelial plasma membranes from crustacean gut, kidney and gills have been shown recently to display an electrogenic 2Na+/1H+ antiporter that differs considerably in its physiological properties from the vertebrate electroneutral 1Na+/1H+ exchange paradigm. In this study, we describe the histological and cytological localization of an antigen associated with invertebrate electrogenic 2Na+/1H+ antiport in lobster (Homarus americanus) tissues using a monoclonal antibody (MAb 11) raised in mice against purified brush border membranes of the hepatopancreatic epithelium. Previous work showed that MAb 11 inhibited electrogenic 2Na+/1H+ and Ca2+/H+ exchange by hepatopancreatic brush border membrane vesicles, but was without effect on Na+-dependent d-glucose transport, suggesting a restricted inhibitory specificity to the cation exchanger. MAb 11 binding occurred at hepatopancreatic epithelial R-cell brush border membranes, at plasma membranes of the antennal gland and gill podocytes, and at vacuolar membranes of hepatopancreatic B- and R-cells, gill nephrocytes and epithelial cells of the antennal gland labyrinth and gill lamellae, as assessed by FITC-labelled secondary antibodies. Control FITC-labelled antibodies raised in mice against vertebrate keratin proteins displayed only weak non-specific binding to the tissues and cells responding intensely to MAb 11, supporting the specific nature of MAb 11 binding to its cognate antigen. The broad histological and cytological distribution of MAb 11 binding to plasma membranes and vacuolar membranes from several lobster organ systems suggests that the physiological activities regulated by its antigen, possibly an element of the invertebrate electrogenic cation exchanger, may be diverse.

Effect of grape seed tannins on the activity of some rat intestinal enzyme activities.

The present study was designed to investigate the effects of grape seed tannins on rat intestinal alkaline phosphatase (AP), sucrase and dipeptidyl peptidase IV (DPP IV) activities. An experiment was performed in vivo by dietary supplementation with 2% tannins; this diet was tested on an experimental group of rats; a control group received a diet without tannins. After 31 days, tannins intake significantly decreased middle-jejunal AP from 123 to 45 mU/mg protein and sucrase activities from 310 to 195 mU/mg protein, while no significant difference appeared at the duodenal stage (p < 0.05). Ileal DPP IV activity was also significantly reduced (p < 0.05) from 190 to 110 mU/mg protein after tannin intake. Using in vitro experiments on purified brush border membranes, AP activity was found to be inhibited by grape tannins; this inhibition was prevented by the detergent Triton X-100. The addition of pancreatic-biliary (PB) juice to the incubation medium prevented or reversed the tannin-inhibited enzyme activity. The present data indicate that in the duodenal lumen, alkalinity and detergency from the PB secretion neutralized the ability of tannins to inactivate brush border hydrolase activities and suggest that enzyme inhibition took place once bile salts were reabsorbed while moving down the gut. This was confirmed by in vitro experiments where sucrase and DPP IV activities inhibited by grape seed tannins were largely recovered after the addition of PB juice to the incubation medium.

Partial purification and characterization of the human placental serotonin transporter

The human placental serotonin transporter was solubilized from purified brush border membranes using digitonin as the solubilizing agent. The solubilizate was subjected to wheat germ agglutinin-Sepharose 6B column chromatography, Centricon-100 ultrafiltration and Sepharose 6B gel filtration to yield a partially purified preparation of the serotonin transporter. Specific binding of the high affinity ligand paroxetine was used to monitor the transporter during the solubilization and the purification steps. The enrichment of paroxetine binding in the final preparation was 51-fold compared to the intact brush border membranes, taking into account the inactivation that occurred during purification. The partially purified transporter exhibited paroxetine binding characteristics which were similar to those of the transporter in intact membranes. The transporter in the partially purified preparation bound paroxetine with a high affinity (dissociation constant, 0.21 nM). The binding was inhibitable by serotonin but not by other monoamines, dopamine and norepinephrine, nor by the serotonin precursor 5-hydroxytryptophan. The antidepressants, imipramine, fluoxetine and desipramine inhibited the binding with a rank order of potency of imipramine = fluoxetine > desipramine. The approximate molecular weight of the transporter was assessed by molecular sieve chromatography on Sepharose 6B and was found to be 300,000. When reconstituted into proteoliposomes, the partially purified transporter was able to catalyse NaCl-dependent serotonin transport in these proteoliposomes. The results of this study show that the human placental serotonin transporter can be solubilized, partially purified and reconstituted in a transport-competent form and, in addition, provide some insight into the protein nature of the transporter.

Mechanism of Transport of Riboflavin in Rabbit Intestinal Brush Border Membrane Vesicles

Uptake of luminal riboflavin (RF) into the absorptive cells of rabbit small intestine was examined using purified brush border membrane vesicle (BBMV) preparations. These preparations were used in order to eliminate the interference of intracellular metabolism that occurs to the RF molecule during absorption. Uptake of RF by BBMV was found to be mainly (> 76%) the result of transport of the vitamin into the intracellular space with less binding to membrane surfaces. All 3H radioactivity that appeared in the intravesicular space after incubation with [3H]RF was found to be in the form of intact RF. Uptake of RF with time was independent of the presence or absence of a Na+ or a K+ gradient (out > in) and occurred without transaccumulation of the substrate in the intravesicular space. Furthermore, changing the incubation buffer pH showed minimal effect on RF uptake. When examined as a function of concentration, the initial rate of RF uptake was found to be saturable both in jejunal and ileal BBMV with an apparent Km of 7.24 +/- 1.06 and 8.88 +/- 0.90 microM and Vmax of 24.31 +/- 1.48 and 34.24 +/- 1.55 pmol/mg protein/5 sec, respectively. Unlabeled RF and the related compounds lumiflavin, 8-aminoriboflavin, isoriboflavin, and lumichrome all inhibited (but to different degrees) the uptake of physiologic concentration of [3H]RF. On the other hand, 8-hydroxyriboflavin, lumazine, and D-ribose all failed to inhibit [3H]RF uptake. Similarly, the membrane transport inhibitors DIDS, SITS, and furosemide all failed to inhibit [3H]RF uptake. The uptake of RF was found to be insensitive to changes in the transmembrane electrical potential, as shown by studies with anion substitution and valinomycin K(+)-induced negative or positive intravesicular potential methodologies. These results indicate that RF uptake by rabbit intestinal BBMV occurs via a carrier-mediated system that is Na+ independent in nature and transports the substrate by an electroneutral process. The role of this system in the overall absorption process of RF is discussed.

A retinyl ester hydrolase activity intrinsic to the brush border membrane of rat small intestine.

Retinol esterified with long-chain fatty acids is a common dietary source of vitamin A. Hydrolysis of these esters in the lumen of the small intestine is required prior to absorption. Bile salt-stimulated retinyl esterase activity was present with purified rat intestinal brush border membrane, with the maximum rate of ester hydrolysis at approximately pH 8, the physiological luminal pH. Taurocholate, a trihydroxy bile salt, stimulated hydrolysis of short-chain fatty acyl retinyl esters more than hydrolysis of long-chain fatty acyl esters. Deoxycholate, a dihydroxy bile salt, primarily stimulated hydrolysis of long-chain esters. Calculated Kms of 0.74 microM for retinyl palmitate (16:0) hydrolysis and 9.6 microM for retinyl caproate (6:0) hydrolysis suggested the presence of two separate activities. Consistent with that, the activity responsible for retinyl caproate hydrolysis could be inactivated to a greater degree than retinyl palmitate hydrolysis by preincubation of the brush border membrane at 37 degrees C for extended times. Brush border membrane from animals who had undergone common duct ligation 48 h prior to tissue collection showed little ability to hydrolyze retinyl caproate but retained 70% of retinyl palmitate hydrolytic activity, compared to sham-operated controls. Thus, two distinguishable retinyl esterase activities were recovered with purified brush border membranes. One apparently originated from the pancreas, was stimulated by trihydroxy bile salts, and preferentially hydrolyzed short-chain retinyl esters, properties similar to cholesterol ester hydrolase, known to bind to the brush border. The other was intrinsic to the brush border, stimulated by both trihydroxy and dihydroxy bile salts, and preferentially hydrolyzed long-chain retinyl esters, providing the majority of activity of the brush border against dietary retinyl esters.

Changes in fatty acid composition during cell differentiation in the small intestine of suckling piglets

Alterations of phospholipid fatty acid composition in the renewing intestine were studied in the infant piglet. 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 mucosa, isolated intestinal cells and purified brush border membranes. Intestinal cells were isolated according 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 those of lactase activity in the corresponding cell homogenates. In cell phospholipids, the relative content of linoleic and linoleic acids increased about 2-fold from crypt base to villus tip. Substantial contents of alkenylacyl glycerophospholipids (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 alterations might be related to the appearance of specific functions.