The Journal of Nutrition Vol. 128 No. 2 February 1998, pp. 224-233

Severe Malnutrition Alters Lipid Composition and Fatty Acid Profile of Small Intestine in Newborn Piglets^1,2

José M. Lopez-Pedrosa^{*, 3, 4}, María I. Torres, María I. Fernández, Antonio Ríos, and Angel Gil^{*, 5}

^* Department of Biochemistry and Molecular Biology and  Department of Cell Biology, University of Granada, 18071 Granada, Spain

	ABSTRACT

Abstract Introduction Methods Results Discussion References

The goal of this study was to evaluate the influence of severe protein-energy malnutrition (PEM) on lipid composition and fatty acid profile in the small intestinal mucosa of lactating pigs. Malnutrition was achieved by 80% protein-energy restriction for 30 d (20% of the food intake in the control group) in 7-d-old newborn piglets. Malnourished piglets had significantly lower concentrations of cholesterol, phospholipid and triglycerides in the jejunum and ileum compared with freely fed controls. Fatty acid composition of the intestinal mucosa was severely affected by malnutrition. A sharp decline in the relative percentages of (n-3) and (n-6) long-chain polyunsaturated fatty acids (LC-PUFA) in malnourished piglets paralleled higher (n-9) fatty acid proportions in the total mucosa, microsomes and phospholipids of the jejunum. The structure of the small intestine was severely affected as assessed by light and electron microscopy, and alkaline phosphatase and disaccharidase activities in the intestinal mucosa were also significantly impaired. Plasma from malnourished piglets had significantly lower concentrations of (n-3) and (n-6) LC-PUFA than that of control piglets; however, the fatty acid composition of red blood cell membrane was unaffected. Our results suggest that early severe PEM dramatically modifies intestinal membrane lipid composition. Changes in the lipid composition of the small intestinal mucosa and in phospholipid distribution as well as in the fatty acid profile may alter membrane fluidity and organization. These alterations appear to affect the activity of membrane-bound hydrolytic enzymes.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Epithelial cells of the gastrointestinal tract undergo biochemical, ultrastructural and morphologic changes during the suckling period, leading to the development of mature mucosa (Henning 1987). The process of maturation represents a coordinated series of events apparently directed toward maximizing the utilization of dietary nutrients. Changes in mucosal lipid composition have been shown to occur during differentiation and migration of cells from the crypt base to the villlus tip in the small intestine (Alessandri et al. 1990). The major compositional changes in the small intestine are increased phospholipid, cholesterol and glycosphingo-lipid contents along the crypt-villus axis. These modifications, together with the changes in the distribution of phospholipid species and fatty acid composition, are associated with a reduction in plasma membrane fluidity and decreased luminal membrane permeability to macromolecules (Meddings and Theisen 1989).

Malnutrition during periods of hyperplastic organ growth slows the rate of cell division in pigs (Pond et al. 1990 and 1996). Protein-energy malnutrition (PEM)⁶ dramatically affects the development of the intestinal mucosa during the postnatal period. A low protein diet causes a reduction in the rate of protein synthesis in most body tissues, with the most marked changes occurring in skin and intestine (Wykes et al. 1996). Biochemical and histologic changes in the gut, observed in malnourished humans and animals, result in a lower intestinal surface, lymphocyte infiltration at the lamina propria, reduced amino acid uptake and changes in the activity of membrane-bound enzymes such as disaccharidases (Butzner and Gall 1990, Gupta et al. 1994). A previous study in our laboratory found that malnutrition induced in nursing piglets by severe dietary restriction reduced the amount of DNA and protein, and lowered segmental disaccharidase and leucine aminopeptidase activities in the small intestine (Nuñez et al. 1996). Many studies have also shown similar metabolic and structural alterations in the small intestine of experimental animals affected by malnutrition (Castillo et al. 1991, Nuñez et al. 1990).

In humans, PEM is often associated with infection, a factor that limits the study of the effects of protein-energy restriction itself on the structure and function of the small intestine. In addition, the availability of small intestine specimens is very limited, even in humans with gastrointestinal diseases. Infant pigs have been used as a model in biomedical and pediatric research because of their physiologic and biochemical similarities to humans during the perinatal period. The pattern of lipids and fatty acids in the piglet intestine resembles that reported for humans (Alessandri et al. 1991).

The effects of early moderate PEM on lipid and fatty acid composition in the liver, plasma and erythrocyte phospholipids in animals and children have been described (Bouzine et al. 1994, Wolff et al. 1984). However, the influence of severe PEM on the lipid composition and fatty acid profile of the small intestinal mucosa remains unknown. The purpose of this study was to evaluate the alterations in lipid composition and the fatty acid pattern in the total mucosa and microsomes from the jejunum and ileum in severely malnourished newborn piglets for 30 d. We also studied the alterations in DNA and protein content of the intestinal mucosa, as well as disaccharidase and alkaline phosphatase activities and the changes in histologic features of the small intestine caused by severe PEM.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Chemicals.  Unless otherwise stated, biochemicals were purchased from Sigma Chemical (St. Louis, MO). All other chemicals were reagent grade or higher and were obtained from commercial sources.

Animals and diets.  Yorkshire piglets, 7 d old, were provided by a certified farm (Ntra. Sra. de las Mercedes, Jaen, Spain). The piglets were randomly assigned to one of two groups: the first (control group, C) (n = 6) was freely fed with recombined milk formula (188 g/L) by nipple, three times a day for a period of 30 d and the daily intake was recorded. When diluted, the formula contained 47.9 g protein, 55.1 g carbohydrate, 68 g fat and a total energy density of 4.28 MJ/L. Another group (malnourished group, M) (n = 6) was fed the same diet, but they received only 20% of the milk intake recorded in C. In addition, they received freely a glucose-saline solution to fully satisfy water requirements. The composition of the glucose-saline solution was as follows: 5 g/L glucose, 3.5 g/L sodium chloride, 1.1 g/L potassium chloride and 2.5 g/L sodium bicarbonate. The daily average of energy supplied by milk formula in group C ranged from 4.07 MJ at the beginning of the study to 9.37 MJ at the end of the experiment; the glucose-saline solution supplied 15 kJ.

The semipurified standard diet used in this study was prepared in accordance with NRC recommendations (Miller and Ullery 1987) by the Department of Research and Development of Abbott Laboratories, Granada, Spain. The chemical and fatty acid composition of the diet is shown in Table 1. All procedures involving piglets were approved by the Animal Care Committee at the University of Granada and conformed with current European Union Regulations on Animal Care for the care and use of animals for research.

Sample collection and analysis.  On d 37 of age, after 16 h of food deprivation, the piglets were bled to death by jugular vein puncture while under anesthesia, and 250 mL of blood was collected in glass bottles with tripotassium EDTA as an anticoagulant. After plasma and red blood cells were separated by centrifugation at 1500 × g for 15 min, erythrocyte membranes were obtained using the method of Burton et al. (1981). Plasma and red blood cell membranes were rapidly frozen in liquid nitrogen, then stored at 80°C until analysis.

Plasma protein was measured using the biuret method (Reinhold et al. 1953). Total plasma albumin was separated from plasma globulins by cellulose acetate electrophoresis. Separated bands stained with Ponceau S were measured by densitometric scanning at 530 nm and quantitated as a function of the total protein value.

The entire small intestine was quickly removed. A 5-cm segment of the small intestine from the ligament of Treitz was selected for histologic analysis. The next 60 cm was considered the proximal jejunum for biochemical measurements. The 60-cm length closest to the ileocecal valve was considered the distal ileum. The intestinal segments were rinsed thoroughly with ice-cold saline solution, opened lengthwise and blotted dry. The mucosa were removed by scrapping the entire luminal surface with a glass coverslip and then weighed. A portion of jejunal mucosa was homogenized with buffer (10 mmol/L HEPES, 0.25 mol/L sucrase, 50 mmol/L NaCl, 20 mmol/L EDTA, 5 mmol/L dithiothreitol and 50 µmol/L leupeptin, pH 7.4) (1:4, wt/vol); the microsomal fraction was prepared by the method of Philip and Saphiro (1979). Briefly, the mucosal homogenate was centrifuged at 5000 × g for 15 min and the supernatant was centrifuged again at 15,000 × g for 15 min. The resulting supernatant was again centrifuged at 105, 000 × g for 60 min. The pellet was resuspended in the homogenizing buffer and stored at 80°C until analysis.

Mucosa from the jejunum and ileum were homogenized in distilled water for protein and enzymatic assays in 50 mmol/L phosphate-buffer, 2 mol/L NaCl and 2 mmol/L EDTA (pH 7.4) for DNA analysis. Concentrations of intestinal mucosa protein and DNA were determined using the methods of Bradford (1976) and Labarca and Paigen (1980), respectively. The activities of sucrase (EC 3.2.1.48), lactase (EC 3.2.1.23) and maltase (EC 3.2.1.20) in mucosa were determined using the method of Dahlqvist (1968); alkaline phosphatase activity (EC 3.1.3.1) was measured according to Goldstein et al. (1970).

Total lipids in jejunal and ileal mucosa were extracted according to the procedure of Kolarovic and Fournier (1986). Jejunal mucosa phospholipids were separated by thin-layer chromatography (Skipsky and Barclay 1969). Cholesterol and triglycerides were measured in the lipid fraction by using enzymatic methods (test kits Chol MPR1, test-combination free cholesterol No. 310328, and triglycerides GPO-PAP Ref. 701882, Boehringer-Mannhein, Mannhein, Germany). Phospholipid content in the mucosa was evaluated by measuring inorganic phosphorous (Zilversmit 1950). Phospholipid species were separated by high performance thin-layer chromatography (HPTLC) and quantitated by densitometry (Macala et al. 1983). Fatty acids from total mucosa, phospholipid and microsomes from mucosa, as well as fatty acids from plasma and red blood cell membranes, were saponified and methylated using the method of Lepage and Roy (1986) and separated and quantified by capillary gas-liquid chromatography. We used a Hewlett-Packard model 5890 gas chromatograph (Hewlett-Packard, Palo Alto, CA) equipped with a flame ionization detector and a 0.25-mm bore, 30-m capillary column filled with SP2330 stationary phase. Fatty acid methyl esters were identified by comparing their retention times with those of authentic standards.

Histologic analysis.  Small intestine samples were examined by light and electron transmission microscopy (ETM). For light microscopy, the samples were fixed in 40 g/L paraformaldehyde in phosphate buffered solution, dehydrated and embedded in paraffin; 5-µm sections were stained with periodic acid-Schiff-alcian blue, pH 2.5. Samples for ETM were fixed in 30 g/L glutaraldehyde in 0.1 mol/L sodium cacodylate buffer, pH 7.3, and postfixed in 15 g/L osmium tetroxide. Finally, the samples were dehydrated in acetone and embedded in Epon 812 resin. Ultrathin sections were doubled-stained with uranyl acetate and lead citrate, and examined under a Zeiss 902 transmission electron microscope (Zeiss, Oberkochen, Germany).

Statistical analysis.  Values in the text are means ± SEM. We tested for homogeneity of variances with Levene's test; in some cases (i.e., fatty acid composition and alkaline phosphatase activity), variances were heteregeneous and data were transformed to natural logarithms. The mean differences between the control and malnourished groups were tested by unpaired Student's t tests. Statistical analyses were performed using the PC-90 version of the BMDP Statistical Software (Los Angeles, CA) (Dixon et al. 1990).

	RESULTS

Abstract Introduction Methods Results Discussion References

The average protein and energy consumed by control and malnourished piglets during the protein-energy restriction period are shown in Figure 1. The 80% reduction in dietary intake in newborn pigs for a period of 30 d led to a significantly lower body weight (9.12 ± 0.75 vs. 4.02 ± 0.27 kg, P < 0.001; C vs. M). This was accompanied by a significantly lower weight per length of both the jejunal and ileal mucosa (jejunum: 175 ± 9 vs. 73 ± 11 mg/cm, P < 0.001; ileum: 169 ± 8 vs. 102 ± 8 mg/cm, P < 0.001; C vs. M).

View larger version (17K): [in this window] [in a new window]   Fig 1. Absolute (upper panel) and relative (lower panel) protein and energy intake in control and protein-energy malnourished piglets. Results are expressed as means ± SEM, n = 6. C: Control group fed an adapted pig milk formula from 7 to 37 d after birth. M: Malnourished group had 20% of the dietary intake of the C group for the same period.

There were no significant differences in total plasma protein (59.7 ± 5.4 vs. 62.0 ± 6.8 g/L), or in plasma albumin (21.6 ± 1.2 vs. 24.4 ± 2.9 g/L) between control and malnourished piglets. The amounts of DNA and protein in mucosa per length were significantly lower in malnourished piglets compared with controls (Table 2). However, the protein-DNA ratio was not affected by PEM in any intestinal segment (not shown). The protein concentration, expressed per intestinal mucosa weight, but not DNA concentration, was lower in the malnourished group than that observed in the control group (jejunum protein: 113 ± 3.3 vs. 84.6 ± 8.2 mg/g, P < 0.05; jejunum DNA: 13.17 ± 0.89 vs. 11.3 ± 0.86; C vs. M). Intestinal disaccharidase and alkaline phosphatase activities generally were lower in malnourished piglets although maltase and alkaline phosphatase activities were not significantly affected in the ileum (Table 2). Nevertheless, specific activities of these enzymes were significantly higher in malnourished piglets than in controls in the jejunal segment; alkaline phosphatase activity was also higher in the ileum (Table 3).

Malnourished piglets had severe histological alterations in the small intestine (Figs. 2 and 3). The mucosa showed atrophy with enterocyte losses and shortened villi. Goblet cells contained lower levels of mucin, and many of them appeared emptied. In addition, the cryptae lumen contained large amounts of glycoproteins (mucin); sections were stained with periodic acid-Schiff-alcian blue (Fig. 2). Ultrastructural examination with ETM showed clear cytoplasmic zones and swollen mitochondria with low cristae. Intercellular spaces were dilated and intraepithelial lymphocyte infiltration was evident (Fig. 3).

View larger version (188K): [in this window] [in a new window]   View larger version (194K): [in this window] [in a new window]   Fig 2. Light micrographs of jejunum from malnourished (M) and control piglets (C). Sections (5 µm) were stained with periodic acid-Schiff-alcian blue. The intestinal mucosa of malnourished piglets showed an atrophic structure with shortened villi; goblet cells contained a low level of mucin and many of them appeared emptied compared with controls. Villi (); goblet cell (). Bars: 100 µm.

View larger version (198K): [in this window] [in a new window]   View larger version (197K): [in this window] [in a new window]   Fig 3. Electron transmission micrographs of enterocyte cytoplasm from control (C) and malnourished piglets (M). The cytoplasm of malnourished piglets shows vesiculation with clear zones (); intraepithelial lymphocyte infiltration was evident compared with controls. Bars: 3 µm.

Cholesterol, phospholipid and triglyceride levels were significantly lower in both intestinal segments of malnourished piglets than in controls (Fig. 4). The cholesterol/phospholipid ratio and the phospholipid composition of the jejunal mucosa were significantly different in the two groups (Table 4). The proportion of lysophosphatidylcholine was higher, whereas the proportions of phosphatidylcholine and phosphatidylinositol were lower in the malnourished group. However, in the ileum, neither the molar ratio of cholesterol to phospholipid nor the distribution of phospholipid species differed significantly between the two groups (Table 4). The jejunum sphingomyelin/phosphatidylcholine ratio was higher in malnourished than in control piglets (0.23 ± 0.03 vs. 0.19 ± 0.01, P < 0.05); no differences were observed for the ileum.

View larger version (16K): [in this window] [in a new window]   Fig 4. Cholesterol (CHOL), phospholipid (PL) and triglyceride (TG) in jejunum and ileum of control (C) and protein-energy malnourished (M) piglets. Results are expressed as means ± SEM, n = 6. C: Control group fed an adapted pig milk formula from 7 to 37 d after birth. M: Malnourished group had 20% of the dietary intake of the C group for the same period. **P < 0.01; ***P < 0.001, significantly different than the C group.

The fatty acid composition of the intestinal mucosa was severely affected in the infant malnourished piglets (Table 5). In the jejunal mucosa, the most notable differences were a higher proportion of oleic acid and lower percentages of arachidonic and total (n-3) long-chain polyunsaturated fatty acids (LC-PUFA), namely, eicosapentaenoic [20:5(n-3)] and docosahexaenoic [22:6(n-3)] acids. In the ileal mucosa, the total saturated fatty acids were significantly lower in malnourished than in control piglets.

To further determine whether the differences in the total lipid fatty acid pattern of the intestinal mucosa reflected specific changes in mucosal phospholipids and microsomes, we analyzed fatty acids in these fractions. Total mucosa, microsomes and mucosa phospholipids in the jejunum exhibited the same fatty acid pattern (Fig. 5). Malnutrition resulted in lower percentages of (n-3) and (n-6) LC-PUFA and concomitantly higher proportions of monounsaturated fatty acids (MUFA). The 18:2(n-6)/20:4(n-6) ratio was significantly higher in malnourished piglets than in controls (Fig. 5).

View larger version (33K): [in this window] [in a new window]   Fig 5. Long-chain (n-6) and (n-3) polyunsaturated fatty acids (LC-PUFA), total monounsaturated fatty acids (MUFA) and linoleic/arachidonic acid ratios [18:2(n-6)/20:4(n-6)] in total mucosa, microsomes and mucosa phospholipids of jejunum of control (C) and protein-energy malnourished (M) piglets. Results are expressed as means ± SEM, n = 6. C: Control group fed an adapted pig milk formula from 7 to 37 d after birth. M: Malnourished group had 20% of the dietary intake of the C group for the same period (n = 6). *P < 0.05, **P < 0.01; *** P < 0.001, significantly different than the C group.

Malnourished piglets had significantly lower levels of saturated fatty acids (SFA), MUFA and (n-3) and (n-6) LC-PUFA in plasma than control piglets (Fig. 6). The fatty acid composition of red blood cell membrane phospholipids was unaffected by malnutrition (results not shown).

View larger version (14K): [in this window] [in a new window]   Fig 6. Levels of total saturated fatty acids (SFA), total monounsaturated fatty acids (MUFA), and long-chain (n-6) and (n-3) polyunsaturated fatty acids (LC-PUFA) in plasma of control and protein-energy malnourished piglets. Results are expressed as means ± SEM in mol/100 mol, n = 6. C: Control group fed an adapted pig milk formula from 7 to 37 d after birth (n = 6). M: Malnourished group had 20% of the dietary intake of the C group for the same period. *P < 0.05, significantly different than the C group.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Reducing the protein-energy intake in piglets to 20% of that of the control group led to severe malnutrition, with the characteristics of infant marasmus. Body weight in malnourished piglets was 50% less than in healthy piglets, and the weights of the small intestine and mucosa were also severely reduced. These results agree with those recently obtained by Nuñez et al. (1996), who showed that restricting the diet in nursing piglets affects body and small intestine weights and rates of growth.

In our experimental model of PEM, there were no significant differences in total plasma protein and albumin between the two groups. A number of studies have found similar results in squirrel monkeys fed low energy diets and in nursing infants with PEM (Ausman et at. 1989, Hayden et al. 1994). Low levels of plasma albumin (22 g/L) have been described in children with kwashiorkor syndrome (Coward and Whitehead 1972); however, healthy piglets have an albumin concentration of 22 g/L at 5 wk of age (Miller and Ullery 1987). Hypoproteinemia and hypoalbuminemia occur more often under conditions of protein restriction than protein-energy restriction. Protein malnutrition in humans and other animals (e.g., rats, rodents or pigs) is consistently associated with lower concentrations of serum total protein and serum albumin, leading to kwashiorkor (Pond et al. 1992, Spiekerman 1993).

The intestine of malnourished piglets contained lower amounts of both protein and DNA per length of mucosa than that of controls, although a protein/DNA ratio close to that of control animals was maintained. Nevertheless, only the protein concentration, but not DNA concentration was lower in the malnourished group than that observed in the control group; these results suggest that the overall processes of protein and DNA synthesis in the small intestine of malnourished piglets are impaired, causing mucosal hypotrophy because of a reduction in the number of cells, rather than a reduction in cell size. There is apparently an adaptation of piglets to protein-energy restriction in which mucosa protein synthesis rate would decrease, thereby permiting the organism to maintain nitrogen balance (Wykes et al. 1996). Pond et al. (1996) previously showed that concentrations of protein and DNA in the liver and jejunum of protein-depleted pigs were lower than in control pigs at 7 wk of age. We observed histological changes, i.e., atrophy with losses and shortening of the villi in the small intestine of malnourished piglets. Previous studies in animals and humans with PEM have demonstrated that histologic alterations in the intestinal mucosa alter disaccharidases, alkaline phosphatase and other enzyme activities in the jejunal and ileal mucosa (Nuñez et al. 1996, Omoike et al. 1990). This pattern, only in jejunum, was confirmed in this study. The segmental activities of disaccharidases and alkaline posphatase were lower in the jejunum of malnourished piglets than in control piglets (Table 2). The high specific activity of the disaccharidases and alkaline posphatase in malnourished piglets suggests that those proteins involved in nutrient digestion are preferentialy maintained relative to other proteins of the intestine. These results agree with those recently obtained by Nuñez et al. (1996), who showed that specific activities of disaccharidases and leucine aminopeptidase in the pig small intestine increase in protein-energy malnutrition. Nuñez et al. (1990) also found that malnutrition associated with chronic diarrhea in rats led to low segmental activities of disaccharidases in the jejunum and ileum, with high specific activities.

Changes in mucosal lipid composition were shown to occur in the small intestine of mammalian neonates during postnatal development. The major compositional changes were an increase in cholesterol and phospholipid contents and a redistribution of phospholipid species within the membrane (Meddings and Theisen 1989). The most striking changes regarding fatty acid composition consisted of a greater incorporation of linoleic and arachidonic acids into total phospholipids of the mucosa (Alessandri et al. 1991). The modification in both cholesterol and phospholipid contents, and in chain length and the degree of saturation of fatty acids in mucosal phospholipid, may have a cumulative effect on the lipid contribution to membrane fluidity (Shu-Heh et al. 1988). In our study, cholesterol, phospholipids and triglycerides were lower in jejunal and ileal mucosa of malnourished piglets than in control animals. A greater reduction in phospholipid than in cholesterol levels in the jejunum led to a higher cholesterol/phospholipid (C/P) ratio in the malnourished group compared with controls. The higher C/P and sphingomyelin/phosphatidylcholine ratios found in the small intestinal mucosa of malnourished piglets suggest that severe alterations of enterocyte membrane fluidity occur in PEM. Previous studies have demonstrated that food deprivation was associated with alterations in cholesterol and phospholipid contents of the intestinal brush border membrane, which resulted in a dramatic modification in membrane fluidity (Keelan et al. 1985). Our results agree with those recently obtained by Gupta et al. (1994), who showed a decrease in the concentrations of cholesterol and phospholipids and an increase in the C/P and sphingomyelin/phosphatidylcholine ratios in the small intestine of monkeys during severe food deprivation (10-12 wk); the changes in lipid composition of the small intestine altered alkaline phosphatase and enterokinase activities in the brush border membrane.

The jejunal mucosa in malnourished piglets was relatively poor in phosphatidylinositol compared with that of controls. Phosphatidylinositol has a unique biochemical function not shared by any other membrane phospholipids; it is involved in the production of diacylglycerol and arachidonate, both second messengers involved in cellular signal transduction (Berridge and Irvine 1984). We speculate that a relatively lower proportion of phosphatidylinositol could affect the responsiveness of the intestine to external stimuli by interfering with transmembrane signaling. Shu-Heh et al. (1988) showed that phosphatidylinositol depletion was associated in the newborn rat with a reduction in alkaline phosphatase activity in the microvillus membrane of the intestine. These findings may help explain why there were parallel changes in the levels of phosphatidylinositol and the activity of alkaline phosphatase in the jejunal mucosa of malnourished piglets (Tables 2 and 4).

The fatty acid composition of the intestine was severely affected by PEM. Malnutrition resulted in lower percentages of (n-6) and (n-3) LC-PUFA, with a parallel increase in MUFA, namely, oleic acid. This fatty acid probably helps to maintain the unsaturation index of the membrane of mucosa cells. The changes in the fatty acid profile of the jejunal mucosa, phospholipids and microsomes in malnourished piglets can thus be attributed to the inhibition of enterocyte desaturase activity. Garg et al. (1990) showed that changes in desaturase activity occurred before alterations in the fatty acid composition of the enterocyte membrane in rats after 24 h of food deprivation, thus supporting the hypothesis that desaturase activity may be inhibited as the primary event. In our study, the linoleic/arachidonic acid ratio for total mucosa, phospholipids and jejunal microsomes, which reflects the overall bioconversion activity of essential fatty acids (Hollman 1986), was higher in malnourished animals than in control animals (Fig. 5). This increase may be the result of alterations in the genetic expression of intestinal desaturase enzymes in response to malnutrition; alternatively, it may be attributed to modulation of their activity by metabolites produced in the malnutrition status. Brenner (1989) reported that severe malnutrition leads to a significant decrease in ⁶-desaturase activity in the liver.

The fatty acid composition of jejunal microsomes closely resembles that of membrane phospholipids and total mucosa of the jejunum. This supports the hypothesis that membrane phospholipids are first synthesized in the microsomes and are then utilized for enterocyte plasma membrane biogenesis in which minor adjustments in composition take place during postnatal maturation and crypt-villus differentiation. The physicochemical properties of the membrane are largely governed by the nature of the fatty acids, cholesterol and phospholipids content, and the distribution of phospholipids. In this study the changes in the C/P and sphingomyelin/phosphatidylcholine ratios and fatty acid profiles may directly affect the activity of membrane-bound enzyme such as alkaline phosphatase and disaccharidases through a change in membrane fluidity. Moreover, they could have negative consequences on cell differentiation and maturation processes, which occur during the postnatal period. In addition, ultrastructural changes in intestinal architecture could be triggered by a decrease in certain prostaglandins involved in the regulation process of mucus secretion (Johansson and Bergstrom 1987) and in the control of gastrointestinal epithelium hyperplasia (Uribe and Johansson 1988). The decrease in LC-PUFA levels, namely, arachidonic and eicosapentaenoic acids, as a consequence of malnutrition could impair the balance between synthesis and degradation of prostaglandins in the intestinal mucosa (Smith et al. 1982). Polyunsaturated fatty acids are also utilized to synthesize phospholipids to form the chylomicron coat required during fat absorption and transport out of the enterocyte.

Plasma fatty acid levels were also severely affected in malnourished piglets. Malnutrition resulted in lower levels of SFA, MUFA, (n-3) and (n-6) LC-PUFA. These levels are consistent with a lower rate of desaturation and elongation of fatty acids in the microsomes of the jejunum and liver in malnourished animals. Some studies of plasma fatty acids in malnourished children showed low levels of both linoleic and arachidonic acids, or arachidonic acid alone, or linoleic acid in kwashiorkor and arachidonic acid in marasmus (Holman et al. 1981, Marin et al. 1991). These apparently contradictory results may reflect different degrees and duration of malnutrition, and associations with other diseases.

The results presented in this study demonstrate that early severe PEM strongly modifies intestinal membrane lipid composition, especially during the rapid growth and development that occur during the postnatal period. Qualitative and quantitative changes in the lipids of the small intestinal mucosa, the pattern of phospholipid distribution and the fatty acid profile may alter membrane fluidity and organization. Collectively, changes in the intestine may contribute in part to the increase in enterocyte plasma membrane permeability and to the reduced activity of membrane-bound hydrolytic enzymes, affect the intestinal transport processes and, finally, they might interfere with receptor-linked signal transduction.

	ACKNOWLEDGMENTS

The authors thank M. L. Jimenez for her excellent technical assistance and M. Ramirez for helpful advice in the statistical analysis. We thank the R&D Department of Abbott Laboratories S.A., Granada, Spain for supplying the diets and allowing us the use of its facilities for biochemical analyses.

	FOOTNOTES

Manuscript received 27 February 1997. Initial reviews completed 14 April 1997. Revision accepted 2 September 1997.

	LITERATURE CITED

Abstract Introduction Methods Results Discussion References

• Alessandri J. M., Arfi T. S., Thieulin C. La muqueuse de l'intestin grêle: évolution de la composition en lipides cellulaires au cours de la différenciation entérocytaire et de la maturation postnatale. Reprod. Nutr. Dev. 1990; 30:551-576
• Alessandri J. M., Guesnet T. S., Arfi T. S., Durand G. Changes in fatty acid composition during cell differention in the small intestine of suckling piglets. Biochim. Biophys. Acta 1991; 1086:340-348 [Medline]
• Ausman L. M., Gallina D. L., Hegsted D. M. Protein-calorie malnutrition in squirrel monkeys: adaptative response to calorie deficiency. Am. J. Clin. Nutr. 1989; 50:19-29 [Abstract/Free Full Text]
• Berridge M. J., Irvine R. F. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature (Lond.) 1984; 312:315-321 [Medline]
• Bouzine M., Prost J., Belleville J. Dietary protein deficiency affects (n-3) and (n-6) polyunsaturated fatty acid hepatic storage and very low density lipoprotein transport in rats on different diets. Lipids 1994; 29:265-272 ^[Medline]
• Bradford M. M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976; 72:248-254 [Medline]
• Brenner, R. R. (1989) Factors influencing fatty acids chain elongation and desaturation. In: The Role of Fats in Human Nutrition, 2nd ed. (Vergroesen, A. J. & Crawford M., eds.), vol. 2, pp. 45-79. Academic Press, London, UK
• Burton G. W., Ingold K. V., Thompson K. E. An improved procedure for the isolation of ghost membranes from human red blood cells. Lipids 1981; 16:946-950 [Medline]
• Butzner J. D., Gall D. G. Impact of refeeding on intestinal development and function in infant rabbits subjected to protein-energy malnutrition. Pediatr. Res. 1990; 27:245-251 [Medline]
• Castillo R. O., Feng J. J., Stevenson D. K., Kwong L. K. Altered maturation of small intestinal function in the absence of intraluminal nutrients: rapid normalization with refeeding. Am. J. Clin. Nutr. 1991; 53:558-561 [Abstract/Free Full Text]
• Chu S. W., Walker A. Development of the gastrointestinal mucosal barrier: changes in phospholipid head groups and fatty acid composition of intestinal microvillus membranes from newborn and adult rats. Pediatr. Res. 1988; 23:439-442 ^[Medline]
• Coward W. A., Whitehead R. G. Changes in serum -lipoprotein concentration during the development of kwashiorkor and in recovery. Br. J. Nutr. 1972; 27:383-394 [Medline]
• Dahlqvist A. Assay of intestinal disaccharidases. Anal. Biochem. 1968; 22:99-107 [Medline]
• Dixon, W. J., Brown, M. B., Engelman, L. & Jennrich, R. I. (1990) BMDP Statistical Software Manual. University of California Press, Berkeley , CA.
• Garg M. L., Keelan M., Thonson A.B.R., Clandinin M. T. Intestinal microsomes: polyunsaturated fatty acid metabolism and regulation of enterocyte transport properties. Can. J. Physiol. Pharmacol. 1990; 68:636-641 [Medline]
• Goldstein R., Klein T., Freier S., Menczel J. Alkaline phosphatase and disaccharidase activities in the rat intestine from birth to weaning. Effect of diet on enzyme development. Am. J. Clin. Nutr. 1970; 24:1224-1231
• Gupta D., Rana S. V., Mehta S., Metha S. K. Effect of protein energy malnutrition on the lipid composition and leucine uptake of small intestinal brush border vesicles of growing rhesus monkeys. Ann. Nutr. & Metab. 1994; 38:97-193 [Medline]
• Hayden J. M., Marten N. W., Strauss D. S. The effect of fasting on insulin-like growth factor-I nuclear transcript abundance in rat liver. Endocrinology 1994; 134:760-768 [Abstract]
• Henning, S. J. (1987) Functional development of gastrointestinal tract. In: Physiology of the Grastrointestinal Tract, Vol. 1 (Johnson, L. R., ed.), pp. 285-300. Raven Press, New York, NY.
• Holman R. T. Nutritional and functional requirements for essential fatty acids. Prog. Clin. Biol. Res. 1986; 222:211-228 [Medline]
• Holman R. T., Johnson S., Mercuri O., Irarte H., Rodrigo A., De Tomás M. E. Essential fatty acid deficiency in malnourished children. Am. J. Clin. Nutr. 1981; 34:1523-1529
• Johansson C., Bergstrom S. Prostaglandins and protection of the gastroduodenal mucosa. Scand. J. Gastroenterol. 1987; 17:21-46
• Keelan, M., Walker, K. & Thomson, A.B.R.L. (1985) Intestinal brush border membrane marker enzymes, lipid composition and villus morphology: effect of fasting and diabetes mellitus in rats. Comp. Biochem. Physiol. 82A: 83-89.
• Kolarovic L., Fournier N. A comparison of extraction methods for the isolation of phospholipids from biological sources. Anal. Biochem. 1986; 156:244-250 [Medline]
• Labarca C., Paigen K. A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 1980; 102:344-352 [Medline]
• Lepage G., Roy C. Direct transesterification of classes of lipids in a one-step reaction. J. Lipid Res. 1986; 27:114-120 [Abstract]
• Macala L. J., Yu R. K., Ando S. Analysis of brain lipids by high performance thin-layer chromatrography and densitometry. J. Lipid Res. 1983; 24:1243-1250 [Abstract]
• Marin C. M., Tomás M. E., Mercuri O., Fernández A., Serres C. Interrelationship between protein-energy malnutrition and essential fatty acid deficiency in nursing infants. Am. J. Clin. Nutr. 1991; 53:466-468 ^{[Abstract/Free Full Text]}
• Meddings J. B., Theisen S. Development of rat jejunum: lipid permeability, physical properties and chemical composition. Am. J. Physiol. 1989; 256:G931-G940 [Abstract/Free Full Text]
• Miller E. R., Ullery D. E. The pig as a model for human nutrition. Annu. Rev. Nutr. 1987; 7:361-382 [Medline]
• Nuñez, M. C., Ayudarte, M. V., Morales, D., Suárez, M. D. & Gil, A. (1990) Effect of dietary nucleotides on intestinal repair in rats with experimental chronic diarrhea. J. Parent. Enteral Nutr. 14: 598-594.
• Nuñez M. C., Bueno J. D., Ayudarte M. V., Almendros A., Rios A., Suárez M. D., Gil A. Dietary restriction induces biochemical and morphometric changes in the small intestine of nursing piglets. J. Nutr. 1996; 126:933-944
• Omoike I., Lindquist B., Abud R., Merrick J., Lebenthal E. The effect of protein-energy malnutrition and refeeding on the adherence of Salmonella typhimarium to small intestinal mucosa and isolated enterocytes in rats. J. Nutr. 1990; 120:404-411
• Philipp B. W., Shapiro D. J. Improved methods for the assay and activation of 3-hydroxy-3-methyglutaryl-CoA reductase. J. Lipid Res. 1979; 20:583-593
• Pond W. G., Ellis K. J., Mersmann H. J., Heath J. P., Krook L. P., Burrin D. G., Dudley M. A., Sheng H.-P. Severe protein deficiency and repletion alter body and brain composition and organ weights in infants pigs. J. Nutr. 1996; 126:290-302
• Pond W. G., Ellis K. J., Shoknecht P. Response of blood serum constituents to production of and recovery from a kwashiorkor-like syndrome in the young pig. Proc. Soc. Exp. Biol. Med. 1992; 200:555-561 [Abstract]
• Pond W. G., Yen J. T., Mersmann H. J., Maurer R. R. Reduced mature size in progeny of swine severely restricted in protein intake during pregnancy. Growth Dev. Aging 1990; 54:77-84 [Medline]
• Reinhold, J. G. (1953) Total protein albumin and globulin. Stand. Methods Clin. Chem. 1:88-97.
• Sakuma K., Ohyama T., Sogawa K., Fujii-Kuriyama Y., Matsumara Y. Low protein-high energy diet induces repressed transcription of albumin mRNA in rat liver. J. Nutr. 1987; 117:1141-1148
• Skipski, V. P. & Barclay, M. (1969) Thin layer chromatography of lipids. In: Methods in Enzymology, vol. XIV (Lowenstein, J. M., ed.), pp. 548-550. Academic Press, New York, NY.
• Smith G. S., Warhurst G., Turnberg L. A. Synthesis and degradation of prostaglandin E2 in the epithelial and subepithelial layers of the rat intestine. Biochim. Biophys. Acta 1982; 713:684-687 [Medline]
• Spiekerman A. M. Proteins used in nutritional assesment. Clin. Lab. Med. 1993; 13:353-369 [Medline]
• Uribe A., Johansson C. Initial kinetic changes of prostaglandin E2-induced hyperplasia of the rat small intestinal epithelium occur in the villous compartments. Gastroenterology 1988; 94:1335-1342 ^[Medline]
• Wykes L. J., Fiorotto M., Burrin D. G., Rosario M., Frazer M. E., Pond W. G., Jahoor F. Chronic low protein intake reduces tissue protein synthesis in a pig model of protein malnutrition. J. Nutr. 1996; 126:1481-1488
• Wolff J. A., Margolis S., Bujdoso-Wolff K., Maclean J. R. Plasma and red blood cell fatty acid composition in children with protein-calorie malnutrition. Pediatr. Res. 1984; 18:162-167 [Medline]
• Zilversmit D. B., Davis A. X. Microdefermination of plasma phospholipids by trichloroacetic acid preciptation. J. Lab. Clin. Med. 1950; 35:155-160 ^[Medline]

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