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*
Department of Biochemistry and Molecular Biology,
Department of Cell Biology, University of Granada and
**
R&D Department, Abbott Laboratories S. A., Granada, Spain
4To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • pigs • polyunsaturated fatty acid • lipid • protein-energy malnutrition • small intestine
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Castillo et al. 1991
).
Long-chain polyunsaturated fatty acids
(LC-PUFA)5
play an important role in the maturation process and in the composition
of enterocytes. The fatty acid profile of differentiating-cell
phospholipids changes in the immature intestine of suckling piglets,
possibly in relation to the appearance of specific functions
(Alessandri et al. 1991
). Moreover, the proportion of
phospholipid species and fatty acid composition as well as changes in
the amount of phospholipids and cholesterol in microvillous membranes
differ in newborn rats and adults (Chu and Walker 1988
).
These differences may account for the greater uptake of antigens and
other mucosal-barrier defects in the intestine of newborns
(Chu and Walker 1988
). Other nutrients such as
cholesterol also influence the physicochemical and functional
properties of membranes. It is worth noting that dietary cholesterol
deprivation alters the biophysical properties of the microvillous
intestinal membrane (Neu et al. 1987
) as well as
disaccharidase activities (Tiruppathi et al. 1985
).
Malnutrition causes biochemical and histologic changes in the gut in
both animals and humans, reducing intestinal surface area and amino
acid uptake as well as augmenting lymphocyte infiltration and altering
the activity of membrane-bound enzymes (Butzner and Gall 1990
, Gupta et al. 1994
). Previous studies in
our laboratory revealed that malnutrition induced by dietary
restriction in nursing piglets severely affected the intestinal
histologic structure. In addition, the amount of DNA and protein, the
content of cholesterol, phospholipids and triglycerides, and the
relative percentages of (n-6) and (n-3) LC-PUFA were reduced in the
jejunal and ileal mucosa; in addition, the segmental disaccharidase and
leucine aminopeptidase activities were depressed in the small intestine
(López-Pedrosa et al. 1998
,
Núñez et al. 1996
).
Because dietary LC-PUFA may influence the recovery of intestinal injury caused by malnutrition, the purpose of this study was to evaluate the effect of feeding a diet containing LC-PUFA of the (n-6) and (n-3) series in the form of phospholipids on the recovery of the damaged intestine in piglets that were malnourished due to severe dietary restriction.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unless otherwise stated, chemicals were purchased from Sigma Chemical (St. Louis, MO). DL-[3H]Methyl-3-hydroxy-3-methylglutaryl-Coenzyme A was purchased from Amersham (Cardiff, UK). 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 as follows: the first group of 12 piglets was freely fed recombined milk formula (188 g/L) by nipple for 30 d. The second group of 12 piglets was fed the same diet, but they received only 20% of the intake recorded in the first group. In addition, they received freely a glucose-saline solution to fully satisfy water requirements. Malnourished and healthy piglets were divided into two subgroups of six and fed for 10 d either an adapted piglet milk formula or the same diet supplemented with a phospholipid concentrate of (n-6) and (n-3) LC-PUFA also containing cholesterol. Thus, the malnourished subgroups were designated malnourished (M) and malnourished-LC-PUFA (M-P), and the control subgroups were designed as control (C) and control-LC-PUFA (C-P). During the recovery period, the energy and protein intake of malnourished piglets increased gradually from d 1 to 4. From that point on, they were freely fed.
The composition of the adapted milk formula has been previously
reported (López-Pedrosa et al. 1998
). The
supplemented formula contained 2.53% phospholipids; the phospholipid
species distribution in the brain phospholipid concentrate used as the
LC-PUFA source has also been reported previously
(Suárez et al. 1996
). Both diets were isocaloric.
The fatty acid compositions of the diets are shown in Table 1. All procedures involving piglets were approved by the Animal Care
Committee at the University of Granada and complied with the current
European Union Regulations on Animal Care for care and use of animals
for research.
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At 47 d of age, after a 16-h period of food deprivation, piglets were bled to death by jugular vein puncture under anesthetic. Blood (250 mL) was collected in glass bottles with tripotassium EDTA as anticoagulant, and plasma was obtained by centrifugation at 1500 x g for 15 min. Plasma was rapidly frozen in liquid nitrogen, then stored at -80°C until analyzed.
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 were considered the proximal jejunum for
biochemical measurements. The 60-cm segment 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 was removed by scraping the entire luminal
surface with a glass coverslip and then weighed. A portion of jejunal
mucosa was homogenized with buffer (10 mmol/L
N-[2-hydroyethyl]piperazine-N'-[2-ethanesulfonic
acid], 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 g
of tissue per 4 mL of buffer); the microsomal fraction was prepared by
the method of Philipp and Saphiro (1979)
.
Mucosa from the jejunum and ileum were homogenized in distilled water
for protein and enzymatic assays, and 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 by 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 the mucosa were
determined with the the method of the Dahlqvist (1968)
,
and alkaline phosphatase activity (EC 3.1.3.1) was measured according
to Goldstein et al. (1970)
.
Intestinal mucosa 3-hydroxy-3-methylglutaril coenzyme A
(HMG-CoA) reductase activity (EC 1.1.1.34) was determined in the
pellet of microsomes as the rate of formation of [3H]
mevalonate from DL-[3H]methyl-HMG-CoA,
according to Philipp and Shapiro (1979)
. Results were
calculated as picomoles of HMG-CoA converted to mevalonate per
milligram of microsomal protein per minute.
Total lipids in jejunal and ileal mucosa were extracted according to
the procedure of Kolarovic and Fournier (1986)
. Jejunal
mucosa phospholipids were separated by TLC (Skipski and Barclay 1969
). Cholesterol and triglycerides were measured in the lipid
fraction using enzymatic methods (cholesterol CHOD-PAP and
triglycerides GPO-PAP test combination, Boehringer-Mannhein,
Mannhein, Germany). Phospholipid content in the mucosa was evaluated by
measuring inorganic phosphorus (Zilversmit 1950
). Fatty
acids from total mucosa, mucosal phospholipids and mucosal microsomes,
as well as fatty acids from plasma, were saponified and methylated by
using the method of Lepage and Roy (1986)
and separated
and quantified by capillary gas-liquid chromatography, as
previously described (López-Pedrosa et al. 1998
).
Histologic analysis.
Histologic analysis of small intestine samples was performed by light
and electron transmission microscopy as previously reported
(Núñez et al. 1996
). Basically, 20 ultrathin
(50 µm) sections of jejunum for each piglet were
analyzed. Ten fields of each section were randomly chosen, and
ultrastructural alterations for each field were considered. The analyst
was not aware of the study groups. Samples for transmission electron
microscopy 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, they were dehydrated in acetone and embedded in Epon 812
resin. Ultrathin sections were double-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. The effects of
malnutrition and type of diet and their interaction on recovery of
intestinal injury were evaluated using a 2 x 2 ANOVA. Homogeneity
of variances was tested by Levene's test. When variances were
heterogeneous, data were transformed to natural logarithms or
reciprocal. If transformations did not equalize variances, the
Brown-Forsythe statistic was used. When a significant effect was
found (P < 0.05), preplanned comparisons were done
using the Bonferroni correction. Statistical analyses were performed
using the PC-90 version of the BMDP Statistical Software (Los Angeles,
CA) (Dixon et al. 1990
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). During the recovery period, nourished piglets consumed
936 ± 105.2 kJ/(kg body wt · d) and 12.2 ± 1.2 g of
protein/(kg body wt · d). During the same period, the intake in the
malnourished group increased gradually from 470 ± 16 kJ/(kg body
wt · d) [5.2 ± 0.2 g of protein/(kg body wt · d)] to
900 ± 99 kJ/(kg body wt · d) [10.5 ± 0.5 g of
protein · /(kg body wt · d)] and was maintained thereafter. Malnourished piglets that recovered consuming the control diet (M) and the LC-PUFA–supplemented diet (M-P) for 10 d weighed less than their corresponding nourished controls (in kg, 4.7 ± 0.2, M; 5.4 ± 0.3, M-P; 12.6 ± 1.3, C; 12.9 ± 1.3, C-P). The M-P group tended to weigh more than M group after 10 d of refeeding (P = 0.13).
Feeding the LC-PUFA–supplemented diet promoted not only body growth but also organ-specific growth. The weight-per-length ratio (mg/cm) of the jejunal mucosa was lower (P < 0.05) in the M group (166 ± 11) than in the M-P group (210 ± 12) and the C group (227 ± 9). No significant differences were found between the M-P (210 ± 12) and C-P (247 ± 15) groups.
Transmission electron micrographs of enterocyte from M and M-P
piglets are shown in Figure 1
. Both M and M-P groups still showed some histologic signs of
malnutrition in comparison to nourished piglets. However, the M-P
group exhibited a recovery in the morphology of microvilli, mitochodria
and cytoplasma. No apparent differences were found between C and
C-P groups (results not shown).
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). In the ileal segment, the DNA and protein contents were higher
in the nourished piglets (C and C-P) than in the malnourished piglets
that recovered consuming the adapted milk formula (M) and the
LC-PUFA–supplemented formula (M-P). The M piglets had lower
cholesterol and phospholipid levels and higher triglycerides levels in
the jejunum than did the M-P group. The lipid composition of ileum
was not affected by dietary lipids.
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The fatty acid composition of jejunal and ileal mucosa, and the (n-6)
and (n-3) LC-PUFA levels of total mucosa, microsomes and
phospholipids of jejunum are shown in Table 3 and Figure 2
, respectively. The fatty acid composition of the intestinal mucosa
in group M differed markedly from that of the M-P and C groups. The
M group had a higher proportion of 18:1(n-9) and lower proportions of
20:4(n-6) and 22:6(n-3) than did the M-P group. There was a
significant interaction between malnutrition and diet for 20:4(n-6) in
total jejunum mucosa whereby the effect of LC-PUFA was not seen in
nourished piglets. Similar differences were also found in microsomes
and phospholipids from jejunal mucosa; the proportions of (n-6) and
(n-3) LC-PUFA were lower in the M than in the M-P group (Fig. 2)
. Piglets in the C-P group had higher levels of 20:4(n-6)
P < 0.06, 22:4(n-6), 22:5(n-6) and 22:6(n-3) in the
jejunal mucosa than did those in the C group (Table 3)
. The fatty acid
composition of the ileal mucosa was less affected either by the
malnutrition process or by the dietary intervention than that of the
jejunal mucosa (Table 3)
.
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).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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,
Núñez et al. 1996
). In this study, we report the recovery of malnourished piglets fed an adapted milk formula or an adapted milk formula containing LC-PUFA from a phospholipid source. These phospholipids were obtained and purified from pig brains and contained a small amount of cholesterol, 0.24 g/100 g (final formula). The small intestine of piglets fed the LC-PUFA–supplemented formula recovered more completely from the histologic lesions and biochemical alterations caused by the malnutrition process than the small intestine of those fed the control formula without LC-PUFA.
The group that recovered with the LC-PUFA–supplemented formula had
higher protein, DNA, cholesterol and phospholipid contents, and a
higher weight:length ratio in the jejunal segment than those that
recovered with the control formula, indicating that growth and
maturation rates of the intestinal mucosa were higher in the M-P
group than in the M group. Other authors have reported that nutrients
stimulate epithelial cell proliferation in the gastrointestinal tract
(Butzner and Gall 1990
, Castillo et al. 1991
). Furthermore, dietary cholesterol influences the
composition and function of intestinal cells (Neu et al. 1987
, Thomson et al. 1993
) and is a basic
component of cell membranes and lipoproteins. Although cholesterol is
not recognized as essential in the diet of young infants, it may
interact with dietary LC-PUFA and participate in lipoprotein
metabolism later in life (Agostini and Riva 1998
). Also,
(n-3) LC-PUFA reportedly have a trophic effect on jejunal mucosa in
rats, associated with increased mucosal surface area; this effect was
accentuated by the presence of dietary cholesterol (Dietschy and
Gamel
). The combination of LC-PUFA, cholesterol and
phospholipids in the diet may account for the results of this study
because M-P piglets could use those dietary lipid components and
save the energy cost of their synthesis for cell proliferation. Light
and electron transmission studies also support the synergistic effect
of dietary LC-PUFA, phospholipids and cholesterol on the recovery
of proximal intestinal mucosa. Jejunal HMG-CoA reductase activity
did not differ in piglets that recovered consuming the adapted milk
formula and those fed the formula containing LC-PUFA and
cholesterol. Thus, the cholesterol synthesis rate seems to be
unaffected by dietary cholesterol intake during recovery from PEM. Our
results agree with those reported by Dietschy and Gamel
(1971)
who found that cholesterol synthesis in the proximal
small intestine of humans was not inhibited by cholesterol intake.
In this study, the effects of dietary lipids were stronger in the jejunal than in the ileal segment, possibly due to the preferential use of nutrients in the former part of the intestine and/or the persistence of bacterial overgrowth caused by malnutrition in this experimental model.
Disaccharidase and alkaline phosphatase activities in the malnourished piglets that recovered consuming the adapted milk formula or the LC-PUFA formula were similar to those found in nourished piglets despite the compositional and histologic alterations of the mucosa. However, ileal maltase and alkaline phosphatase activities were not fully restored in recovered piglets, supporting the finding that the ileum was less affected by dietary nutrients that was the jejunum.
Jejunal triglyceride content was 1.5-fold higher in piglets that
recovered consuming the adapted formula than in those recovered with
the LC-PUFA formula, indicating that LC-PUFA phospholipids
and/or cholesterol may play an important role in intestinal lipid
metabolism. The jejunal triglyceride accumulation in the M group could
be attributable to the concomitant decrease in the phospholipid
content. Jenkins et al. (1983)
suggested that
triglycerides accumulate in tissues as a result of many nutritional
factors, such as deficiencies of protein, certain amino acids or
cholesterol, or inadequate levels of labile methyl groups. Jejunal
steatosis could also be due to an impairment of lipoprotein synthesis
and secretion from intestinal cells. Phospholipids are the main
component of the lipoprotein surface, and hence a reduction in the
content of intestinal phospholipids could lead to alterations of
lipoprotein conformational structure and secretion. Tso et al. (1984)
reported that the infusion of esterified fatty acids in
the form of phospholipids instead of triglycerides enhances the
formation and secretion of VLDL into lymph as the major vehicle for
transporting lipids. Feldman et al. (1983)
demonstrated
that cholesterol and triglycerides differentially affect particle size
of intestinal lymph lipoproteins. With greater cholesterol absorption,
more lipids were carried by VLDL; this contrasts with the preferential
rise in chylomicroms in which more triglycerides were absorbed. This
study provides evidence that dietary phospholipids and cholesterol may
have a synergistic effect on transport of triglycerides through
intestinal VLDL production.
Jejunal mucosa and microsomes of malnourished piglets fed the
LC-PUFA–supplemented formula showed a fatty acid composition closer to
that of C-P piglets in contrast to those fed the adapted formula
without LC-PUFA. This finding indicates that dietary (n-3) and
(n-6) LC-PUFA were efficiently taken up and acylated into membrane
phospholipids of jejunal cells. This may further explain the repair of
histologic damage caused by malnutrition. In fact, it has been reported
that LC-PUFA may promote fusion of rough endoplasmic-reticulum
membranes (Paiement et al. 1994
). This could be related
to the presence of clear areas in the transmission electron micrographs
of the jejunum from piglets that recovered consuming the adapted milk
formula; such areas nearly disappeared in the group that recovered
consuming the LC-PUFA formula. The appearance of clear areas in
cells is usually related to loss of endoplasmic reticulum and Golgi
complexes. In addition, enterocyte differentiation involves an
increasing incorporation of (n-6) fatty acids, which is controlled by
the type of dietary fat (Alessandri et al. 1993
). The
LC-PUFA phospholipids added to the experimental formula in this
study contained both (n-3) and (n-6) fatty acids in a ratio that allows
the incorporation of both series into cell membranes during intestinal
repair, which also may explain the improvement of the mitochondrial
structure. However, the presence of a significant interaction between
malnutrition and diet for 20:4(n-6) in jejunum mucosa means that
dietary 20:4(n-6) supplementation affected malnourished and healthy
piglets differently. We have previously reported that 
-6-desaturase
activity (in terms of precursor:product ratio) of jejunum was severely
affected by PEM (López-Pedrosa et al. 1998
).
Arachidonic acid may play an important role during the recovery process
either as a component of membrane lipids or as a precursor of bioactive
compounds.
Finally, differences in plasma fatty acid composition generally were
consistent with those found in the jejunal mucosa, which also suggests
that piglets recovered with a LC-PUFA free formula cannot
synthesize enough LC-PUFA to repair the damaged tissues. Studies of
plasma fatty acids in malnourished children have shown low levels of
LC-PUFA as well (Holman et al. 1981
, Marin et al. 1991
). Given that the pig is a good model for human
nutrition (Miller and Ullrey 1987
), malnourished infants
may suffer histologic and compositional alterations similar to those
described here and could benefit from comparable dietary lipid
intervention.
In conclusion, the results presented in this study show that dietary LC-PUFA as phospholipids with a (n-6):(n-3) ratio of 1.9 enhance the recovery of the damaged small intestine after PEM induced by severe dietary restriction. In this study, we did not attempt to compare LC-PUFA sources as triglycerides or phospholipids. Thus, we cannot exclude the possibility that some of the effects found in intestinal repair may be affected not only by LC-PUFA themselves but by the form in which they are esterified (triglycerides or phospholipids). In addition, the residual content of cholesterol and/or other components (such as hydrophilic head groups of phospholipids, sphyngo- and glycolipids) in the LC-PUFA source may also be important for intestinal recovery. This dietary intervention promoted intestinal cell growth, normalized the lipid and fatty acid composition of jejunum and reduced the histologic alterations caused by malnutrition. Further research is required to evaluate the separate effects of dietary LC-PUFA, cholesterol and other lipid components on the histology and biochemistry of the small intestine in humans.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 J. M. L.-P. was the recipient of a
fellowship (Ayudas para el Intercambio de Personal Investigador entre
Indsutrias y Centros Públicos de Investigación) provided by
the Spanish Ministry of Education.
3 Current address: Research Department, Abbott
Laboratories, Camino de Purchil, 68, 18004 Granada, Spain.
5 Abbreviations used: C, control
subgroup; C-P, control LC-PUFA subgroup; HMG-CoA,
3-hydroxy-3-methylglutaryl coenzyme A; LC-PUFA, long-chain
polyunsaturated fatty acids; M, malnourished subgroup; M-P,
malnourished LC-PUFA subgroup; PEM, protein-energy
malnutrition; PL, phospholipid; MUFA, monounsaturated fatty acids; TG,
triglycerides.
Manuscript received November 18, 1998. Initial review completed December 24, 1998. Revision accepted March 2, 1999.
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S. A. Mathews, W. T. Oliver, O. T. Phillips, J. Odle, D. A. Diersen-Schade, and R. J. Harrell Comparison of Triglycerides and Phospholipids as Supplemental Sources of Dietary Long-Chain Polyunsaturated Fatty Acids in Piglets J. Nutr., October 1, 2002; 132(10): 3081 - 3089. [Abstract] [Full Text] [PDF]
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G. S. Patten, M. Y. Abeywardena, E. J. McMurchie, and A. Jahangiri Dietary Fish Oil Increases Acetylcholine- and Eicosanoid-Induced Contractility of Isolated Rat Ileum J. Nutr., September 1, 2002; 132(9): 2506 - 2513. [Abstract] [Full Text] [PDF]
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L. Amate, A. Gil, and M. Ramirez Dietary Long-Chain Polyunsaturated Fatty Acids from Different Sources Affect Fat and Fatty Acid Excretions in Rats J. Nutr., December 1, 2001; 131(12): 3216 - 3221. [Abstract] [Full Text] [PDF]
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L. Amate, A. Gil, and M. Ramírez Feeding Infant Piglets Formula with Long-Chain Polyunsaturated Fatty Acids as Triacylglycerols or Phospholipids Influences the Distribution of These Fatty Acids in Plasma Lipoprotein Fractions J. Nutr., April 1, 2001; 131(4): 1250 - 1255. [Abstract] [Full Text]
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