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Effects of dietary polyamines at physiologic doses in early-weaned piglets
Nutrition 25 (2009) 940–946
Basic nutritional investigation
www.nutritionjrnl.com
Effects of dietary polyamines at physiologic doses in early-weaned piglets Marı´a Sabater-Molina, Ph.Da, Elvira Larque´, Ph.D.a,*, Francisco Torrella, Ph.D.b, Javier Plaza, Maa, Teresa Lozano, Ph.D.c, Antonio Mun˜oz, Ph.D.d, and Salvador Zamora, Ph.D.a a Department of Physiology, Faculty of Biology, University of Murcia, Murcia, Spain Department of Microbiology, Faculty of Biology, University of Murcia, Murcia, Spain c Department of Cell Biology, Faculty of Biology, University of Murcia, Murcia, Spain d Department of Animal Production, Veterinary School, University of Murcia, Murcia, Spain b
Manuscript received July 18, 2008; accepted January 22, 2009
Abstract
Objective: Polyamines are essential for many cell functions, and they form part of the composition of maternal milk; despite this, their addition to infant formulas is currently under evaluation. The aim of the present study was to evaluate the effects of milk formulas designed to resemble sow milk supplemented with polyamines at maternal physiologic milk doses on the gut maturation of early-weaned piglets. Methods: We fed 30 newborn piglets with maternal milk (n ¼ 10), a control milk formula (n ¼ 10), or a milk formula supplemented with polyamines (5 nmol/mL of spermine and 20 nmol/mL of spermidine, n ¼ 10) for 13 d (day 2 after birth through day 15). Several growth and intestinal development parameters were measured. Results: The piglets fed the formula containing polyamine at physiologic doses showed significantly increased crypt depth in the small intestine compared with those fed with the control formula. Villus length was correlated to crypt depth. Although there were no differences in the disaccharidase activities between the animals fed the two formulas, alkaline phosphatase and g-glutamyl transferase activities tended to be higher in the jejunum of those fed the polyamine-supplemented diet. Dietary polyamines did not significantly modify the gut mucosal concentrations of putrescine, spermine, or spermidine. Conclusion: Milk formulas supplemented with polyamines at maternal milk physiologic doses slightly enhanced gut growth and maturation in neonatal piglets. Ó 2009 Elsevier Inc. All rights reserved.
Keywords:
Polyamines; Human milk; Small intestine; Gut development; Piglets
Introduction Neonatal life, when only one type of food is the source of nutrition, is a very vulnerable period. Although several factors are involved in infants’ health, breast milk is considered the optimal source of nutrition during the first 6 mo of life. The nutritional components of human milk may influence intestinal maturation in neonates [1,2], and milk polyamines have been related to gut maturation and development in several studies [2–5]. Nevertheless, these components have not yet been incorporated into infant formulas because more This study was supported in part by a grant from DANONE S.A., Spain. *Corresponding author. Tel.: þ34-968-363-942; fax: þ34-968-363-963. E-mail address: [email protected] (E. Larque´). 0899-9007/09/$ – see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2009.01.017
studies are needed to clarify their role during this early stage of life. Polyamines (putrescine, spermidine, and spermine) are aliphatic molecules present in the cells of all organisms [6,7]. Dietary polyamines contribute significantly to the luminal gastrointestinal polyamine pool [8–10]. Physiologic intracellular polyamine levels are essential for many cell functions, such as protein, RNA, and DNA synthesis, and the stabilization of DNA and chromatin structure [11]. In addition, polyamines may modify the immune response, block calcium ion channels, and regulate apoptosis [12] and are involved in cell differentiation and proliferation [13]. In suckling rats, the oral administration of high doses of polyamines was correlated with the early appearance of morphologic and biochemical modifications typical of the mature intestine [3,5,10].
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In contrast, polyamine-deficient diets resulted in a significant hypoplasia of the small intestinal and colonic mucosa [14]. Although the importance of luminal gastrointestinal polyamines is well documented, little is known about their effects at physiologic levels. Polyamines are present in appreciable amounts in human milk [15–18]. Maternal milk polyamine levels are higher during the first week of lactation [15], which raises a question of the physiologic effect of these substances on neonates. At present, infant formulas are not supplemented with any exogenous source of polyamines, and their polyamine levels are 10 times lower than in human milk [19]. Because milk polyamines could exert stimulatory effects on postnatal growth and gut maturation in lactation [15–18], their addition to infant formulas could be of major importance for the newborn. The purpose of the present study was to determine the effects of neonatal milk formulas supplemented with polyamines, at the physiologic levels found in maternal milk, on gut development, by evaluating mucosal morphology, enzymatic activities, and polyamine levels in the small intestine of early-weaned piglets.
Materials and methods Animals and diets Thirty newborn piglets (Landrace by Large White) were provided by the veterinary farm of the University of Murcia (Murcia, Spain). They were matched by gender. Twenty piglets were nursed by sows until 2 d of age, after which they were randomly allocated into one of two groups (10 animals/group): control formula group (n ¼ 10) and polyamine-supplemented formula group (n ¼ 10). The piglets for these two groups came from the same litter as those from the maternal milk group. The piglets were housed in cages provided with attached spot heat lamps and fed ad libitum every 3 h for 13 d. The rest of the animals (n ¼ 10) continued to be nursed by sows until 15 d of age (sow milk group). The study was approved by the animal care committee at the University of Murcia and conformed to the European Union Regulation of Animal Care for the care and use of animals for research. The milk formula was designed to resemble sow milk in its macronutrient composition and to meet National Research Council nutrient requirements for the growing piglet [20]. The ingredients for the preparation of the milk formula were 118 g of cow’s milk solids (3.6% fat), 137 g of calcium caseinate, 153 g of demineralized milk whey, 451 g of cream (35% fat), 116 g of olive oil, 182 g of coconut oil, 36 g of soybean oil, 3.5 g of lecithin, 206 g of lactose, 37 g of a mineral complex, and 3.3 g of a vitamin complex (Piensos CARN, Murcia, Spain) per kilogram. The nutrient composition of the milk formula is presented in Table 1. The formulas were dissolved in warm water at a concentration of 200 g/L. No detectable polyamine levels were found in the control
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Table 1 Nutrient composition of milk formula in percentage of dry mass Composition (%) Raw protein Lysine Fat Lactose Vitamins* Minerals* Fatty acids (% total weight) 4:0 6:0 8:0 10:0 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 C20
27.05 2.14 33.22 29.96 5.0 4.76 0.0319 0.0159 1.0781 1.0863 8.9509 3.7651 31.0650 0.0262 4.8008 35.1280 12.5001 0.988 0.1183
* The mineral and vitamin mixture contained (milligrams per kilogram of diet): dibasic calcium phosphate 22,240, calcium citrate tetrahydrate 5070, sodium phosphate dibasic dodecahydrate 5060, manganese sulfate 1530, ferrous lactate 1380, potassium sulfate 920, copper sulfate 10.7, manganese sulfate 32.6, potassium iodate 0.8, zinc sulfate 5.24, thiamine 2, riboflavin 3, pyridoxine HCl 3, nicotinic acid 30, calcium pantothenate 20, folic acid 1, biotin 0.8, cyanobalamin 0.025, retinol acetate 0.069, cholecalciferol 0.0093, DL-tocopherol 50, phylloquinone 0.15, ascorbic acid 700.
milk formula for three replicates. The polyamine-supplemented formula contained 5 nmol/mL of spermine (Sigma Chemical Co., St. Louis, MO, USA), and 20 nmol/mL of spermidine (Sigma Chemical Co.), reflecting the polyamine levels detected in sow milk. The polyamine dose used was calculated from the mean concentration of polyamines in 10 milk samples from all the different sows (Table 2). Numerous factors modify the amount of milk polyamines, such as genetic factors, the preweaning period, environmental factors and, especially, the nutritional state and diet [2]. It was therefore important that the sows used in the present study were subjected to the same conditions of feeding, light, and water availability as those used for the quantification of milk polyamine concentration. Dissection protocol At 15 d of age, fasting piglets, deprived of food for at least 8 h, were anesthetized by retro-ocular injection with a 50:50 mixture of ketamine:propofol (1 mL/kg). The abdominal wall was opened and the entire gastrointestinal tract was removed. Using scissors, the mesentery was cut, and five intestinal tissue samples, each 1 cm in length, were removed at a point 10%, 25%, 50%, 75%, and 90% of the length of the small intestine and kept in Bouin fixative until analysis. In addition, mucosa samples from the jejunum and ileum were removed by scraping the entire luminal surface with a glass
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Table 2 Polyamine concentration in milk samples from 10 different sows Day of lactation
Putrescine (nmol/mL)
Spermidine (nmol/mL)
Spermine (nmol/mL)
4 (n ¼ 3) 5 (n ¼ 1) 6 (n ¼ 1) 7 (n ¼ 2) 11 (n ¼ 1) 13 (n ¼ 1) 15 (n ¼ 1) Mean 6 SEM
0.94 0.00 5.87 0.31 0.41 0.70 0.72 1.28 6 0.77
21.33 17.26 17.58 27.73 21.77 30.59 12.06 21.19 6 2.41
11.47 2.78 10.19 2.33 2.59 3.56 1.17 4.87 6 1.57
coverslip over an ice-cold Petri dish, frozen immediately in liquid nitrogen, and stored at 80 C. The piglets were euthanized by an intracardial injection of sodium pentobarbital (1 mL/kg). Analytical methods The microscopical morphologic measurements of the gut were performed with the five intestinal tissue samples collected at the sites mentioned above in the small intestine. After fixing the tissue samples in Bouin liquid (75 mL of picric acid water saturated solution, 25 mL of formaldehyde, and 5 mL of acetic acid), a portion of each sample was embedded in paraffin wax using standard techniques. Two transverse sections were selected from each sample, stained with hematoxylin and eosin, and examined with an optical microscope (Axioskop, Zeiss, Germany). Villus length and crypt depth were measured using an image processing program (Microm Image Processing 4.5, Consulting Image Digital, Barcelona, Spain). The length and depth of at least 10 villi and crypts were measured, calculating the mean villus length and the mean crypt depth. In addition, the number of cells per length of villus was estimated from 10 villi in each histologic section [21,22]. Mucosal samples were used to determine disaccharidase, alkaline phosphatase, and g-glutamyl transferase activities and polyamine levels. After homogenization of the jejunum mucosa samples in saline solution (1:10, w:v) and centrifugation at 1500 3 g for 20 min, the supernatants were analyzed for total protein content according to the method of Bradford [23], disaccharidase (i.e., maltase, sucrase, and lactase) according to the spectrophotometric method of Dahlqvist [24], alkaline phosphatase activity according to Rosalki et al. [25], and g-glutamyl transferase activity using the method of Szasz [26], and the Biosystems diagnostic kit (Barcelona, Spain). The polyamine content in jejunum and ileum mucosa samples was quantified according to a modified version of the method described by Seiler [27]. Briefly, mucosa samples were homogenized (1:10, w:v) in 5-sulfosalicylic acid (0.2 g/mL) and kept frozen overnight at 80 C. After centrifugation at 6700 3 g for 20 min, 50 mL of 1,6-diaminohexane (internal standard), 200 mL of saturated sodium
carbonate, and 400 mL of dansyl chloride (10 mg/mL in acetone) were successively added to 100 mL of the supernatant. This dansylation mixture was incubated overnight at room temperature. Dansylated derivatives were then extracted with 1.5 mL of cyclohexane and subsequently dried before being dissolved in 100 mL of the injection medium (i.e., acetonitrile:methanol 3:2, v:v). Dansylated polyamines were quantified by high-performance liquid chromatography using a reverse-phase column (Nova-Pak C18, Waters, Barcelona, Spain). We used a two-phase gradient, starting with 57% phase A (phase A: acetonitrile:methanol 3:2; phase B: Milli Q water), reaching 100% phase A after a 53-min run. Polyamine standards (putrescine dihydrochloride, spermidine trihydrochloride, and spermine tetrahydrochloride) were purchased from Sigma Chemical Co. Determination of polyamines in sow milk and formulas Polyamines in sow milk and formulas were analyzed using the AccQ-Fluor Reagent Kit (Waters), which uses 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate as derivation reagent. Ten milk samples were obtained from a group of sows on different days during the lactation period (Table 2). The samples were frozen at 80 C until polyamine analysis was performed. Two hundred microliters of 5-sulfosalicylic solution (0.2 g/mL) and 50 mL of 1,6-diaminohexane (internal standard) were successively added to 1-mL milk samples and then kept frozen overnight at 20 C. After centrifugation at 6700 3 g for 20 min, the resulting precipitate was removed. The supernatant was centrifuged again at 6700 3 g for 10 min, and the supernatant was passed through a 0.45-mm Millipore filter before derivation. Then 70 mL of borate buffer (pH 8.8) and 20 mL of AccQ Fluor (Waters) were added to 100 mL of a polyamine-containing sample. The mixture was incubated in a water bath for 10 min at 55 C. Derivatized polyamines were determined by high-performance liquid chromatography using a reverse-phase column (Nova-Pak C18, Waters). We used a two-phase gradient, starting with 25% phase A (phase A: 20 mM sodium acetate; phase B: methanol), reaching 100% phase A after a 30-min run. Polyamine standards (putrescine dihydrochloride, spermidine trihydrochloride, and spermine tetrahydrochloride) were purchased from Sigma Chemical Co. Statistical analyses The results are expressed as mean 6 standard error of the mean. Analysis of variance was performed to analyze differences among the three groups of animals with a posteriori Bonferroni’s test. The correlations between the histologic parameters and enzymatic activities, on the one hand, and polyamine concentration, on the other, were evaluated using Pearson’s correlation test. Differences were considered statistically significant at P < 0.05. All statistical analyses were carried out using SPSS 12.0 for Windows (SPSS Inc., Chicago, IL, USA).
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Table 3 Histologic measurements and enzymatic activities in the small intestine of 15-d-old piglets maintained on three different diets* Sow milk (n ¼ 10)
Control (n ¼ 10)
Polyamines (n ¼ 10)
145.2 6 5.9 147.7ab 6 7.9 148.2ab 6 5.6
145.7 6 7.9 142.6b 6 6.4 143.4b 6 3.7
162.5 6 7.6 164.6a 6 6.2 162.5a 6 5.1
0.161 0.043 0.040
149.2 6 9.5 140.1 6 9.2 140.0 6 9.4 141.5 6 7.3
125.1 6 6.7 129.4 6 6.8 133.7 6 8.2 127.7 6 5.9
142.4 6 7.4 138.2 6 4.9 140.6 6 8.3 140.4 6 3.1
0.130 0.553 0.827 0.184
361.4 6 17.6 419.2 6 27.4 390.3 6 19.4
386.9 6 35.7 410.8 6 22.5 393.2 6 27.7
418.4 6 19.8 385.4 6 17.4 401.9 6 12.9
0.116 0.117 0.915
463.6 6 27.3 528.1 6 40.7 452 6 20.6 481.2a 6 21.6 54.1 6 18.8 537.8 6 124.2 82.8 6 15.9 21.6a 6 6.8 218.5 6 67.8
368.7 6 36.9 411.9 6 34.2 390.6 6 25.1 393.1b 6 25.9 52.9 6 7.9 732.1 6 150.1 72.7 6 8.8 12.4ab 6 2.4 284.9 6 71.7
387.8 6 21.6 405.6 6 35.4 421.1 6 21.7 404.8ab 6 20.7 64.2 6 16.9 815.2 6 199.7 53.7 6 9.8 4.9b 6 1.0 243.6 6 37.4
0.077 0.784 0.163 0.022 0.873 0.267 0.238 0.030 0.567
P
y
Crypt depth (mm) Jejunum 10% 25% Mean Ileum 50% 75% 90% Mean Villus length (mm)y Jejunum 10% 25% Mean Ileum 50% 75% 90% Mean g-Glutamyl transferase (nmol $ min1 $ mg1 protein) Alkaline phosphatase (nmol $ min1 $ mg1 protein) Maltase (U/mg protein) Sucrase (U/mg protein) Lactase (U/mg protein)
* Mean 6 SEM. Values on the same row with different superscript letters were significantly different (P < 0.05). y Values 10% and 25% refer to the jejunum region, and 50%, 75%, and 90% refer to the ileum.
Results The amount of milk ingested by the polyamine group was similar to that of the control group (147 6 4.5 versus 131 6 6.3 mL $ kg1 $ d1). There were also no differences in the weight of these piglets (3.1 6 0.14 versus 3.1 6 0.27 kg, respectively). Piglets fed the polyaminesupplemented formula had a significantly greater crypt depth in the jejunum (Table 3) and in the whole of the small intestine than those fed the control formula (Fig. 1). There were no significant differences in villus length between
these animals (Table 3 and Figure 1), although villus length in the jejunum was significantly correlated with crypt depth (R ¼ 0.489, P ¼ 0.002) in this region of maximum nutrient uptake. The number of cells per villus length in the jejunum was larger in the polyamine-supplemented group than in the controls, although the differences were not statistically significant (55.34 6 1.15 versus 51.91 6 2.88 cells/villus length, respectively); nevertheless, the number of cells/villus length was correlated with the villus length in the entire small intestine (R ¼ 0.670, P ¼ 0.001).
Histological measurements 500
Sow Milk
450
Control
400
Polyamine
350
µm
300 250 200
ab
150
b
a
100 50 0 VILLUS LENGTH
CRYPT DEPTH
Fig. 1. Mean crypt depth and mean villus length calculated from histologic cuts obtained at 10%, 25%, 50%, 75%, and 90% along the entire length of the small intestine of early-weaned piglets maintained on different diets. Bars with different superscript letters were significantly different at P < 0.05.
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Table 4 Polyamine concentrations in jejunum and ileum mucosa of 15-d-old piglets maintained on three different diets* Sow milk (n ¼ 10) Jejunum (nmol/mL) Putrescine Spermidine Spermine Ileum (nmol/mL) Putrescine Spermidine Spermine
Control (n ¼ 10)
Polyamines (n ¼ 10)
P
1.9 6 0.92 40.4 6 5.13 62.7 6 7.25
3.4 6 0.87 30.5 6 2.33 71.6 6 4.24
2.6 6 1.16 28.6 6 2.26 71.1 6 3.53
0.589 0.093 0.414
5.94a 6 1.71 53.12a 6 5.45 69.76 6 5.82
1.55b 6 0.53 32.06b 6 2.79 73.54 6 2.46
3.96ab 6 1.12 33.92b 6 3.08 59.43 6 5.94
0.045 0.001 0.139
* Mean 6 SEM. Values on the same row with different superscript letters were significantly different (P < 0.05).
The alkaline phosphatase activity in the jejunum tended to be higher in the group fed polyamines than in the other two groups (P ¼ 0.267; Table 3). The activity of this enzyme was correlated with g-glutamyl transferase activity, and both were correlated with the number of cells/villus length in this region of the intestine (R ¼ 0.564, P ¼ 0.01, and R ¼ 0.642, P ¼ 0.002, for alkaline phosphatase and g-glutamyl transferase, respectively). There were no differences in disaccharidase activities between the animals fed the two formulas, although sucrase activity was significantly higher in the sow milk group (Table 3). The concentrations of polyamines in the small intestine mucosa are presented in Table 4. Dietary polyamines did not significantly modify the mucosal concentration of putrescine, spermine, or spermidine. Putrescine and spermidine levels in the ileum were significantly higher in the sow milk group than in animals fed the formulas, but there were no differences in spermine concentrations (Table 4). Discussion We report for the first time that feeding newborn piglets with a milk formula containing polyamines at physiologic maternal milk doses significantly increases the crypt depth in the small intestine of the animals unlike in piglets fed the control formula. Moreover, villus length was positively correlated with crypt depth. The present results are in agreement with observations of Lo¨ser et al. [14] who reported the beneficial effects of polyamine supplementation at physiologic doses on the weight of the small intestinal mucosa and the protein content of the organ in suckling rats. Many studies have evaluated the effects of oral non-physiologic amounts of individual polyamines on gut histology [10,28– 30], although the different polyamines do not have identical biological activity. Grant et al. [29] did not find differences in jejunal villus height or crypt depth despite using high concentrations of putrescine in piglets from 2 to 14 d of age. However, Cheng et al. [28] observed increased crypt depth and decreased villus height in the jejunum of piglets supplemented with high doses of spermine. In the present study, piglets fed a polyamine-supplemented formula tended to show higher levels of alkaline phosphatase activity in the jejunum mucosa than those
groups of animals fed the control formula or maternal milk, although the differences were not statistically significant. In addition, the activities of alkaline phosphatase and g-glutamyl transferase enzymes were positively correlated with the number of cells per villus length in this region of the intestine, which indicates that histologic changes were related to the enzymatic activities. In the brush-border membranes, most of the digestive enzymes (lactase, sucrase, maltase, aminopeptidase, and alkaline phosphatase) are glycoproteins whose activity changes a great deal at weaning time to enable the animal to cope with the adult solid diet [31]. Spermidine levels have been reported to be significantly higher in weaned than in suckling rats [32], suggesting that the increase in polyamine levels at weaning time might be partly responsible for the natural maturation of digestive enzymes of the brush border through glycoprotein fucosylation processes [32]. However, Peulen et al. [33] reported contradictory results for alkaline phosphatase activity in the gut of suckling rats that had received spermine orally at non-physiologic doses for a short time. They found an increase in this enzymatic activity in the jejunum, but not in the ileum, of suckling rats after 3 d of spermine supplementation [33]. These researchers suggested that spermine ingestion might increase alkaline phosphatase expression, translation, or activity in the jejunum. However, in another study [34], the same investigators reported decreased alkaline phosphatase activity in the jejunum of sucking rats, whereas no changes were observed in weaned rats; this was explained as being because the upper part of the villus, where this enzyme is mainly located, could be removed as a result of higher cell turnover [34]. The maturity degree of the small intestine could be behind this process, and the effect of exogenous polyamines seems limited. We found no differences in disaccharidase activities between the animals fed formulas. Several investigators have reported that orally administered polyamines induce changes in these intestinal enzyme activities, similar to those occurring during normal maturation, although always using nonphysiologic doses of polyamines [4,33–36]. Dufour et al. [3] showed that orally administered spermine and spermidine in rats decreased lactase activity, whereas sucrase and maltase activities increased. These results were also confirmed by other researchers [4,28,33,37–39]. In suckling rats, a dose-dependent response of microvillus enzymes (lactase,
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sucrase, and maltase) was reported at high levels of spermine administration, although these changes were undetectable at physiologic levels of spermine intake [4]. Dietary polyamines did not significantly affect the jejunum mucosa concentration of putrescine, spermine, or spermidine, which could be explained 1) by the fact that polyamines are rapidly taken up from the small intestinal lumen, from which they pass into the blood, and 2) because they are fairly regulated. Biol N’Garagba et al. [32] determined putrescine levels in the small intestine of rats during the 24 h after its uptake and demonstrated that putrescine levels increased gradually until they reached a maximum 4 h after treatment, before decreasing to the basal level. No similar kinetic studies were made for spermine and spermidine. In addition, putrescine seems to behave differently from spermine and spermidine; the gut putrescine content in suckling rats was not related to the amount of putrescine consumed during a 4-d period, whereas significant increases were found for the other polyamines consumed [32]; these investigators suggested that the catabolic pathway of putrescine may be more active than those of spermine and spermidine [32]. This hypothesis was also supported by Bardocz et al. [8,40] who showed that 1 h after administration, 80% of the putrescine had been converted to other polyamines and non-polyamine metabolites. For spermine and spermidine, however, about 70–80% of the intragastrically intubated dose remained in the original form [8]. Lo¨ser et al. [14] observed no significant changes in the concentrations of polyamines in the small intestine mucosa of rats fed polyamines at physiologic doses for 26 wk. Regarding non-physiologic doses, Grant et al. [29] showed that dietary putrescine (25 g/kg of dry diet) in newborn piglets increased the mucosal concentration of this polyamine, whereas spermine and spermidine concentrations remained unchanged. Other studies that evaluated postintake levels of spermine observed no change in their concentration in the mucosa of the small intestine [34,41]. The results concerning the polyamine content of the gut resulting from the diet are very controversial. Although the piglets in the present study were deprived of food for at least 8 h before sacrifice, the polyamine metabolism seemed to be very well regulated. In the ileum, putrescine and spermidine concentrations were significantly higher in animals fed sow milk than in animals fed formulas. Sow milk composition affects gut microflora of neonates in a different way than infant formulas and promotes anaerobic microbiota in the cecum. Because bacteroides, fusobacteria, and anaerobic cocci are able to synthesize large amounts of putrescine and spermidine [42], a contamination of the ileum by part of the cecal microbiota might explain the results concerning polyamine in the ileum. Regarding the effects of polyamines on the development and maturation of the small intestine, our results suggest that the oral administration of polyamines at physiologic doses slightly improves the development and growth of the small intestine. Positive changes were observed in several biochemical and morphologic markers associated with gut
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maturation, such as a trend toward higher specific enzymatic activities (alkaline phosphatase and g-glutamyl transferase), and significant histologic changes in the jejunum region such as crypt depth. However, in this study, ingested spermine and spermidine failed to enhance intestinal disaccharidase activities and villus length, possibly because the period of polyamine supplementation was short compared with the overall time of gut maturation in piglets. In conclusion, our results demonstrate that polyamine ingestion at physiologic doses by early-weaned piglets significantly affects the morphology of the small intestine but not all biochemical parameters associated with it, suggesting that dietary polyamines might be regarded as beneficial compounds for small intestinal growth and development. The impact on jejunum structural development needs to be further evaluated. Polyamines from the diet may be important in supplementing endogenous levels, although further research is required to investigate the role and dose-dependent effects of polyamines from foods and the potential benefits to be gained from increasing their levels in infant formulas to mimic those of breast milk. Acknowledgments The authors acknowledge the expert technical assistance of Drs. Guillermo Ramis Vidal and Jose´ Salvador Martinez Martinez and the collaboration of Almudena Haro Revenga and Bele´n Morales Pe´rez. References [1] Picciano MF. Nutrient composition of human milk. Pediatr Clin North Am 2001;48:53–67. [2] Baro´ L, Jimenez J, Martı´nez-Ferez, Boza JJ. Bioactive compounds derived from human milk. Ars Pharm 2001;42:21–8. [3] Dufour C, Dandrifosse G, Forget P, Vermesse F, Romain N, Lepoint P. Spermine and spermidine induce intestinal maturation in the rat. Gastroenterology 1988;95:112–6. [4] Buts JP, Keyser ND, Kolanowski J, Sokal EM, van Hoof F. Maturation of villus and crypt cell functions in rat small intestine: role of dietary polyamines. Dig Dis Sci 1993;38:1091–8. [5] Kaouass M, Deloyer P, Wery I, Dandrifosse G. Analysis of structural and biochemical events occurring in the small intestine after dietary polyamine ingestion in suckling rats. Dig Dis Sci 1996;41:1434–44. [6] Pegg E, McCann PP. Polyamine metabolism and function. Am J Physiol Cell Physiol 1982;243:C212–21. [7] Tabor CW, Tabor H. Polyamines. Annu Rev Biochem 1984; 53:749–90. [8] Bardocz S, Duguid TJ, Brown DS, Grant G, Pusztai A, White A, Ralph A. The importance of dietary polyamines in cell regeneration and growth. Br J Nutr 1995;73:819–28. [9] Lo¨ser C, Torff L, Folsch UR. Uptake of extracellular dietary putrescine is an important regulatory mechanism of intracellular polyamine metabolism during camostate-induced pancreatic growth in rats. Dig Dis Sci 1997;42:503–13. [10] Dorhout B, van Faassen A, van Beusekom CM, Kingma AW, de Hoog E, Nagel GT, et al. Oral administration of deuterium-labelled polyamines to sucking rat pups: luminal uptake metabolic fate and effects on gastrointestinal maturation. Br J Nutr 1997;78:639–54. [11] Matthews HR. Polyamines chromatin structure and transcription. Bioessays 1993;15:561–6.
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[12] Farriol M, Segovia T, Venereo Y, Orta X. Importance of the polyamines: review of the literature. Nutr Hosp 1999;14:101–13. [13] McCormack SA, Johnson LR. Role of polyamines in gastrointestinal mucosal growth. Am J Physiol Gastrointest Liver Physiol 1991; 260:G795–G806. [14] Lo¨ser C, Eisel A, Harms D, Fo¨lsch UR. Dietary polyamines are essential luminal growth factors for small intestinal and colonic mucosal growth and development. Gut 1999;44:12–6. [15] Romain N, Dandrifosse G, Jeusette F, Forget P. Polyamine concentration in rat milk and food human milk and infant formulas. Pediatr Res 1992;32:58–63. [16] Pollack PF, Koldovsky O, Nishioka K. Polyamine in human and rat milk and in infant formulas. Am J Clin Nutr 1992;56:371–5. [17] Motyl T, Płoszaj T, Wojtasik A, Kukulska W, Podgurniak M. Polyamines in cow’s and sow’s milk. Comp Biochem Physiol 1995; 111(Suppl B):427–33. [18] Kelly D, Smyth JA, McCracken KJ. Digestive development of the early-weaned pig. Br J Nutr 1991;65:169–80. [19] Buts JP, De Keyser N, De Raedemaeker L, Collette E, Sokal EM. Polyamine profiles in human milk, infant artificial formulas, and semi-elemental diets. J Pediatr Gastroenterol Nutr 1995;21:44–9. [20] Subcommittee on Swine Nutrition, Committee on Animal Nutrition, National Research Council. Nutrient requirements of swine. 10th ed. Washington, DC: National Academy Press; 1998. [21] Nabuurs MJ, Hoogendoorn A, van Zijderveld FG, van der Klis JD. A long-term perfusion test to measure net absorption in the small intestine of weaned pigs. Res Vet Sci 1993;55:108–14. [22] Van Dijk AJ, Niewold TA, Margry RJ, van den Hoven SG, Nabuurs MJ, Stockhofe-Zurwieden N, Beynen AC. Small intestinal morphology in weaned piglets fed a diet containing spray-dried porcine plasma. Res Vet Sci 2001;71:17–23. [23] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54. [24] Dahlqvist A. Method for assay of intestinal disaccharidases. Anal Biochem 1964;7:18–25. [25] Rosalki SR, Foo AY, Burlina A, Prellwitz W, Stieber P, Neumeier D, et al. Multicenter evaluation of iso-ALP test kit for measurement of bone alkaline phosphatase activity in serum and plasma. Clin Chem 1993;39:648–52. [26] Szasz G. Methods of enzymatic analysis. 2nd English ed. New York: Academic Press; 1974. p. 717. [27] Seiler N. Liquid chromatographic methods for assaying polyamines using prechromatographic derivatization. Methods Enzymol 1983; 94:10–25.
[28] Cheng ZB, Li DF, Xing JJ, Guo XY, Li ZJ. Oral administration of spermine advances intestinal maturation in sucking piglets. J Anim Sci 2006;82:621–6. [29] Grant AL, Thomas JW, King KJ, Liesman JS. Effects of dietary amines on small intestinal variables in neonatal pigs fed soy protein isolate. J Anim Sci 1990;68:363–71. [30] Peulen O, Pirlet C, Klimek M, Goffinet G, Dandrifosse G. Comparison between the natural postnatal maturation and the spermine-induced maturation of the rat intestine. Arch Physiol Biochem 1998;106:46–55. [31] Henning SJ. Postnatal development: coordination of feeding, digestion, and metabolism. Am J Physiol Gastrointest Liver Physiol 1981; 241:G199–214. [32] Biol N’Garagba MC, Greco S, George P, Hugueny I, Louisot P. Polyamine participation in the maturation of glycoprotein fucosylation, but not sialylation, in rat small intestine. Pediatr Res 2002;51:625–34. [33] Peulen O, Gharbi M, Powroznik B, Dandrifosse G. Differential effect of dietary spermine on alkaline phosphatase activity in jejunum and ileum of unweaned rats. Biochimie 2004;86:487–93. [34] Peulen O, Deloyer P, Dandrifosse G. Short-term effects of spermine ingestion on the small intestine: a comparison of suckling and weaned rats. Reprod Nutr Dev 2004;44:353–64. [35] Kaouass M, Deloyer P, Dandrifosse G. Intestinal development in suckling rats: direct or indirect spermine action? Digestion 1994;55:160–7. [36] ter Steege JC, Buurman WA, Forget PP. Spermine induces maturation of the immature intestinal immune system in neonatal mice. J Pediatr Gastroenterol Nutr 1997;25:332–40. [37] Wild GE, Daly AS, Sauriol N, Bennett G. Effect of exogenously administered polyamine on the structural maturation and enzyme ontogeny of the postnatal rat intestine. Biol Neonate 1993;63:246–57. [38] Harada E, Hashimoto Y, Syuto B. Orally administered spermine induces precocious intestinal maturation of macromolecular transport and disaccharidase development in suckling rats. Comp Biochem Physiol A Physiol 1994;109:667–73. [39] Dorhout B, Van Beusekom CM, Huisman M, Kingma AK, De Hoog E, Rudy Boersma E, Muskiet FAJ. Estimation of twenty-four hour polyamine intake from mature human milk. J Pediatr Gastroenterol Nutr 1996;23:298–302. [40] Bardo´cz S, Grant G, Brown DS, Pusztai A. Putrescine as a source of instant energy in the small intestine of the rat. Gut 1998;42:24–8. [41] Loret S, Brolet P, Pierzynowski S, Gouders I, Klimek M, Danielson V, et al. Pancreatic exocrine secretions as a source of luminal polyamines in pigs. Exp Physiol 2000;85:301–8. [42] Noack J, Kleessen B, Proll J, Dongowski G, Blaut M. Dietary guar gum and pectin stimulate intestinal microbial polyamine synthesis in rats. J Nutr 1998;128:1385–91.
Basic nutritional investigation
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Effects of dietary polyamines at physiologic doses in early-weaned piglets Marı´a Sabater-Molina, Ph.Da, Elvira Larque´, Ph.D.a,*, Francisco Torrella, Ph.D.b, Javier Plaza, Maa, Teresa Lozano, Ph.D.c, Antonio Mun˜oz, Ph.D.d, and Salvador Zamora, Ph.D.a a Department of Physiology, Faculty of Biology, University of Murcia, Murcia, Spain Department of Microbiology, Faculty of Biology, University of Murcia, Murcia, Spain c Department of Cell Biology, Faculty of Biology, University of Murcia, Murcia, Spain d Department of Animal Production, Veterinary School, University of Murcia, Murcia, Spain b
Manuscript received July 18, 2008; accepted January 22, 2009
Abstract
Objective: Polyamines are essential for many cell functions, and they form part of the composition of maternal milk; despite this, their addition to infant formulas is currently under evaluation. The aim of the present study was to evaluate the effects of milk formulas designed to resemble sow milk supplemented with polyamines at maternal physiologic milk doses on the gut maturation of early-weaned piglets. Methods: We fed 30 newborn piglets with maternal milk (n ¼ 10), a control milk formula (n ¼ 10), or a milk formula supplemented with polyamines (5 nmol/mL of spermine and 20 nmol/mL of spermidine, n ¼ 10) for 13 d (day 2 after birth through day 15). Several growth and intestinal development parameters were measured. Results: The piglets fed the formula containing polyamine at physiologic doses showed significantly increased crypt depth in the small intestine compared with those fed with the control formula. Villus length was correlated to crypt depth. Although there were no differences in the disaccharidase activities between the animals fed the two formulas, alkaline phosphatase and g-glutamyl transferase activities tended to be higher in the jejunum of those fed the polyamine-supplemented diet. Dietary polyamines did not significantly modify the gut mucosal concentrations of putrescine, spermine, or spermidine. Conclusion: Milk formulas supplemented with polyamines at maternal milk physiologic doses slightly enhanced gut growth and maturation in neonatal piglets. Ó 2009 Elsevier Inc. All rights reserved.
Keywords:
Polyamines; Human milk; Small intestine; Gut development; Piglets
Introduction Neonatal life, when only one type of food is the source of nutrition, is a very vulnerable period. Although several factors are involved in infants’ health, breast milk is considered the optimal source of nutrition during the first 6 mo of life. The nutritional components of human milk may influence intestinal maturation in neonates [1,2], and milk polyamines have been related to gut maturation and development in several studies [2–5]. Nevertheless, these components have not yet been incorporated into infant formulas because more This study was supported in part by a grant from DANONE S.A., Spain. *Corresponding author. Tel.: þ34-968-363-942; fax: þ34-968-363-963. E-mail address: [email protected] (E. Larque´). 0899-9007/09/$ – see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2009.01.017
studies are needed to clarify their role during this early stage of life. Polyamines (putrescine, spermidine, and spermine) are aliphatic molecules present in the cells of all organisms [6,7]. Dietary polyamines contribute significantly to the luminal gastrointestinal polyamine pool [8–10]. Physiologic intracellular polyamine levels are essential for many cell functions, such as protein, RNA, and DNA synthesis, and the stabilization of DNA and chromatin structure [11]. In addition, polyamines may modify the immune response, block calcium ion channels, and regulate apoptosis [12] and are involved in cell differentiation and proliferation [13]. In suckling rats, the oral administration of high doses of polyamines was correlated with the early appearance of morphologic and biochemical modifications typical of the mature intestine [3,5,10].
M. Sabater-Molina et al. / Nutrition 25 (2009) 940–946
In contrast, polyamine-deficient diets resulted in a significant hypoplasia of the small intestinal and colonic mucosa [14]. Although the importance of luminal gastrointestinal polyamines is well documented, little is known about their effects at physiologic levels. Polyamines are present in appreciable amounts in human milk [15–18]. Maternal milk polyamine levels are higher during the first week of lactation [15], which raises a question of the physiologic effect of these substances on neonates. At present, infant formulas are not supplemented with any exogenous source of polyamines, and their polyamine levels are 10 times lower than in human milk [19]. Because milk polyamines could exert stimulatory effects on postnatal growth and gut maturation in lactation [15–18], their addition to infant formulas could be of major importance for the newborn. The purpose of the present study was to determine the effects of neonatal milk formulas supplemented with polyamines, at the physiologic levels found in maternal milk, on gut development, by evaluating mucosal morphology, enzymatic activities, and polyamine levels in the small intestine of early-weaned piglets.
Materials and methods Animals and diets Thirty newborn piglets (Landrace by Large White) were provided by the veterinary farm of the University of Murcia (Murcia, Spain). They were matched by gender. Twenty piglets were nursed by sows until 2 d of age, after which they were randomly allocated into one of two groups (10 animals/group): control formula group (n ¼ 10) and polyamine-supplemented formula group (n ¼ 10). The piglets for these two groups came from the same litter as those from the maternal milk group. The piglets were housed in cages provided with attached spot heat lamps and fed ad libitum every 3 h for 13 d. The rest of the animals (n ¼ 10) continued to be nursed by sows until 15 d of age (sow milk group). The study was approved by the animal care committee at the University of Murcia and conformed to the European Union Regulation of Animal Care for the care and use of animals for research. The milk formula was designed to resemble sow milk in its macronutrient composition and to meet National Research Council nutrient requirements for the growing piglet [20]. The ingredients for the preparation of the milk formula were 118 g of cow’s milk solids (3.6% fat), 137 g of calcium caseinate, 153 g of demineralized milk whey, 451 g of cream (35% fat), 116 g of olive oil, 182 g of coconut oil, 36 g of soybean oil, 3.5 g of lecithin, 206 g of lactose, 37 g of a mineral complex, and 3.3 g of a vitamin complex (Piensos CARN, Murcia, Spain) per kilogram. The nutrient composition of the milk formula is presented in Table 1. The formulas were dissolved in warm water at a concentration of 200 g/L. No detectable polyamine levels were found in the control
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Table 1 Nutrient composition of milk formula in percentage of dry mass Composition (%) Raw protein Lysine Fat Lactose Vitamins* Minerals* Fatty acids (% total weight) 4:0 6:0 8:0 10:0 12:0 14:0 16:0 16:1 18:0 18:1 18:2 18:3 C20
27.05 2.14 33.22 29.96 5.0 4.76 0.0319 0.0159 1.0781 1.0863 8.9509 3.7651 31.0650 0.0262 4.8008 35.1280 12.5001 0.988 0.1183
* The mineral and vitamin mixture contained (milligrams per kilogram of diet): dibasic calcium phosphate 22,240, calcium citrate tetrahydrate 5070, sodium phosphate dibasic dodecahydrate 5060, manganese sulfate 1530, ferrous lactate 1380, potassium sulfate 920, copper sulfate 10.7, manganese sulfate 32.6, potassium iodate 0.8, zinc sulfate 5.24, thiamine 2, riboflavin 3, pyridoxine HCl 3, nicotinic acid 30, calcium pantothenate 20, folic acid 1, biotin 0.8, cyanobalamin 0.025, retinol acetate 0.069, cholecalciferol 0.0093, DL-tocopherol 50, phylloquinone 0.15, ascorbic acid 700.
milk formula for three replicates. The polyamine-supplemented formula contained 5 nmol/mL of spermine (Sigma Chemical Co., St. Louis, MO, USA), and 20 nmol/mL of spermidine (Sigma Chemical Co.), reflecting the polyamine levels detected in sow milk. The polyamine dose used was calculated from the mean concentration of polyamines in 10 milk samples from all the different sows (Table 2). Numerous factors modify the amount of milk polyamines, such as genetic factors, the preweaning period, environmental factors and, especially, the nutritional state and diet [2]. It was therefore important that the sows used in the present study were subjected to the same conditions of feeding, light, and water availability as those used for the quantification of milk polyamine concentration. Dissection protocol At 15 d of age, fasting piglets, deprived of food for at least 8 h, were anesthetized by retro-ocular injection with a 50:50 mixture of ketamine:propofol (1 mL/kg). The abdominal wall was opened and the entire gastrointestinal tract was removed. Using scissors, the mesentery was cut, and five intestinal tissue samples, each 1 cm in length, were removed at a point 10%, 25%, 50%, 75%, and 90% of the length of the small intestine and kept in Bouin fixative until analysis. In addition, mucosa samples from the jejunum and ileum were removed by scraping the entire luminal surface with a glass
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Table 2 Polyamine concentration in milk samples from 10 different sows Day of lactation
Putrescine (nmol/mL)
Spermidine (nmol/mL)
Spermine (nmol/mL)
4 (n ¼ 3) 5 (n ¼ 1) 6 (n ¼ 1) 7 (n ¼ 2) 11 (n ¼ 1) 13 (n ¼ 1) 15 (n ¼ 1) Mean 6 SEM
0.94 0.00 5.87 0.31 0.41 0.70 0.72 1.28 6 0.77
21.33 17.26 17.58 27.73 21.77 30.59 12.06 21.19 6 2.41
11.47 2.78 10.19 2.33 2.59 3.56 1.17 4.87 6 1.57
coverslip over an ice-cold Petri dish, frozen immediately in liquid nitrogen, and stored at 80 C. The piglets were euthanized by an intracardial injection of sodium pentobarbital (1 mL/kg). Analytical methods The microscopical morphologic measurements of the gut were performed with the five intestinal tissue samples collected at the sites mentioned above in the small intestine. After fixing the tissue samples in Bouin liquid (75 mL of picric acid water saturated solution, 25 mL of formaldehyde, and 5 mL of acetic acid), a portion of each sample was embedded in paraffin wax using standard techniques. Two transverse sections were selected from each sample, stained with hematoxylin and eosin, and examined with an optical microscope (Axioskop, Zeiss, Germany). Villus length and crypt depth were measured using an image processing program (Microm Image Processing 4.5, Consulting Image Digital, Barcelona, Spain). The length and depth of at least 10 villi and crypts were measured, calculating the mean villus length and the mean crypt depth. In addition, the number of cells per length of villus was estimated from 10 villi in each histologic section [21,22]. Mucosal samples were used to determine disaccharidase, alkaline phosphatase, and g-glutamyl transferase activities and polyamine levels. After homogenization of the jejunum mucosa samples in saline solution (1:10, w:v) and centrifugation at 1500 3 g for 20 min, the supernatants were analyzed for total protein content according to the method of Bradford [23], disaccharidase (i.e., maltase, sucrase, and lactase) according to the spectrophotometric method of Dahlqvist [24], alkaline phosphatase activity according to Rosalki et al. [25], and g-glutamyl transferase activity using the method of Szasz [26], and the Biosystems diagnostic kit (Barcelona, Spain). The polyamine content in jejunum and ileum mucosa samples was quantified according to a modified version of the method described by Seiler [27]. Briefly, mucosa samples were homogenized (1:10, w:v) in 5-sulfosalicylic acid (0.2 g/mL) and kept frozen overnight at 80 C. After centrifugation at 6700 3 g for 20 min, 50 mL of 1,6-diaminohexane (internal standard), 200 mL of saturated sodium
carbonate, and 400 mL of dansyl chloride (10 mg/mL in acetone) were successively added to 100 mL of the supernatant. This dansylation mixture was incubated overnight at room temperature. Dansylated derivatives were then extracted with 1.5 mL of cyclohexane and subsequently dried before being dissolved in 100 mL of the injection medium (i.e., acetonitrile:methanol 3:2, v:v). Dansylated polyamines were quantified by high-performance liquid chromatography using a reverse-phase column (Nova-Pak C18, Waters, Barcelona, Spain). We used a two-phase gradient, starting with 57% phase A (phase A: acetonitrile:methanol 3:2; phase B: Milli Q water), reaching 100% phase A after a 53-min run. Polyamine standards (putrescine dihydrochloride, spermidine trihydrochloride, and spermine tetrahydrochloride) were purchased from Sigma Chemical Co. Determination of polyamines in sow milk and formulas Polyamines in sow milk and formulas were analyzed using the AccQ-Fluor Reagent Kit (Waters), which uses 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate as derivation reagent. Ten milk samples were obtained from a group of sows on different days during the lactation period (Table 2). The samples were frozen at 80 C until polyamine analysis was performed. Two hundred microliters of 5-sulfosalicylic solution (0.2 g/mL) and 50 mL of 1,6-diaminohexane (internal standard) were successively added to 1-mL milk samples and then kept frozen overnight at 20 C. After centrifugation at 6700 3 g for 20 min, the resulting precipitate was removed. The supernatant was centrifuged again at 6700 3 g for 10 min, and the supernatant was passed through a 0.45-mm Millipore filter before derivation. Then 70 mL of borate buffer (pH 8.8) and 20 mL of AccQ Fluor (Waters) were added to 100 mL of a polyamine-containing sample. The mixture was incubated in a water bath for 10 min at 55 C. Derivatized polyamines were determined by high-performance liquid chromatography using a reverse-phase column (Nova-Pak C18, Waters). We used a two-phase gradient, starting with 25% phase A (phase A: 20 mM sodium acetate; phase B: methanol), reaching 100% phase A after a 30-min run. Polyamine standards (putrescine dihydrochloride, spermidine trihydrochloride, and spermine tetrahydrochloride) were purchased from Sigma Chemical Co. Statistical analyses The results are expressed as mean 6 standard error of the mean. Analysis of variance was performed to analyze differences among the three groups of animals with a posteriori Bonferroni’s test. The correlations between the histologic parameters and enzymatic activities, on the one hand, and polyamine concentration, on the other, were evaluated using Pearson’s correlation test. Differences were considered statistically significant at P < 0.05. All statistical analyses were carried out using SPSS 12.0 for Windows (SPSS Inc., Chicago, IL, USA).
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Table 3 Histologic measurements and enzymatic activities in the small intestine of 15-d-old piglets maintained on three different diets* Sow milk (n ¼ 10)
Control (n ¼ 10)
Polyamines (n ¼ 10)
145.2 6 5.9 147.7ab 6 7.9 148.2ab 6 5.6
145.7 6 7.9 142.6b 6 6.4 143.4b 6 3.7
162.5 6 7.6 164.6a 6 6.2 162.5a 6 5.1
0.161 0.043 0.040
149.2 6 9.5 140.1 6 9.2 140.0 6 9.4 141.5 6 7.3
125.1 6 6.7 129.4 6 6.8 133.7 6 8.2 127.7 6 5.9
142.4 6 7.4 138.2 6 4.9 140.6 6 8.3 140.4 6 3.1
0.130 0.553 0.827 0.184
361.4 6 17.6 419.2 6 27.4 390.3 6 19.4
386.9 6 35.7 410.8 6 22.5 393.2 6 27.7
418.4 6 19.8 385.4 6 17.4 401.9 6 12.9
0.116 0.117 0.915
463.6 6 27.3 528.1 6 40.7 452 6 20.6 481.2a 6 21.6 54.1 6 18.8 537.8 6 124.2 82.8 6 15.9 21.6a 6 6.8 218.5 6 67.8
368.7 6 36.9 411.9 6 34.2 390.6 6 25.1 393.1b 6 25.9 52.9 6 7.9 732.1 6 150.1 72.7 6 8.8 12.4ab 6 2.4 284.9 6 71.7
387.8 6 21.6 405.6 6 35.4 421.1 6 21.7 404.8ab 6 20.7 64.2 6 16.9 815.2 6 199.7 53.7 6 9.8 4.9b 6 1.0 243.6 6 37.4
0.077 0.784 0.163 0.022 0.873 0.267 0.238 0.030 0.567
P
y
Crypt depth (mm) Jejunum 10% 25% Mean Ileum 50% 75% 90% Mean Villus length (mm)y Jejunum 10% 25% Mean Ileum 50% 75% 90% Mean g-Glutamyl transferase (nmol $ min1 $ mg1 protein) Alkaline phosphatase (nmol $ min1 $ mg1 protein) Maltase (U/mg protein) Sucrase (U/mg protein) Lactase (U/mg protein)
* Mean 6 SEM. Values on the same row with different superscript letters were significantly different (P < 0.05). y Values 10% and 25% refer to the jejunum region, and 50%, 75%, and 90% refer to the ileum.
Results The amount of milk ingested by the polyamine group was similar to that of the control group (147 6 4.5 versus 131 6 6.3 mL $ kg1 $ d1). There were also no differences in the weight of these piglets (3.1 6 0.14 versus 3.1 6 0.27 kg, respectively). Piglets fed the polyaminesupplemented formula had a significantly greater crypt depth in the jejunum (Table 3) and in the whole of the small intestine than those fed the control formula (Fig. 1). There were no significant differences in villus length between
these animals (Table 3 and Figure 1), although villus length in the jejunum was significantly correlated with crypt depth (R ¼ 0.489, P ¼ 0.002) in this region of maximum nutrient uptake. The number of cells per villus length in the jejunum was larger in the polyamine-supplemented group than in the controls, although the differences were not statistically significant (55.34 6 1.15 versus 51.91 6 2.88 cells/villus length, respectively); nevertheless, the number of cells/villus length was correlated with the villus length in the entire small intestine (R ¼ 0.670, P ¼ 0.001).
Histological measurements 500
Sow Milk
450
Control
400
Polyamine
350
µm
300 250 200
ab
150
b
a
100 50 0 VILLUS LENGTH
CRYPT DEPTH
Fig. 1. Mean crypt depth and mean villus length calculated from histologic cuts obtained at 10%, 25%, 50%, 75%, and 90% along the entire length of the small intestine of early-weaned piglets maintained on different diets. Bars with different superscript letters were significantly different at P < 0.05.
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Table 4 Polyamine concentrations in jejunum and ileum mucosa of 15-d-old piglets maintained on three different diets* Sow milk (n ¼ 10) Jejunum (nmol/mL) Putrescine Spermidine Spermine Ileum (nmol/mL) Putrescine Spermidine Spermine
Control (n ¼ 10)
Polyamines (n ¼ 10)
P
1.9 6 0.92 40.4 6 5.13 62.7 6 7.25
3.4 6 0.87 30.5 6 2.33 71.6 6 4.24
2.6 6 1.16 28.6 6 2.26 71.1 6 3.53
0.589 0.093 0.414
5.94a 6 1.71 53.12a 6 5.45 69.76 6 5.82
1.55b 6 0.53 32.06b 6 2.79 73.54 6 2.46
3.96ab 6 1.12 33.92b 6 3.08 59.43 6 5.94
0.045 0.001 0.139
* Mean 6 SEM. Values on the same row with different superscript letters were significantly different (P < 0.05).
The alkaline phosphatase activity in the jejunum tended to be higher in the group fed polyamines than in the other two groups (P ¼ 0.267; Table 3). The activity of this enzyme was correlated with g-glutamyl transferase activity, and both were correlated with the number of cells/villus length in this region of the intestine (R ¼ 0.564, P ¼ 0.01, and R ¼ 0.642, P ¼ 0.002, for alkaline phosphatase and g-glutamyl transferase, respectively). There were no differences in disaccharidase activities between the animals fed the two formulas, although sucrase activity was significantly higher in the sow milk group (Table 3). The concentrations of polyamines in the small intestine mucosa are presented in Table 4. Dietary polyamines did not significantly modify the mucosal concentration of putrescine, spermine, or spermidine. Putrescine and spermidine levels in the ileum were significantly higher in the sow milk group than in animals fed the formulas, but there were no differences in spermine concentrations (Table 4). Discussion We report for the first time that feeding newborn piglets with a milk formula containing polyamines at physiologic maternal milk doses significantly increases the crypt depth in the small intestine of the animals unlike in piglets fed the control formula. Moreover, villus length was positively correlated with crypt depth. The present results are in agreement with observations of Lo¨ser et al. [14] who reported the beneficial effects of polyamine supplementation at physiologic doses on the weight of the small intestinal mucosa and the protein content of the organ in suckling rats. Many studies have evaluated the effects of oral non-physiologic amounts of individual polyamines on gut histology [10,28– 30], although the different polyamines do not have identical biological activity. Grant et al. [29] did not find differences in jejunal villus height or crypt depth despite using high concentrations of putrescine in piglets from 2 to 14 d of age. However, Cheng et al. [28] observed increased crypt depth and decreased villus height in the jejunum of piglets supplemented with high doses of spermine. In the present study, piglets fed a polyamine-supplemented formula tended to show higher levels of alkaline phosphatase activity in the jejunum mucosa than those
groups of animals fed the control formula or maternal milk, although the differences were not statistically significant. In addition, the activities of alkaline phosphatase and g-glutamyl transferase enzymes were positively correlated with the number of cells per villus length in this region of the intestine, which indicates that histologic changes were related to the enzymatic activities. In the brush-border membranes, most of the digestive enzymes (lactase, sucrase, maltase, aminopeptidase, and alkaline phosphatase) are glycoproteins whose activity changes a great deal at weaning time to enable the animal to cope with the adult solid diet [31]. Spermidine levels have been reported to be significantly higher in weaned than in suckling rats [32], suggesting that the increase in polyamine levels at weaning time might be partly responsible for the natural maturation of digestive enzymes of the brush border through glycoprotein fucosylation processes [32]. However, Peulen et al. [33] reported contradictory results for alkaline phosphatase activity in the gut of suckling rats that had received spermine orally at non-physiologic doses for a short time. They found an increase in this enzymatic activity in the jejunum, but not in the ileum, of suckling rats after 3 d of spermine supplementation [33]. These researchers suggested that spermine ingestion might increase alkaline phosphatase expression, translation, or activity in the jejunum. However, in another study [34], the same investigators reported decreased alkaline phosphatase activity in the jejunum of sucking rats, whereas no changes were observed in weaned rats; this was explained as being because the upper part of the villus, where this enzyme is mainly located, could be removed as a result of higher cell turnover [34]. The maturity degree of the small intestine could be behind this process, and the effect of exogenous polyamines seems limited. We found no differences in disaccharidase activities between the animals fed formulas. Several investigators have reported that orally administered polyamines induce changes in these intestinal enzyme activities, similar to those occurring during normal maturation, although always using nonphysiologic doses of polyamines [4,33–36]. Dufour et al. [3] showed that orally administered spermine and spermidine in rats decreased lactase activity, whereas sucrase and maltase activities increased. These results were also confirmed by other researchers [4,28,33,37–39]. In suckling rats, a dose-dependent response of microvillus enzymes (lactase,
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sucrase, and maltase) was reported at high levels of spermine administration, although these changes were undetectable at physiologic levels of spermine intake [4]. Dietary polyamines did not significantly affect the jejunum mucosa concentration of putrescine, spermine, or spermidine, which could be explained 1) by the fact that polyamines are rapidly taken up from the small intestinal lumen, from which they pass into the blood, and 2) because they are fairly regulated. Biol N’Garagba et al. [32] determined putrescine levels in the small intestine of rats during the 24 h after its uptake and demonstrated that putrescine levels increased gradually until they reached a maximum 4 h after treatment, before decreasing to the basal level. No similar kinetic studies were made for spermine and spermidine. In addition, putrescine seems to behave differently from spermine and spermidine; the gut putrescine content in suckling rats was not related to the amount of putrescine consumed during a 4-d period, whereas significant increases were found for the other polyamines consumed [32]; these investigators suggested that the catabolic pathway of putrescine may be more active than those of spermine and spermidine [32]. This hypothesis was also supported by Bardocz et al. [8,40] who showed that 1 h after administration, 80% of the putrescine had been converted to other polyamines and non-polyamine metabolites. For spermine and spermidine, however, about 70–80% of the intragastrically intubated dose remained in the original form [8]. Lo¨ser et al. [14] observed no significant changes in the concentrations of polyamines in the small intestine mucosa of rats fed polyamines at physiologic doses for 26 wk. Regarding non-physiologic doses, Grant et al. [29] showed that dietary putrescine (25 g/kg of dry diet) in newborn piglets increased the mucosal concentration of this polyamine, whereas spermine and spermidine concentrations remained unchanged. Other studies that evaluated postintake levels of spermine observed no change in their concentration in the mucosa of the small intestine [34,41]. The results concerning the polyamine content of the gut resulting from the diet are very controversial. Although the piglets in the present study were deprived of food for at least 8 h before sacrifice, the polyamine metabolism seemed to be very well regulated. In the ileum, putrescine and spermidine concentrations were significantly higher in animals fed sow milk than in animals fed formulas. Sow milk composition affects gut microflora of neonates in a different way than infant formulas and promotes anaerobic microbiota in the cecum. Because bacteroides, fusobacteria, and anaerobic cocci are able to synthesize large amounts of putrescine and spermidine [42], a contamination of the ileum by part of the cecal microbiota might explain the results concerning polyamine in the ileum. Regarding the effects of polyamines on the development and maturation of the small intestine, our results suggest that the oral administration of polyamines at physiologic doses slightly improves the development and growth of the small intestine. Positive changes were observed in several biochemical and morphologic markers associated with gut
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maturation, such as a trend toward higher specific enzymatic activities (alkaline phosphatase and g-glutamyl transferase), and significant histologic changes in the jejunum region such as crypt depth. However, in this study, ingested spermine and spermidine failed to enhance intestinal disaccharidase activities and villus length, possibly because the period of polyamine supplementation was short compared with the overall time of gut maturation in piglets. In conclusion, our results demonstrate that polyamine ingestion at physiologic doses by early-weaned piglets significantly affects the morphology of the small intestine but not all biochemical parameters associated with it, suggesting that dietary polyamines might be regarded as beneficial compounds for small intestinal growth and development. The impact on jejunum structural development needs to be further evaluated. Polyamines from the diet may be important in supplementing endogenous levels, although further research is required to investigate the role and dose-dependent effects of polyamines from foods and the potential benefits to be gained from increasing their levels in infant formulas to mimic those of breast milk. Acknowledgments The authors acknowledge the expert technical assistance of Drs. Guillermo Ramis Vidal and Jose´ Salvador Martinez Martinez and the collaboration of Almudena Haro Revenga and Bele´n Morales Pe´rez. References [1] Picciano MF. Nutrient composition of human milk. Pediatr Clin North Am 2001;48:53–67. [2] Baro´ L, Jimenez J, Martı´nez-Ferez, Boza JJ. Bioactive compounds derived from human milk. Ars Pharm 2001;42:21–8. [3] Dufour C, Dandrifosse G, Forget P, Vermesse F, Romain N, Lepoint P. Spermine and spermidine induce intestinal maturation in the rat. Gastroenterology 1988;95:112–6. [4] Buts JP, Keyser ND, Kolanowski J, Sokal EM, van Hoof F. Maturation of villus and crypt cell functions in rat small intestine: role of dietary polyamines. Dig Dis Sci 1993;38:1091–8. [5] Kaouass M, Deloyer P, Wery I, Dandrifosse G. Analysis of structural and biochemical events occurring in the small intestine after dietary polyamine ingestion in suckling rats. Dig Dis Sci 1996;41:1434–44. [6] Pegg E, McCann PP. Polyamine metabolism and function. Am J Physiol Cell Physiol 1982;243:C212–21. [7] Tabor CW, Tabor H. Polyamines. Annu Rev Biochem 1984; 53:749–90. [8] Bardocz S, Duguid TJ, Brown DS, Grant G, Pusztai A, White A, Ralph A. The importance of dietary polyamines in cell regeneration and growth. Br J Nutr 1995;73:819–28. [9] Lo¨ser C, Torff L, Folsch UR. Uptake of extracellular dietary putrescine is an important regulatory mechanism of intracellular polyamine metabolism during camostate-induced pancreatic growth in rats. Dig Dis Sci 1997;42:503–13. [10] Dorhout B, van Faassen A, van Beusekom CM, Kingma AW, de Hoog E, Nagel GT, et al. Oral administration of deuterium-labelled polyamines to sucking rat pups: luminal uptake metabolic fate and effects on gastrointestinal maturation. Br J Nutr 1997;78:639–54. [11] Matthews HR. Polyamines chromatin structure and transcription. Bioessays 1993;15:561–6.
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M. Sabater-Molina et al. / Nutrition 25 (2009) 940–946
[12] Farriol M, Segovia T, Venereo Y, Orta X. Importance of the polyamines: review of the literature. Nutr Hosp 1999;14:101–13. [13] McCormack SA, Johnson LR. Role of polyamines in gastrointestinal mucosal growth. Am J Physiol Gastrointest Liver Physiol 1991; 260:G795–G806. [14] Lo¨ser C, Eisel A, Harms D, Fo¨lsch UR. Dietary polyamines are essential luminal growth factors for small intestinal and colonic mucosal growth and development. Gut 1999;44:12–6. [15] Romain N, Dandrifosse G, Jeusette F, Forget P. Polyamine concentration in rat milk and food human milk and infant formulas. Pediatr Res 1992;32:58–63. [16] Pollack PF, Koldovsky O, Nishioka K. Polyamine in human and rat milk and in infant formulas. Am J Clin Nutr 1992;56:371–5. [17] Motyl T, Płoszaj T, Wojtasik A, Kukulska W, Podgurniak M. Polyamines in cow’s and sow’s milk. Comp Biochem Physiol 1995; 111(Suppl B):427–33. [18] Kelly D, Smyth JA, McCracken KJ. Digestive development of the early-weaned pig. Br J Nutr 1991;65:169–80. [19] Buts JP, De Keyser N, De Raedemaeker L, Collette E, Sokal EM. Polyamine profiles in human milk, infant artificial formulas, and semi-elemental diets. J Pediatr Gastroenterol Nutr 1995;21:44–9. [20] Subcommittee on Swine Nutrition, Committee on Animal Nutrition, National Research Council. Nutrient requirements of swine. 10th ed. Washington, DC: National Academy Press; 1998. [21] Nabuurs MJ, Hoogendoorn A, van Zijderveld FG, van der Klis JD. A long-term perfusion test to measure net absorption in the small intestine of weaned pigs. Res Vet Sci 1993;55:108–14. [22] Van Dijk AJ, Niewold TA, Margry RJ, van den Hoven SG, Nabuurs MJ, Stockhofe-Zurwieden N, Beynen AC. Small intestinal morphology in weaned piglets fed a diet containing spray-dried porcine plasma. Res Vet Sci 2001;71:17–23. [23] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54. [24] Dahlqvist A. Method for assay of intestinal disaccharidases. Anal Biochem 1964;7:18–25. [25] Rosalki SR, Foo AY, Burlina A, Prellwitz W, Stieber P, Neumeier D, et al. Multicenter evaluation of iso-ALP test kit for measurement of bone alkaline phosphatase activity in serum and plasma. Clin Chem 1993;39:648–52. [26] Szasz G. Methods of enzymatic analysis. 2nd English ed. New York: Academic Press; 1974. p. 717. [27] Seiler N. Liquid chromatographic methods for assaying polyamines using prechromatographic derivatization. Methods Enzymol 1983; 94:10–25.
[28] Cheng ZB, Li DF, Xing JJ, Guo XY, Li ZJ. Oral administration of spermine advances intestinal maturation in sucking piglets. J Anim Sci 2006;82:621–6. [29] Grant AL, Thomas JW, King KJ, Liesman JS. Effects of dietary amines on small intestinal variables in neonatal pigs fed soy protein isolate. J Anim Sci 1990;68:363–71. [30] Peulen O, Pirlet C, Klimek M, Goffinet G, Dandrifosse G. Comparison between the natural postnatal maturation and the spermine-induced maturation of the rat intestine. Arch Physiol Biochem 1998;106:46–55. [31] Henning SJ. Postnatal development: coordination of feeding, digestion, and metabolism. Am J Physiol Gastrointest Liver Physiol 1981; 241:G199–214. [32] Biol N’Garagba MC, Greco S, George P, Hugueny I, Louisot P. Polyamine participation in the maturation of glycoprotein fucosylation, but not sialylation, in rat small intestine. Pediatr Res 2002;51:625–34. [33] Peulen O, Gharbi M, Powroznik B, Dandrifosse G. Differential effect of dietary spermine on alkaline phosphatase activity in jejunum and ileum of unweaned rats. Biochimie 2004;86:487–93. [34] Peulen O, Deloyer P, Dandrifosse G. Short-term effects of spermine ingestion on the small intestine: a comparison of suckling and weaned rats. Reprod Nutr Dev 2004;44:353–64. [35] Kaouass M, Deloyer P, Dandrifosse G. Intestinal development in suckling rats: direct or indirect spermine action? Digestion 1994;55:160–7. [36] ter Steege JC, Buurman WA, Forget PP. Spermine induces maturation of the immature intestinal immune system in neonatal mice. J Pediatr Gastroenterol Nutr 1997;25:332–40. [37] Wild GE, Daly AS, Sauriol N, Bennett G. Effect of exogenously administered polyamine on the structural maturation and enzyme ontogeny of the postnatal rat intestine. Biol Neonate 1993;63:246–57. [38] Harada E, Hashimoto Y, Syuto B. Orally administered spermine induces precocious intestinal maturation of macromolecular transport and disaccharidase development in suckling rats. Comp Biochem Physiol A Physiol 1994;109:667–73. [39] Dorhout B, Van Beusekom CM, Huisman M, Kingma AK, De Hoog E, Rudy Boersma E, Muskiet FAJ. Estimation of twenty-four hour polyamine intake from mature human milk. J Pediatr Gastroenterol Nutr 1996;23:298–302. [40] Bardo´cz S, Grant G, Brown DS, Pusztai A. Putrescine as a source of instant energy in the small intestine of the rat. Gut 1998;42:24–8. [41] Loret S, Brolet P, Pierzynowski S, Gouders I, Klimek M, Danielson V, et al. Pancreatic exocrine secretions as a source of luminal polyamines in pigs. Exp Physiol 2000;85:301–8. [42] Noack J, Kleessen B, Proll J, Dongowski G, Blaut M. Dietary guar gum and pectin stimulate intestinal microbial polyamine synthesis in rats. J Nutr 1998;128:1385–91.