Analysis of PEG 400 and 4000 in urine for gut permeability assessment using solid phase extraction and gel permeation chromatography with refractometric detection.

We developed a treatment of urine samples allowing the analysis of two intestinal permeability markers: polyethylene glycol (PEG) 400 (highly diffusible; basal permeability indicator) and PEG 4000 (poorly diffusible; indicator of an abnormal increase of permeability) by a unique gel permeation chromatography (GPC) with refractometric detection. Urinary PEG were extracted using a mixed-bed resin composed of C2 and C18 layers. Permeability mean values determined in 11 human healthy subjects were 24.20 +/- 9.30% and 0.12 +/- 0.08% for, respectively, PEG 400 and 4000. The percentage of the PEG 4000 permeability value to the one of PEG 400 corresponded to an intestinal permeability index (IPI) of 0.52 +/- 0.35 expressing a low diffusion of this poorly permeability marker.

Spermine induces precocious development of the spleen in mice.

Spermine is a low molecular weight polyamine involved in the postnatal maturation of the gut. When it is administered orally to suckling rats, it induces maturation of the intestinal tract (liver, pancreas and small intestine). Here we show that this polyamine is able to induce precocious intestinal and splenic development in suckling mice. In fact, in 15-day-old mice which had received spermine orally twice daily for 3 days we observed an increase in the ratio of white pulp surface to total spleen surface in comparison with untreated mice. The two macrophage subsets of the marginal zone and the B-cell population were more developed and reached the development level of 5- or 10-week-old mice. The proliferation rate of B-cells was increased by spermine administration to pups. These observations suggest that spermine might play a role in immune system development; further investigation of its effects are intended, namely the evaluation of its capacity to enhance defence during the neonatal period.

Spermine-Induced alteration of small intestine in suckling rat: involvement of apoptosis or Zn2+ enzymes?

Polyamines are of great importance in several physiological processes, such as cell proliferation and differentiation. The ingestion of spermine by suckling rats induces precocious maturation of their small intestine. Shortly after ingestion, spermine produces cell elimination at the villous top. The origin of this exfoliation was investigated to determine whether it was due to apoptosis. Wistar rats were orally treated with spermine. Apoptosis was analyzed in their small intestine by Tdt-mediated dUTP-fluorescein nick-end labeling reaction, caspase-3-like analysis, and DNA laddering. Polyamine content was measured by HPLC. The intestinal transitory alteration appeared as soon as 2 hr after spermine administration. Apoptosis events increased strongly at the same moment in the small intestine. They were evidenced by Tdt-mediated dUTP-fluorescein nick-end labeling analysis, DNA laddering, and caspase-3-like activity. Changes observed are consistent with apoptosis, but caspase inhibitor did not reduce intestinal alteration, as did Zn2+ chelator.

Dietary polyamines and non-neoplastic growth and disease.

This review presents the data that are now available concerning the effects of dietary polyamines at either postnatal or adult stages in non-neoplastic growth and disease. Polyamines provided by food have a potential role in growth and development of the digestive system in neonatal mammals (and fishes). In humans, this property could be of importance in preventing the appearance of food allergies. Dietary polyamines also seem necessary for the maintenance of normal growth and general properties of adult digestive tract. Their possible therapeutic effects have been investigated in gastric, intestinal, and, more recently, whole-body healing.

Dietary fructans modulate polyamine concentration in the cecum of rats.

Nondigestible but fermentable dietary fructans such as oligofructose exert many effects on gut physiology through their fermentation end products such as short-chain fatty acids. Could other metabolites be produced in the gut and contribute to the physiologic effects of dietary fructans? The aim of the study was to evaluate the influence of oligofructose on putrescine, spermidine and spermine concentrations in the cecum, the portal vein and the liver of rats and to assess their involvement in cecal enlargement and the modulation of hepatic lipid metabolism. Putrescine, spermidine and spermine were quantified by HPLC in samples obtained from male Wistar rats fed a nonpurified standard diet (controls) or the same diet enriched with 10 g/100 g oligofructose (OFS) for 4 wk. OFS-fed rats had significantly greater cecal content and tissue weights. OFS almost doubled the concentration of putrescine in the cecal contents. The concentration of all three polyamines in the cecal tissue was significantly greater than in controls. The concentration of spermidine in portal plasma was lower in rats fed OFS, whereas the treatment did not affect the polyamine concentrations in the liver. The fermentation of dietary fructans contributed to an increase in the concentration of putrescine in the gut without modifying putrescine concentration in either the portal blood or liver. Moreover, the greater levels of polyamines in cecal tissue may be related to the cell proliferation resulting from OFS fermentation in the gut.

Pancreatic exocrine secretions as a source of luminal polyamines in pigs.

The goal of the present study was twofold: (1) to detect the possible storage of dietary polyamines (PAs) in various tissues and (2) to investigate the role of dietary PAs in the differentiation of the pig intestinal epithelium. A first experimental series was designed to assess the accumulation of either milk PAs (mostly spermidine) or orally administered spermine (SPM) in piglet red blood cells (RBCs) and plasma, a preliminary stage in their distribution to growing and storage organs. Though PA concentrations of piglet RBCs and plasma were generally significantly higher than their sow counterparts, our experimental conditions failed to demonstrate that this increase could stem from ingested PAs. A second experimental series dealt with the determination of disaccharidase specific activities in proximal and distal parts of piglet gut on the 26th and 29th days after birth (preweaning time). In agreement with observations made previously on rat pups, we observed an increase in maltase specific activity (SA) at the end of the suckling period (the observed increase in sucrase SA was not significant). However, orally administered SPM did not affect this activity. Compared to the constant protein concentrations observed in both parts of the gut, the pancreatic protein content decreased sharply between the 26th and 29th postnatal days. At the same time pancreatic concentrations of spermidine (SPD) also decreased, suggesting that some pancreatic PAs were released as the organ secreted its proteins. In accordance with this hypothesis, we recorded SPM and SPD in pancreatic juice. The increases in PA concentrations seemed to follow the protein secretion pattern (i.e. PA concentrations reached a maximal value when the protein concentration was highest). The presence of PAs in pancreatic juice could be indicative of a control mechanism exerted by the pancreas on PA-induced growth and differentiation of porcine intestinal epithelium.

Are milk polyamines preventive agents against food allergy?

Insufficient polyamine intake could play a role in the induction of sensitization to dietary allergens. This proposal is based essentially on investigations made in sucking rats and in children. In sucking rats it has been established that oral administration of spermine can induce all the modifications occurring in the digestive tract at weaning. In the intestine events occur in two phases. The early event consists of desquamation of the epithelium resulting from an activation of apoptosis. The late event appears to involve an hormonal cascade in which adrenocorticotropic hormone, cytokines, bombesin and corticosterone are included. Observations in human subjects show that: (1) the spermine and spermidine concentrations are generally lower in infant formulas than in human breast milk. Mothers seem consistently to have relatively high or relatively low concentrations of spermine and spermidine in their milk. These individual variations may be due to diet, lifestyle or genetic background; (2) the probability of developing allergy can reach 80 % if the mean spermine concentration in the milk is lower than 2 nmol/ml milk. It is approximately 0 % if the mean spermine concentration is higher than 13 nmol/ml milk; (3) preliminary results show that the intestinal permeability to macromolecules differs in premature babies when they are fed on breast milk compared with infant formulas (J Senterre, J Rigo, G Forget, G Dandrifosse and N Romain, unpublished results). This difference does not seem to be present when powdered milk is supplemented with polyamines at the concentration found in breast milk; (4) spermine increases proliferation and differentiation of lymphocytes isolated from the tonsils of children.

Cyclosporine A inhibits partially spermine-induced differentiation but not cell loss of suckling rat small intestine.

The polyamines are of great importance in several biological processes, such as cell proliferation, and differentiation. The ingestion of spermine by suckling rats induces the precocious maturation of their small intestine. This phenomenon is preceded by a cell elimination at the villus tip. We hypothesize that these two phenomena could be mediated by the immune system and thus inhibited by an immunosuppressive agent such as cyclosporine A. Cyclosporine A inhibits, at least partially, the spermine-induced increase of the maltase- and sucrase-specific activities in the small intestine but failed to inhibit lactase-specific activity decrease and cell loss. Spermine does not act by the same mechanism in differentiation and in cell loss. Moreover, spermine acts in a different way on lactase-specific activity compared to maltase- or sucrase-specific activity. We hypothesize that spermine acts on differentiation by a T-cell/IL-2-dependent mechanism.

[Statement on polyamines]. / Le point sur les polyamines.

Polyamines are ubiquitous substances. Their intracellular concentration is controlled quickly and rigorously by extremely sophisticated systems. It depends on metabolism and cellular permeability. Polyamines act as structural and functional elements in the cell (nucleic acid conformation, cytoskeleton, radioprotection, apoptosis, proliferation and differentiation of cells...). They also play a role in various diseases (origin of food allergy, cancers...). They present a great therapeutic interest (oncology, molecular transfer to cell nucleus, transfer across the blood-brain barrier, parasitosis, effects on NMDA and GABA receptors in the central nervous system...).

Comparison between the natural postnatal maturation and the spermine-induced maturation of the rat intestine.

In the suckling rats, orally provided spermine induced structural and biochemical changes in the intestine, which are characteristics of the postnatal maturation. This induced maturation was compared to that occurring spontaneously. Eight mumol spermine were administered orally once a day, for one or three days, to suckling rats which were 11 days old at the beginning of the experiment. The animals were killed 0, 2, 4, 6, 8, 10 hours or 3 days after the first treatment. Control rats from the same litter were treated in the same way but received only the vehicle. In order to complete the study of the naturally occurring maturation, another group of rats was killed when they were 12, 13, 15, 16, 17, 18, 19, 20, 21 or 30 days old. Animal and intestine weights were measured. Disaccharidase specific activity, and protein, DNA and RNA contents were estimated in the small intestine. Histological and ultrastructural aspects of the intestinal mucosa were examined. For all these parameters, the maturation induced by spermine ingestion appeared close to that occurring naturally at weaning. Consequently, dietary spermine induces all the morphological and biochemical modifications characterizing the intestinal postnatal maturation in the suckling rat suggesting a role of the polyamines in the naturally occurring processes.

Effect of spermine administration on pancreatic maturation in unweaned rats.

The effect of oral administration of spermine on pancreatic maturation was investigated in the suckling rat. The treatment consisted of 0.3-0.4 mmol spermine kg-1 body weight given orally once a day for 3 days starting at day 11 after birth. Spermine administration does not adversely affect the growth of the pancreas (wet weight, protein and DNA contents remain unchanged). The proliferating cell nuclear antigen (PCNA) index decreases significantly in spermine-treated rats, indicating that spermine slows down the proliferation rate of the organ. The enzymatic activities of trypsin, chymotrypsin and alpha-amylase are increased significantly in the pancreas of spermine-treated rats. The morphology of the organ seems affected as shown by hematoxylin-eosin staining: a cytoplasm indicative of higher synthetic activity is visible after spermine treatment. We conclude that spermine treatment of unweaned rats can induce precocious biochemical and morphological maturation of the exocrine pancreas, pushing the organ forward in the process of differentiation (closer to the adult stage).

[Necrolytic migratory erythema]. / L'érythème nécrolytique migrateur.

Necrolytic migratory erythema (NME) is generally associated with glucagonoma. It waxes and wanes by successive relapses and remissions. The clinical and microscopical diagnosis is complex. In addition to glucagonoma treatments, the administration of corticoids, aminoacids, zinc or essential fatty acids can be helpful. There exist several etiological hypotheses for NME. These are based on modifications of pancreatic enzyme activities and on variations of aminoacids, fatty acids, zinc or glucagon concentrations.

The relationship between spermine content of human milk during the first postnatal month and allergy in children.

DESIGN: Qualitative case study and mathematical model. SETTING: Belgium. OBJECTIVES: To evaluate the correlation between the polyamine mean concentration of the milk drunk during the first postnatal month and the appearance of allergy in children who drank this milk. RESULTS: A model that describes the dependence of the allergy appearance with the spermine mean concentration of milk drunk during the first postnatal month was established. CONCLUSIONS: This model shows that 5.02 nmol ml(-1) of spermine is a critical value to prevent the appearance of allergy.

Involvement of bombesin in spermine-induced corticosterone secretion and intestinal maturation in suckling rats.

In this study we investigated whether brain-gut peptides are implicated in the activation of the hypophysial-adrenal axis (HAA) in suckling rats treated orally with spermine. The first group of rats received i.p. injections of bombesin, vasoactive intestinal polypeptide (VIP), somatostatin or neurotensin, starting on day 11 of life, and killed on day 14. The small intestine was removed and analysed for its content of proteins, DNA, polyamines and for its specific activity (SA) of disaccharidases. The second group of rats received one of the hormones cited above and was killed 45 min after the treatment for determination of corticosterone plasma concentration. Rats of the third group were adrenalectomised then treated with bombesin as the first group. The fourth group of rats was orally treated with spermine and sacrificed 2, 3, 4, 6 and 8 h thereafter for analysis of plasma and intestinal concentrations of bombesin. The i.p. injection of bombesin increased the sucrase and maltase SA in the whole small intestine, while it decreased the lactase SA in the distal part. Intestinal weight and length, contents of DNA, protein, spermidine and spermine, and corticosterone plasma levels were enhanced by bombesin treatment. Somatostatin, neurotensin and VIP were ineffective on all the parameters studied. Adrenalectomy, in bombesin-treated rats, decreased the sucrase and maltase SA in the whole intestine, and decreased the lactase SA in the proximal intestine. It has no effect on intestinal weight and length, and protein content. Oral administration of spermine had no effect on plasma concentration of bombesin, whereas it decreased the content of this peptide in the whole small intestine. It is possible that bombesin may control intestinal development in suckling rats and be a link between the ingestion of spermine and the liberation of corticosterone by the adrenal glands.

Role of interleukin-1 beta, interleukin-6, and TNF-alpha in intestinal maturation induced by dietary spermine in rats.

In the present investigation, the authors aimed to evaluate the role of cytokines in intestinal postnatal maturation induced by dietary polyamines. Neonatal rats were administered either saline (8 mumol) orally. Spermine increased interleukin-1 beta (IL-1 beta), IL-6, and TNF-alpha plasma concentration. The maximum concentrations of IL-1 beta, IL-6, and TNF-alpha were, respectively, observed at 4, 4, and 8 h posttreatment. Intraperitoneal (i.p.) injection of IL-1 beta increased the specific activity of sucrase in whole small intestine, whereas the specific activities of maltase and lactase were significantly enhanced only in the jejunum. IL-6 elicited sucrase and increased maltase specific activity in the whole small intestine, but lactase specific activity was not affected. TNF-alpha had no effect on sucrase and maltase specific activity, but a slight augmentation of lactase specific activity was detected in the jejunum. Spermine and spermidine content in the intestine was increased by i.p. injection of IL-1 beta and IL-6. Corticosterone secretion was elevated by single i.p. injection of IL-1 beta, IL-6, or TNF-alpha. These findings suggest that spermine could induce postnatal intestinal development and corticosterone secretion through a cytokine-dependent mechanism.

Exogenous spermine induces maturation of the liver in suckling rats.

In the present study, we investigated the effects of spermine on postnatal liver maturation in suckling rats. The animals were given spermine either per os (8 micromol) or by intraperitoneal injection (1 micromol), once daily for three or five days. The percentage of liver cells in different cell cycle phases and of diploid cells in the parenchyma was estimated. The protein content, ornithine aminotransferase (OAT) activity, and content of DNA polyamines and receptors for polymeric immunoglobulins (RPI) were also measured in liver extracts. The ingestion of spermine had the following effects: the percentage of the cells in S and G2M phases of the cell cycle diminished the percentage of diploid cells increased the content of polymeric immunoglobulin receptors increased; the OAT activity increased; the contents of putrescine and spermidine decreased and almost reached adult values; and the spermidine/spermine ratio became similar to that observed in the liver of adult rats. These phenomena were detected 40 hours after the beginning of oral spermine treatment. The intraperitoneal injection of spermine had no effect on the OAT activity, but it decreased the spermidine content and enhanced the spermine content. Our data demonstrated for the first time that dietary polyamines play a role in the initiation of liver postnatal maturation in suckling rats.

Polyamine and intestinal properties in adult rats.

We questioned whether polyamines coming from the diet or produced by intestinal microflora or by intracellular metabolism influence intestinal functions. Therefore, we compared pathogen-free rats and germ-free rats receiving a diet with low polyamine content and either treated or not treated with difluoromethylornithine (DFMO) and/or methylglyoxal bis (guanylhydrazone) (MGBG). Wet weight, protein content, DNA content, sucrase (EC 3.2.1.48), maltase (EC 3.2.1.20) and lactase (EC 3.2.1.23) specific activities, amounts of putrescine, spermidine and spermine were measured in the mucosa of the proximal and distal intestine. Body weight was also determined. Rats without microflora had a higher specific activity of maltase and higher amounts of spermidine and spermine but lower lactase specific activity than pathogen-free animals; the low-polyamine diet given to germ-free rats had little effect on the functional variables measured (decrease of maltase and lactase specific activities) and did not modify the amounts of polyamines. DFMO and/or MGBG administered to germ-free rats receiving a low-polyamine diet induced modifications of most of the variables studied. Body weight and wet weight of proximal and distal intestine decreased, disaccharidase specific activities decreased, and amounts of polyamines changed according to the inhibitor used. Thus, our results showed that the deprivation of polyamine supply from microflora or from the diet failed, under our experimental conditions, to affect the intestinal properties analysed but exogenous and endogenous polyamine restriction altered general properties of the organism as well as intestinal functions.

Analysis of structural and biochemical events occurring in the small intestine after dietary polyamine ingestion in suckling rats.

In the present investigation, we analyzed the mechanism involved in spermine-induced intestinal maturation in suckling rats. Spermine was given orally to suckling pups and biochemical as well as morphological parameters were studied at different times after the beginning of the treatment. Eight hours after administration, spermine produced cell elimination at the villus tops and a decrease in intestinal DNA and protein content. In parallel, protein and DNA concentration and disaccharidase activity were enhanced in the chyme. These transitory alterations were not induced by growth inhibition, as DNA synthesis was not modified, although a brief decrease in protein synthesis was observed. Spermine was not metabolized in cytotoxic products: rat pretreatment with MDL72527 (an inhibitor of polyamine oxidase) did not avoid the decrease in disaccharidase activity and in DNA and protein content. Three days after treatment, sucrase and maltase activity was higher in rats treated with spermine and MDL72527 than that in animals receiving spermine alone. Lactulose or acetylspermine ingestion induced intestinal maturation. Our data suggest that dietary polyamines exert a direct and specific maturational effect on rat small intestine and that an early decrease in lactase activity plays an important role in this phenomenon.

Effects of a single dose of orally-administered spermine on the intestinal development of unweaned rats.

Investigations were undertaken to obtain information on the mechanism by which orally administered spermine induces postnatal maturation in the rat intestine. Suckling rats ingested one dose of spermine (8 mumol) then were sacrificed at different intervals. -A. Proximal and distal parts of the intestine were homogenised. -B. A modification of the Wieser's technique was used to isolate cell fractions from the proximal mucosa. Wet weight and length of intestine; protein content, DNA amount, disaccharidase activity, polyamine amounts in intestinal and cellular extracts were measured. Spermine ingestion induced two phases of events: first, a cellular desquamation then a new cell differentiation. In the isolated epithelial cells, two and four hours after spermine ingestion, modifications in lactase and maltase specific activity were recorded, as were variations in spermine, spermidine and putrescine content. These observations clarify the cellular and molecular events of the intestinal development occurring after spermine ingestion and open new research perspectives.