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.

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.

Antitumor activity of the polyamine analog N(1), N(11)-diethylnorspermine against human prostate carcinoma cells.

BACKGROUND: Recent studies indicate that N-terminally bis-ethylated-polyamine analogs have significant antitumor activity in several human solid-tumor models. In this study, the in vitro and in vivo antitumor potential of the polyamine analog N(1), N(11)-diethylnorspermine (DENSpm) in human prostate carcinoma cells was examined. METHODS: The antiproliferative and biochemical effects of DENSpm were tested in four human prostate cancer cell lines, i.e., PC-3, TSU-pr1, DU-145, and JCA-1. The in vivo antitumor potential was explored in two groups of nude mice bearing small or more developed xenografts of the DU-145 cell line. The mice were treated with 40 mg/kg DENSpm, three times per day for two cycles of 6 days, on days 1-6 and 8-13. RESULTS: In vitro studies showed that all four tested human prostate carcinoma cell lines were sensitive to DENSpm in micromolar concentrations. In tumor-bearing mice, DENSpm clearly prevented tumor growth in both size groups, which became significant after day 17. Treatment with DENSpm evoked intracellular accumulation of the analog and various regulatory responses, e.g., downregulation of the polyamine biosynthesis, the induction of the catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT), and the depletion or decrease of natural polyamines. The cellular sensitivity to growth inhibition by DENSpm only correlated with the degree of ODC inhibition and SSAT induction. CONCLUSIONS: DENSpm has sustained inhibitory effects on the growth of human prostate carcinoma cells in vitro as well in vivo. This polyamine analog may provide a new tool in the chemotherapy of prostate cancers with various phenotypes.

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.

[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...).

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.

Spermine-induced precocious intestinal maturation in suckling rats: possible involvement of glucocorticoids.

The mechanism(s) involved in the spermine-induced precocious postnatal maturation of the intestine in the unweaned rat was examined. Spermine given orally to 11-day-old rats stimulated ACTH and corticosterone secretion. Maximum serum levels of ACTH and corticosterone were observed between 4 and 6 h after spermine ingestion and were five- and sevenfold greater respectively than those of control rats receiving saline alone. Intraperitoneal injection of the polyamine had no effect on corticosterone production. Repeated intraperitoneal administration of gastrin, cholecystokinin, glucagon(1-37) and secretin to 11-day-old rats had no effect on the specific activity of intestinal disaccharidases. These data indicate that (1) the hypophysial-adrenal axis is implicated in the postnatal development of the gastrointestinal tract induced by spermine and (2) spermine affects ACTH and corticosterone secretion indirectly, probably by stimulating the release of gastrointestinal hormone(s).

Intestinal development in suckling rats: direct or indirect spermine action?

The present investigation addresses the question of whether spermine orally given to unweaned rats directly or indirectly exerts its effects on the intestinal brush border disaccharidases and if the adrenal gland secretions play a role in this phenomenon. The results showed that spermine, surgically placed in the lower part of the distal small intestine, induced sucrase, stimulated maltase-specific activity and decreased lactase-specific activity in both proximal and distal segments of the small intestine. Introduction of spermine into the lumen of the large intestine stimulated the specific activities of disaccharidases in the whole small intestine. Intraperitoneal injection had no effect except a slight reduction of lactase-specific activity in the distal intestine. Adrenalectomy prevented the oral effect of spermine on sucrase- and maltase-specific activity but not on lactase-specific activity. Addition of spermine to intestinal explants in organ cultures fails to reproduce any of these effects. It even reduced maltase-specific activity. These findings suggest that dietary polyamines have either direct and indirect effects on properties of rat immature intestine.

Reversibility of spermine-induced intestinal maturation in the rat.

In the present investigation, the reversibility of spermine-induced precocious intestinal maturation was studied. Neonatal rats received either saline or spermine (4 mumol, twice daily) solution orally on the 11th and 12th postnatal day. They were killed on the 13th, 14th, 15th, 16th, and 17th postnatal days. After the small bowel was removed, it was either divided into three equal parts or prepared for electrophoretic analysis. Histological examination, protein content measurement, and disaccharidase activity estimation were performed on each part of the intestine. Spermine administration was shown to induce structural and mucosal enzyme changes characteristic of postnatal maturation. This phenomenon, which was generally clearly observed in 13- and 14-day-old rats, then became less apparent in 15- and 16-day-old animals. Differences were noted according to the segment of intestine or the biochemical parameter analyzed. When rats were 17 days old, no significant differences generally existed between control and spermine-treated rats. If the 140- to 150-kDa proteins, isolated by electrophoresis, are assumed to represent the subunits of the sucrase-isomaltase complex, the results obtained indicate that spermine induces a modification of the concentration of this complex. When compared to values obtained in adult rats, the concentration of the complex was approximately three times higher in spermine-treated 13-day-old rats, while no differences were found in spermine-treated 14-day-old rats. Further, similar concentrations were found in control and spermine-treated rats with an age of 17 days. These results suggest that spermine-induced precocious intestinal maturation is reversible when spermine treatment is stopped.