POLYAMINES IN MILK

Jean-Paul BUTS
Division of Pediatric Gastroenterology and Nutrition, Department of Pediatrics,
Cliniques Universitaires St-Luc, Catholic University of Louvain, Brussels, Belgium.

The diamine putrescine and the polyamines spermidine and spermine are ubiquitous substances found virtually in all cells from higher prokaryotes and eukaryotes (1-5). These highly charged, low molecular weight and polycationic substances (Figure 1) are essential for cell growth and differentiation and their intracellular concentrations increase during periods of rapid cell proliferation (6-8). To meet the needs of division processes and protein synthesis, the intracellular concentration of polyamines are critically regulated by several enzymes including meanly ornithine decarboxylase (ODC) and S-adenosyl-methionine decarboxylase (SAM-DC). As shown in Figure 2, in the "de novo" synthesis pathway, ODC catalyzes the decarboxylation of the aminoacid ornithine to form putrescine. This is the first rate-limiting step in polyamine synthesis (9,10). The synthesis of ODC activity and of ODC mRNA can be enhanced by several hormones and growth factors including insulin (11,12), gastrin (13), nerve growth factor (12) and epidermal growth factor (14). To the reverse inhibition of ODC by a-difluoromethylornithine, an irreversible inhibitor of the enzyme, without the concurrent administration of polyamines, leads to severe depletion of cellular polyamines and in turn to cessation of DNA synthesis and cell division (15). Since the turnover of ODC is the fastest among eukaryotic enzymes, any change in the rate of ODC synthesis will be rapidly transmitted to the amount of enzyme protein (16) and thus to polyamine synthesis. In cell culture, it has been demonstrated that putrescine and spermidine levels regulate ODC mRNA translation (17). Increasing polyamine levels exert a feedback regulation on ODC activity by a direct inhibition of ODC mRNA translation (17). Thus, it appears that polyamines by means of various feedback mechanisms control their own cellular levels. The complexity of the regulation of polyamine metabolism is illustrated by the observations that ODC is subject to transcriptional (18), translational (19) as well as to post-translational controls (20). A second rate-limiting enzyme in polyamine synthesis is S-adenosyl methionine decarboxylase (SAM DC) which decarboxylates S-adenosyl methionine, providing aminopropyl groups for spermidine and spermine synthesis (21). Putrescine is converted into spermidine by the action of an aminopropyltransferase called spermidine synthase (22). A second aminopropyltransferase, termed spermine synthase, adds an additional propylamine moiety to spermidine forming spermine (22). Regarding the catabolic pathway, putrescine can be catabolized by several enzymes, the much frequently analyzed being diamine oxidase (23,24). Putrescine is also a precursor in the biosynthesis of gamma-aminobutyric acid (GABA) via a pathway that does not involve L-glutamic acid (25). GABA formation from putrescine represents a major process in some developing neural tissues such as the embryonic chick retina (25).

Rapidly dividing tissues such as the intestinal epithelium are also dependent upon exogenous sources of polyamines supplied by food, secretions and microbial flora (28,29). Because the apical surfaces of intestinal cells are exposed to variable luminal contents, this tissue has an unique advantage for regulating its polyamine levels. It was previously believed that polyamines are synthesized by every cell when required. Recent evidence has shown that the diet can theoretically supply sufficient amounts of polyamines to support at least intestinal cell renewal and growth. In a recent analysis of different foods consumed by british adults (30), the average daily polyamine consumption was calculated to be in the range of 350-500 mmol per person per day which represents an amount 1000 times higher than the amount of polyamines provided by 100 ml human milk (31). The major sources of putrescine were fruit, cheese and non-green vegetables. All foods contributed similar amounts of spermidine to the diet, although levels were generally higher in green vegetables. Meat was the richest source of spermine. Subsequent experiments in rats revealed that during their intestinal absorption, endoluminal polyamines are partly metabolized. More than 80% of putrescine can be converted into other polyamines including cadaverin and into non-polyamine metabolites, mostly to aminoacids. The enzyme responsible for controlling the bio availability of putrescine is diamine oxidase (DAO) (Figure 2). For spermidine and spermine, however, about 70-80% of the intragastrically intubated dose remained in the original molecular form.

Human milk contains substantial quantities of polyamines mainly spermine and spermidine, with much less putrescine. Although these substances have been detected in samples of human milk as well as in the milk of a number of mammalian species including rats (37,38), cows (39) and pigs (39,40) quantification by two-dimensional gel chromatography (41) or by high performance liquid chromatography (HPLC) methods (31,37,38,40) has provided a wide range of data. For instance, Pollack et al (38) reported mean concentrations of spermidine in human milk ranging from 7.3 to 351 nmol/dl while for the same period of lactation (0-15 days) Romain et al (37) found mean spermidine concentrations to be 3 to 4 times higher (Table 1). Using a high sensitive and reproductible HPLC method, we found (31) mean spermidine concentrations in human milk of 220 " 20 nmol/dl which are quite equivalent to the concentrations measured by Pollack et al (38). The profile of concentrations established for the three polyamines in human milk appears to be the same for the three studies (31,37,38) that used HPLC methods and is

spermine , spermidine , putrescine with a constant spermidine/spermine ratio of 0.78-0.86. These figures differ noticeably from the data reported by Sanguansermsi et al (41). Using gel chromatography, these authors were the first to show the presence of polyamines in human milk (41). However, compared with more sensitive HPLC methods, their measurements showed much lower spermine and spermidine levels and higher putrescine and cadaverine levels. Also, the ratio of spermidine/spermine ranged from 3.7 to 6.6 which contrasts with the ratio of 0.78-0.86 established by 3 other studies (31,37,38). Many factors can affect the composition of milk including genetic influences, circadian rhythms, nutritional status, dietary intakes, duration of lactation, bacterial contamination, environmental influences and degree of milk accumulation in the breast (42,43). We attempted to take into account most of these factors in our study (31). All the samples were collected by standardized procedures at the same time of the day from the right breast. These were no reported problems of engorgements and expression was done at the end of the feeding period. All milk samples were analyzed for bacterial contamination. Because milk contains polyamine oxidases that can induce a decrease in polyamine concentration over time (44) we took special care to maintain our samples on ice for a very short period of time after which the assays were immediately started (31). The accuracy of polyamine measurements can be tested by adding to milk, samples of known amounts of spermidine and spermine handled in exactly the same fashion as were the samples. Using this method, recovery rates averaged 90% of the amount of polyamines added with an efficiency of the dansylation ranging between 70 and 86% compared with commercial dansyl derivates of high purity grade (31). A relevant finding of our study is that spermine and spermidine concentrations markedly increased during the first 3 days of lactation, reaching plateau levels that were 12 and 8 times higher respectively than the levels of these substances measured by day 0 postpartum (Figure 3).

Similar increases in spermine and spermidine concentrations have been noted by others in human milk (37) but they were not found to be relevant because of transient declines at day 4 and 5 postpartum or lack of day-to-day monitoring (37,38). Likewise, Motyl et al (39) found a profile of polyamines in cow's and sow's milk, similar to human milk (spermine , spermidine , putrescine) and documented peak concentrations of polyamines occurring in the colostrum and milk between the first and 3rd week of lactation in cow and sow respectively. Also, these authors found a significant and positive correlation between milk yield and spermidine secretion in cow as well as between the number of piglets fed and milk spermidine concentration.

Table 1. Polyamine concentrations (nmol/dl) measured in human milk at the end of the

(p + 0.05) in second age formulas in concordance with their higher protein content ( +8.2%, p + 0.05).

A relevant finding of our assays is the very high concentration of polyamines in semi-elemental diets prepared by hydrolytic procedures using crude extracts of pancreatic enzymes (Pepti Junior^R Nutricia). Such diets represent a major source of polyamines for infants and have a polyamine profile similar to human milk (spermine , spermidine , putrescine) but with individual concentrations in spermine and spermidine exceeding those of human milk. Compared with first age infant formulas, the mean concentration of spermine and of spermidine were 39 and 6 times higher, respectively in these semi-elemental diets than in first-age formulas, whereas putrescine levels remained almost equivalent in all types of milk tested (Figure 4).

Standard powdered formulas contain intact cow's milk proteins while in semi-elemental diets, original proteins have been hydrolyzed by crude extracts of pancreatic enzymes or by purified enzymes resulting in small peptides (mol weight + 6000 d) and aminoacids. The high polyamine content of semi-elemental diets cannot derive from the hydrolysis of the original protein because polyamines are not constituents of polypeptides and their synthesis is critically regulated by specific enzymes and substrates. In fact, semi-elemental diets such as Pepti Junior^R (Nutricia) are prepared by hydrolysis of the original protein using a whole lyophilized extract of pancreatic enzymes including trypsin, chemotrypsin, elastase, carboxypeptidases, DNases and RNases. These whole pancreatic extracts are extremely concentrated in polyamines as are in vivo exocrine pancreatic secretions. Practically, an amount of whole pancreatic enzyme extract is added industrially to the milk extract, and after hydrolysis is complete, the mixture is filtered to retain high-molecular weight peptides ( , 10000 d), allowing small peptides, aminoacids and polyamines to pass in the filtrate and to accumulate in the final milk product. Thus, the high content of polyamines detected in such semi-elemental diets would derive from the pancreatic extracts added to the milk and not from the hydrolytic procedure of basic milk proteins. This explanation is further supported by the observation that partially hydrolyzed milk formulas such as NAN HA^R (Nestlé, Vevey, Switzerland) with basic milk proteins hydrolyzed by one or two purified enzymes (trypsin, chemotrypsin) contain spermine and spermidine at concentrations equal to those of first-age formulas (37).

Recently, we have analyzed the role of yeast polyamines as potential mediators in the trophic intestinal response observed after oral administration of Saccharomyces boulardii to human volunteers (49) or to growing rats (5). In weanling rats, a daily dose of 100 mg of lyophilized S. boulardii produced significant increases in sucrase and maltase activities (5). This dose corresponded to a total oral load of 678 nmol of polyamines per day (spermidine : 376 " 32, spermine : 293 " 26, putrescine : 9.5 " 1.4 nmol/100 mg).

Further analysis of mucosal and endoluminal fluid contents in polyamines revealed that the yeast exerted trophic effects on the small intestine and enhanced enzyme expression by endoluminal release of their spermine and spermidine content. These studies confirmed that for the rat as little as 500 to 600 nmol polyamines can affect the maturation of intestinal cells. Furthermore, increases in microvillous enzyme activities were observed in adult human volunteers after 5 days of an oral dose of 1 g Saccharomyces boulardii, corresponding to a daily oral intake of 6.7 mmol of polyamines (49). Assuming that neonates and young infants consume 600-700 ml of human milk per day, a single deduction from our data indicate that this volume would represent an oral load of $ 3.5 mmol polyamines per day which theoretically could be biologically active (Figure 5).

Figure 1. Chemical structure of the diamine "putrescine" and of the polyamines

"spermidine and spermine"

Figure 3. Changes in polyamine concentrations in human milk during the first postnatal
week of lactation. (n = number of samples collected from individual mothers).

Figure 4. Comparison of polyamine concentrations in semi-elemental diets, human milk
and infant artificial formulas.

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