High-Dose Spermidine Supplementation Does Not Increase Spermidine Levels in Blood Plasma and Saliva of Healthy Adults: A Randomized Placebo-Controlled Pharmacokinetic and Metabolomic Study.

(1) Background: Spermidine is a biogenic polyamine that plays a crucial role in mammalian metabolism. As spermidine levels decline with age, spermidine supplementation is suggested to prevent or delay age-related diseases. However, valid pharmacokinetic data regarding spermidine remains lacking. Therefore, for the first time, the present study investigated the pharmacokinetics of oral spermidine supplementation. (2) Methods: This study was designed as a randomized, placebo-controlled, triple-blinded, two-armed crossover trial with two 5-day intervention phases separated by a washout phase of 9 days. In 12 healthy volunteers, 15 mg/d of spermidine was administered orally, and blood and saliva samples were taken. Spermidine, spermine, and putrescine were quantified by liquid chromatography-mass spectrometry (LC-MS/MS). The plasma metabolome was investigated using nuclear magnetic resonance (NMR) metabolomics. (3) Results: Compared with a placebo, spermidine supplementation significantly increased spermine levels in the plasma, but it did not affect spermidine or putrescine levels. No effect on salivary polyamine concentrations was observed. (4) Conclusions: This study's results suggest that dietary spermidine is presystemically converted into spermine, which then enters systemic circulation. Presumably, the in vitro and clinical effects of spermidine are at least in part attributable to its metabolite, spermine. It is rather unlikely that spermidine supplements with doses <15 mg/d exert any short-term effects.

Detection of Thermospermine and Spermine by HPLC in Plants.

Thermospermine, a structural isomer of spermine, is widely spread in the plant kingdom and has recently been shown to play a key role in the repression of xylem differentiation in vascular plants. However, a standard high-performance liquid chromatography (HPLC) protocol for detecting polyamines as their dansyl derivative cannot distinguish themospermine from spermine. These isomers become separated from each other after benzoylation. In this chapter, we describe a simple protocol for extraction, benzoylation, and HPLC detection of thermospermine and spermine with other polyamines from plant material.

Brewing effect on levels of biogenic amines in different coffee samples as determined by LC-UV.

Eight biogenic amines (spermine, spermidine, putrescine, histamine, tyramine, phenylethylamine, cadaverine and serotonin) were determined by LC-UV after derivatization with dansyl-chloride in both ground coffee and coffee beverages obtained by different methods. In ground coffee, the most relevant amine was PUT, followed by SPD, HIS, TYR, CAD, SPM, PHE, and SER, with the total BAs content decreasing as the roasting degree increased. In coffee brews, the order was PUT, SPM, TYR, CAD, SPD, PHE, HIS, and SER, but at a very low level in comparison with the amount of BAs determined in roasted ground coffee. Beverages prepared by espresso, capsule, and pod machines had the lowest BAs contents, as a result of the thermal and physical stress imposed on ground coffee by these methods, while mocha contained the highest BAs amounts owing to lower pressure and longer brewing time.

Polyamine profile in ovine and caprine colostrum and milk.

The objective of this study was to monitor the post-partum variation of polyamine content, in ovine and caprine milk, from indigenous Greek breeds. Twenty samples of ewe and 20 samples of goat colostrum and milk were collected at the 1st, 2nd, 3rd, 4th, 5th and 15th day post-partum. Putrescine, spermidine and spermine were measured as dansylated derivatives by high-performance liquid chromatography. Putrescine was the least concentrated of these substances in both milk types. Spermidine was the prevailing polyamine in caprine samples, reaching levels up to 4.41 µmol/l on the 3rd day post-partum. In ovine milk, the profile of the mean concentrations showed greater levels of spermine than spermidine, except for the 5th day post-partum. These data suggest that goat colostrum and ewe milk (15th day) could be considered as good natural sources for these bioactive growth factors, and may become useful raw materials for designing tailored dairy products for specific population groups.

Comprehensive analysis of Cirsium spinosissimum Scop., a wild alpine food plant.

Plants which have been traditionally eaten by alpine populations may provide new opportunities of agricultural development for mountain regions. In this context we investigated the chemical composition of Cirsium spinosissimum (Asteraceae), a perennial thistle. Its receptacles were eaten by shepherds in Valais (Switzerland). Extracts of aerial parts were subjected to a comprehensive metabolite profiling, using a dereplication platform, combining HPLC-PDA-MS and offline microprobe NMR analysis. Twenty compounds, including various phenolic glycosides, a monoterpene lactone, a spermine derivative, and fatty acids, could be identified online, or after targeted isolation. The total phenolic content was determined, and the major flavonoids were quantitatively assessed in fresh receptacles by HPLC-PDA analysis. In addition, substances relevant for nutrition, such as ß-carotene, fatty acids, ascorbic acid, and minerals, were quantified. The ethanolic extract of the receptacles showed no sign of cytotoxicity when tested in Caco-2 cells.

Effect of MeJA treatment on polyamine, energy status and anthracnose rot of loquat fruit.

The effect of methyl jasmonate (MeJA) on changes in polyamines content and energy status and their relation to disease resistance was investigated. Freshly harvested loquat fruit were treated with 10 µmol l(-1) MeJA and wound inoculated with Colletotrichum acutatum spore suspension (1.0 × 10(5) spores ml(-1)) after 24h, and then stored at 20 °C for 6 days. MeJA treatment significantly reduced decay incidence. MeJA treated fruit manifested higher contents of polyamines (putrescine, spermidine and spermine) compared with the control fruit, during storage. MeJA treatment also maintained higher levels of adenosine triphosphate, and suppressed an increase in adenosine monophosphate content in loquat fruit. These results suggest that MeJA treatment may inhibit anthracnose rot by increasing polyamine content and maintaining the energy status.

Hippocampal polyamine levels and transglutaminase activity are paralleling spatial memory retrieval in the C57BL/6J mouse.

Although a potential role for polyamines and transglutaminases (TGs) in memory mechanisms have been proposed, hippocampal spermine (SPM) and spermidine (SPD) levels as well as transamidating activity of TG in spatial memory have not been addressed yet. It was therefore the aim of the study to assess hippocampal polyamines and TG activity at the probe trial in a spatial memory paradigm. C57BL/6J mice (20 animals per group) were used for the experiments and divided into a trained and a yoked (untrained) group. The Morris water maze (MWM) was selected as the memory test, animals were sacrificed within 5 min following the probe trial and hippocampi were taken for biochemical analysis. SPD and SPM levels were assessed by an analytical procedure according to Gismondi et al. Transamidating activity of TG was determined following the method described by Chung and Folk using [14C] methylamine as substrate. γ-(Glutamyl)-polyamine levels were evaluated by ion exchange chromatography according to Folk et al. Animals learned the task in the MWM as latencies and pathlengths were significantly reduced. At the probe trial mice showed significantly higher preference for the target quadrant. Free SPD and SPM levels were manifold decreased in the trained as compared to the yoked group. Transamidating activity of TG was fourfold increased in trained as compared to yoked controls. γ-(Glutamyl)-SPD was comparable while γ-(glutamyl)-SPM was significantly higher in the trained group. The findings show a potential role for polyamines, their derivative γ-(glutamyl)-SPM and transamidating activity of TG at memory retrieval or formation. Results from this study are extending and knowledge on polyamines and report for the first time involvement of γ-(glutamyl)-SPM and transamidating activity of TG that may form the basis for future neurochemical and pharmacological studies and indeed, modulation of polyamine and TG activity has been already proposed as a tentative therapeutical concept.

Quantitative analysis of plant polyamines including thermospermine during growth and salinity stress.

Arabidopsis thaliana was thought to contain two spermine synthase genes, ACAULIS 5 (ACL5) and SPMS. Recent investigations, however, revealed that the ACL5 gene encodes thermospermine synthase. In this study, we have established a simple method to separate two isomers of tetraamine, spermine and thermospermine, in extracts from plant tissues of less than 500 mg. Polyamines (PAs) extracted from plant tissues were benzoylated, and the derivatives were completely resolved by high-performance liquid chromatography on a C18 reverse-phase column, by eluting with 42% (v/v) acetonitrile in water in an isocratic manner at 30 degrees C and monitoring at 254 nm. The relevance of the method was confirmed by co-chromatography with respective PAs and by the PA analysis of the single- and double-mutants of acl5 and spms, which could not synthesize thermospermine and/or spermine, respectively. Furthermore, with this method, we monitored the thermospermine contents in various tissues of A. thaliana and found that stems and flowers contain two- to three-fold more thermospermine compared to whole seedlings and mature leaves. The presence of thermospermine was confirmed in Oryza sativa and Lycopersicon pesculentum. Finally we addressed whether salinity stress changes the contents of PAs including thermospermine in Arabidopsis.

Polyamines in tea processing.

The distribution of dietary polyamines, putrescine, spermidine and spermine, was determined during processing of Camellia sinensis. Black tea manufacture is carried by a series of processes on fresh tea leaves involving withering, rolling, fermentation, drying and sieving. The aim of this research was to determine the effect of tea processing on the polyamine content in relation with antioxidant enzymes, superoxide dismutase, lipid peroxidase and glutathione peroxidase. Before processing, the spermine content was much higher than the putrescine and spermidine content in green tea leaves. Spermine was significantly decreased during processing while the putrescine and spermine contents increased during withered and rolling and decreased in the following stages. The superoxide dismutase activity increased at the withering stage and declined during processing. The transcript level of the polyamine biosynthesis-responsible enzyme ornithine decarboxylase was reduced during each processing step. This study reveals the importance of protection of nutritional compounds that are essential for health during the manufacturing process.

[Content comparison of polyamines compounds in Penis et Testis Cervi].

OBJECTIVE: To compare the content of the main polyamines compositions in 10 samples of Penis et Testis Cervi from different species and habitats. MEHTODs: The putrescine spermidine and spermine in Penis et Testis Cervi were determined by HPLC. RESULTS: The contents of total Polyamine and 3 kinds of polyamines compositions in all samples were different. CONCLUSION: The content of the main polyamines compositions can be used to control the quality of Penis et Testis Cervi.

Dihydrocaffeoyl polyamines (kukoamine and allies) in potato (Solanum tuberosum) tubers detected during metabolite profiling.

Four related phenolic amides previously undescribed from the species were revealed during metabolic profiling of potato (Solanum tuberosum) tubers. N(1),N(12)-Bis(dihydrocaffeoyl)spermine (kukoamine A) and N(1),N(8)-bis(dihydrocaffeoyl)spermidine were positively identified by comparison with authentic standards, while the structures N(1),N(4),N(12)-tris(dihydrocaffeoyl)spermine and N(1),N(4),N(8)-tris(dihydrocaffeoyl)spermidine are proposed for the other two metabolites. Each amide was present at several tens of micrograms per gram of dry matter. Several of these compounds were subsequently detected in other solanaceous species, such as tomato (Lycopersicon esculentum) and Nicotiana sylvestris. They appeared not to be present in Arabidopsis thaliana or Beta vulgaris. Bis(dihydrocaffeoyl)spermine isomers have previously been identified in only a single plant, the Chinese medicinal species Lycium chinense (Solanaceae), where they may account for some of the described biological activity. The other compounds have not until now been reported in vivo, though some of the equivalent hydroxycinnamoyl derivatives are known. The surprising discovery of kukoamine and allies in a range of solanaceous species including potato, a common food crop that has a long history of scientific investigation, provides exemplary evidence for the potential of the nontargeted techniques of metabolomics in studying plant metabolites.

Biogenic amines in restructured beef steaks as affected by added walnuts and cold storage.

This article evaluates changes in biogenic amines and how these relate to microbiological growth in chilled, fresh restructured beef steaks containing transglutaminase as a cold binding agent and different amounts of walnut. Added walnut and chilling favored higher total and lactic acid bacteria counts during storage, whereas Enterobacteriaceae were not affected. The highest initial biogenic amine concentrations were identified as spermidine, spermine, and tyramine. Both added walnut and cold storage generally favored the formation of amines (tyramine, histamine, putrescine, and cadaverine), which was more obviously apparent by the end of the storage period. Agmatine, on the other hand, was not generally affected by the walnut.

Quantitation of polyamines in hypothalamus and pituitary of female and male developing rats.

The quantitation of four polyamines in hypothalamus and pituitary is studied in male and female developing rats using an improved high-performance liquid chromatography method. In the hypothalamus, putrescine (PUT) reaches the highest concentration (nmol/mg protein) on day 6. It shows the lowest value in comparison with any other polyamine. Spermidine (SPD) is high during the first postnatal days. Spermine (SPM) fluctuates, and agmatine (AGM) is highest during the first week. SPD, SPM and AGM are lower in females. In the pituitary, PUT, SPD and AGM are high during the first week. SPM remains constant and it is higher in males. AGM is higher in males only on day 1. PUT shows the lowest concentration of all. Concentrations of PUT, SPD and SPM are higher in the pituitary; AGM is higher in the hypothalamus. alpha-Difluoromethylornithine (a specific and irreversible inhibitor of ornithine decarboxylase) decreases PUT and SPD, increased SPM and AGM remain unchanged in the hypothalamus and pituitary. Thus, each polyamine has its own pattern in hypothalamus and in pituitary during development in males and females; these changes could be related to the hypothalamic control of pituitary secretion of hormones related to reproduction in mammals.

Nucleotide and polyamine levels in colostrum and mature milk in relation to maternal atopy and atopic development in the children.

The prophylactic benefit of breastfeeding against atopic disease is still controversial. It seems to be limited to infants with genetic propensities to allergy in combination with late solid food introduction. Lower levels of n-3 polyunsaturated fatty acids in human milk have been related to atopy in children, stressing a non-specific role of nutritional components in the development of atopy. Nucleotides and polyamines have been related to intestinal integrity and immune function in infancy. The main sources of these nutrients are human milk nucleotides and polyamines early in life. Our aim was to study the composition of nucleotides and polyamines in colostrum and mature milk from atopic and non-atopic mothers and the relationship to sensitization against egg, milk or cat in their children during the first year of life. The nucleotide/nucleoside and polyamine levels were measured by HPLC in colostrum and in milk at 3 mo of lactation from mothers of 21 atopic and 14 non-atopic children. Among the mothers, 10 were atopic and 25 non-atopic. The nucleotides cytidine monophosphate (CMP), uridine monophosphate (UMP), adenosine monophosphate (AMP) and guanosine monophosphate (GMP) and the nucleosides cytidine and uridine were detected in human milk. In colostrum, CMP dominated, and the levels increased in mature milk, while the levels of the other compounds remained constant. The nucleotide/nucleoside composition was similar in colostrum from all mothers independent of the development of sensitization in their babies, except for the higher cytidine levels in mature milk from atopic mothers of atopic babies, as compared to healthy mothers of atopic babies. The polyamine levels were similar in colostrum from atopic and non-atopic mothers. However, putrescine and spermine levels were lower in mature milk from atopic mothers than non-atopic mothers. No relationship was found between milk putrescine and spermine levels and development of atopy in the children. In conclusion, low levels of human milk putrescine and spermine seem to be related to maternal atopy.

Biogenic amines and microbial quality of sprouts.

Changes in the biogenic amine content relative to microbial activities in mung bean, lentil and radish sprouts were investigated in prepacked and "home-grown" products. Biogenic amines were determined by ion-exchange chromatography. The major groups of micro-organisms were enumerated by aerobic plate count procedures, using universal and selective media. Putrescine, cadaverine, spermidine, agmatine and spermine were detected in different concentrations, depending on the type of sprouts. In prepacked retail products the total biogenic amine content was higher than in home-grown samples (mung bean 106 micrograms/g compared to 87 micrograms/g; lentil 316 micrograms/g compared to 181 micrograms/g; radish 1486 micrograms/g compared to 252 micrograms/g). It is concluded that sprouting time and storage conditions play a major part in the hygienic quality of legume sprouts.

Biogenic amines in silage. 1. The occurrence of biogenic amines in silage.

In laboratory silages made from orchardgrass, red clover and oats, significant levels of toxic biogenic amines were found. The most widespread were putrescine and cadaverine, concentrations of spermidine, spermine and histamine were ten times lower on average. Of the amines considered, putrescine content seems to be the most sensitive indicator of the extent of putrefaction in silages. A relationship between the putrescine contents and the degree of proteolysis was found. The biogenic amine content is closely connected with both the variety of crop ensiled and even more so with the method and the efficiency of preservation. Neither the application of formic acid nor wilting suppressed the formation of any of the amines in silages selectively. Formic acid suppressed the total concentration of amines to 23% as compared with controls. The highest levels of amines were found in orchardgrass and oat silages; smaller amounts were detected in clover silages. In the case of very poorly preserved silage 100-150 g of toxic amines can be consumed by a cow in a day. This could have a deleterious effect on the physiological condition and the performance of livestock.

Change of polyamine content in mouse skin by leupeptin, a protease inhibitor, during early stage of tumorigenesis.

The effect of a microbial protease inhibitor, leupeptin, on the content of polyamine in the mouse skin was examined during the early stage of tumorigenesis induced by a single application of 7,12-dimethylbenz[a]anthracene (DMBA) and repeated application of croton oil thereafter. Polyamine content in the skin was measured at 3, 5, 7, and 9 weeks during tumorigenesis. The mice with no visible tumor were selected for measurement of polyamine content at 9 weeks. Mice were left untreated for at least 1 week before measurement of polyamine. Polyamine in the skin was extracted with ice-cold 0.4N HClO4 and separated into putrescine, spermidine, and spermine fractions through CM-cellulose column. Polyamine concentration was determined by fluorometry with fluorescamine. Group A mice painted with croton oil 3 times a week did not develop tumors. Group B mice painted with a single DMBA developed skin tumors, and group C mice painted with a single DMBA and croton oil 3 times a week showed higher development of skin tumors than group B. Group D mice treated as group C and then painted with leupeptin about 2 hr after croton oil treatment. Animals in groups B, C, and D had higher spermidine content as group A at 3 and 5 weeks. Content of spermidine in group B decreased at 7 and 9 weeks compared with group C which had a high content throughout the time tested. Leupeptin treatment in group D inhibited spermidine content in the skin after 7 weeks without affecting until 5 weeks.