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Concentration of biologically active polyamines in rabbit meat, liver and kidney after slaughter and their changes during meat storage and cooking
Meat Science 90 (2012) 796–800
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Concentration of biologically active polyamines in rabbit meat, liver and kidney after slaughter and their changes during meat storage and cooking Eva Dadáková, Tamara Pelikánová, Pavel Kalač ⁎ Department of Applied Chemistry, Faculty of Agriculture, University of South Bohemia, 370 05 České Budějovice, Czech Republic
a r t i c l e
i n f o
Article history: Received 5 January 2011 Received in revised form 28 April 2011 Accepted 2 November 2011 Keywords: Dietary polyamines Spermidine Spermine Rabbit Meat Liver
a b s t r a c t The concentration of putrescine (PUT), spermidine (SPD) and spermine (SPM) was determined in chilled meat and kidneys of 18 rabbits and in liver of 12 animals 24 h after slaughter. Very low PUT concentrations were detected only in kidneys. Mean SPD levels were 2.2, 2.2, 61.7 and 32.7 mg kg− 1 in saddle, leg, liver and kidneys, respectively. The respective SPM concentrations were 14.7, 8.0, 115 and 88.4 mg kg− 1. SPD and SPM losses of about one third of the initial levels were apparent in saddles stored at −18 °C for 8 months. Losses of both polyamines of about 15–20% of the initial concentrations were found in saddles stored aerobically at +2 °C for up to 9 days. Stewing of saddles caused significant SPD and SPM losses of about 20–25%, while upon roasting and panroasting without oil a decrease of about 50% of the initial concentration was observed. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Polyamines putrescine (PUT; H2N[CH2]4NH2), spermidine (SPD; H2N [CH2]3NH[CH2]4NH2) and spermine (SPM; H2N[CH2]3NH[CH2]4NH [CH2]3NH2) are widespread in living organisms. These compounds are fully protonated under physiological conditions and can react as polyvalent cations with numerous cell constituents, such as nucleic acids, ATP, specific proteins and phospholipids. Polyamines are essential for cell growth and participate in numerous physiological processes (Agostinelli et al., 2010; Igarashi & Kashiwagi, 2010). Due to different biological roles and biosynthesis pathways, polyamines started to be separated from their traditional classification within biogenic amines during the 1990s. Putrescine, structurally a diamine, is classified as a biogenic amine and also as a polyamine, being the precursor of spermidine and spermine. The polyamine pool in human body is maintained by three primary sources: i) endogenous (de novo) biosynthesis, ii) production by intestinal bacteria, and iii) dietary intake. According to Bardócz (1995), diet provides a larger daily quantity of polyamines than does endogenous biosynthesis. Unfortunately, daily cellular requirements for polyamines have not yet been determined.
⁎ Corresponding author. Fax: + 420 385 310 405. E-mail address: [email protected] (P. Kalač). 0309-1740/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2011.11.017
Recent studies have suggested that reducing the level of polyamines in cells may help to slow down cancer processes. One direction in cancer therapy research is to limit the intake of dietary polyamines (Cipolla, Havouis, & Moulinoux, 2007, 2010). However, dietary polyamines may be required in wound healing and for growth, maturation and regeneration of the intestinal mucosa. Main roles of polyamines in health and disease have been reviewed several times (Dandrifosse, 2009; García-Faroldi, Sánchez-Jiménez, & Fajardo, 2009; Larqué, Sabater-Molina, & Zamora, 2007; Moinard, Cynober, & de Bandt, 2005; Soda, 2010). Information on the concentration of polyamines in foods and beverages is needed for assessing their dietary intake. Higher SPM concentrations, as compared to SPD concentrations, are usual in foods of animal origin, mainly in muscles and inner organs, while the opposite is observed in foods of plant origin (for reviews see Kalač, 2006, 2010; Kalač & Krausová, 2005). Unlike PUT, dietary SPD and SPM originate from raw materials as intimate cell constituents and their production by microbial activity in foods is limited if any. Polyamine levels are high in young and metabolically active tissues and organs (Nishimura, Shiina, Kashiwagi, & Igarashi, 2006). Consumption of rabbit meat is recommended by nutritionists as it is low in fat and easily digestible (Dalle Zotte & Szendrö, 2011). To the best of our knowledge, only a single report on polyamine concentrations in rabbit meat has been published (Cipolla et al., 2007). Therefore, the objective of this study was to determine polyamine concentration in raw rabbit meat, liver and kidneys and to evaluate
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changes in polyamine levels in meat under various storage conditions and cooking treatments. 2. Materials and methods 2.1. Sampling Rabbit carcasses were purchased in two batches (10 and 8 heads) from a large-scale producer during February 2010. The rabbits of Hyplus® breed, reared in cages, were fed ad libitum granules containing mainly hot-air-dried lucerne (80% w/w) plus barley bran. The rabbits were slaughtered at the age of 85 days. The carcasses were cooled to 3 °C during 3.5 h in a cooling tunnel and immediately transported to the laboratory. Mean weight of the carcasses with head and kidneys was 1561 ± 148 g, that of liver and both kidneys 63.7 ± 10.5 g and 15.6 ± 3.5 g, respectively. Meat samples were separated from saddle (m. gluteus medius) and leg (m. tensor fasciae latae). About a half of liver and both kidneys were used for polyamine determination. The analyses started 22–24 h after slaughter. Complete saddle muscles were separated for storage and cooking experiments. 2.2. Storage conditions Tested storage conditions simulated usual practice in households. The effect of meat storage at −18± 1 °C on polyamine concentrations was tested in an experiment using saddles of four rabbits. Each of separated saddle halves was packaged in a bag made of high-density polyethylene (HDPE; foil thickness 0.017 mm), frozen in a household freezer and then stored. Prior to analyses, the samples were allowed to thaw at 21–22 °C for 2 h. The analyses were carried out on day 0 (24 h after slaughter) and then after 3-month and 8-month storage. The effect of cold-storage was tested in second experiment using saddles of four rabbits 24 h after slaughter (storage day 0). A part of the meat was used for determination of the initial polyamine concentrations; the other parts were packaged in polyethylene bags (the same as in frozen-storage) and then stored at + 2 ± 0.5 °C. The analyses were carried out on days 5 and 9.
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Retentions of the polyamines were calculated according to Murphy, Criner, and Gray (1975). True retention should measure the proportion of the SPD or SPM remaining in the cooked meat in relation to the amount originally present in a given weight of the meat before cooking. True retention (TR) was calculated as follows: TRð% Þ ¼ PA concentration per kg of cooked meat meat weight ðkgÞafter cookingÞ= PA concentration per kg of raw meat weight ðkgÞ of raw meatÞ 100:
Apparent retention (AR) was calculated as ratio of the SPD or SPM concentration in the cooked meat without discard to the PA concentration in the raw meat, with both values expressed on dry basis: ARð% Þ ¼ ½PA concentration per kg of cooked meatðdry basisÞ= ½PA concentration per kg of raw meat ðdry basisÞ 100
2.4. Analytical methods All chemicals used were of analytical grade. Dry matter content was determined by drying of a homogenised sample in an oven at 105 °C until constant weight. The analytical procedure used for polyamines determination was described in details in a previous paper (Dadáková, Křížek, & Pelikánová, 2009). Briefly, acid extracts were prepared by homogenising of 40 ± 1 g of meat or liver sample or both kidneys with 100 ml of 0.6 M perchloric acid for 3 min. The slurry was centrifuged at 1800 g for 10 min. The supernatant was then filtered through a paper filter, which was washed with HClO4, and the volume was recorded. The extracted polyamines were derivatised with dansyl chloride. 1,7-Heptandiamine was used as an internal standard. An ultra-performance liquid chromatography (UPLC) method was used for the separation and quantitation. Chromatograms of a standard mixture of amines and extracts from saddle and kidneys are shown as Figs. 1–3, respectively. 2.5. Quality control The polyamines were identified by matching their retention times with those of standard compounds. The identity of the polyamines was verified by standard addition of all polyamines.
2.3. Cooking treatments Third experiment simulated usual culinary processing of rabbit meat in Central Europe. Both halves of saddle from nine rabbits were used. The saddles packaged in polyethylene foil (thickness 0.017 mm) aged at + 2 ± 0.5 °C for six days after slaughter. Three different treatments were then carried out in triplicates: • Stewing: both saddle halves were cut into cubes of about 1.5 × 1.5 × 1.5 cm, water was added to the meat (meat/water 4/1, w/w) and the mixture was sealed in a polyethylene bag. The bag was immersed in boiling water for 30 min. The inner temperature was measured by a puncture thermometer (accuracy ± 0.5 °C, Amarell Electronic, Germany). The temperature reached its maximum of 97 °C after 16–18 min. The bags were then cooled under tap water to air temperature and both cubes and broth were used for polyamine and dry matter determinations; • Roasting: uncut halves of saddle were roasted in an oven at 190 °C in a porcelain bowl covered with an aluminium foil for 45 min and then without the foil for 20 min to evaporate the gravy. About 25 ml water was initially added to prevent burning; • Pan-roasting without oil: complete halves of saddle were tenderised to slices 1–1.5 cm thick. The slices were roasted in a steel pan with a teflone surface. No oil or water was added. The slices were roasted for 8 and 12 min on one and the opposite side, respectively. The inner temperature reached 90 °C.
Fig. 1. Separation of a standard mixture of polyamines and biogenic amines as dansylamides. Peaks numbering and amine concentrations (mg l−1; in bracket): 1 — tryptamine (8.96); 2 — phenylethylamine (9.08); 3 — putrescine (8.90); 4 — cadaverine (9.04); 5 — histamine (9.56); 6 — internal standard 1,7-heptandiamine (9.12); 7 — tyramine (9.19); 8 — spermidine (8.95); 9 — spermine (9.21).
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Tukey's test using the statistical tools of MS Office Excel and Statistica 5.1 (StatSoft, Tulsa, USA). A significance level of P b 0.05 was used in all statistical tests. 3. Results and discussion The presence of none of the biogenic amines mentioned in Section 2.5. was detected. 3.1. Polyamines in meat and inner organs 24 h after slaughter
Fig. 2. Chromatogram of polyamine determination in a rabbit saddle. Peak numbers as in Fig. 1.
Fig. 3. Chromatogram of polyamine determination in rabbit kidney. Peak numbers as in Fig. 1.
Repeatability of the polyamine determination was tested by ten analyses of a rabbit saddle aged for four days. The values were 4.7 and 4.3% for SPD and SPM, respectively, at mean concentrations of 2.32 and 16.2 mg kg − 1, respectively. The recoveries were 91 and 95% for SPD and SPM, respectively, at concentrations of 2.4 and 17.1 mg kg − 1 of added SPD and SPM, respectively. Limits of quantification (LOQ), calculated for the signal-to-noise ratio of 10, were 1.2 and 3.7 mg kg − 1 fresh meat for SPD and SPM, respectively. Longtime stability of the analytical method was confirmed by repeated analyses of a mixed solution of standards as a control sample. The stable solution was analysed regularly for 15 months and results were plotted as Shewhart control charts. Moreover, the analytical procedure enabled simultaneous determination of the biogenic amines histamine, tyramine, phenylethylamine, cadaverine and tryptamine with LOQ of 1.9, 3.3, 2.5, 1.9 and 2.9 mg kg − 1, respectively. 2.6. Statistical methods Normal distribution of data was proved. Statistical significance of changes in the polyamine concentrations was tested by Student's test of means and by regression analysis (ANOVA) followed by
Polyamine concentrations in rabbit saddle and leg meat, liver and kidneys are given in Table 1. Putrescine was detected only in kidney samples with mean and maximum value of 3.5 and 5.3 mg kg − 1, respectively. Dietary intake of putrescine seems to be of limited health implications due to its low levels as compared with some other food items (e.g. citrus fruits or ketchup; Kalač & Krausová, 2005) and to generally low consumption of rabbit kidneys. Concentrations of SPD and SPM varied widely in meat and particularly in the organs. All differences of SPD or SPM concentrations between the analysed materials were statistically significant (P b 0.05) with the exception of that between SPD levels in saddle and leg meats. A statistically significant correlation between SPD and SPM concentrations was found only in kidneys. The only reported mean concentrations of SPD and SPM in unspecified rabbit leg meat were 7.6 and 15.4 mg kg − 1, respectively (Cipolla et al., 2007). The spermine levels reported by Cipolla et al. (2007) are nearly twice and those of SPD more than three times higher than our data (Table 1). In comparison with meats of various animals (Table 2), SPD concentrations determined in this work are comparable, whereas SPM values are lower. It is somewhat surprising because meats of very young rabbits were analysed. As reported by Nishimura et al. (2006), polyamine concentrations in tissues and organs of mice decrease with ageing. Nevertheless, we found lower SPM concentrations in lamb leg meat than in the corresponding mutton (Table 2). Thus, some other factor(s) probably affect polyamine levels in muscles. Very high SPD and particularly SPM concentrations have been reported in liver of various animals (Table 2). The determined SPD concentration of 61.7 ± 14.3 mg kg− 1 (Table 1) is comparable with data reported for chicken liver, higher than in porcine, sheep, lamb and roe deer livers, but lower than in bovine liver. Spermidine concentration of 37.2 ± 12.4 mg kg− 1 was determined in liver of zoologically related European brown hare 6 h post mortem (Paulsen et al., 2008). Spermine concentration of 115±19.5 mg kg− 1 in rabbit liver (Table 1) is very comparable with values reported for brown hare, pigs, roe deer and chicken liver, but higher than in bovine liver (Table 2). In rabbit kidneys, concentrations of SPD and SPM were 32.7 ± 9.3 and 88.4 ± 22.3 mg kg − 1, respectively (Table 1). The value for SPD is higher than data of Table 2, while that for SPM is comparable with the concentrations reported for brown hare and roe deer. Thus, both rabbit liver and kidneys belong among food items with very high concentrations of SPM and SPD. 3.2. Polyamines in frozen meat Changes in SPD and SPM concentrations were determined in four samples of saddles after 3 and 8 months storage (Table 3). It was found that dry matter content decreased only slightly. The retentions were not thus calculated. Both SPD and SPM concentrations decreased significantly during the initial 3 months of storage with a higher rate for SPD than for SPM. The decrease continued also during further 5 months. After the storage period, relative losses of both polyamines were about 30–35% of the initial concentration in fresh meat.
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Table 1 Polyamine concentrations (mg kg− 1) in rabbit meat, kidney (n = 18) and liver (n = 12) 24 h after slaughter. Material
n1
Putrescine Mean
Saddle Leg Liver Kidney
– – – 3.5
0 0 0 18
Spermidine SD
Range
– – – 0.7
Mean c
– – – 2.6–5.3
2.2 2.2c 61.7a 32.7b
Spermine SD 0.4 0.6 14.3 9.3
Range
Mean c
1.7–3.1 1.2–3.2 44.1–88.7 15.1–51.0
14.7 8.0d 115a 88.4b
SD
Range
1.6 0.7 19.5 22.3
12.2–17.7 6.8–9.3 92.2–157 55.5–124
n = number of analysed samples; n1 = number of samples with putrescine concentration above limit of quantification 2.1 mg kg− 1; SD = standard deviation. Different superscript letters in a column indicate significant difference (P b 0.05).
Table 2 Literature data on the concentration of polyamines (mg kg− 1) in fresh meat, liver and kidney of various animals. Putrescine concentrations were quantifiable only rarely by the used analytical methods. Product
n
Spermidine
Spermine
Mean
SD
Mean
Reference
SD
Meat Beef sirloin
63
–
–
21.7
5.8
Beef rump Pork loin Pork leg Mutton leg
57 15 15 20
– – – 5.6
– – – 3.5
22.0 26.1 28.4 24.7
5.6 7.0 8.5 6.8
Mutton breast Lamb leg Lamb breast Chicken breast
19 20 20 20
4.0 4.2 5.1 4.8
1.6 1.6 0.9 1.7
17.2 17.2 16.5 36.8
2.6 2.2 3.2 5.9
Chicken thigh
20
10.2
2.2
38.0
3.7
Liver Steer
58
122
82.0
43.1
26.5
Barrow Pig
19 20
32.1 31.2
11.6 1.1
115 95.1
49.4 17.6
Sheep Lamb Roe deer
22 31 39
13.5 16.8 10.7
7.9 5.9 5.3
128 78.6 79.9
69.4 26.2 15.0
Brown hare Chicken
20 38 20
37.2 56.9 48.7
12.4 15.1 8.8
111 120 133
24.9 43.0 18.0
Kidney Pig
40
9.4
3.4
53.1
14.0
Roe deer Brown hare
20 39 20
21.3 10.7 24.4
7.8 5.3 4.1
64.5 79.9 82.8
23.2 15.0 13.8
Krausová, Kalač, Křížek, and Pelikánová (2006b) Krausová et al. (2006b) Krausová et al. (2006b) Krausová et al. (2006b) Dadáková, Pelikánová, and Kalač (2011) Dadáková et al. (2011) Dadáková et al. (2011) Dadáková et al. (2011) Kozová, Kalač, and Pelikánová (2009b) Kozová et al. (2009b)
Krausová, Kalač, Křížek, and Pelikánová (2006a) Krausová et al. (2006a) Fuchs, Bauer, and Paulsen (2009) Dadáková et al. (2011) Dadáková et al. (2011) Paulsen, Dicakova, and Bauer (2008) Paulsen et al. (2008) Krausová et al. (2006a) Kozová et al. (2009b)
Kozová, Kalač, and Pelikánová (2008) Fuchs et al. (2009) Paulsen et al. (2008) Paulsen et al. (2008)
n = number of analysed samples; SD = standard deviation.
Storage at + 2 ± 0.5 °C Initial concentration After 5 days After 9 days
Dry matter
Spermidine
26.43a ± 0.91 26.74a ± 0.83 26.92a ± 0.97
2.31a ± 0.24 1.57b ± 0.27 1.50c ± 0.19
26.85a ± 0.86 27.07a ± 0.98 27.28a ± 0.82
2.20a ± 0.22 1.63a ± 0.43 1.75a ± 0.57
3.3. Polyamines in cold-stored meat It was found that dry matter content changed only slightly during these experiments. The retentions were not thus calculated. Losses of both polyamines were about 20–25% of their initial levels in fresh saddles. While the SPM decrease was significant, the situation for SPD was contentious probably due to increasing analytical error at its very low concentrations (Table 3). The losses were higher than those observed in beef loin (Kozová et al., 2009a), pork loin (Krausová et al., 2008) or chicken breast (Kozová et al., 2009b) but comparable with losses in mutton legs (Dadáková et al., 2011) stored under similar experimental conditions. Even higher SPM losses were observed in six-day-aged saddles prior to the cooking treatments (Table 4). The effects of ageing are thus variable. Different microbial development seems to be one of the possible reasons for this observation. 3.4. Polyamines in cooked meat Dry matter content increased considerably in all three cooking treatments (Table 4) and the changes were taken into consideration
Table 4 Changes in dry matter content and polyamine concentrations in rabbit saddles after three cooking treatments and data on retention. Values of three samples. Parameter
Table 3 Changes of dry matter (%) and polyamine concentrations (mg kg− 1) during storage of rabbit saddles at − 18 °C (n = 4) and at + 2 ± 0.5 °C (n = 4).
Storage at − 18 °C Initial concentration After 3 months After 8 months
A similar trend of polyamine level decrease was observed during storage of frozen pig liver (Krausová, Kalač, Křížek, & Pelikánová, 2007). However, polyamine levels increased moderately in pork loin (Krausová, Kalač, Křížek, & Pelikánová, 2008) and chicken thighs (Kozová et al., 2009b), while initially increased and then significantly decreased in frozen beef loin (Kozová, Kalač, & Pelikánová, 2009a) and in mutton loin (Dadáková et al., 2011). To our knowledge, there is no information on the mechanism(s) of the polyamine losses during storage of frozen foods.
Spermine 13.8a ± 1.2 11.5b ± 1.3 9.7c ± 1.6
15.5a ± 1.2 11.5b ± 1.1 13.7a,b ± 2.1
Different superscript letters in a column for each type of storage indicate significant difference (P b 0.05).
Dry matter (%) After 6-day ageing After cooking Spermidine (mg kg− 1 fresh/cooked matter) 24 h after slaughter After 6-day ageing After cooking True retention (%) Apparent retention (%) Spermine (mg kg− 1 fresh/cooked matter) 24 h after slaughter After 6-day ageing After cooking True retention (%) Apparent retention (%)
Stewing
Roasting
Pan-roasting without oil
25.32 ± 0.33 34.04 ± 1.40
25.42 ± 0.49 44.53 ± 0.46
25.46 ± 0.24 43.25 ± 3.19
2.21 ± 0.47 2.56 ± 0.21 2.71 ± 0.02 72.3a ± 2.3 79.1a ± 4.1
2.10 ± 0.19 3.19 ± 0.62 2.80 ± 0.48 53.8b ± 3.2 50.3b ± 4.7
2.52 ± 0.51 2.75 ± 0.82 2.24 ± 0.73 51.2b ± 9.3 47.7b ± 8.0
14.32 ± 1.73 8.01 ± 0.66 9.22 ± 1.61 77.9a ± 5.0 85.2a ± 6.2
14.96 ± 1.26 9.43 ± 2.35 8.09 ± 0.78 53.7b ± 8.5 50.4b ± 9.2
14.85 ± 2.63 7.69 ± 0.61 6.98 ± 0.85 57.6b ± 7.7 53.7b ± 7.0
Different superscript letters in the rows of retentions indicate significant differences (P b 0.05).
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for the calculation of true and apparent retentions. No detectable levels of polyamines were determined in the broth and drippings. Data on polyamine concentrations before and after cooking and on relative changes caused by various cooking conditions are given in Table 4. According to Murphy et al. (1975), the true retention reflects losses of a constituent in cooked foods more accurately than the apparent retention. The lowest decrease of both SPD and SPM, about 20–25% of the level in aged saddles, was observed in stewed meat. Significantly higher (P b 0.05) were the losses of both polyamines in roasted and pan-roasted-without-oil meat. The decrease in the latter two treatments was about 50%. The results are well comparable with common trends resulting from similar experiments with pork loin (Krausová et al., 2008), mutton leg (Dadáková et al., 2011) and chicken breast (Kozová et al., 2009b). Thus, similarly as in other tested meats, rabbit meat roasting or pan-roasting without oil cause higher losses of both SPD and SPM than stewing (and very probably also than cooking). 3.5. Fate of polyamines lost during storage or cooking Unfortunately, information on SPM and SPD catabolic pathways in food matrices is lacking. The thoroughly described polyamine catabolism in living mammalian organs (Seiler, 2004) can be applied to post-mortem processes in meat and food inner organs to only a limited extent. There has emerged a fear of aldehydes (particularly of acrolein) produced during polyamine degradation for the development and progression of several grave neurodegenerative diseases (Wood, Khan, & Moskal, 2007) and brain infarction (Saiki et al., 2011). It is possible that the primary amino groups of the polyamines react, under the conditions of roasting and pan-roasting without oil, with glucose in the Maillard-type reaction. Méndez and Leal (2004) reported that putrescine, cadaverine, spermidine and spermine can compete with the free primary amino groups of amino acids (particularly L-lysine) for glucose under in vitro conditions. Spermine was observed to be the most efficient. 3.6. Exposure assessment The estimated polyamine intake from a serving of 100 g stewed or pan-roasted rabbit saddle meat is about 0.27 and 0.22 mg SPD, and 0.92 and 0.70 mg SPM, respectively (data from Table 4). These values account for only a low proportion (2–7%) of the estimated daily intake of these two polyamines. For example, the mean daily intake of 12.6 and 11.0 mg of SPD and SPM, respectively, has been reported for the United Kingdom, Italy, Spain, Finland, Sweden and the Netherlands (Ralph, Englyst, & Bardócz, 1999). The respective values adapted for Japan are 12.0 and 7.9 mg (Nishibori, Fujihara, & Akatuki, 2007) and for the USA convenient sample diet 7.9 and 7.2 mg (Zoumas-Morse et al., 2007). It can be assumed that the polyamine intake from cooked rabbit liver would be considerably higher than that from meat. Acknowledgement The authors acknowledge the financial support of the project MSM 6007665806 of the Czech Ministry of Education, Youth and Sports. References Agostinelli, E., Marques, M. P. M., Calheiros, R., Gil, F. P. S. C., Tempera, G., Viceconte, N., et al. (2010). Polyamines: Fundamental characters in chemistry and biology. Amino Acids, 38, 393–403. Bardócz, S. (1995). Polyamines in food and their consequences for food quality and human health. Trends in Food Science and Technology, 6, 341–346.
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(1975). Comparisons of methods for calculating retentions of nutrients in cooked foods. Journal of Agricultural and Food Chemistry, 23, 1153–1157. Nishibori, N., Fujihara, S., & Akatuki, T. (2007). Amounts of polyamines in foods in Japan and intake by Japanese. Food Chemistry, 100, 491–497. Nishimura, K., Shiina, R., Kashiwagi, K., & Igarashi, K. (2006). Decrease in polyamines with aging and their ingestion from food and drink. Journal of Biochemistry, 139, 81–90. Paulsen, P., Dicakova, Z., & Bauer, F. (2008). Biogenic amines and polyamines in liver, kidney and spleen of roe deer and European brown hare. European Food Research and Technology, 227, 209–213. Ralph, A., Englyst, K., & Bardócz, S. (1999). Polyamine content of the human diet. In S. Bardócz, & A. White (Eds.), Polyamines in health and nutrition (pp. 123–137). London: Kluwer Acad. Publ. Saiki, R., Park, H., Ishii, I., Yoshida, M., Nishimura, K., Toida, T., et al. (2011). Brain infarction correlates more closely with acrolein than with reactive oxygen species. Biochemical and Biophysical Reseach Communications, 404, 1044–1049. Seiler, N. (2004). Catabolism of polyamines. Amino Acids, 26, 217–233. Soda, K. (2010). Polyamine intake, dietary pattern, and cardiovascular disease. Medical Hypotheses, 75, 2099–2301. Wood, P. L., Khan, M. A., & Moskal, J. R. (2007). The concept of “aldehyde load” in neurodegenerative mechanisms: Cytotoxicity of the polyamine degradation products hydrogen peroxide, acrolein, 3-aminopropanal, 3-acetamidopropanal and 4-aminobutanal in a retinal ganglion cell line. Brain Research, 1145, 150–156. Zoumas-Morse, C., Rock, C. L., Quintana, E. L., Neuhouser, M. L., Gerner, E. W., & Meyskens, F. L. (2007). Development of a polyamine database for assessing dietary intake. Journal of the American Dietetic Association, 107, 1024–1027.
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Concentration of biologically active polyamines in rabbit meat, liver and kidney after slaughter and their changes during meat storage and cooking Eva Dadáková, Tamara Pelikánová, Pavel Kalač ⁎ Department of Applied Chemistry, Faculty of Agriculture, University of South Bohemia, 370 05 České Budějovice, Czech Republic
a r t i c l e
i n f o
Article history: Received 5 January 2011 Received in revised form 28 April 2011 Accepted 2 November 2011 Keywords: Dietary polyamines Spermidine Spermine Rabbit Meat Liver
a b s t r a c t The concentration of putrescine (PUT), spermidine (SPD) and spermine (SPM) was determined in chilled meat and kidneys of 18 rabbits and in liver of 12 animals 24 h after slaughter. Very low PUT concentrations were detected only in kidneys. Mean SPD levels were 2.2, 2.2, 61.7 and 32.7 mg kg− 1 in saddle, leg, liver and kidneys, respectively. The respective SPM concentrations were 14.7, 8.0, 115 and 88.4 mg kg− 1. SPD and SPM losses of about one third of the initial levels were apparent in saddles stored at −18 °C for 8 months. Losses of both polyamines of about 15–20% of the initial concentrations were found in saddles stored aerobically at +2 °C for up to 9 days. Stewing of saddles caused significant SPD and SPM losses of about 20–25%, while upon roasting and panroasting without oil a decrease of about 50% of the initial concentration was observed. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Polyamines putrescine (PUT; H2N[CH2]4NH2), spermidine (SPD; H2N [CH2]3NH[CH2]4NH2) and spermine (SPM; H2N[CH2]3NH[CH2]4NH [CH2]3NH2) are widespread in living organisms. These compounds are fully protonated under physiological conditions and can react as polyvalent cations with numerous cell constituents, such as nucleic acids, ATP, specific proteins and phospholipids. Polyamines are essential for cell growth and participate in numerous physiological processes (Agostinelli et al., 2010; Igarashi & Kashiwagi, 2010). Due to different biological roles and biosynthesis pathways, polyamines started to be separated from their traditional classification within biogenic amines during the 1990s. Putrescine, structurally a diamine, is classified as a biogenic amine and also as a polyamine, being the precursor of spermidine and spermine. The polyamine pool in human body is maintained by three primary sources: i) endogenous (de novo) biosynthesis, ii) production by intestinal bacteria, and iii) dietary intake. According to Bardócz (1995), diet provides a larger daily quantity of polyamines than does endogenous biosynthesis. Unfortunately, daily cellular requirements for polyamines have not yet been determined.
⁎ Corresponding author. Fax: + 420 385 310 405. E-mail address: [email protected] (P. Kalač). 0309-1740/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2011.11.017
Recent studies have suggested that reducing the level of polyamines in cells may help to slow down cancer processes. One direction in cancer therapy research is to limit the intake of dietary polyamines (Cipolla, Havouis, & Moulinoux, 2007, 2010). However, dietary polyamines may be required in wound healing and for growth, maturation and regeneration of the intestinal mucosa. Main roles of polyamines in health and disease have been reviewed several times (Dandrifosse, 2009; García-Faroldi, Sánchez-Jiménez, & Fajardo, 2009; Larqué, Sabater-Molina, & Zamora, 2007; Moinard, Cynober, & de Bandt, 2005; Soda, 2010). Information on the concentration of polyamines in foods and beverages is needed for assessing their dietary intake. Higher SPM concentrations, as compared to SPD concentrations, are usual in foods of animal origin, mainly in muscles and inner organs, while the opposite is observed in foods of plant origin (for reviews see Kalač, 2006, 2010; Kalač & Krausová, 2005). Unlike PUT, dietary SPD and SPM originate from raw materials as intimate cell constituents and their production by microbial activity in foods is limited if any. Polyamine levels are high in young and metabolically active tissues and organs (Nishimura, Shiina, Kashiwagi, & Igarashi, 2006). Consumption of rabbit meat is recommended by nutritionists as it is low in fat and easily digestible (Dalle Zotte & Szendrö, 2011). To the best of our knowledge, only a single report on polyamine concentrations in rabbit meat has been published (Cipolla et al., 2007). Therefore, the objective of this study was to determine polyamine concentration in raw rabbit meat, liver and kidneys and to evaluate
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changes in polyamine levels in meat under various storage conditions and cooking treatments. 2. Materials and methods 2.1. Sampling Rabbit carcasses were purchased in two batches (10 and 8 heads) from a large-scale producer during February 2010. The rabbits of Hyplus® breed, reared in cages, were fed ad libitum granules containing mainly hot-air-dried lucerne (80% w/w) plus barley bran. The rabbits were slaughtered at the age of 85 days. The carcasses were cooled to 3 °C during 3.5 h in a cooling tunnel and immediately transported to the laboratory. Mean weight of the carcasses with head and kidneys was 1561 ± 148 g, that of liver and both kidneys 63.7 ± 10.5 g and 15.6 ± 3.5 g, respectively. Meat samples were separated from saddle (m. gluteus medius) and leg (m. tensor fasciae latae). About a half of liver and both kidneys were used for polyamine determination. The analyses started 22–24 h after slaughter. Complete saddle muscles were separated for storage and cooking experiments. 2.2. Storage conditions Tested storage conditions simulated usual practice in households. The effect of meat storage at −18± 1 °C on polyamine concentrations was tested in an experiment using saddles of four rabbits. Each of separated saddle halves was packaged in a bag made of high-density polyethylene (HDPE; foil thickness 0.017 mm), frozen in a household freezer and then stored. Prior to analyses, the samples were allowed to thaw at 21–22 °C for 2 h. The analyses were carried out on day 0 (24 h after slaughter) and then after 3-month and 8-month storage. The effect of cold-storage was tested in second experiment using saddles of four rabbits 24 h after slaughter (storage day 0). A part of the meat was used for determination of the initial polyamine concentrations; the other parts were packaged in polyethylene bags (the same as in frozen-storage) and then stored at + 2 ± 0.5 °C. The analyses were carried out on days 5 and 9.
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Retentions of the polyamines were calculated according to Murphy, Criner, and Gray (1975). True retention should measure the proportion of the SPD or SPM remaining in the cooked meat in relation to the amount originally present in a given weight of the meat before cooking. True retention (TR) was calculated as follows: TRð% Þ ¼ PA concentration per kg of cooked meat meat weight ðkgÞafter cookingÞ= PA concentration per kg of raw meat weight ðkgÞ of raw meatÞ 100:
Apparent retention (AR) was calculated as ratio of the SPD or SPM concentration in the cooked meat without discard to the PA concentration in the raw meat, with both values expressed on dry basis: ARð% Þ ¼ ½PA concentration per kg of cooked meatðdry basisÞ= ½PA concentration per kg of raw meat ðdry basisÞ 100
2.4. Analytical methods All chemicals used were of analytical grade. Dry matter content was determined by drying of a homogenised sample in an oven at 105 °C until constant weight. The analytical procedure used for polyamines determination was described in details in a previous paper (Dadáková, Křížek, & Pelikánová, 2009). Briefly, acid extracts were prepared by homogenising of 40 ± 1 g of meat or liver sample or both kidneys with 100 ml of 0.6 M perchloric acid for 3 min. The slurry was centrifuged at 1800 g for 10 min. The supernatant was then filtered through a paper filter, which was washed with HClO4, and the volume was recorded. The extracted polyamines were derivatised with dansyl chloride. 1,7-Heptandiamine was used as an internal standard. An ultra-performance liquid chromatography (UPLC) method was used for the separation and quantitation. Chromatograms of a standard mixture of amines and extracts from saddle and kidneys are shown as Figs. 1–3, respectively. 2.5. Quality control The polyamines were identified by matching their retention times with those of standard compounds. The identity of the polyamines was verified by standard addition of all polyamines.
2.3. Cooking treatments Third experiment simulated usual culinary processing of rabbit meat in Central Europe. Both halves of saddle from nine rabbits were used. The saddles packaged in polyethylene foil (thickness 0.017 mm) aged at + 2 ± 0.5 °C for six days after slaughter. Three different treatments were then carried out in triplicates: • Stewing: both saddle halves were cut into cubes of about 1.5 × 1.5 × 1.5 cm, water was added to the meat (meat/water 4/1, w/w) and the mixture was sealed in a polyethylene bag. The bag was immersed in boiling water for 30 min. The inner temperature was measured by a puncture thermometer (accuracy ± 0.5 °C, Amarell Electronic, Germany). The temperature reached its maximum of 97 °C after 16–18 min. The bags were then cooled under tap water to air temperature and both cubes and broth were used for polyamine and dry matter determinations; • Roasting: uncut halves of saddle were roasted in an oven at 190 °C in a porcelain bowl covered with an aluminium foil for 45 min and then without the foil for 20 min to evaporate the gravy. About 25 ml water was initially added to prevent burning; • Pan-roasting without oil: complete halves of saddle were tenderised to slices 1–1.5 cm thick. The slices were roasted in a steel pan with a teflone surface. No oil or water was added. The slices were roasted for 8 and 12 min on one and the opposite side, respectively. The inner temperature reached 90 °C.
Fig. 1. Separation of a standard mixture of polyamines and biogenic amines as dansylamides. Peaks numbering and amine concentrations (mg l−1; in bracket): 1 — tryptamine (8.96); 2 — phenylethylamine (9.08); 3 — putrescine (8.90); 4 — cadaverine (9.04); 5 — histamine (9.56); 6 — internal standard 1,7-heptandiamine (9.12); 7 — tyramine (9.19); 8 — spermidine (8.95); 9 — spermine (9.21).
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Tukey's test using the statistical tools of MS Office Excel and Statistica 5.1 (StatSoft, Tulsa, USA). A significance level of P b 0.05 was used in all statistical tests. 3. Results and discussion The presence of none of the biogenic amines mentioned in Section 2.5. was detected. 3.1. Polyamines in meat and inner organs 24 h after slaughter
Fig. 2. Chromatogram of polyamine determination in a rabbit saddle. Peak numbers as in Fig. 1.
Fig. 3. Chromatogram of polyamine determination in rabbit kidney. Peak numbers as in Fig. 1.
Repeatability of the polyamine determination was tested by ten analyses of a rabbit saddle aged for four days. The values were 4.7 and 4.3% for SPD and SPM, respectively, at mean concentrations of 2.32 and 16.2 mg kg − 1, respectively. The recoveries were 91 and 95% for SPD and SPM, respectively, at concentrations of 2.4 and 17.1 mg kg − 1 of added SPD and SPM, respectively. Limits of quantification (LOQ), calculated for the signal-to-noise ratio of 10, were 1.2 and 3.7 mg kg − 1 fresh meat for SPD and SPM, respectively. Longtime stability of the analytical method was confirmed by repeated analyses of a mixed solution of standards as a control sample. The stable solution was analysed regularly for 15 months and results were plotted as Shewhart control charts. Moreover, the analytical procedure enabled simultaneous determination of the biogenic amines histamine, tyramine, phenylethylamine, cadaverine and tryptamine with LOQ of 1.9, 3.3, 2.5, 1.9 and 2.9 mg kg − 1, respectively. 2.6. Statistical methods Normal distribution of data was proved. Statistical significance of changes in the polyamine concentrations was tested by Student's test of means and by regression analysis (ANOVA) followed by
Polyamine concentrations in rabbit saddle and leg meat, liver and kidneys are given in Table 1. Putrescine was detected only in kidney samples with mean and maximum value of 3.5 and 5.3 mg kg − 1, respectively. Dietary intake of putrescine seems to be of limited health implications due to its low levels as compared with some other food items (e.g. citrus fruits or ketchup; Kalač & Krausová, 2005) and to generally low consumption of rabbit kidneys. Concentrations of SPD and SPM varied widely in meat and particularly in the organs. All differences of SPD or SPM concentrations between the analysed materials were statistically significant (P b 0.05) with the exception of that between SPD levels in saddle and leg meats. A statistically significant correlation between SPD and SPM concentrations was found only in kidneys. The only reported mean concentrations of SPD and SPM in unspecified rabbit leg meat were 7.6 and 15.4 mg kg − 1, respectively (Cipolla et al., 2007). The spermine levels reported by Cipolla et al. (2007) are nearly twice and those of SPD more than three times higher than our data (Table 1). In comparison with meats of various animals (Table 2), SPD concentrations determined in this work are comparable, whereas SPM values are lower. It is somewhat surprising because meats of very young rabbits were analysed. As reported by Nishimura et al. (2006), polyamine concentrations in tissues and organs of mice decrease with ageing. Nevertheless, we found lower SPM concentrations in lamb leg meat than in the corresponding mutton (Table 2). Thus, some other factor(s) probably affect polyamine levels in muscles. Very high SPD and particularly SPM concentrations have been reported in liver of various animals (Table 2). The determined SPD concentration of 61.7 ± 14.3 mg kg− 1 (Table 1) is comparable with data reported for chicken liver, higher than in porcine, sheep, lamb and roe deer livers, but lower than in bovine liver. Spermidine concentration of 37.2 ± 12.4 mg kg− 1 was determined in liver of zoologically related European brown hare 6 h post mortem (Paulsen et al., 2008). Spermine concentration of 115±19.5 mg kg− 1 in rabbit liver (Table 1) is very comparable with values reported for brown hare, pigs, roe deer and chicken liver, but higher than in bovine liver (Table 2). In rabbit kidneys, concentrations of SPD and SPM were 32.7 ± 9.3 and 88.4 ± 22.3 mg kg − 1, respectively (Table 1). The value for SPD is higher than data of Table 2, while that for SPM is comparable with the concentrations reported for brown hare and roe deer. Thus, both rabbit liver and kidneys belong among food items with very high concentrations of SPM and SPD. 3.2. Polyamines in frozen meat Changes in SPD and SPM concentrations were determined in four samples of saddles after 3 and 8 months storage (Table 3). It was found that dry matter content decreased only slightly. The retentions were not thus calculated. Both SPD and SPM concentrations decreased significantly during the initial 3 months of storage with a higher rate for SPD than for SPM. The decrease continued also during further 5 months. After the storage period, relative losses of both polyamines were about 30–35% of the initial concentration in fresh meat.
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Table 1 Polyamine concentrations (mg kg− 1) in rabbit meat, kidney (n = 18) and liver (n = 12) 24 h after slaughter. Material
n1
Putrescine Mean
Saddle Leg Liver Kidney
– – – 3.5
0 0 0 18
Spermidine SD
Range
– – – 0.7
Mean c
– – – 2.6–5.3
2.2 2.2c 61.7a 32.7b
Spermine SD 0.4 0.6 14.3 9.3
Range
Mean c
1.7–3.1 1.2–3.2 44.1–88.7 15.1–51.0
14.7 8.0d 115a 88.4b
SD
Range
1.6 0.7 19.5 22.3
12.2–17.7 6.8–9.3 92.2–157 55.5–124
n = number of analysed samples; n1 = number of samples with putrescine concentration above limit of quantification 2.1 mg kg− 1; SD = standard deviation. Different superscript letters in a column indicate significant difference (P b 0.05).
Table 2 Literature data on the concentration of polyamines (mg kg− 1) in fresh meat, liver and kidney of various animals. Putrescine concentrations were quantifiable only rarely by the used analytical methods. Product
n
Spermidine
Spermine
Mean
SD
Mean
Reference
SD
Meat Beef sirloin
63
–
–
21.7
5.8
Beef rump Pork loin Pork leg Mutton leg
57 15 15 20
– – – 5.6
– – – 3.5
22.0 26.1 28.4 24.7
5.6 7.0 8.5 6.8
Mutton breast Lamb leg Lamb breast Chicken breast
19 20 20 20
4.0 4.2 5.1 4.8
1.6 1.6 0.9 1.7
17.2 17.2 16.5 36.8
2.6 2.2 3.2 5.9
Chicken thigh
20
10.2
2.2
38.0
3.7
Liver Steer
58
122
82.0
43.1
26.5
Barrow Pig
19 20
32.1 31.2
11.6 1.1
115 95.1
49.4 17.6
Sheep Lamb Roe deer
22 31 39
13.5 16.8 10.7
7.9 5.9 5.3
128 78.6 79.9
69.4 26.2 15.0
Brown hare Chicken
20 38 20
37.2 56.9 48.7
12.4 15.1 8.8
111 120 133
24.9 43.0 18.0
Kidney Pig
40
9.4
3.4
53.1
14.0
Roe deer Brown hare
20 39 20
21.3 10.7 24.4
7.8 5.3 4.1
64.5 79.9 82.8
23.2 15.0 13.8
Krausová, Kalač, Křížek, and Pelikánová (2006b) Krausová et al. (2006b) Krausová et al. (2006b) Krausová et al. (2006b) Dadáková, Pelikánová, and Kalač (2011) Dadáková et al. (2011) Dadáková et al. (2011) Dadáková et al. (2011) Kozová, Kalač, and Pelikánová (2009b) Kozová et al. (2009b)
Krausová, Kalač, Křížek, and Pelikánová (2006a) Krausová et al. (2006a) Fuchs, Bauer, and Paulsen (2009) Dadáková et al. (2011) Dadáková et al. (2011) Paulsen, Dicakova, and Bauer (2008) Paulsen et al. (2008) Krausová et al. (2006a) Kozová et al. (2009b)
Kozová, Kalač, and Pelikánová (2008) Fuchs et al. (2009) Paulsen et al. (2008) Paulsen et al. (2008)
n = number of analysed samples; SD = standard deviation.
Storage at + 2 ± 0.5 °C Initial concentration After 5 days After 9 days
Dry matter
Spermidine
26.43a ± 0.91 26.74a ± 0.83 26.92a ± 0.97
2.31a ± 0.24 1.57b ± 0.27 1.50c ± 0.19
26.85a ± 0.86 27.07a ± 0.98 27.28a ± 0.82
2.20a ± 0.22 1.63a ± 0.43 1.75a ± 0.57
3.3. Polyamines in cold-stored meat It was found that dry matter content changed only slightly during these experiments. The retentions were not thus calculated. Losses of both polyamines were about 20–25% of their initial levels in fresh saddles. While the SPM decrease was significant, the situation for SPD was contentious probably due to increasing analytical error at its very low concentrations (Table 3). The losses were higher than those observed in beef loin (Kozová et al., 2009a), pork loin (Krausová et al., 2008) or chicken breast (Kozová et al., 2009b) but comparable with losses in mutton legs (Dadáková et al., 2011) stored under similar experimental conditions. Even higher SPM losses were observed in six-day-aged saddles prior to the cooking treatments (Table 4). The effects of ageing are thus variable. Different microbial development seems to be one of the possible reasons for this observation. 3.4. Polyamines in cooked meat Dry matter content increased considerably in all three cooking treatments (Table 4) and the changes were taken into consideration
Table 4 Changes in dry matter content and polyamine concentrations in rabbit saddles after three cooking treatments and data on retention. Values of three samples. Parameter
Table 3 Changes of dry matter (%) and polyamine concentrations (mg kg− 1) during storage of rabbit saddles at − 18 °C (n = 4) and at + 2 ± 0.5 °C (n = 4).
Storage at − 18 °C Initial concentration After 3 months After 8 months
A similar trend of polyamine level decrease was observed during storage of frozen pig liver (Krausová, Kalač, Křížek, & Pelikánová, 2007). However, polyamine levels increased moderately in pork loin (Krausová, Kalač, Křížek, & Pelikánová, 2008) and chicken thighs (Kozová et al., 2009b), while initially increased and then significantly decreased in frozen beef loin (Kozová, Kalač, & Pelikánová, 2009a) and in mutton loin (Dadáková et al., 2011). To our knowledge, there is no information on the mechanism(s) of the polyamine losses during storage of frozen foods.
Spermine 13.8a ± 1.2 11.5b ± 1.3 9.7c ± 1.6
15.5a ± 1.2 11.5b ± 1.1 13.7a,b ± 2.1
Different superscript letters in a column for each type of storage indicate significant difference (P b 0.05).
Dry matter (%) After 6-day ageing After cooking Spermidine (mg kg− 1 fresh/cooked matter) 24 h after slaughter After 6-day ageing After cooking True retention (%) Apparent retention (%) Spermine (mg kg− 1 fresh/cooked matter) 24 h after slaughter After 6-day ageing After cooking True retention (%) Apparent retention (%)
Stewing
Roasting
Pan-roasting without oil
25.32 ± 0.33 34.04 ± 1.40
25.42 ± 0.49 44.53 ± 0.46
25.46 ± 0.24 43.25 ± 3.19
2.21 ± 0.47 2.56 ± 0.21 2.71 ± 0.02 72.3a ± 2.3 79.1a ± 4.1
2.10 ± 0.19 3.19 ± 0.62 2.80 ± 0.48 53.8b ± 3.2 50.3b ± 4.7
2.52 ± 0.51 2.75 ± 0.82 2.24 ± 0.73 51.2b ± 9.3 47.7b ± 8.0
14.32 ± 1.73 8.01 ± 0.66 9.22 ± 1.61 77.9a ± 5.0 85.2a ± 6.2
14.96 ± 1.26 9.43 ± 2.35 8.09 ± 0.78 53.7b ± 8.5 50.4b ± 9.2
14.85 ± 2.63 7.69 ± 0.61 6.98 ± 0.85 57.6b ± 7.7 53.7b ± 7.0
Different superscript letters in the rows of retentions indicate significant differences (P b 0.05).
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for the calculation of true and apparent retentions. No detectable levels of polyamines were determined in the broth and drippings. Data on polyamine concentrations before and after cooking and on relative changes caused by various cooking conditions are given in Table 4. According to Murphy et al. (1975), the true retention reflects losses of a constituent in cooked foods more accurately than the apparent retention. The lowest decrease of both SPD and SPM, about 20–25% of the level in aged saddles, was observed in stewed meat. Significantly higher (P b 0.05) were the losses of both polyamines in roasted and pan-roasted-without-oil meat. The decrease in the latter two treatments was about 50%. The results are well comparable with common trends resulting from similar experiments with pork loin (Krausová et al., 2008), mutton leg (Dadáková et al., 2011) and chicken breast (Kozová et al., 2009b). Thus, similarly as in other tested meats, rabbit meat roasting or pan-roasting without oil cause higher losses of both SPD and SPM than stewing (and very probably also than cooking). 3.5. Fate of polyamines lost during storage or cooking Unfortunately, information on SPM and SPD catabolic pathways in food matrices is lacking. The thoroughly described polyamine catabolism in living mammalian organs (Seiler, 2004) can be applied to post-mortem processes in meat and food inner organs to only a limited extent. There has emerged a fear of aldehydes (particularly of acrolein) produced during polyamine degradation for the development and progression of several grave neurodegenerative diseases (Wood, Khan, & Moskal, 2007) and brain infarction (Saiki et al., 2011). It is possible that the primary amino groups of the polyamines react, under the conditions of roasting and pan-roasting without oil, with glucose in the Maillard-type reaction. Méndez and Leal (2004) reported that putrescine, cadaverine, spermidine and spermine can compete with the free primary amino groups of amino acids (particularly L-lysine) for glucose under in vitro conditions. Spermine was observed to be the most efficient. 3.6. Exposure assessment The estimated polyamine intake from a serving of 100 g stewed or pan-roasted rabbit saddle meat is about 0.27 and 0.22 mg SPD, and 0.92 and 0.70 mg SPM, respectively (data from Table 4). These values account for only a low proportion (2–7%) of the estimated daily intake of these two polyamines. For example, the mean daily intake of 12.6 and 11.0 mg of SPD and SPM, respectively, has been reported for the United Kingdom, Italy, Spain, Finland, Sweden and the Netherlands (Ralph, Englyst, & Bardócz, 1999). The respective values adapted for Japan are 12.0 and 7.9 mg (Nishibori, Fujihara, & Akatuki, 2007) and for the USA convenient sample diet 7.9 and 7.2 mg (Zoumas-Morse et al., 2007). It can be assumed that the polyamine intake from cooked rabbit liver would be considerably higher than that from meat. Acknowledgement The authors acknowledge the financial support of the project MSM 6007665806 of the Czech Ministry of Education, Youth and Sports. References Agostinelli, E., Marques, M. P. M., Calheiros, R., Gil, F. P. S. C., Tempera, G., Viceconte, N., et al. (2010). Polyamines: Fundamental characters in chemistry and biology. Amino Acids, 38, 393–403. Bardócz, S. (1995). Polyamines in food and their consequences for food quality and human health. Trends in Food Science and Technology, 6, 341–346.
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