J. Trace Elem. Med. Biol. VoL. 17 (4) 307-311 (2004) http://www.eLsevier-deutsch[a nd.

de/jtemb

Pork meat: A good source of selenium?

Susanne B[igeLI,*, Brittmarie SandstriJmlt and Leif H. Skibsted 2
i Research Department of Human Nutrition, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark 2Department of Dairy and Food Science, Centre for Advanced Food Studies, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark

Received Mai 2003 Accepted September 2003

Abstract
The knowledge about Se bioavaiLabiLity from animal food is sparse. This study was therefore initiated in order to evacuate the bioavaiLabi[ity of Se from pork meat in humans. TweLve maCevolunteers (age 21-30 years) participated in a randomised crossover study with strictly controLLed diet containing 170 g pig meat/lO MJ per day and 106 + 13 pg Se/d for 3 x 3 weeks. CompLete faecaL and urinary coLLectionswere made during the Cast week of each period. BioavaiLabiLity was evaCuatedfrom absorption and retention of Se and changes in plasma Se concentration and blood g[utathione peroxidase (GSHPx) activity. The apparent absorption of Se was 94 _+2% (100 + 13 pg/d). Faecal and urinary excretion were 7 + 1 pg/d and 39 + 21 pg/d, respectively, resulting in a retention of 61 + 24 IJg/d. The diet intervention did not affect pLasma Se concentration and GSHPxactivity. Absorption and retention of Se from pig meat were high suggesting a high avaiLabiLity. However, the avaiLabiLity of pig meat Se for bLood Se protein appears to be Low. Key words: selenium, pork meat, humans, bioavaiLabiLity, absorption, gtutathione peroxidase

Introduction
SeLenium plays a central roLe in human antioxidative defence (I). Earlier observations suggest that a high Se intake reduce the risk for certain types of cancer (2) and cardiovascular disease (3). Se intake from foods varies substantially between and within populations depending on the soil Se content, with a direct impact on the Se content of plant foods and indirectly on animal foods derived from Locally produced crops. The Nordic countries, together with New Zealand, are Low Se areas due to the ancient type of bed-rock (4). Addition of Se to fertilizers has been shown to increase Se content of Finnish foods and to improve Se status in the Finnish population (5). Similarly, the Se content of animal feed affects the Se concentration of e.g. milk (6). The absorption, metabolism and final utilization of Se for vital body functions are dependent on the chemical form of Se. Se in cereals occurs mainly as Se-methionine (Se-meth), but inorganic forms and unknown Se compounds may also be present (7). Animals and humans are able to incorporate Se-meth non-specifically into proreins. Thus, part of Se in animal food might be in the form of Se-meth. However, it has been suggested that the main form of Se in food of animal origin is se[enocysteine present in the enzyme gLutathione peroxidase (GSHPx) and other se[enoenzymes (7). In addition, Se in animal foods is present in other organic forms bound to proteins or amino acids. However, the relative composition of Se compounds in foods is not known in details. Based on data from animal experiments, Se from animal and seafood has been judged as poorly available (4). There is, however, no generally accepted method for
0946-672X/04/17/04-307 $ 30,00/0

*Correspondence to: Susanne B[ige[, Research Department of Human Nutrition, The Royal Veterinary and AgricuLturaLUniversity, Ro[ighedsve] 30, DK-1958 Frederiksberg C, Denmark, Phone: (+45) 3528 2490, Fax: (+45) 3528 2483, E-maiL: shb@kvLdk Reprints not avaltabte Sponsorship: This research was funded by the Commission of the European Communities Program Dietox: Dietary treatment and oxidative stability of muscle and meat products: Nutritive value, sensory quality and safety (Air III-CT-92-1577). It does not necessarily reflect its views and in no way anticipates the Commissions future policy in this area.

308 s. BiJge[, B. Sandstri~m and L. H. Skibsted

assessment of bioavailabitity of Se in humans. A functiona[ assay, restoration of GSHPx activity in blood proteins, has been used to assess different Se supplements (4, 8, 9) mainly L-Se-meth, selenite, se[enate and Se yeast (:tO, :l:t, 12, :13, 14, :t5). However, to be useful this approach can only be used for populations with initially Low Se status. In Se-replete subjects retention of Se in body fluids or tissues or in total body may provide additional information about the potential value of a Se food source. The aim of this study was to evaluate the bioavailabi[ity of Se from pig meat. The data are derived from a study demonstrating that changes in pig feed composition can be used to alter pig fat composition in a way that lowers blood cholesterol concentrations (:16). Providing Se in pig meat has a high availability, pig meat would be a valuable food with regard to prevention of chronic diseases. etc. The experimental diets in the three periods were identical, except for the pig group used. The intake of pork meat and fat was 256 g/lO MJ (170 g meat and 86 g fat). Duplicate portions were taken of each diet in each period and analysed for Se content. The analysed Se content is given in Table i.

Urine sampling
24-h urine was collected from day :t5 to day 2:1 in each period in acid washed plastic bottles containing 10 mL of 1 moL/L HN03. Paraaminobenzoic acid (PABA) (3 80 mg) was consumed at the same days as the urine collection.

Faeces sampling
Faeces were collected in acid washed plastic containers at day :15-21 i.e. the Last week of each intervention period. Radio-opaque faecal markers (Marker capsules; Dunn Nutrition Centre, Cambridge, England), 3 20 per day were consumed, two days prior to, and during the faecal collection.

Materials and methods

Subjects
Twelve men (age 21-30 years) volunteered for the study. Exclusion criteria were family disposition for coronary heart disease, serum cholesterol > 8 rumor/L, cigarette smoking and high physical activity. The subjects were art apparently healthy and none of them used any medication. Height, weight and body mass index (BMI) of the subjects were 182 + 8.6 cm; 72.7 + 8.7 kg and 21.8 _+1.7 kg/cm 2, respectively (see also 16).

BLoodsampUng
Venous blood samples were collected after a minimum of 12 h fasting and 10 min supine rest. The subjects abstained from alcohol for at least 24 h and from physical activity for at Least 36 h before sampling.

Analysis
Duplicate portions of the intervention diet served or distributed at the department were collected, homogenized and [yophitised. Faeces were freeze dried and homogenized and then pooled corresponding to approximately 3 days according to the content of faecal markers. Portions of urine and freeze-dried food and faeces were analysed in duplicate for their content of Se. Urine was also analysed for PABA (19). All glassware was washed in hydrochloric acid and rinsed in deionised water before use. Food was acid decomposed according to (20). PLasma Se was measured by ftameless-AAS (Varian, Victoria, Australia) and dietary Se was measured by flame[ess-AAS with standard addition and Zeeman background correction (Perkin Elmer Zeeman/3030, Perkin ELmer, CA, USA). Standard reference material (1548 Total diet, National Institute of Standards and Technology, Gaithersburg, USA) was analysed along with the diet samples. The analysed Se concentration of the standard material was 0.251 IJg/g compared to a certified value of 0.245 pg/g. Se in faeces was measured by hydride generation ICP-MS (Perkin-Elmer FIAS 200, MG ELemental PQ2+ turbo, Perkin ELmer, CA, USA) after microwave digestion (Perkin ELmer "Mu[tiwave", Perkin Elmer, CA, USA) with concentrated HNO3. Sample responses were compared to standard concentration responses prepared in a similar acid matrix. A certified reference material (BCR No. 184, Commission of the European Communities, Community Bureau of Reference-BCR, Brussels, Belgium) was measured along with the faecal samples. The analysed Se concentration of the certified material was 0.18 4- 0.07 IJg/g (n = 4) compared to the certified value of 0.183 + 0.012 pg/g. Se concentration in urine was measured by hydride generation ICPMS (Perkin-ELmer FIAS 200, MG ELemental PQ2 + turbo,

Study design
Meat and meat products from three pig feed treatments were served for three weeks each in a randomised crossover design. The experimental periods were separated by at least four weeks during which the subjects consumed their habitual diets. BLood samples were taken on two consecutive days, before and at the end of each dietary period. Energy intakes were individualized based on gender, age, body weight and habitual physical activity (17). At[ foods were weighed on precision sca[es, and all meals were prepared in individual portions at the department. Breakfast, snacks and weekend meals were prepacked and consumed at home, whereas other meats were consumed at the department under supervision.

Pig treatment
Female pigs (Danish Landrace x Danish Yorkshire) were randomly assigned to A) basal feed (barkey, wheat, soybean, 2% fat), B) basal feed + 6% rapeseed oil and C) basal feed + 6Io rapeseed oil + 200 mg vitamin E per kg feed. The Se content was 0.26 mg/kg in the basal feed and 0.27 mg/kg in the rapeseed feeds. The animals were given ad [ibftum access to feed and water. The animals were kept on the feeding regime from 25 to :tOO kg tire weights (slaughter). For more information on pig breeding and treatment see 08).

Experimental diet
A detailed description of the experimental diets is given in (16). In short, the pork meat and fat was blended and incorporated into minced meat dishes, sausages, pates
J. Trace Elem. Med. Biol. 17/4 (2004)

Pork selenium availability

Perkin Elmer, CA, USA) after wet-digestion of urine sampies in concentrated HNO3 and HC[. Sample responses were compared to standard concentration responses prepared in a similar acid matrix. The detection limit was 0.01 mg/kg. The activity of GSHPx was measured on a Cobas Mira (Roche, Basel, Switzerland) as the oxidation of NADPH using tertiary butyLhydroperoxide as a substrate (21). The results were expressed as units per gram of haemoglobin. Haemoglobin was measured by a Cobas Minos (ABX, Montpe[tier, France).

309

Results
The average Se intake for the three dietary periods was 106 + 13 pg (Table 1). The diets made from pig lines fed rapeseed oil without addition of vitamin E (diet B) had a lower concentration of Se and the diet made from pig lines fed rapeseed oil with addition of vitamin E (diet C) a higher concentration of Se than the basal feed pig line diet (diet A). The feeding regime resulted in differences in fatty acid composition of the pig fat (16). Saturated, monounsaturated and polyunsaturated fatty acid contributed with 3 8 0 , 4 5 % and 141oof the fat energy in diet A, 30%, 47/o and 20% in diet B and 281o, 47/o and 21% in diet C. No differences were observed in Se faecal and urinary excretion in the three dietary periods (Table 1). The faecal Se excretion was low, approximately 7 pg/d giving an apparent absorption of 94 + 2/o (100 + 13 #g/d), Urinary Se excretion corresponded to 37 _+ 20% (37 _+ 20 #g/d) of the apparently absorbed Se. More than 50% of intake (61 +_ 24 pg/d) was retained, with the highest retention from the diet with highest Se content (diet C). Plasma Se concentrations (TabLe 2) were sEightEy higher after intake of diets from pigs fed rapeseed oil without addition of vitamin E (diet B) than after intake of the

Ethics The subjects were given oral and written information about aims and procedure of the study. The protocol was approved by the Local Research Ethics Committee for Copenhagen and Frederiksberg (KF-01-161/95). Statistics Measurements taken before each intervention were analysed using a one-tailed ANOVA to ensure that there was no carry-over effect. There was no carry-over effect and the effect of the diets was evaluated by ANOVA of end values followed by Student's t-test for paired data when ANOVA indicated significant difference.

Table 1. Intake, excretion and retention of selenium in 12 men fed diets with meat and fat from pigs fed three different types of fodder. Values are mean 4- SD. Diet A Diet B Diet C Mean

Intake, #g/day (#mo[/day) Urinary excretion, #g/day (#tool/day) Fecal excretion, #g/day (#tool/day) Apparent absorption% Apparent absorption, pg/day (IJmo[/day) Total excretion, #g/day (IJmo[/day) Retention, #g/day (pmo[/day)

102 + 5a (1.3 + 0.06) 40 __25 (0.5 4-0.3) 74-2 (0.09 4- 0.02) 93 4-1 96 + 5a (1.2 4. 0.06) 47 _+25 (0.59 4- 0.3) 56 4- 24a (0.7 0.3)

93 4- 5 b (1.2 4-0.06) 37 4- 19 (0.46 4-0.24) 64-1 (0.08 _+0.01) 94 4-2 87 4. 4 b (1.1 4. 0.05) 43 4-20 (0.54 4-0.26) 50 4- 20a (0.64 + 0.25)

122 _+6c (1.5 4. 0.08) 40 + 22 (0,51 4- 0.27) 74-1 (0.08 _+0.02) 95 4-1 116 4. 6c (1.47 4. 0.08) 47 4- 22 (0.59 4- 0.28) 76 4- 22 b (0.96 4- 0.28)

106 4- 13 (1.3 4. 0.16) 39 4- 21 (0.49 _+0.27) 74-1 (0.08 4- 0.02) 94 4-2 100 4. 13 (1.27 4-0.16) 45 4- 22 (0.57 4- 0,28) 61 4- 24 (0.77 4- 0.3)

Values with different Letters (a, b, c) are significantly (p < 0.05) different.

TaMe 2. Blood glutathione peroxidase activity (GSHPx), plasma Se concentration (P-Se) at start and/or end of each 3 week dietary period. Values are mean + SD.
Diet A Diet B Diet C

start
GSH-Px (U/g Hb) P-Se (# mo[/L) 49 _+7.5

end
47 4- 7.4 1.06 _+0.2a

start
49 _ _ _ 6.3

end
48 4. 6.6 1.12 + 0.1b

start
49 _+9.7

end
48 _+6.8 1.08 4. 0.i a

Values with different letters (a, b) are significantly different. 3. TraceE[em. Med. BioL 17/4 (2004)

31.0 s. BiJgel, B. Sandstr6m and L. H. Skibsted

other two pig meat diets. However, compared to initial plasma Se concentrations (90 lag/L) no changes were observed. The GSHPxactivity at the start and end of each dietary period did not differ and there was no effect of type of diet (Table 2). ipating in this study already was saturated, which together with the relatively short intervention period explain the lack of response in GSHPx activity. Some of the absorbed Se appears to have been excreted. At low Se intakes urine excretion is we[[ below 40 pg/d (25). In an earlier study with subjects from the same population baseline urine excretion was 23 pg/d (20). Intake of 135 pg Se/d as either Se-rich bread or meat resu[ted in urinary excretion of 47 pg/d and 40 pg/d, respectively (23), while selenite supplementation with 200 pg/d resulted in a higher urinary excretion of 127 pg/d (26). In this study the majority of absorbed Se was retained in the body. Assuming that Se retention over the total three week period was similar to the last week, over I mg Se was retained in each period. This should be compared to a total body Se content in the order of 2-20 mg (27, 28). The high retention of Se from pork meat could indicate a low Se status of the participating subjects not revealed in plasma Se concentrations and GSHPx activity. Long-term studies in deplete, as well as replete subjects are needed to eva[uate the value of pork meat for improvement of Se status. The different pig feeding strategies used in the main study (16) resulted in differences in Se content of the otherwise identical diets. This may have been due to differences in feed intake and growth rate induced by the larger amount of fat in the rapeseed-based feed (18). The resulting differences in fatty acid composition of the human diets, with a higher content of polyunsaturated fatty acids in diets B and C, appear not perse to affect Se absorption or retention. In conclusion, Se from pig meat is retained in the human body but seems not to affect either plasma Se concentration or GSHPx activity in erythrocytes. These results highlight the need for alternative or combined approaches for estimates of the nutritional value of different food Se sources.

Discussion
The results from this study suggest a low bioavailability of Se from pig meat for blood Se proteins and indicate that Se in pork meat is not in the form of Se-meth. Despite a high absorption and retention, the plasma Se concentration and the red blood cell GSHPx activity were not affected by the diet intervention. The habitual Se intake of the Danish population is in the order of 51 pg/d (56 _+ 28 pg/lO M3) based on duplicate portion data (22). Due to the inadequacy of available food tables no attempt was made in this study to calculate each individual's habitual Se intake. However, a brief dietary interview suggests food habits close to the average Danish population. Thus, the diet intervention periods increased the Se intake 2-3 times the normal intake without any changes in plasma Se and GSHPx. Previous studies have reported different effects of an increased Se intake on plasma Se concentrations depending on Se status, chemical form of Se and the doses of Se ingested (10-14, 20, 23). In all studies in which Se-yeast, Se-wheat, L-Se-meth or selenite (10-14, 23) have been given as a supplement (100-200 pg/d), plasma Se concentrations have been significantly increased independent of initial plasma Se concentrations. When selenate (200 pg/d) (12), or Se-rich fish (115 pg/d) (13) were administered to Se-replete subjects (initia[ plasma Se : 1.4 pmo[/ L) plasma Se did not increase, while a small but significant increase in plasma Se was observed after intake of shrimps (127 pg Se/d) in an apparently Se-replete group of subjects (initial plasma Se = 1.2 pmol/L) (20). The increases in plasma Se have been observed within 2-3 weeks of increased intake and with doses ranging from 50-200 pg Se/d. An increased GSHPxactivity in erythrocytes has only been observed after 17 weeks of supplementation with 100 pg Se/d as Se-meth or selenite (10). The other studies have probably been either too short (less than 11 weeks of supplementation) or the initial Se status of the subjects has been too good (initial plasma Se > 0.9 pmol/L) for changes in GSHPxactivity to be expected. The lack of effect of a relatively high intake of Se on plasma Se in this study indicates that Se in pork meat is not in the form of selenite or Se-meth. At [east two distinct pools of body Se have been suggested (24). Se-meth enters a non-exchangeable pool and is handled as methionine, thereby increasing body stores and plasma concentrations in direct proportion to its intake. Other forms of Se enter a selenite-exchangeable metabolic pool (Se-EMP) that is used for the synthesis of GSHPx and other functional selenoproteins. If GSHPx capacity is saturated, surplus Se from the Se-EMP appears to be stored in the liver or excreted. It is reasonable to assume from the initial plasma Se concentrations that GSHPx activity of the subjects particJ. TraceElem. Med.Biol. 17/4 (2004)

Acknowledgement
We thank all the participating subjects for their cooperation and enthusiasm. We thank our own staff who prepared the foods, sampled, prepared and analysed blood, food, urine and faeces (dietician Hanne Jensen, the staff of the metabolic kitchen (Berit Hoie[t and her assistants) and the laboratory technicians Hanne Lysdal Petersen and Majbritt Schwaner). We also thank Malcolm Baxter and Helen Crews from CSL Food Science, Norwich, UK, for analysing Se in urine and faeces and Anette Almgren, Sweden for analysing Se in the diet.

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