Separation of soluble selenium compounds in muscle from seven animal species using size exclusion chromatography and inductively coupled plasma mass spectrometry

Abstract

To study the role of selenium compounds for the quality and nutritional value of meat, speciation of selenium compounds was performed in two muscles from each of seven animal species (chicken, turkey, duck, ostrich, lamb, cattle and pig). Soluble selenium compounds were separated by size-exclusion chromatography (SEC) and detected by inductively coupled plasma-mass spectrometry (ICP-MS). Four selenium peaks were found in the muscle extracts by SEC, with partition coefficients (K_av) of 0.02, 0.44, 0.74 and 0.86, respectively. The second and third peaks had a chromatographic mobility approximately corresponding to that of tetrameric glutathione peroxidase and selenoprotein W, respectively, and they contained 68–100% of the recovered selenium in different muscles. The distribution of selenium among the four peaks varied considerably in muscles from different species, the second peak accounting for 28–71% of the recovered selenium and the third peak for 17–72%. These differences in selenium distribution among animal species are discussed in relation to meat quality and nutritional value.

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Introduction

Selenium is an essential trace element for both humans and animals, and selenium deficiency causes a variety of symptoms in different species. Many studies on selenium in foods have concerned the total amount of this element, but only few have assessed the occurrence of different selenium compounds. Several specific selenoproteins containing selenocysteine have been demonstrated in mammals^1,2 in addition to unspecific selenium-containing proteins (containing selenomethionine), and low-molecular-weight selenium compounds.

The variation in selenium forms has several biological and nutritional implications and different seleno-compounds have different bioavailability in humans.^3,4 Moreover, organically bound selenium has been considered as more bioavailable than inorganic forms in cattle and lamb.^5–7 The occurrence of different selenium forms in meat may not only affect its nutritional value but may also be important for meat quality and stability. One important family of selenoproteins in meat are the glutathione peroxidases (GSHPx) functioning as antioxidative enzymes.⁸ Meat from different animal species and muscles within a species varies in oxidative stability.⁹ However, little is known of the role of selenium compounds for this variation.^8,10,11

A major advance for the speciation of selenium in foods has been the use of inductively coupled plasma mass spectrometry (ICP-MS) as an on-line detection technique, in combination with different separation methods.^12,13 The use of size exclusion chromatography (SEC) coupled to either atom absorption spectrophotometry or ICP-MS allows an estimation of the molecular size and amounts of selenium-containing proteins and other compounds.^14–16 Other chromatographic methods have been used for the separation of low-molecular-weight selenium compounds.^17–21 Previously only few studies have been performed using the ICP-MS technique for selenium speciation in foods.^16,22 Some studies on selenium compounds in fish have been done, using SEC or high pressure liquid chromatography (HPLC) as separation techniques.^14–16,21 Other studies on the distribution of selenium in selenium-enriched vegetables have been described,^23,24 but the information on selenium speciation in meat is scarce.

The aim of this investigation was to separate soluble seleno-compounds of muscles from seven species used for meat production, to study the differences in selenium distribution by use of SEC and on-line detection with ICP-MS, and to compare the patterns of selenium distribution among animal species. The results were evaluated in relation to total selenium content and GSHPx activity.

Material and methods

Chemicals

Tris(hydroxymethyl)-aminomethane, ammonium acetate, methanol (pro analysi), and selenite standard solution (1000 mg l⁻¹ in 0.5 mol l⁻¹ nitric acid) were obtained from Merck (Darmstadt, Germany). Superdex 200 10/30 columns were used for SEC (24 ml; Amersham Pharmacia Biotech, Uppsala, Sweden). The columns were calibrated using a marker kit obtained from Sigma Chemical Co (MW GF-200, St. Louis, USA), cyanocobalamin and aprotinin. Selenomethionine (min. 98%) and bovine cellular GSHPx were obtained from Sigma Chemical Co (St. Louis, USA), sodium selenite (p.a.) from Merck (Darmstadt, Germany) and sodium selenate from BDH Chemicals (Poole, UK).

Sample collection

Two types of muscle from each of chicken, duck and turkey (M. pectoralis (breast); M. gastrocnemius and M. peroneus longus (thigh)), ostrich (steak and fillet) and cattle (M. longissimus dorsi (LD) and M. psoas major (PM)) were obtained at local slaughterhouses. Pig and lamb muscles (LD and PM) were obtained at a local butcher. In most cases each pair of muscles was taken from the same animal (n = 5; for bovine muscles n = 4), and the samples were immediately vacuum packed and stored at −20 °C. Breast from chicken and turkey, thigh from duck, and LD from lamb, cattle and pig were considered as glycolytic muscles, and the other muscles from those species as oxidative ones.

Sample preparation

After thawing, the samples were kept on ice during the procedure. All visible fat was removed prior to grinding of the meat through a plate with holes 6 mm in diameter. For each muscle type 5 g from each animal was pooled together to one sample. The samples were suspended (1 ∶ 4) with Tris acetate buffer (20 mmol l⁻¹ Tris, 0.15 mol l⁻¹ ammonium acetate; pH 7.5) and homogenised in an Ultra Turrax apparatus (Janke & Kunkel KG, Breisgau, Germany) for 20 s. After centrifugation at 5000g for 20 min at 4 °C (Beckman GPR, Beckman, Palo Alto, CA, USA), the supernatant was filtered twice (Whatman 41 paper; Whatman, Maidstone, UK and Millex LCR filter unit, 0.22 µm; Millipore, Billerica, MA, USA) and then stored at −20 °C until analysis.

Size exclusion chromatography (SEC)

The SEC columns used separated proteins in the molecular weight range 10–600 kDa. The eluent (Tris–acetate buffer containing 3% MeOH (v/v)) was pumped through the column with a flow of 0.75 ml min⁻¹. The column outlet was connected to an UV spectrometer (Waters, Milford, MA, USA) adjusted to 280 nm followed by an ICP-MS (see below). Compensation for the time delay between the two detectors was done in the chromatograms presented. The muscle supernatant samples were injected through a 500 µl sample loop. All samples were analysed in duplicate and usually the data from the duplicates agreed within 10%. The mobility of different reference seleno compounds was studied using a mixture of bovine cellular GSHPx, selenomethionine, selenate and selenite. The columns were regularly washed according to the recommendations of the manufacturer to remove adsorbed material.

Inductively coupled plasma mass spectrometry (ICP-MS)

The determination of selenium was performed by ICP-MS (PQ2+ from Fisons Elemental, Winsford, Cheshire, UK) as previously described.¹⁶ A water chilled spray chamber (Scott 7C), a PTFE V-groove nebulizer and nickel sampler and skimmer cones were used (Thermo Elemental, Winsford, Cheshire, UK). The gas flows were 13.0 l min⁻¹ for the cooling gas, 1.1 l min⁻¹ for the auxiliary gas and 0.96 l min⁻¹ for nebulizer gas. The forward power was 1350 W, and the reflected power <5 W. The samples were analysed in peak-jumping mode for ⁷⁷Se and ⁸²Se (3 points per peak, 50 ms dwell time) in the time resolved analysis mode (TRA).

The linearity of the selenium response was assessed by injecting selenium standard solutions (0, 2.5, 5.0, 10.0 µg l⁻¹) using a second loop (500 µl) attached distal to the column (r² = 0.99; y = 635.2x). The 5.0 µg l⁻¹ Se standard was also injected in the second loop simultaneously with the sample injection in all runs. Peaks containing only one of the selenium isotopes were not considered as selenium peaks. Data obtained for ⁸²Se were used for quantification and they were expressed as percentage of recovered selenium in the different peaks. The limit of detection calculated as 3 × standard deviation of a blank was 0.012 µg l⁻¹. Data on GSHPx activity, total selenium and soluble selenium in muscle were taken from parallel studies.^8,11

Statistical methods

The significance of differences between groups was calculated using the Mann-Whitney test. Correlations were computed by the Spearman method.

Results

Soluble selenium compounds in muscle

Four major peaks detected as selenium-containing compounds by ICP-MS were found in the muscle extracts separated by SEC (Figs. 1–7). The K_av for the first selenium peak (1) was <0.04, i.e. the void volume of the column (V₀) corresponding to an apparent molecular weight of more than 200 kDa. The partition coefficient K_av was calculated as (V_e − V₀)/(V_t − V₀) where V_e is the elution volume and V_t the total volume. The selenium is probably bound to proteins because of the high molecular weight of the peak. This minor selenium peak (1) was most clearly seen in muscles from chicken (Fig. 1), and it could also be seen in most other muscles except for the bovine muscles and lamb PM.

Fig. 1 SEC-ICP-MS chromatogram of chicken breast (a) and chicken thigh (b) supernatant. The black line shows the m/z 82 and the dotted line the UV absorbance (280 nm). The first peak shows a 5 µg l⁻¹ selenium standard, injected at the same time as sample. * indicates a non-selenium peak since no m/z 77 peak was seen.

Fig. 2 SEC-ICP-MS chromatogram of turkey breast (a) and turkey thigh (b) supernatant. The black line shows the m/z 82 and the dotted line the UV absorbance (280 nm). Other symbols as in Fig. 1.

Fig. 3 SEC-ICP-MS chromatogram of duck breast (a) and duck thigh (b) supernatant. The black line shows the m/z 82 and the dotted line the UV absorbance (280 nm). Other symbols as in Fig. 1.

Fig. 4 SEC-ICP-MS chromatogram of ostrich steak (a) and ostrich fillet (b) supernatant. The black line shows the m/z 82 and the dotted line the UV absorbance (280 nm). Other symbols as in Fig. 1.

Fig. 5 SEC-ICP-MS chromatogram of lamb LD (M. longissimus dorsi) (a) and lamb PM (M. psoas major) (b) supernatant. The black line shows the m/z 82 and the dotted line the UV absorbance (280 nm). Other symbols as in Fig. 1.

Fig. 6 SEC-ICP-MS chromatogram of bovine LD (M. longissimus dorsi) (a) and bovine PM (M. psoas major) (b) supernatant. The black line shows the m/z 82 and the dotted line the UV absorbance (280 nm). Other symbols as in Fig. 1.

Fig. 7 SEC-ICP-MS chromatogram of pig LD (M. longissimus dorsi) (a) and pig PM (M. psoas major) (b) supernatant. The black line shows the m/z 82 and the dotted line the UV absorbance (280 nm). Other symbols as in Fig. 1.

The second peak (2) eluted at a K_av of 0.42–0.46 and its peak maximum was equivalent to a molecular weight of 65–90 kDa. This peak was found in all species. The third peak (3) eluted at a K_av of 0.71–0.76 corresponding to a calculated molecular weight of approx. 5 kDa and was also observed in every species. Peak (3) was in some cases not clearly separated from peak (4), which eluted at K_av 0.84–0.88 indicating a molecular weight of approx. 2 kDa. This minor selenium peak (4) thus contained low-molecular-weight selenium compounds and was only observed in chicken and ostrich muscles and in duck breast (Figs. 1, 3a and 4).

To acquire more information on the identity of the different selenium peaks, a mixture of cellular GSHPx, selenomethionine, selenite and selenate was analysed (Fig. 8). Peak (2) co-eluted with the GSHPx peak, which had a K_av of 0.44. All low-molecular-weight-selenium reference compounds (K_av = 0.83) corresponded to peak (4) in the chromatograms.

Fig. 8 SEC-ICP-MS chromatogram (⁸²Se) of a selenium mixture containing bovine cGSHPx, selenomethionine, selenite and selenate (black line). The dotted line shows injected pure selenomethionine in another run. The first peak shows a 5 µg l⁻¹ selenium standard, injected at the same time as the mixture.

Distribution of selenium compounds in different species and muscles

Peak (2) and (3) together contained 73–100% of the recovered selenium (Table 1) and the distribution of selenium in different muscles within a species were found to be quite similar. However, the distribution of selenium among the four peaks varied considerably among muscles from different species. Peak (2) accounted for 28–71% of the recovered selenium and peak (3) for 17–72%. The proportion of selenium in peak (2) (38–72%) in bird muscles was higher than that in peak (3) (17–47%) and the opposite were found in cattle, lamb and pig (peak 2, 28–49%; peak 3, 51–72%; P < 0.001 bird samples vs. non-bird samples using the Mann-Whitney test). Another distinguishing feature was that the fourth peak (4) eluting near the total volume of the column was found in almost all bird muscles (Figs. 1–4) but not in cattle, lamb and pig muscles (Figs. 5–7). Peak (1) was only observed with certainty in chicken muscle (Fig. 1).

Table 1 Percentage of recovered soluble selenium distributed among the different selenium peaks from two types of muscles in seven animal species. Glycolytic muscles: breast (chicken, turkey), thigh (duck), LD (lamb, cattle and pig), ostrich steak. Oxidative muscles: thigh (chicken, turkey), breast (duck), PM (lamb, cattle and pig), ostrich fillet

Species	Peak 1		Peak 2		Peak 3		Peak 4
	Mean Kav = 0.02		Mean Kav = 0.45		Mean Kav = 0.73		Mean Kav = 0.86
	Mw range >200 kDa		Mw range 12–>200 kDa		Mw range 2–12 kDa		Mw range <2 kDa.
	Glycolytic (%)	Oxidative (%)	Glycolytic (%)	Oxidative (%)	Glycolytic (%)	Oxidative (%)	Glycolytic (%)	Oxidative (%)
a Visible but not measurable.b nd: not detectable.
Chicken	8	14	49	38	32	35	11	12
Turkey	^a	^a	58	53	30	47	12	nd^b
Duck	^a	^a	71	72	18	17	11	8
Ostrich	^a	^a	50	54	38	46	11	^a
Lamb	^a	nd^b	43	49	57	51	nd^b	nd^b
Cattle	nd^b	nd^b	35	35	65	65	nd^b	nd^b
Pig	^a	^a	32	28	68	72	nd^b	nd^b

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Discussion

Soluble seleno compounds in muscle

In parallel reports the activity of GSHPx, the content of selenium and the percentage of soluble selenium in these samples were given^8,11 and the data are summarised in Fig. 9. GSHPx activity varied more among species (approx. 10-fold) than the total selenium content (2–3-fold). The percentage of soluble selenium ranged from 31 to 76%. The present study was performed to further compare the pattern of selenium compounds in different muscles.

Fig. 9 GSHPx activity (U g⁻¹) (a), tissue selenium (ng g⁻¹) (b) and percentage of soluble selenium (c) in two muscles from seven different species. The data are expressed as mean and SD. Symbols used for the significance of differences between the muscle data for a given species: ***P < 0.001; **P < 0.01; *P < 0.05. Additional symbols: ■ Thigh (chicken, turkey), breast (duck), PM (cattle, lamb), oxidative muscles; □ Breast (chicken, turkey), thigh (duck), LD (cattle, pig, lamb), glycolytic muscles; ▧ Fillet (ostrich); ▨Steak (ostrich).

The main forms of selenium in mammalian tissue are either selenomethionine unspecifically incorporated into proteins, or specific selenoproteins containing selenocysteine. Several other compounds may occur but our interpretation was based on the existence of these two major forms. The chromatographic profiles for selenium as protein-bound selenomethionine would be expected to mainly follow the total protein concentration, i.e. total methionine concentration. In all samples selenium peak (2) (65–90 kDa) was related to a major UV peak, and also any occurring selenium peaks (1) and (4) coincided with such UV peaks. However, for several other high-molecular weight UV peaks there was very low selenium content, which suggests that only a low proportion of the soluble selenium in these meat extracts was protein-bound selenomethionine.

The major selenium peaks (2) and (3) may largely represent specific selenoproteins. The maximal selenium intensity in peak 2 would be expected to include the tetrameric GSHPx:s, having molecular weights in the range 80–90 kDa. In accordance with this, injected pure GSHPx had an estimated molecular size of 78 kDa. The contribution of GSHPx to peak (2) is also supported by the possible association between the selenium content in this peak and GSHPx activity (see below). The broad calculated size range for peak (2) (12–200 kDa) may also include any possible thioredoxin reductase, selenoprotein P and other selenoproteins.

Selenium peak (3) coincided with very low UV absorbance indicating that most of it constituted specific selenoproteins. Similar findings were previously reported in heart and muscle from lamb, and the corresponding selenium compound had an estimated molecular weight of 10 kDa²⁵ and was later identified as selenoprotein W (9.5 kDa).²⁶ Although the maximal selenium intensity in peak (3) had an estimated molecular weight of only approx. 5 kDa, selenoprotein W and similar small selenoproteins^2,27 are most probably included in peak (3) since the entire peak had a calculated mass range of 2–12 kDa. It must be realized that the calculated molecular weights in this range are only approximate.

Only low selenium amounts were found in low-molecular weight compounds close to the total column volume. This may include seleno amino acids and inorganic compounds. Selenomethionine, selenite and selenate were not however, separated in this system. The low proportion of free seleno amino acids indicates that possible proteolysis during sample preparation and storage had little influence on the results, if any. However, it cannot be excluded that any low-molecular-weight selenium compounds were adsorbed to macromolecules. Studies of hydrolysed samples from other tissues have shown the presence of several small selenium compounds including unidentified peaks.^19,22 Future studies are needed to unravel if such compounds are also present in meat.

We used the soluble fraction from muscle tissue accounting for 31–76% of total selenium. Further solubilisation of bound selenium can be achieved by using enzymatic and other extraction techniques.^21,28–30 In this manner it may be possible to develop methods to characterize insoluble and particle-bound selenium compounds in foods, part of which is probably non-specifically bound as selenomethionine in many proteins. Another methodological issue in selenium speciation is the stability of different compounds. We used material that had been frozen but found a good agreement between duplicate chromatograms. Previous studies have shown that selenium compounds can be either labile or stable during storage in a freezer, one important factor being the surrounding matrix.^31,32

Selenium distribution pattern in different animal species

The distribution of muscle selenium thus varied among the species in several respects. Previous studies have shown that duck muscles have the highest activity of GSHPx and pig muscles the lowest activity among the species studied.^8,11 In accordance with this finding, pig muscle had the smallest selenium content in peak (2) and duck muscle the largest. There was, however, no significant correlation between the selenium content of peak (2) and GSHPx activity. This study thus provides new information about the distribution of high- and low- molecular weight selenium in meat from different species, but further studies are needed for additional identification of the different seleno compounds in the peaks using complementary techniques.^13,19

Meat quality and nutritional value

GSHPx is one of the selenoproteins in meat functioning as an antioxidative enzyme contributing to the oxidative defence in tissues.³³ Muscle fibres can be categorized into different metabolic types and a factor possibly influencing the susceptibility of muscle to lipid oxidation is the relative proportions of oxidative and glycolytic fibres. Previous studies have showed a higher GSHPx activity in oxidative (thigh) compared to glycolytic (breast) muscles in chicken and turkey^11,34,35 and therefore it was of interest to evaluate any differences in the distribution of selenium compounds between these two types of muscles. Since peak (2) contained GSHPx, it would be expected to be a higher in oxidative muscles. However, we found that the distribution of selenium in the different muscles within the same species was quite similar. The oxidative muscle in lamb had a tendency towards a higher proportion of selenium in peak (2), but the opposite finding was made in chicken and turkey.

Further studies are necessary to measure the contribution of other selenoproteins to the selenium peaks and another interesting approach in the future would be to correlate data from speciation studies with data on selenium bioavailability from different foods.

Meat and poultry are an important source of selenium in Sweden, accounting for about 21% of the mean intake.³⁶ It could be calculated that pork would supply a mean of approximately 5 µg of selenium per day, the corresponding figures for beef and poultry being 3 and 4 µg per day. Comparing these values with those for the recommended intake of selenium, 50 µg per day for men and 40 µg per day for women, meat can be seen to be a significant source of dietary selenium. Of the persons who abstain from eating meat, the vegans represent a particular interesting group. In a recent study these persons were found to have as low a selenium intake as 10–12 µg per day,³⁷ whereas persons who were omnivores had a threefold higher intake. It is pertinent in this connection to note that vegans also refrain from eating milk products, egg and fish. A more relevant group for evaluating the influence of meat elimination on selenium status would be that of the lactovegetarians. In a study of healthy persons who switched from a mixed to a lactovegetarian diet, a significant decline in nutritional status for selenium could be observed,³⁸ again indicating the significant role of meat selenium in human nutrition.

Acknowledgements

The authors would like to thank Ms Lisbet Persson and Mr Stig Juhlén for kind help with sample acquisition. We acknowledge the contribution of the late Dr Andrejs Schütz at the initiation of the study. The financial support from the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (project no. 50.0330), the J. Andersson Foundation and Lund University Hospital is gratefully acknowledged.

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Footnote

† Results from this study were presented at Euroanalysis-12, Dortmund, Germany, September 2002.

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