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ANIMAL NUTRITION |
Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100094
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: Amino acids • Bioavailability • Chelation • Complexes • Minerals • Superoxide Dismutase
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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; Henry et al., 1989; Henry et al., 1992). Others (Baker and Halpin, 1987; Scheideler, 1991) reported no differences in bioavailability among organic and inorganic Mn sources. The chemical characteristics of organic Mn sources tested and the criterion used in bioavailability assays may explain the discrepancies in relative bioavailability estimates among the research reports.
Chemical characteristics considered important in predicting the bioavailability of chelated and complexed metals include the chelation effectiveness (strength of bonds between an organic ligand and a metal) and the percentage of organic ligand that remains bound to the metal under physiological pH conditions. Neither the AOAC (1995) nor AAFCO (2001) have approved definitive methods to test the degree of chelation or complex bonding of a mineral element to an organic ligand in commercial feed ingredient samples. To our knowledge, no experiment has directly compared the chemical characteristics of organic Mn sources with their relative bioavailability values for broilers.
The objectives of the current studies were to evaluate chemical characteristics of commercial organic Mn sources and correlate these data with relative bioavailability estimates based on tissue Mn concentration, Mn superoxide dismutase (SOD) activity, and MnSOD messenger RNA level in heart following additions of Mn to chick diets.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Mineral and Amino Acid Analysis
Mineral concentrations in Mn sources, diets and tissue samples were determined by inductively coupled argon plasma spectroscopy (model 9000; Thermal Jarrell Ash, Waltham, MA). Approximately 0.2 g of each Mn source was weighed in triplicate and digested with 8 mL of HNO3 and 0.5 mL of HClO4 at 200°C in a 50-mL calibrated flask until the solution cleared and then condensed to approximately 0.2 mL, and diluted 1:50 with deionized water before analysis. Validation of the mineral analysis was conducted using bovine liver powder as a standard reference material (National Institute of Standards and Technology, Beijing, China).
Amino acids were analyzed in duplicate using an amino acid analyzer (model L-8500A, Hitachi, Chyoudaku, Japan). The methionine and cysteine concentrations were determined using performic acid oxidation with the acid hydrolysis-sodium metabisulfite method (AOAC, 2000). A 50-mg sample was hydrolyzed at 0°C in performic acid (a mixture of 30% hydrogen peroxide and 88% methanoic acid as a 1:9 vol/vol) for 16 h, and then sodium metabisulfite was added to the hydrolyzed sample to decompose the performic acid. The solution was stirred 15 min to liberate SO2 and hydrolyzed at 110°C in 6 N HCl for 22 to 24 h. After hydrolysis, the sample was vacuum-dried and adjusted to pH 2.2 with 2 M NaOH and reconstituted in pH 2.2 citrate buffer. Validation was conducted using reference material containing 50 nM standard cysteic acid and methionine sulfone (National Institute of Standards and Technology, Beijing, China). Tryptophan was analyzed by a base hydrolysis method (National Institute of Standards and Technology, 2000). A 50-mg sample was hydrolyzed at 110 ± 1°C in 4 N lithium hydroxide at 7 Pa for 20 h and then neutralized with 6 N HCl and diluted with pH 4.3 citrate buffer before tryptophan analysis. Another 50-mg sample was hydrolyzed at 110 ± 1°C in 6 N HCl for 22 to 24 h, vacuum dried, and reconstituted in pH 2.2 citrate buffer for analysis of other amino acids. Validation of amino acid analysis was conducted using reference material containing 50 nM each L-amino acid (National Institute of Standards and Technology, Beijing, China).
Solubility and Chelation Properties
Manganese solubility of all Mn sources was determined in triplicate by mixing a 0.2-g sample with 100 mL of neutral ammonium citrate (NAC), 2% (wt/vol) citric acid (CA), 0.4% HCl (wt/vol), or deionized H2O. Mixtures were stirred constantly while incubating at 37°C for 1 h, and then filtered through Whatman No. 42 filter paper (Whatman, Clifton, NJ; Watson et al., 1970). The Mn content of the filtrates was analyzed after proper dilution with deionized H2O. The Mn in the filtrates was assumed to be soluble, and the values obtained were expressed as a percentage of total Mn in the source.
Solubility of all Mn sources was also measured in 0.1 M K2HPO4-KH2PO4 buffer (pH 5.0) and in 0.2.0 M HCl-KCl buffer (pH 2.0). A 0.2-g sample was weighed in duplicate, mixed with 100 mL of buffer and incubated at 39°C in a water bath with constant shaking for 12 h and then filtered through ashless paper (Whatman 42) for mineral analysis (Brown and Zeringue, 1994; Cao et al., 2000).
Soluble fractions from solubility tests (pH 2.0 and 5.0 buffers) were evaluated by gel filtration chromatography for structural integrity as described by Brown and Zeringue (1994) to determine the percentage of complex or chelate in each solution. The chromatographic column (1.5 cm x 19 cm) was packed with Biogel P-2, a polyacrylamide size exclusion gel with a fractionation range of 100 to 1,800 Da (catalog No. 150-4114; Bio-Rad, Rockville Center, NY) according to manufacturer’s instructions. Aliquots (0.20 mL) of filtrates were loaded onto the column and eluted with the same buffer that was used in the solubility test. Eluent fractions (0.7 mL) were collected and analyzed for Mn by inductively coupled argon plasma spectroscopy. The presence of amino acids or related proteinaceous material was detected qualitatively using a modified ninhydrin procedure (Moore and Stein, 1954). The capacity of this chromatographic column to separate free metal ions, amino acids and small peptides, and metal chelates or complexes based on molecular size was verified as described by Brown and Zeringue (1994).
Because of assay costs, the structural characteristics of only four organic Mn sources (Mn Met E, Mn Pro B, Mn AA B, and Mn AA C) and reagent-grade DL-methionine were measured. A 0.2-g sample was mixed with potassium bromide and pressed into a disc. The structural characteristics were determined using infrared spectroscopy (IR-435; Shimadu, Kyoto, Japan).
The chelation effectiveness of organic Mn sources was determined as described by Holwerda et al. (1995) using polarography with a hanging mercury drop electrode (Ag/AgCl reference electrode, Potentiostat/Galvanostat model 283; EG & G Inc., Gaithersburg, MD). Saturated solutions for each organic Mn source were prepared in 50 mL of deionized H2O, and final pH was measured. The saturated solution was diluted 1:100 in pH 8.0, 0.1 M N, N-bis (2-hydroxyethyl)-2-amino ethanesufulfonic acid, a noncomplexing buffer for anaerobic electrochemical measurements with a nitrogen purge. Molar metal concentration was determined from the catholic wave height (0.1 M Mn sulfate standard). The half-wave potential (E1/2) and the shift in half-wave potential (
E1/2) were measured and used to calculate the chelation quotient (Qf), which is a quantitative measure of chelation or complex strength.
Broiler Chick Bioassay and Test Diets
A total of 546 1-d-old Arbor Acres commercial male broilers were used to determine the relative bioavailability of the Mn sources. The birds were randomly allotted by weight to six replicate cages (seven chicks per cage) for each of 13 treatment groups in a completely randomized design involving a 4 x 3 factorial arrangement of treatments (four sources of Mn x three levels of added Mn plus a control with no added Mn). Based on the assumption that the chelation strength of organic Mn sources was highly correlated with chemical characteristics and to bioavailability, three organic Mn sources with different chelation strengths were selected in this trial. The three Mn sources chosen included Mn-Met E with a weak chelation strength (Qf value = 3.2, 8.27% Mn), Mn-AA B with a moderate chelation strength (Qf value = 45.3, 6.48% Mn), and Mn-AA C with a strong chelation strength (Qf value = 115.4, 7.86% Mn). The dietary treatments included the basal diet supplemented with 60, 120, or 180 mg Mn/kg using reagent-grade MnSO4 or one of the three organic Mn sources. The basal corn/soybean meal diets (Table 1) contained by analysis 23.00 mg Mn/kg for starter (d 1 to 21) and 20.95 mg Mn/kg for grower (d 22 to 42) diets. Diets were formulated so that all other nutrients met or exceeded requirements for broilers (NRC, 1994). Chicks were housed in electrically heated, thermostatically controlled cages with fiberglass feeders and a 24-h constant-light schedule. The birds were allowed ad libitum access to feed and tap water that contained no detectable Mn. Chicks were managed according to guidelines suggested by Arbor Acres Farm in Beijing. At 21 and 42 d of age, feed consumption was recorded for each cage, and three birds from each cage were selected according to average body weight of the cage, weighed individually, and killed by cervical dislocation. The heart was excised, a subsample was frozen (–20°C) for Mn and MnSOD activity analysis, and a second subsample was frozen in liquid nitrogen for assays of MnSOD gene expression. The left leg was excised and frozen in an individual heat-sealed polyethylene bag. Incidence of leg abnormalities was calculated as a percentage of chicks within each cage with visual swelling at the tibiotarsus joint (Luo et al., 1991).
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MnSOD Activity
Activity of MnSOD was measured by the nitrite method as described by Oyanagui (1984). Duplicate samples (approximately 0.4 g) of heart tissue were homogenized with a glass-Teflon tissue grinder in 10% (vol/vol) cold saline for 5 min, treated with an ultrasonic wave cell grinder (JY92-11; Ningbo, Jiangsu, China) for 1 min (1 s, with 2-s interval), and then centrifuged (1,500 x g, 15 min, 4°C). The supernatant was diluted 50-fold with deionized H2O. The final assay solution contained hydroxylamine (0.2 mM), xanthine oxidase (1.25 mU/mL), hypoxanthine (0.2 mM), EDTA (10–4 M EDTA-Na2), and the samples were incubated with or without KCN (1 mM) at pH 8.2, 40°C for 30 min. Diazo dye-forming reagent (300 µg/mL sulfanilic acid, 5 µg/mL N-1-naphthylethylenediamine, and 16.7% acetic acid) was added and optical density was measured at 550 nm. The MnSOD activity in the heart was expressed as nitrite units (NU) per gram of fresh weight, and one NU was defined as the amount of the enzyme needed to obtain 50% inhibition of nitrite formation.
Heart RNA Extraction and MnSOD mRNA Analysis
Total RNA in heart tissue was isolated using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. Concentrations of total RNA were estimated by measuring UV light absorbance at 260 nm (Ultrospec III; Perkin Elmer Cetus, Norwalk, CT). Heart MnSOD mRNA abundance was determined from samples using a semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) method. Reverse transcription was performed using the SuperScript II first-strand synthesis system for RT-PCR kit (Invitrogen Life Technologies) and oligo(dT)12–18 as a primer. Oligonucleotide primers used for amplification were as follows:
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The size of reaction products was 400 bp for MnSOD and 648 bp for ß-actin, which was used as an internal control in all reactions. Amplification was completed at an annealing temperature of 58°C, 45 s for 20 cycles. For each reaction, 10 µL of reaction product was size fractionated on 2% agarose gel in 1x Tris-borate/EDTA buffer, and bands were visualized with ethidium bromide staining. A digital image of the gel was obtained with an imager (AlphaImager 2200 and 1220 Document & Analysis Systems; Alpha Innotech Co., San Leandro, CA), and band intensities were quantified using gel analysis software (Biot Software System, Beijing, China). Data were presented as the ratio between band intensity of MnSOD mRNA and ß-actin mRNA. Each PCR reaction was performed in duplicate on two individual preparations of reverse-transcribed cDNA.
Statistical Analysis
The chick bioassay involved a randomized complete block design with 13 dietary treatment groups and six replicate cages for each treatment. Dietary treatments included a control diet with no added Mn and 12 diets in a factorial arrangement with four Mn sources, each added to provide three dietary levels of Mn. Data from the chick trial were subjected to two-way ANOVA using the GLM procedure of SAS (Release 6.03; SAS Inst. Inc., Cary, NC), with a model that included Mn source, added Mn level as main effects, and the interaction of Mn source and level. Cage was the experimental unit and was based on the average of seven chicks in the starter period and four chicks in the grower period. Inferences about main effects (Mn level) were based on orthogonal comparisons for linear and quadratic responses of dependent variables to independent variables. A sin–1 transformation was applied to data on the incidence of leg abnormalities before statistical analysis. Multiple linear regression equations were calculated by least squares using the GLM procedure of SAS. Relative bioavailability values were determined using MnSO4 as the standard source by slope ratio comparisons from multiple linear regression (Littell et al., 1995, 1997). Because feed intake differences among treatments could affect Mn intake, regressions were calculated using dietary added Mn intake (based on Mn assays of diets) as the independent variable rather than added Mn concentrations (Wedekind et al., 1992, 1994; Boling et al., 1998). Slope ratios and their standard errors were estimated using the method of error propagation as described by Littell et al. (1995). Differences among sources were determined by differences in their respective regression coefficients. Actual probability levels are reported for all main effects and interactions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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). Some sources also contained variable amounts of other macro- or microelements. For example, Mn Met A and Mn Met D contained 1.16 and 2.47% Fe, respectively, and Mn AA A contained 1.14% Cu, and Mn AA B contained 5.32% Zn. The variation in elemental concentrations is most likely due to the carrier material used in preparation of the Mn source.
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). Total amino acid content of Mn Met B was greatest with 50.6%, and least in Mn AA D with 1.5%, respectively. Methionine content of the four Mn Met complexes were greater than values of other organic Mn sources. This was probably related to the ingredients used as ligands in the manufacture of the Mn products.
Solubility and Chelation Properties
At concentrations of 1 mg of Mn product per milliliter of solvent and physiological temperatures, most organic Mn sources were highly soluble in NAC, CA, and 0.4% HCl with more than 95% solubility (data not shown). The only exception was the solubility (90.3%) of Mn-AA D in 0.4% HCl. Solubility of organic Mn sources in deionized H2O varied from 5.8 to 97.9%, probably related to the insolubility of carrier materials.
At pH 2.0, solubilities of organic Mn sources were more than 95% except for Mn Pro B and Mn AA D, which were only 74.1 and 85.3% soluble, respectively (data not shown). The solubility of Mn sources in pH 5.0 buffer varied from as low as 24.5% to as high as 99.1%.
The capacity of the gel filtration column to separate molecules of different sizes, such as free metal ions and metals, in intact chelates was confirmed. Herein, ionic Mn eluted in fractions 39 to 49 (Figure 1). The Mn-EDTA chelate eluted in fractions 25 to 31, along with a small amount of ionic Mn in fractions 40 to 49. Methionine eluted in fractions 32 to 36. Chromatographs revealed that all Mn from organic products eluted in the same range of fractions as Mn from MnSO4 for filtrates at pH 2.0 and 5.0 (Figures 2 and 3), indicating that none of the materials remained chelated under these pH conditions.
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). One weak peak was at a wavelength of approximately 2,100 cm–1, representing the amino (–NH2) group of amino acids. The strong peak at a wavelength near 3,000 cm–1 corresponds to the carboxyl (-COOH) group of amino acids. The spectra of the four organic Mn products displayed some differences (Figure 4), but the weak characteristic peak (2,100 cm–1) of –NH2 from methionine had disappeared in all sources and only a moderate peak was detected at a wavelength of 3,300 to approximately 3,400 cm–1, corresponding to carboxyl bonds. The latter bonds are consistent with complex or chelate bonds between Mn and amino acids in the organic Mn products.
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). The Mn concentration in a saturated solution also differed considerably from 0.019 M to 0.579 M, but Mn concentration was not directly associated with pH. Chelation strength of the organic Mn products, presented as Qf-values, changed from 1.9 to 115.4. All five Mn methionine products (Mn Met A to E), and Mn AA D and Mn AA E contained weak chelated Mn, with Qf-values that ranged from 1.9 to 4.7, Mn AA C displayed strong chelation, with a Qf-value of 115.4, and Mn Pro A, Mn Pro B, Mn AA A and Mn AA B showed moderate chelation, with Qf-values between 10 and 100.
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). The Mn source, added Mn level, or an interaction between Mn source and level did not affect (P > 0.13) daily feed intake, gain, or feed conversion of chicks during either experimental period (Table 5). Incidence of leg abnormality during the grower phase was not influenced (P > 0.58) by Mn source or the interaction between Mn source and added Mn level. However, the incidence of leg abnormalities decreased (P < 0.07) as added Mn level increased. Differences in the incidence of leg abnormality can be attributed to responses of birds fed the control diet. No differences were detected (P > 0.36) among the various supplemental levels.
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). As added Mn level increased, bone Mn, heart Mn, and MnSOD activity in heart at both 21 and 42 d increased linearly (P < 0.001). Heart MnSOD mRNA of birds at 21 d increased (P < 0.001) linearly as Mn levels increased. The MnSOD mRNA content of heart tended to be affected by Mn source (P < 0.10), with birds fed Mn AA B having the largest linear (P < 0.07) response to dietary Mn. No differences were detected (P > 0.76) among other supplemental Mn sources in heart MnSOD mRNA content. The trends observed in all heart tissue responses of broilers fed the Mn AA B source are consistent with this source being more bioavailable than the other sources.
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). On both d 21 and 42, the coefficient of determination (R2) for fit to a linear model was greatest for bone ash Mn (R2 = 0.89 and 0.63), followed in decreasing sequence by heart Mn concentration (R2 = 0.80 and 0.64) and heart MnSOD activity (R2 = 0.67 and 0.44), indicating that the response criteria on d 42 were less well related to dietary Mn than on d 21. Consequently, the d-21 responses were used to calculate relative bioavailability estimates. The R2 for MnSOD mRNA in heart on d 21 was 0.71 but was not determined on d 42.
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). When the response for MnSO4 was set at 100%, the estimated relative bioavailability values for Mn Met E, Mn AA B, and Mn AA C were 95, 96, and 93% for bone ash Mn concentration; 110, 127, and 114% for heart Mn concentration; and 97, 129, and 112% for heart MnSOD activity, respectively. There were no differences (P > 0.14) among slopes for respective sources, but Mn AA B and Mn AA C, and especially Mn AA B tended to be more available than MnSO4. When relative bioavailability was estimated based on heart MnSOD mRNA, the values for Mn Met E, Mn AA B, and Mn AA C were 99, 133, and 113%, respectively. The slope for Mn AA B was greater (P < 0.05) than that for other Mn sources, but there were no differences among other slopes. Based on the slopes of responses to Mn level, heart MnSOD mRNA responses were more sensitive than MnSOD activity or Mn concentration in heart (Table 7). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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, a complex or chelate must contain at least a 1:1 molar ratio of ligand and metal. If the average molecular weight of an amino acid is 140 and considered to be available for chelation, the sources Mn Met B, Mn Met C, Mn AA A, Mn AA B, and Mn AA C provided a molar ratio of amino acid to metal greater than 1:1 (Table 2) and, thus, probably contained a large amount of chelated or complexed Mn. The other sources contained a ratio of amino acid to metal of less than 1:1 and probably contained only partial chelated or complexed Mn. However, the results from mineral and amino acid analysis should be interpreted with caution, because a product consisting of a simple mixture of amino acid with a metal salt would contain the same ratio of amino acid to metal as a complex or chelate. A simple analysis of the mineral and amino acid composition does not provide an indication of the amount of product that is actually chelated or an indication of the chelation bond strength.
The chelation strength of an organic mineral source and its behavior under physiological conditions is critical in determining the value of products used as supplements in animal nutrition. Applying the classification proposed by Holwerda et al. (1995) to the results of the present experiments suggests that the chelation strength of seven organic Mn products (Mn Met A to Mn Met E and Mn AA D and E) with Qf-values lower than 10 were weak; four organic Mn sources (Mn Pro A, Mn Pro B, Mn AA A, and Mn AA B) with their chelation quotients between 10 and 100 were moderate; and Mn AA C with a Qf-value greater than 100, was strong.
Solubility tests in NAC, CA, HCl, and deionized H2O have been used to assess the bioavailability of inorganic Mn sources. Several researchers (Watson et al., 1971; Black et al., 1984) reported that the solubility of inorganic Mn products in NAC was closely associated with their bioavailability values for chicks and sheep. Henry et al. (1987), however, found no relationship between solubility of Mn monoxide or dioxide sources and their bioavailability values. For organic mineral products, solubility is partly a function of the ligand; thus, the solubility results may be misleading (Johnson and Socha, 1998). In the present experiment, no relationship between solubility and bioavailability were observed.
Brown and Zeringue (1994), Cao et al. (2000), and Guo et al. (2001) determined the solubility of organic metal sources at concentrations ranging from 0.125 to 12.5 mg of product/mL in buffers at pH 2.0 and 5.0. However, in the present experiment, the solubility was determined only at a concentration of 2 mg of product/mL buffer. This lower concentration was chosen to mimic the concentration in feed. Free Mn in solution was not measured because Mn-selective electrodes are not available.
Although the results from infrared spectroscopy and polarography implied that complexes or chelates were present in the organic Mn products, no peaks for complexes or chelates were found in any of the organic Mn filtrates from buffers at pH 2.0 or 5.0. The observation that none of the material remained chelated in buffers at pH 2.0 and pH 5.0 did not prove that the organic mineral products completely dissociated in animal’s gastrointestinal tract. Many other components in chyme may play important roles in mineral digestion and absorption. Whether the organic metal source is absorbed intact and then used by tissues in vivo remains to be determined.
For assessment of bioavailability, tissue accumulation of trace minerals has been considered to be a sensitive criterion. Bone Mn has been used as the criterion for bioavailability assays of Mn sources, but the estimated bioavailability value differs among experiments. Bioavailability values decreased when the level of dietary supplemental Mn increased in experiments conducted with similar designs (Black et al., 1984; Henry et al., 1986). Black et al. (1984) reported that bone Mn increased linearly as added dietary Mn concentration increased. Based on multiple linear regression of bone Mn, the bioavailability value of Mn from MnO was 60% relative to Mn sulfate monohydrate when chicks were fed corn/soybean meal diets (containing 116 mg Mn/kg) supplemented with 1,000, 2,000, or 4,000 mg Mn/kg. In a similar study, Henry et al. (1986) reported that the bioavailability value from multiple linear regression of bone was 81% for MnO for chicks fed corn/soybean meal diets (containing 35 mg Mn/kg) supplemented with 40, 80, or 120 mg Mn/kg. Although there was no difference among experiments between the average bioavailability values of Mn from MnO based on criteria for liver or kidney tissue concentrations, the bone Mn bioavailability value changed by 20%. Luo (1994) compared the effects of dietary Mn levels on estimates of bioavailability values in a single experiment. Bioavailability values of MnO and MnO2 estimated from diets with added Mn at 20 to 120 mg/kg were 20 and 40% higher than estimates derived from diets with added Mn at 1,000 to 4,000 mg/kg. In the current experiment, dietary added Mn levels ranged from 60 to 180 mg/kg because these levels reflect concentrations commonly found in broiler diets.
Some bioavailability values of organic Mn sources have been determined based on bone Mn accumulation in experiments with high supplemental dietary Mn. Henry et al. (1989) reported that the estimated bioavailability based on bone Mn accumulation was 108 for Mn Met relative to MnSO4 when chicks were fed corn/soybean meal diets (93 mg/kg) supplemented with 700 to 2,100 mg/kg Mn, indicating that Mn from Mn Met was significantly more available than that from MnSO4. However, in the present experiment and in earlier work reported by Baker and Halpin (1987) and Scheideler (1991), no differences were detected among bioavailability values of organic Mn sources based on bone Mn accumulation relative to Mn sulfate or Mn oxide.
Heart Mn was used for the bioavailability assay with lambs because it best fit a linear model when dietary Mn was added at 500 to 4,000 mg/kg (Black et al., 1985). In the present studies, heart Mn responded to dietary Mn and was similar to bone Mn responses for chicks, but the bioavailability estimates for both measures failed to distinguish differences between supplemental Mn sources.
The Mn-containing enzyme, MnSOD, functions as a free radical scavenger. The MnSOD activity was a sensitive criterion for assessing Mn status and requirements for mice (De Rosa et al., 1980), rats (Paynter, 1980), and chicks (Luo, 1994). However, the estimated bioavailability values based on MnSOD herein showed no differences among various Mn sources, indicating that MnSOD activity in heart lacked the sensitivity required to detect differences among Mn sources.
The MnSOD gene in E. coli is regulated transcriptionally (Touati, 1988) and posttranslationally (Privalle and Fridovich, 1992) in a metal-dependent fashion. The mode of regulation in MnSOD gene expression in eukaryon is not clear even though many investigations have focused on MnSOD gene structure and regulator sequences (Duttary et al., 1997). Mice that are Mn-deficient have a low Mn concentration, low MnSOD activity, and low MnSOD mRNA in liver (Borrello et al., 1992). The lower MnSOD mRNA probably resulted from the down-regulation at the (pre)-transcriptional level. No information on the poultry MnSOD gene has been reported. The MnSOD cDNA of chicks was cloned, and the sequence was analyzed in our laboratory (Bu et al., 2001). In the current experiment, MnSOD mRNA levels in heart increased linearly as dietary Mn levels increased, suggesting that dietary Mn significantly affected heart MnSOD gene transcription. Heart concentration of MnSOD mRNA was a more sensitive criterion than MnSOD activity in heart or other indices for estimation of bioavailability of organic Mn sources. The difference between MnSOD activity and MnSOD mRNA in distinguishing organic Mn sources could be explained by observations reported by Warner et al. (1991). These authors showed that MnSOD activity and mRNA in human pulmonary adenocarcinoma cells increased significantly in a dose- and time-dependent manner. The MnSOD activity was increased 3-fold and mRNA 20-fold after a 48-h incubation with TNF
at a level of 25 ng/mL. The greater-fold increase in mRNA compared with activity, along with the observations in the current experiment, are consistent with MnSOD mRNA being a sensitive criterion for assessment of Mn bioavailability.
Any chemical differences found among sources by various laboratory procedures are meaningless unless concurrent, measurable improvements of bioavailability can also be detected. The chelation strength of an organic mineral source and its behavior under physiological conditions are perceived as key points in determining the value of products used as supplements in animal nutrition. In the present studies, three organic Mn sources had similar solubility in buffers at pH 2 or 5, but provided weak, moderate, and strong complex strength formation quotients based on polarography. The regression of bioavailability on chemical characteristics was not calculated, but the results are consistent with an effect of complex strengths of these organic Mn sources on their relative bioavailability values for broilers. The Mn AA B source with a moderate complex strength was the most available, and the Mn AA C source with the strong complex strength had a tendency to be more available than the Mn Met E source with the weak complex strength numerically, whereas the Mn Met E source was similar to Mn sulfate. The current results show that use of chemical characteristics as an indicator of bioavailability should be approached with caution. Further experiments are required to verify use of complex strength as an indicator of bioavailability with a wider range of organic Mn sources.
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Footnotes |
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2 Current address: Hebei Vocation-Technical Teachers College, Changli 066600, P. R. China.
4 Current address: Dept. Anim. Sci., University of Wisconsin, Madison 53706-1284.
3 Correspondence: Nutrition Div., Inst. Anim. Sci., Chinese Acad. of Agric. Sci., No. 2 Yuanmingyuan West Road, Haidian (phone: 0086-10-62816012; fax: 0086-10-62810184; e-mail: wlysz{at}263.net or wlysz{at}public.bta.net.cn).
Received for publication October 16, 2003. Accepted for publication May 3, 2004.
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increase MnSOD expression: Protection against oxidant injury. J. Physiol. 260:L296–L301.
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