Effect of Oxidation of Dietary Proteins with Performic Acid on True Ileal

Jan 8, 2014 - ABSTRACT: The study examined the impact of oxidation with performic acid on the true ileal amino acid digestibility. (TIAAD) of seven di...
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Effect of Oxidation of Dietary Proteins with Performic Acid on True Ileal Amino Acid Digestibility As Determined in the Growing Rat Shane M. Rutherfurd,* Carlos A. Montoya, and Paul J. Moughan Riddet Institute, Massey University, Palmerston North, New Zealand ABSTRACT: The study examined the impact of oxidation with performic acid on the true ileal amino acid digestibility (TIAAD) of seven dietary protein sources. TIAAD of both the unoxidized and oxidized protein sources was determined in the growing rat and compared. After oxidation, TIAAD was 30 and 58% higher across amino acids (P < 0.001) for the more poorly digestible protein sources, zein and blood meal respectively, 6−16% lower (P < 0.05) for the more highly digestible protein sources (casein, soy protein isolate, and beef muscle protein), and generally unchanged ( 0.05; *, 0.05 > P > 0.01. dDifference was calculated according to eq 3. eMean true ileal amino acid digestibility across all examined amino acids.

reported to be small ( 0.05; * 0.05 > P > 0.01. dDifference was calculated according to eq 3. e Mean true ileal amino acid digestibility across all examined amino acids.

Table 4. Mean (n = 8) True Ileal Amino Acida Digestibility (TIAAD) of Unoxidized and Performic Acid-Oxidized Blood Meal TIAAD (%) unoxidized

oxidized

overall SEb

significancec

differenced (%)

aspartic acid threonine serine glutamic acid alanine valine isoleucine leucine phenylalanine histidine lysine arginine

54.4 63.7 64.2 58.8 65.2 64.8 76.6 65.3 69.1 70.0 66.2 66.5

78.0 81.3 81.0 79.0 84.2 88.5 85.9 89.3 88.5 87.9 84.7 83.8

4.81 4.08 4.16 4.22 2.87 3.12 2.80 2.72 2.81 2.79 2.91 2.83

** * * ** ** *** * *** *** *** *** ***

43 28 26 34 29 37 12 37 28 26 28 26

mean digestibilitye

65.4

84.4

a

Methionine, cysteine, tyrosine, and tryptophan were not included in the analysis because they were destroyed during the oxidation process. bOverall standard error of the unoxidized and oxidized blood meal. c*, 0.05 > P > 0.01; **, 0.01 > P > 0.001; ***, P < 0.001. dDifference was calculated according to eq 3. eMean true ileal amino acid digestibility across all examined amino acids.

amount of the determined amino acids present in the protein sources after performic acid treatment was due to the oxidation of those amino acids. Oxidation appeared to be incomplete for methionine in beef muscle protein (2.1% of the original methionine was present after oxidation) and for tyrosine in beef muscle protein, blood meal, lactalbumin, and zein (1.2−2.4% of the original tyrosine was present after oxidation). The recovery of histidine after performic acid oxidation was also low for beef muscle protein, gelatin, blood meal, lactalbumin, and zein with recoveries of 27, 65, 59, 31, and 13%, respectively. The recoveries of all of the remaining determined amino acids were virtually complete, suggesting that no modification of the latter amino acids occurred during performic acid oxidation, although it is possible that oxidized amino acids were present but that any altered amino acids reverted back to their parent form during HCl hydrolysis. True ileal amino acid digestibility was determined for both the unoxidized and oxidized protein sources, and these data are presented in Tables 2−8. Digestibility values for glycine were

omitted because the enzyme-hydrolyzed protein (casein)/ ultrafiltration method used to determine endogenous ileal amino acids flows, which are in turn used to calculate true ileal amino acid digestibility, underestimates endogenous ileal flows for glycine because a significant amount of the endogenous glycine in the gastrointestinal tract is conjugated to bile salts, the latter of which are excluded from the endogenous protein fraction during ultrafiltration of the digesta.16 For almost all of the amino acids examined in lactalbumin (Table 2) and gelatin (Table 3), histidine being the exception for gelatin and aspartic acid being the exception for lactalbumin, there was no difference (P > 0.05) between true ileal amino acid digestibility of the protein sources prior to and after treatment with performic acid. However, for the oxidized lactalbumin, the true ileal amino acid digestibility values obtained for one replicate were quite different from the others within the treatment group. The digestibility values for the latter replicate, which were not outliers based on the Grubb test and were therefore not excluded from the statistical analysis, may explain why large 702

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Table 5. Mean (n = 8) True Ileal Amino Acida Digestibility (TIAAD) of Unoxidized and Performic Acid-Oxidized Zein TIAAD (%) unoxidized

oxidized

overall SEb

significancec

differenced (%)

aspartic acid threonine serine glutamic acid alanine valine isoleucine leucine phenylalanine histidine arginine

47.7 42.6 45.4 49.6 86.9 54.5 47.8 53.8 53.0 45.1 57.9

83.9 81.3 85.5 90.8 93.1 91.5 90.3 94.4 92.1 64.8 88.2

3.18 3.88 3.13 2.68 3.07 2.77 3.03 2.45 2.54 7.27 3.55

*** *** *** *** NS *** *** *** *** NS ***

76 91 88 83

mean digestibilitye

53.1

86.9

68 89 75 74 53

a

Methionine, cysteine, tyrosine, and tryptophan were not included in the analysis because they were destroyed during the oxidation process. Lysine was not determined because the lysine content of zein is negligible. bOverall standard error of the unoxidized and oxidized zein. cNS, not significant, P > 0.05; ***, P < 0.001. dDifference was calculated according to eq 3. eMean true ileal amino acid digestibility across all examined amino acids.

Table 6. Mean (n = 8) True Ileal Amino Acida Digestibility (TIAAD) of Unoxidized and Performic Acid-Oxidized Beef Muscle Protein TIAAD (%) unoxidized

oxidized

overall SEb

significancec

differenced (%)

aspartic acid threonine serine glutamic acid alanine valine isoleucine leucine phenylalanine histidine lysine arginine

86.0 88.3 91.4 91.2 94.2 93.2 94.6 94.6 94.4 95.0 94.5 94.2

60.4 55.1 70.4 79.8 85.5 81.5 83.7 86.7 87.0 65.2 89.9 90.4

2.52 2.88 2.18 1.52 0.89 1.33 1.10 1.00 0.98 2.18 0.70 0.77

*** *** *** *** *** *** *** *** *** *** *** ***

−30 −38 −23 −12 −9 −13 −12 −8 −8 −31 −5 −4

mean digestibilitye

92.6

78.0

a

Methionine, cysteine, tyrosine, and tryptophan were not included in the analysis because they were destroyed during the oxidation process. bOverall standard error of the unoxidized and oxidized beef muscle protein. c***, P < 0.001. dDifference was calculated according to eq 3. eMean true ileal amino acid digestibility across all examined amino acids.

numerical differences observed between the true ileal amino acid digestibility for the unoxidized and oxidized lactalbumin were not statistically significant. For blood meal (Table 4) and zein (Table 5), true ileal amino acid digestibility was higher (P > 0.05) after treatment with performic acid for all of the amino acids for blood meal and for all of the amino acids except alanine and histidine for zein. For beef muscle protein (Table 6), SPI (Table 7), and casein (Table 8), true ileal amino acid digestibility was lower (P > 0.001) after treatment with performic acid for all of the amino acids examined, with the exception of serine for casein. Overall, treatment with performic acid led to an increase in the mean true ileal amino acid digestibility across amino acids of 63% for zein (Table 5) and 29% for blood meal (Table 4), a decrease in the mean true ileal amino acid digestibility across amino acids of 16% for beef muscle protein (Table 6), 15% for SPI (Table 7), and 6% for casein (Table 8) and had little effect on the mean true ileal amino acid digestibility of lactalbumin (Table 2) and gelatin (Table 3).

A correlation analysis was also conducted comparing true ileal amino acid digestibility prior to performic acid treatment and the percentage change in true ileal amino acid digestibility after performic acid treatment across the seven protein sources for each of the amino acids individually and across all amino acids collectively. A negative correlation (P < 0.01, r = −0.90 to −0.98) was observed for each of the amino acids individually and also for the amino acids collectively. True ileal amino acid digestibility prior to performic acid treatment and the percentage change in true ileal amino acid digestibility after performic acid treatment were plotted for all of the amino acids collectively, and a quadratic model fitted the data well (P < 0.001, adjusted R2 = 0.90; Figure 1).



DISCUSSION Performic acid is a powerful oxidizing agent and was chosen in the present study to provide conditions that would lead to extensive, but defined, modifications of selected amino acids, namely, cysteine, methionine, tyrosine, and tryptophan.24 In the 703

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Table 7. Mean (n = 8) True Ileal Amino Acida Digestibility (TIAAD) of Unoxidized and Performic Acid-Oxidized Soy Protein Isolate TIAAD (%) unoxidized

oxidized

overall SEb

significancec

differenced (%)

aspartic acid threonine serine glutamic acid alanine valine isoleucine leucine phenylalanine histidine lysine arginine

88.2 85.0 92.7 94.4 91.4 90.8 92.7 91.1 94.3 96.0 99.1 96.9

78.5 54.6 69.5 79.8 77.8 79.0 84.6 86.8 88.8 68.4 85.3 88.6

0.86 1.94 1.17 0.69 1.97 1.06 0.77 0.97 0.60 2.31 1.17 0.57

*** *** *** *** *** *** *** *** *** *** *** ***

−11 −36 −25 −15 −15 −13 −9 −5 −6 −29 −14 −9

mean digestibilitye

92.7

78.5

a

Methionine, cysteine, tyrosine, and tryptophan were not included in the analysis because they were destroyed during the oxidation process. bOverall standard error of the unoxidized and oxidized soy protein isolate. c***, P < 0.001. dDifference was calculated according to eq 3. eMean true ileal amino acid digestibility across all examined amino acids.

Table 8. Mean (n = 8) True Ileal Amino Acida Digestibility (TIAAD) of Unoxidized and Performic Acid-Oxidized Casein TIAAD (%) unoxidized

oxidized

overall SEb

significancec

differenced (%)

aspartic acid threonine serine glutamic acid alanine valine isoleucine leucine phenylalanine histidine lysine arginine

90.2 88.2 77.3 89.7 92.1 91.6 87.5 96.5 98.4 99.3 96.5 95.2

78.6 80.4 75.7 85.3 81.3 87.8 83.2 91.8 94.0 95.9 92.5 91.8

1.22 1.30 1.60 0.95 0.94 0.74 1.05 0.48 0.40 0.51 0.42 0.72

*** *** NS *** *** *** *** *** *** *** *** ***

−13 −9

mean digestibilitye

91.9

86.5

−5 −12 −4 −5 −5 −4 −3 −4 −4

a

Methionine, cysteine, tyrosine, and tryptophan were not included in the analysis because they were destroyed during the oxidation process. bOverall standard error of the unoxidized and oxidized casein. cNS, not significant, P > 0.05; ***, P < 0.001. dDifference was calculated according to eq 3. e Mean true ileal amino acid digestibility across all examined amino acids.

presence of performic acid, cysteine is oxidized to cysteic aci 25 and any disulfide bonds present will be inevitably broken, which will in turn cause the affected proteins to unfold.26 Methionine is quantitatively oxidized to methionine sulfone in the presence of performic acid,25,27 tyrosine undergoes halogenation, and the indole ring of tryptophan is destroyed.28 In the present study, the oxidation of methionine, tyrosine, and tryptophan was evaluated on the basis of the disappearance of the original amino acids after performic acid treatment rather than quantitation of the modified amino acids themselves. The negligible recoveries of methionine, tyrosine, and tryptophan after performic acid treatment, as determined using conventional amino acid analysis, suggest that the latter amino acids were almost completely modified. In addition, the poor recoveries of histidine suggest that this amino acid also was modified, at least to some degree. The concentrations of cysteine in the unoxidized and oxidized protein sources were not determined because cysteine is commonly determined

following performic acid oxidation of the protein, and any unoxidized cysteine oxidized to cysteic acid as part of the amino acid analysis procedure would not be distinguishable from cysteic acid present due to the original performic acid treatment used to chemically modify the dietary protein sources. However, given that cysteine is particularly sensitive to oxidation, it is likely that performic acid treatment would have resulted in the complete oxidation of cysteine to cysteic acid in the oxidized protein sources.29 The protein sources used in the present study were chosen to represent both poorly and highly digestible protein sources. Zein and blood meal were poorly digestible (mean digestibility across amino acids was 53 and 65% for zein and blood meal, respectively), lactalbumin and gelatin were digested to an intermediate degree (mean digestibility across amino acids was 85 and 88% for lactalbumin and gelatin, respectively), and casein, beef muscle protein, and SPI were the most digestible proteins (mean digestibility across amino acids ranged from 92 704

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Figure 1. Relationship between the true ileal amino acid digestibility (TIAAD) of the seven protein sources prior to oxidation and the percentage change in TIAAD after oxidation. Methionine, cysteine, tyrosine, tryptophan, and histidine were not included in the analysis because they were destroyed (or partially destroyed in the case of histidine) during the oxidation process. Percentage change (%) was calculated according to eq 3. The differences within individual amino acids for each protein source are presented in Tables 2−8.

to 93% for the latter three protein sources). Interestingly, for the poorly digested protein sources, performic acid treatment led to a quantitatively large increase in the overall true ileal amino acid digestibility, whereas for the moderately digested proteins performic acid treatment had little impact on true ileal amino acid digestibility and for the highly digested protein sources true ileal amino acid digestibility generally decreased after performic acid treatment. The rate and extent to which dietary proteins are digested in the gastrointestinal tract are dependent on many factors including the structure of the dietary protein, the presence of antinutritional factors, digesta viscosity, and enzyme to substrate ratio within the gastrointestinal tract. In the context of the present study protein structure is likely to have had the greatest impact on the extent of protein digestion. Proteins possess primary (the amino acid sequence), secondary (α-helix and β-sheet), and tertiary (the three-dimensional folding of the protein chain) structures, all of which can affect the efficacy of proteases in the digestive tract. Given the range of cleavage site specificities of the proteases in the gastrointestinal tract, it is expected that primary structure would have only a relatively minor influence on the overall extent of protein digestion in the small intestine. In contrast, the presence of secondary and tertiary structures in dietary proteins may be of greater significance for digestion. Deshpande and Damodaran30 demonstrated that by changing the tertiary structure, but not the secondary structure, using heat treatment, the in vitro digestion of phaseolin was markedly increased. Samadi et al.31 reported a strong correlation between the ratio of α-helix to βsheet structures in heated canola meal proteins and the digestion of protein in the bovine rumen and intestine. Moreover, Carbonaro et al.32 reported a strong inverse correlation between in vitro digestibility and the relative amount of β-sheets across a range of unprocessed and processed protein-containing foods. The latter workers suggested that the β-sheet structure present in the unprocessed foods and the intermolecular β-sheet aggregates induced by

processing negatively affected digestibility and indeed were major factors in this regard. In the present study, the performic acid oxidation would be expected to denature the proteins, leading to an increase in the digestibility of the protein source overall. However, for highly digestible proteins such as those present in casein and SPI, any increase in digestibility due to denaturation will be small. While for poorly digested proteins, the increase in digestibility will be much greater. In the present study, performic acid oxidation led to a marked increase in the true ileal amino acid digestibility of zein and blood meal, both of which were poorly digested prior to oxidation. The proteolytic endopeptidases such as pepsin, trypsin, and chymotrypsin do not cleave proteins at random but rather cleave peptides adjacent to specific amino acids. For example, pepsin has a preference for large uncharged residues, whereas trypsin is specific for arginine and lysine and chymotrypsin has a preference for hydrophobic, preferably aromatic, residues.33 When amino acids within a protein source are chemically modified, proteases present in the gastrointestinal tract are often less effective, leading to the presence of relatively large indigestible peptides (limit peptides) at the terminal ileum, which would in turn lead to lower true ileal amino acid digestibility.34 Although denaturation of the dietary proteins may explain the increase in true ileal amino acid digestibility for zein and blood meal, it does not explain the decrease in the true ileal amino acid digestibility of casein, SPI, and beef muscle protein. Conversely, although the formation of limit peptides may explain the decrease in the true ileal amino acid digestibility of casein, SPI, and beef muscle protein, it does not explain the increase in true ileal amino acid digestibility for zein and blood meal. Consequently, it is hypothesized that the change in true ileal amino acid digestibility of the dietary protein sources after performic acid oxidation observed in the present study was due to the relative contribution of the two opposing processes: (1) increased digestion due to the denaturation of the proteins and (2) decreased digestibility due to the formation of indigestible 705

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Notes

limit peptides. It is hypothesized that denaturation was the predominant factor for zein and blood meal and that the presence of limit peptides was the predominant factor for casein, SPI, and beef muscle protein. For lactalbumin and gelatin, it is conjectured that the relative contributions of the two opposing factors were similar. Although the analysis of digesta material for the presence of limit peptides can be carried out using LC-MS, the latter analysis is not trivial and therefore was not done as part of this preliminary study. However, more work in this area is warranted. It is also possible that oxidation of the dietary protein sources affected the endogenous protein secretion into the gastrointestinal tract, which could in turn affect the determination of true ileal amino acid digestibility given that endogenous ileal amino acid flows were used to correct apparent ileal amino acid digestibility values to true values. However, in the present study, modulation of the endogenous ileal amino acid flows due to alimentation with oxidized versus unoxidized protein sources did not appear to be a factor. The evidence for this is given below. Zein contains negligible amounts of lysine and tryptophan, and as such the total ileal flows of the latter two amino acids for the rats given the unoxidized and oxidized zeinbased diets were equivalent to endogenous ileal flows. The endogenous ileal lysine and tryptophan flows were not different (P > 0.05) between the unoxidized and oxidized zein (313 ± 30.1 and 327 ± 35.1 mg/kg DMI for lysine in the unoxidized and oxidized zein, respectively, and 171 ± 16.7 and 203 ± 13.4 mg/kg DMI for tryptophan in the unoxidized and oxidized zein, respectively). Similarly for gelatin, which is almost completely devoid of tryptophan, the endogenous ileal tryptophan flows were not different between the unoxidized (321 ± 15.6 mg/kg DMI) and oxidized (299 ± 19.7 mg/kg DMI) gelatins. The present study represents a preliminary investigation into the impact of oxidation of proteins on protein digestion using an in vivo (rat) model. Only the net effects of oxidation on true ileal amino acid digestibility were examined, and as a result the mechanisms by which performic acid oxidation altered protein digestion can only be surmised. Nevertheless, the results suggest that protein oxidation affected protein digestion in the gastrointestinal tract, possibly by more than one mechanism, most likely (1) denaturation of the protein and (2) formation of limit peptides, and that the overall effect of oxidation on digestibility as a whole is a result of the balance of these individual effects. The study provides insights as to how oxidation affects the digestion of proteins in the gastrointestinal tract, and further studies elucidating the structural changes that take place in proteins in relation to true ileal amino acid digestibility and using oxidation conditions relevant to those used for the processing of food proteins are warranted.



The authors declare no competing financial interest.



ACKNOWLEDGMENTS We acknowledge Maggie L. Zou, Russell K. Richardson, and Chanapha Sawatdeenaruenat for their expertise in conducting the chemical analyses.



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AUTHOR INFORMATION

Corresponding Author

*(S.M.R.) Mailing address: Riddet Institute, Massey University, Private Bag 11222, Palmerston North, New Zealand. Phone: +64 6 3505894. Fax: +64 6 3505655. E-mail: s.m.rutherfurd@ massey.ac.nz. Funding

The work was supported by a Centre of Research Excellence fund from the Tertiary Education Commission and the Ministry of Education, New Zealand. 706

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