Nantawat Tatiyaborworntham

Mass spectrometric characterization and redox instability of turkey and chicken myoglobins as induced by unsaturated aldehydes.

Turkey and chicken myoglobins (Mbs) were isolated, purified, and characterized using electrospray ionization mass spectrometry (ESI-MS), and the effect of unsaturated aldehydes (nonenal and hexenal) on their redox stability was investigated in vitro. The deconvoluted spectra from ESI-MS exhibited a molecular mass of 17291 Da for both turkey and chicken Mbs. Significant homogeneity in the fragmentation pattern of both Mbs was indicated by ESI-MS/MS. Both turkey and chicken oxymyoglobins (OxyMbs) were more prone to oxidation at pH 5.8 than at pH 7.4. Metmyoglobin formation was greater in the presence of unsaturated aldehydes than controls (P < 0.05). The results demonstrated that both turkey and chicken Mbs have identical molecular mass and that the effects of alpha,beta-unsaturated aldehydes on their redox stability are consistent with those of mammalian livestock Mbs.

Myoglobin and haemoglobin-mediated lipid oxidation in washed muscle: observations on crosslinking, ferryl formation, porphyrin degradation, and haemin loss rate.

Reduced trout haemoglobin (Hb) is a mixture of oxy- and deoxy-Hb at pH 6.3. Addition of oxy/deoxyHb to washed muscle resulted in detectable ferryl Hb while adding bovine oxyHb, trout metHb, or bovine metHb did not. Trout metHb promoted lipid oxidation more rapidly than bovine metHb, attributable to lower haemin affinity in fish Hbs. Protoporphyrin IX degradation was prevalent during trout and bovine Hb-mediated lipid oxidation. Caffeic acid prevented porphyrin degradation and lipid oxidation. Crosslinked myoglobin (Mb) promoted lipid oxidation more effectively than metMb. Fish metMb released haemin more readily than mammalian metMb at pH 5.5. These studies suggest haemin dissociation from metHb causes formation of free radicals that degrade protoporphyrin and cause lipid oxidation, and appreciable quantities of deoxyHb are needed to generate ferryl Hb oxidant. Crosslinking appears to facilitate Mb-mediated lipid oxidation in washed muscle yet haemin release can occur from fish metMb at low pH.

Redox instability and hemin loss of mutant sperm whale myoglobins induced by 4-hydroxynonenal in vitro.

The effects of 4-hydroxy-2-nonenal (HNE) on redox stability of Oxy- and Deoxy- wild-type (WT) and recombinant sperm whale myoglobins (P88H/Q152H, L29F, H97A, and H64F) and hemin loss from Met-myoglobin (Mb) were investigated. HNE induced greater redox instability in WT and mutant Mbs compared to controls (p < 0.05). The extent of HNE-induced OxyMb oxidation was lesser in L29F (p < 0.05) and greater in H97A and P88H/Q152H than in WT (p < 0.05). H64F DeoxyMb was more redox stable than WT DeoxyMb in the presence of HNE (p < 0.05). HNE alkylation occurred exclusively on histidine residues, and histidine 48 was alkylated in all sperm whale myoglobins. HNE alkylation accelerated the protoporphyrin moiety loss only in H97A. Met- forms of WT and L29F but not Deoxy- or Oxy- forms released hemin during storage. Primary structure strongly influenced Mb redox stability in the presence of reactive secondary lipid oxidation products.

Effect of 4-hydroxy-2-nonenal on myoglobin-mediated lipid oxidation when varying histidine content and hemin affinity

The compound 4-hydroxy-2-nonenal (HNE) dissolved in water was examined to remove potential effects of using ethanol to solubilize the aldehyde such as altering protein structure or redox properties of myoglobin (Mb). HNE became covalently bound to sperm whale Mb at up to five sites based on ESI-MS analysis. Adducted Mb promoted lipid oxidation in washed muscle more effectively than non-adducted Mb. Alkylation of P88H/Q152HMb with HNE accelerated metMb formation and subsequent lipid oxidation. P88H/Q152HMb exposed to HNE enhanced lipid oxidation compared to wild-type Mb exposed to HNE. Results using H97A Mb suggested that the combination of HNE and low hemin affinity facilitated rapid decomposition of preformed lipid hydroperoxides to secondary lipid oxidation products. HNE and HHE (4-hydroxy-2-hexenal) facilitated Mb-mediated lipid oxidation similarly. The potential mechanisms by which Mb binding of α,β-unsaturated aldehydes affect Mb oxidation and the onset of lipid oxidation are discussed.

Assessing Low Redox Stability of Myoglobin Relative to Rapid Hemin Loss from Hemoglobin

Bovine myoglobin (Mb) auto-oxidized 11-fold faster at pH 5.7 compared to bovine hemoglobin (Hb). Replacement of Ser(F7) in bovine Mb with positively charged or large apolar residues decreased auto-oxidation rates (2- to 4-fold) in comparison with wild-type Mb (P < 0.05). However, the same substitutions increased hemin loss rate (15- to 28-fold), indicating that hydrogen bonding between Ser(F7) and the heme-7-propionate is critical for stabilizing protoporphyrin in the globin. The anchoring of Ser(F7) to the heme-7-propionate in the proximal pocket of Mb is suggested to expose the distal pocket to solvent molecules that accelerate auto-oxidation. The rate of hemin loss from metHb at pH 5.7 was 68-fold faster compared to metMb. The ability of Ser(F7) and His(FG3) in Mb to form stabilizing contacts with the heme-7-propionate maintains hemin within the globin whereas Leu(F7) and Leu(FG3) of Hb cannot form stabilizing contacts which results in low hemin affinity. MetHb promoted lipid oxidation more effectively in washed muscle at pH 5.7 compared to metMb (P < 0.05). The greater ability of bovine metHb to promote lipid oxidation is likely due to its enhanced rate of hemin dissociation compared to bovine metMb.

Role of Cytochrome P450 Hydroxylase in the Decreased Accumulation of Vitamin E in Muscle from Turkeys Compared to that from Chickens

Turkeys and chickens reared to 5 weeks of age and fed diets with feedstuffs low in endogenous tocopherols were examined. Treatments included feed supplemented with RRR (natural source vitamin E) alpha tocopheryl acetate (AcT, 35 mg/kg feed) and all-racemic (synthetic vitamin E) AcT (10 and 58 mg/kg feed). Alpha tocopherol hydroxylase activity was greater in liver microsomes prepared from turkeys compared to that from chickens (p < 0.01). Alpha and gamma tocopherol metabolites were higher in turkey bile than in chicken when assessing the RRR AcT diet and the all-racemic AcT diet at 58 mg/kg feed (p < 0.01). Turkey cytochrome P450 2C29 was increased relative to its chicken ortholog on the basis of RNA-Seq transcript abundance (p < 0.001) and activity-based protein profiling (p < 0.01) of liver tissue. Alpha tocopherol concentrations in plasma, liver, and muscle from turkey were lower than the respective tissues from chicken (p < 0.05). Lipid oxidation was greater in turkey thigh than in chicken (p < 0.05). These results suggest that elevated tocopherol metabolism by cytochrome P450 hydroxylase(s) in turkeys contributes to the decreased accumulation of alpha tocopherol in turkey tissues compared to that of chickens.

The effects of HNE on ovine oxymyoglobin redox stability in a microsome model

The effect of 4-hydroxy-2-nonenal (HNE), a secondary lipid oxidation product, on ovine myoglobin (Mb) redox stability was investigated. HNE increased oxymyoglobin (OxyMb) oxidation under all pH/temperature conditions studied. Mono-, di- and tri-HNE adducts were detected by ESI-Q-TOF MS analysis. Sites of adduction, His 120, His 25 and His 65, were determined by ESI-CID-MS/MS analysis. The relationship between ovine Mb (with/without HNE) and lipid oxidation was also studied in a microsome model in the presence of α-tocopherol. Surprisingly, preincubation of Mb with HNE did not affect subsequent Mb redox stability in the microsome model (P<0.05). Microsomes with elevated concentrations of α-tocopherol delayed lipid and Mb oxidations relative to controls. HNE-treated ovine Mb caused greater lipid oxidation compared to control ovine Mb in control microsomes (P<0.05). This study demonstrated an interaction between ovine Mb oxidation and lipid oxidation.

Hemolysis, tocopherol, and lipid oxidation in erythrocytes and muscle tissue in chickens, ducks, and turkeys

&NA; Muscle from turkeys is more sensitive to lipid oxidation during post mortem storage compared with that of chicken and duck which may involve increased lysis of turkey erythrocytes that releases hemoglobin oxidant. Three separate experiments were conducted to study characteristics of chicken, duck, and turkey erythrocytes in which dietary tocopherols were standardized. In Experiment I, tocopherol, fatty acid composition, and lipid oxidation capacity were measured in erythrocytes from chickens, ducks, and turkeys. Tocopherol content was greater in chicken erythrocytes compared with that of duck and turkey (P < 0.05). Oleic and linoleic acid content was higher in chicken erythrocytes compared with that of turkey (P < 0.05). Lipid oxidation capacity of erythrocytes in washed turkey muscle (WTM) at pH 5.8 ranked chicken > duck > turkey (P < 0.05). In Experiment II, hemolysis was measured in erythrocytes from turkeys and chickens. Detergent‐induced hemolysis (pH 7.4) was on average 12‐fold greater for turkey erythrocytes compared with that of chicken (P < 0.05). In Experiment III, the ability of lysed and non‐lysed erythrocytes to promote lipid oxidation was examined. Lysed erythrocytes promoted lipid oxidation in WTM more effectively than intact erythrocytes (P < 0.05). Reasons that turkey erythrocytes were more labile to detergent‐induced hemolysis whereas chicken erythrocytes more effectively promoted lipid oxidation in the WTM model system are discussed. These studies describe variation in chemical and physical properties of erythrocytes from chickens, ducks, and turkeys that can influence progression of lipid oxidation in poultry muscle.

Mechanisms involved in hemoglobin‐mediated oxidation of lipids in washed fish muscle and inhibitory effects of phospholipase A2

BACKGROUND Hemoglobin (Hb) is a lipid oxidation promoter in fish muscle. Phospholipase A2 (PLA2; EC 3.1.1.4) is linked to an increased resistance to lipid oxidation of frozen-thawed cod fillets via an unknown mechanism. The present study aimed to investigate the mechanism of Hb-mediated lipid oxidation with a focus on ferryl Hb and methemoglobin (metHb), the pro-oxidative Hb species, and to examine how porcine pancreatic PLA2 inhibits Hb-mediated lipid oxidation in washed cod muscle (WCM). Lipid hydroperoxides (LOOHs) and thiobarbituric acid reactive substances (TBARS) were measured as primary and secondary lipid oxidation products, respectively. The formation of metHb and ferryl Hb was also monitored. RESULTS Ferryl Hb and metHb formed during the Hb-mediated lipid oxidation. PLA2 inhibited the formation of LOOHs and TBARS and suppressed the formation of metHb and ferryl Hb. WCM was pre-oxidized by hemin to increase the amount of LOOHs. PLA2 promoted the depletion of LOOHs in the pre-oxidized WCM with limited TBARS formation at the expense of the heme moiety of Hb. CONCLUSION The results of the present study suggest that ferryl Hb may play a role in Hb-mediated lipid oxidation and that PLA2 from pig pancreas may work together with Hb as a novel antioxidant with an ability to remove pre-formed LOOHs from a lipid substrate.