A highly sensitive method for the micro-determination of lipid hydroperoxides by potentiometry

A modified method for peroxide value (POV) determination of lipids was developed through the application of potentiometry to conventional POV tests such as the official method of the Japan Oil Chemists’ Society (JOCS). The new method permits a simple and reliable determination of low hydroperoxide levels in the initial stages of lipid autoxidation when only very small amounts of sample are available, even when those levels are measured on less than 10 mg of lipid. Using the present method, hydroperoxide levels as low as 20 nanoequivalents (neq) were determined with reasonable precision. This method is applicable to all lipids tested including oils and fats, free fatty acids, phospholipids, glycolipids and cholesterol esters.     

Quantitative determination of total lipid hydroperoxides by a flow injection analysis system

A flow injection analysis (FIA) system coupled with a fluorescence detection system using diphenyl-1-pyrenylphosphine (DPPP) was developed as a highly sensitive and reproducible quantitative method of total lipid hydroperoxide analysis. Fluorescence analysis of DPPP oxide generated by the reaction of lipid hydroperoxides with DPPP enabled a quantitative determination of the total amount of lipid hydroperoxides. Use of 1-myristoyl-2-(12-((7-nitro-2-1,3-benzoxadiazol-4-yl)amino) dodecanoyl)-sn-glycero-3-phosphocholine as the internal standard improved the sensitivity and reproducibility of the analysis. Several commercially available edible oils, including soybean oil, rapeseed oil, olive oil, corn oil, canola oil, safflower oil, mixed vegetable oils, cod liver oil, and sardine oil were analyzed by the FIA system for the quantitative determination of total lipid hydroperoxides. The minimal amounts of sample oils required were 50 μg of soybean oil (PV=2.71 meq/kg) and 3 mg of sardine oil (PV=0.38 meq/kg) for a single injection. Thus, sensitivity was sufficient for the detection of a small amount and/or low concentration of hydroperoxides in common edible oils. The recovery of sample oils for the FIA system ranged between 87.2±2.6% and 102±5.1% when PV ranged between 0.38 and 58.8 meq/kg. The CV in the analyses of soybean oil (PV=3.25 meq/kg), cod liver oil (PV=6.71 meq/kg), rapeseed oil (PV=12.3 meq/kg), and sardine oil (PV=63.8 meq/kg) were 4.31, 5.66, 8.27, and 11.2%, respectively, demonstrating sufficient reproducibility of the FIA system for the determination of lipid hydroperoxides. The squared correlation (r ²) between the FIA system and the official AOCS iodometric titration method in a linear regression analysis was estimated at 0.9976 within the range of 0.35−77.8 meq/kg of PV (n=42). Thus, the FIA system provided satisfactory detection limits, recovery, and reproducibility. The FIA system was further applied to evaluate changes in the total amounts of lipid hydroperoxides in fish muscle stored on ice.     

Coloring conditions of thiobarbituric acid test for detecting lipid hydroperoxides

The coloring reaction of the thiobarbituric acid test for hydroperoxides was completely inhibited by the addition of EDTA. Therefore, it was necessary to add a metal salt to the reaction mixture to complete the reaction and also to add an antioxidant to prevent autoxidation when unoxidized unsaturated fatty acids co-exist. The optimal pH of the reaction was found at 3.6 using glycine-hydrochloric acid buffer.     

Study of oxidation by chemiluminescence. IV. Detection of low levels of lipid hydroperoxides by chemiluminescence

Sodium hypochlorite (NaOCl) induced decomposition of organic hydroperoxides gave strong chemiluminescence. Chemiluminescence intensity reached its maximum a few seconds after the addition of sodium hypochlorite and decreased to the background level in three min. Good linear relationships were observed between total chemiluminescence counts in three min and the amounts of hydroperoxides. This chemiluminescence method can be applied to the detection of low levels of lipid hydroperoxides.     

Physico-chemical treatment of pulping effluent: Characterization of flocs and sludge generated after treatment

The present study deals with the treatment of simulated pulping effluent (pH = 11, and total organic carbon (TOC) = 1900 mg/L) by acid precipitation and coagulation-flocculation processes. The maximum TOC removal (= 4845 mg/g Al³⁺) from the effluent was found with Al₂(SO₄)₃ coagulant. Addition of sufloc (a flocculant) improved the sludge settling significantly. Hydrolyzed mono- and polynuclear species generated from Fe and Al coagulants could have resulted in TOC removal. Thermal analysis of sludge showed release of gases like CO, CO₂, HCHO, CH₃SH and SO₂ during combustion. Afterwards, the dissolved metal species in treated wastewater samples should be removed.     

Formation of dityrosine and other fluorescent amino acids by reaction of amino acids with lipid hydroperoxides

Formation of fluorescence by the reaction of various amino acids with lipid hydroperoxides,i.e., linoleic acid 13-monohydroperoxide, methyl linoleate 13-monohydroperoxide and phosphatidylcholine hydroperoxide, in the presence of methemoglobin was investigated. Two types of fluorescence were produced: fluorescent dityrosine (3,3′-dityrosine) from tyrosine, and unidentified fluorophores with α- and ε-amino groups of various amino acids. While the former was stable after treatment with borohydride, the latter fluorophores were readily destroyed. The rate of dityrosine formation was rapid, and the yield of dityrosine was dependent on the concentrations of tyrosine and the lipid hydroperoxides. Butylated hydroxytoluene and tocopherol inhibited the formation of dityrosine, but did not affect the formation of fluorophores on the amino groups. Dityrosine appears to be formed by radical reaction of the lipid hydroperoxides, while the other fluorophores seem to be created by nonradical mechanisms.     

Preparation and purification of lipid hydroperoxides from arachidonic and γ-linolenic acids

Commercial soybean lipoxygenase may be used under carefully controlled reaction conditions to give high yields of lipid hydroperoxides. Lipid hydroperoxides so derived from γ-linolenic or arachidonic acid may be purified by high pressure liquid chromatography. Thus, commercial lipoxygenase serves as a viable source for 100 mg quantities of lipid hydroperoxides.     

Limitations of the method using peroxidase activity of hemoglobin for detecting lipid hydroperoxides

The method using peroxidase activity of hemoglobin (Hb) for the determination of lipid peroxides was examined by using pure methyl linoleate hydroperoxides, trilinoleoylglycerol hydroperoxides and egg yolk phosphatidylcholine hydroperoxides as substrates and tetramethyl benzidine as electron donor for the peroxidase reaction of Hb. The reactivities of these substrates were quite varied. Furthermore, some electron donors were tested for peroxidase activity of Hb, but none showed a complete reduction of methyl linoleate hydroperoxides. From these results, it seems the Hb method needs to be carefully applied to biological materials that contain mixtures of different types of lipid classes.     

Characterization of wet microalgal cells pretreated with steam for lipid extraction

Steam pretreatment was employed to disrupt microalgal cells for lipids extraction.Effects of steam pre-treatment on microstructure of microalgal cells were investigated through scanning electron microscopy(SEM) and transmission electron microscopy (TEM).Effect of treatment on lipid extraction was also stud-ied.Microalgal cell walls were distorted after steam pretreatment due to the hydrolysis of organic macro-molecules contained in cell wall.Maximum curvature was increased from 1.88 × 10-6 m-1 to 1.43 × 10-7 m-1 after treatment with the steam at 130 ℃.The fractal dimension of microalgal cells increased from 1.25 to 1.30 after pretreatment for 15 min,and further increased to 1.47 when the pre-treatment time was increased to 60 min.Increased steam pretreatment temperature and time enhanced the hydrolysis of organic macromolecules,and finally destroyed microalgal cell walls at pretreatment temperature of 130 ℃ and pretreatment time of 60 min.Lipid extracted from wet microalgal was signif-icantly increased (2.1-fold) after pretreatment.     

Development of a flow injection chemiluminescent assay for the quantification of lipid hydroperoxides

An automated flow injection chemiluminescence (FICL) system for measuring lipid hydroperoxide (LOOH) concentrations in oils was developed. Initially, a crude oil-in-water emulsion (formed by mixing solvent-diluted oil with the aqueous-based CL compound, luminol, and the catalyst for the reaction, cytochrome c) was tested. The assay was rapid (60 samples per hour), reproducible (CV no greater than 10%, n=3) and had a low sample requirement (1 mg of oil) because of its high sensitivity (0.5 nmol LOOH). CL intensity was influenced by the amount and type of oil under analysis. Owing to these factors, quantitative data were attainable only with a uniform oil concentration and with a calibrant derived from an oil equivalent to that under analysis. This method yielded quantitative data in good agreement with an iodometric titration assay for LOOH (r=0.9204). A refinement of the first method consisted of replacing the luminol and cytochrome c CL compounds with lucigenin, resulting in an assay insensitive to α-tocopherol. A monophasic reaction solution was devised to remove the effect of turbidity; however, the CL signal was still influenced by oil type. Therefore, quantitative data were still attainable only when the same type of oil was used for calibration.     

Fluorescent image analysis of lipid hydroperoxides in fish muscle with 3-perylene diphenylphosphine

A fluorescent image analysis method was developed to evaluate lipid hydroperoxide formation in fish muscle. The lipid hydroperoxides generated in white and dark fish muscles during storage at 5–6°C oxidized 3-perylene diphenylphosphine located in the tissue to yield the fluorescent derivative, 3-perylene diphenylphosphine oxide (3-PeDPPO). 3-PeDPPO thus obtained was determined by digital fluorescent image analysis. The 3-PeDPPO fluorescence intensity of white and dark muscle increased during low-temperature storage (0–24 h) and was clearly correlated with total lipid hydroperoxide levels in muscle extracts, which were determined by using HPLC based on a triphenylphosphine oxidation method (R ²=0.954). These results suggest that 3-PeDPPO fluorescence, coupled with fluorescent image analysis, is a novel tool for direct determination of lipid hydroperoxides in fish muscle without a need for extraction of lipid.     

Kinetic study of the prooxidant effect of tocopherol. Hydrogen abstraction from lipid hydroperoxides by tocopheroxyls in solution

A kinetic study of the prooxidant effect of vitamin E (tocopherol, TocH) has been carried out. The rates of hydrogen abstraction (k₋₁) from methyl linoleate hydroperoxide (ML-OOH) by α-tocopheroxyl (α-Toc^.) (1) and eight types of alkyl substituted Toc^. radicals, (2–9) in benzene solution have been determined spectrophotometrically. The results show that the rate constants decrease as the total electron-donating capacity of the alkyl substituents on the aromatic ring of Toc^. increases. The k₋₁ value (5.0×10⁻¹M⁻¹s⁻¹) obtained for α-Toc^. (1) was found to be about seven orders of magnitude lower than the k₁ value (3.2×10⁶M⁻¹s⁻¹) for the reaction of α-TocH with peroxyl radical, which is well known as the usual radical-scavenging reaction of α-TocH. The above reaction rates (k₋₁) obtained were compared with those (k₃) of methyl linoleate with Toc^. (1–9) in benzene solution. The rates (k₋₁) were found to be about six times larger than those (k₃) of the corresponding Toc^.. The results suggest that both reactions may relate, to the prooxidant effect of α-TocH at high concentrations in foods and oils. The effect of the phytyl side chain on the reaction rate, of Toc^. in micellar dispersions has also been studied. We have measured the rate constant, k₋₁, for the reaction of phosphatidylcholine hydroperoxide with a Toc^. radical in benzene,tert-butanol and in Triton X-100 micellar dispersions, and compared the observed k₋₁ values with the corresponding values for ML-OOH.     

Mechanisms of tubulin modification by phosphatidylcholine hydroperoxides

The interaction of lipid peroxides with cellular proteins has been postulated to contribute to cellular aging. A potential target for such effects is tubulin, the building block of microtubules. We examined the concentration-dependent effects of phosphatidylcholine hydroperoxides on the ability of tubulin to polymerize into microtubules. The results demonstrated that even very low concentrations of peroxides were sufficient to interfere with the tubulin and, therefore, the microtubule function. Decreased tubulin activity (as measured by tubulin GTPase activity) showed correlation with the modification of methionine and cysteine in tubulin and a change in the tubulin conformational state as indicated by fluorescence and ultraviolet spectroscopic measurements. As no effect on electric conductivity was observed, indicating that modulation of ionic binding was not involved, the interaction mechanism may be a hydrophobic one.     

Characterization of hair lipid images by argon sputter etching-scanning electron microscopy

Hair lipid images, as visualized by argon sputter etching-scanning electron microscopy (ASE-SEM), reveal convex structures with a stitch pattern (SP) at the cell membrane complex (CMC) in the transverse hair plane. Based on interindividual variation, different features of the convex SP were classified into Types 0 to 4 with the corresponding scores 0 to 4. Observations using hair fibers collected from 27 Japanese females revealed significant positive correlations between the scores and the levels of exogenous lipids, which suggests that exogenous lipids internalized at the CMC predominantly constitute the convex SP. Intraindividual variation with different levels of exogenous lipids among hair fibers derived from individual females may be relevant to the uneven physicochemical properties of hair fibers on the scalp. Observations of 380 hair fibers collected from Japanese (Mongoloid), German and American (Caucasoid) females aged 3 to 77 yr demonstrated similar age-related changes in the lipid images, which represent an increase and then a decrease in levels of exogenous lipids with increasing age. This suggests that age-related changes in exogenous lipids are attributable to alterations in sebum excreted during aging and that this elicits age-related changes in physical parameters, which affect human hair texture.     

Quantification of carbonyls produced by the decomposition of hydroperoxides

Carbonyls produced by the decomposition of cyclohexene hydroperoxide and various hydroperoxides of linoleic and linolenic acids and their methyl esters were determined by gas chromatography of the 2,4,6-trichlorophenylhydrazones. The effect of temperature, iron and copper ions, ethanol and several antioxidants on the rate of decomposition, the nature of the products and their yield was observed. The hydroperoxides of methyl esters decomposed more slowly than those of free fatty acids. Ethanol slowed, and metal ions accelerated the rates of decomposition. Metal ions, especially copper, increased the yield and complexity of the carbonyls formed, but ethanol decreased carbonyl yields. Antioxidants and decomposition temperatures changed the relative yields of carbonyls produced. The 9- and 13-hydroperoxides of linoleic acid gave similar carbonyls, but those of linolenic acid did not. The carbonyl mixtures produced from autoxidized fatty acid methyl esters were more complex than those produced from lipoxygenase-treated fatty acids.     

Effects of membrane charges and hydroperoxides on Fe(II)-supported lipid peroxidation in liposomes

The processes in producing a lag phase in Fe²⁺-supported lipid peroxidation in liposomes were investigated. Incorporation of phosphatidylserine (PS) or dicetyl phosphate (DCP) into phosphatidylcholine [PC(A)] liposomes, which have arachidonic acid, produced a marked lag phase in Fe²⁺-supported peroxidation, where PS was more effective than DCP. Phosphatidylcholine dipalmitoyl [PC(DP)] with a net-neutral charge was still effective in producing a lag phase, though weak. Increasing concentrations of PS, DCP, and PC(DP) prolonged the lag period. Initially after adding Fe²⁺, slight oxygen consumption occurred in PC(A)/PS liposomes including hydroperoxides, followed by a lag phase. An increase in the hydroperoxide resulted in a shortening of the lag period. The initial events of Fe²⁺ oxidation accompanied by oxygen consumption were dependent on the hydroperoxide content, but significant changes in diene conjugation and hydroperoxide levels at this stage were not found. The molar ratios of both dis-appeared Fe²⁺ and consumed O₂ to preformed hydroperoxide in liposomes with or withouttert-butylhydroxytoluene were constant, regardless of the different amounts of lipid hydroper-oxides. The antioxidant completely inhibited the propagation of lipid peroxidation in the lipid phase, following a lag phase. In a model system containing 2,2′-azobis (2-amidinopropane) dihydrochloride, Fe²⁺ were consumed. We suggest that Fe²⁺ retained at a high level on membrane surfaces play a role in producing a lag phase following the terminating behavior of a sequence of free radical reactions initiated by hydroperoxide decompositin, probably by intercepting peroxyl radicals.     

Reduction of fatty acid hydroperoxides by human parotid saliva

Arachidonic acid hydroperoxide (15-hydroperoxyeicosatetraenoic acid; 15-HPETE) was introduced into human parotid saliva and incubated at 37°C. Straight phase high-performance liquid chromatography analysis of the reaction mixture showed that 15-HPETE was detoxified to its reduced form, 15-hydroxyeicosatetraenoic acid, in the presence of glutathione. Therefore, it is concluded that human parotid saliva possesses fatty acid hydroperoxide-reducing ability. However, its effectiveness was found to be lower than that of blood plasma.     

Detection and measurement of hydroperoxides by near infrared spectrophotometry

Summary Near-infrared spectra have been measured on a group of hydroperoxides of fatty acid esters and related substances. Only those substances having an −OOH group were found to absorb at 1.46 and 2.07 μ. Dialkyl peroxides and ozonized unsaturated substances had no such maxima in their near infrared spectra although they had high iodometric peroxide values. In a study of the thermal decomposition of methyl oleate hydroperoxide and a study of the autoxidation of methyl linoleate, the intensity of absorption at 1.46 and 2.07 μ paralleled the iodometric peroxide value. This work was aided by grants-in-aid from the Hormel Foundation, the Atomic Energy Commission (Contract AT-11-1-108), the Office of Naval Research (Contract N8 onr66218), the National Live Stock and Meat Board, and the National Diary Council. Hormel Institute Publication No. 170.