植物膜脂过氧化水平硫代巴比妥酸测定法中的干扰因素

硫代巴比妥酸反应物质(TBARS)是表示植物组织膜脂过氧化水平的最常用指标。干旱条件下植物组织中TBARS含量增加。一般认为TBARS主要是丙二醛(MDA),实际上TBA能与包括某些可溶性糖在内的多种醛类物质反应,并可能对TBA-MDA反应造成干扰。干旱条件下植物组织中可溶性糖大量增加,在这种条件下TBARS含量能否     

硫代巴比妥酸（TBA）法检测脂质过氧化水平的探讨

表示脂质过氧化反应有多种指标,如测定丙二醛产生(下称TBA法)、氧的吸收、共轭双键形成、乙烷释放等。在这些指标中,TBA法由于操作简便而广为应用。此法最早见于Wilbur等,他们发现人工氧化的     

视网膜组织脂质过氧化反应产物的测定——硫代巴比妥酸荧光法

作者研究了家兔视网膜组织脂质过氧化反应产物的硫代巴比妥酸(TBA)荧光测定法;测定了正常家兔视网膜组织的TBA值;并对视网膜铁锈症与脂质过氧化的关系进行了讨论。正常家兔视网膜组织的TBA值为：0.092±0.02nmol MDA／mg。实验结果表明,视网膜铁锈症可使视网膜组织中脂质过氧化产物显著增高。     

植物组织内丙二醛的分离与鉴定

丙二醛(MDA)含量变化是广为应用的膜脂过氧化指标之一。植物组织内MDA的水平一般是用硫代巴比妥酸(TBA)显色后得到的TBA值来衡量。但一些研究表明MDA-TBA反应存在一些干扰因素,在某些条件下甚至会造成假象,以至得出错误结论。本实验应用薄层层析法对MDA进行分离与鉴定,为植物组织内MDA的存在提供了直接的证据。     

亚铁盐诱发谷氨酸降解的自由基机理

铁是生物体中一种极为重要的金属元素,但从铁蛋白释放的铁,在特定情况下可诱发体內不饱和脂肪酸发生过氧化反应.Gutteridge报道了离体实验中亚铁盐尚能降解几种常见氨基酸和糖,生成丙二醛(MDA)样物质,因而可与硫代巴比妥酸(TBA)发生反应,生成粉红色产物.我们观察了亚铁盐诱发谷氨駿降解,并对其自由基机理进行了探讨.     

金属硫蛋白拮抗同型半胱氨酸对血管内皮细胞的脂质过氧化作用

目的:观察金属硫蛋白(metallothionein,MT)对同型半胱氨酸(homocysteine Hcy)损伤血管内皮细胞的拮抗作用及机制.方法:培养液中分别加入Hcy及Hcy MT孵育血管内皮细胞,自动生化分析仪测培养液乳酸脱氢酶(LDH)活性,硫代巴比妥酸法测细胞丙二醛(MDA)含量.结果:同型半胱氨酸增加内皮细胞LDH及蛋白漏出,并使细胞MDA含量明显升高.MT呈浓度依赖性地抑制Hcy所致的血管内皮细胞损伤及MDA增高.结论:MT拮抗Hcy对血管内皮细胞的脂质过氧化损伤.     

脂质体中卵磷脂的氧化产物与溶血的关系

卵磷脂是构成脂质体膜的基本骨架,其化学性质不稳定,易氧化水解,氧化产物直接影响脂质体的包封率,且增加毒性。作者曾用氧化指数作为检测卵磷脂氧化的质量指标,但不理想,无法定量,且易受脂质体中其它组分干扰。根据卵磷脂氧化产物丙二醛(MDA)在酸性条件下可与硫巴比妥(TBA)反应,生成红色颜色(TBA-Pigment),在535nm处有特异吸     

椰子种皮油提取物对低密度脂蛋白氧化的抑制作用

本文研究了椰子种皮油提取物对低密度脂蛋白氧化的抑制作用,以水溶性VE(Trolox)作为对照,测定椰子种皮油提取物的总酚含量,总抗氧化能力,检测提取物对低密度脂蛋白的氧化易感性,硫代巴比妥酸反应产物(TBARS)值及铜络合能力的影响。结果表明,椰子种皮油提取物的总酚含量为68.6 mg/g,总抗氧化能力也随着浓度增大而加强,说明该提取物具有较好的抗氧化活性。浓度为0.5 mg/mL的提取物能将低密度脂蛋白的延滞时间延长了两倍,丙二醛(MDA)含量也明显下降,表明椰子种皮油提取物对低密度脂蛋白的氧化具有一定的抑制作用。     

锌_7-与镉_7-金属硫蛋白对羟自由基损伤大鼠红细胞膜保护作用的比较

用电子自旋共振自旋标记物氮氧自由基硬脂酸和马来酰亚胺标记大鼠红细胞膜脂和膜蛋白,测定膜脂流动性和膜蛋白构象改变,以硫代巴比妥酸法测定脂质过氧化产物丙二醛含量．结果表明,锌7-与镉7-金属硫蛋白对羟自由基引起的膜脂流动性减低、脂质过氧化反应增强双膜蛋白构象改变有明显抑制作用,而且,前者的作用明显强于后者．     

连翘叶黄酮的体外抗氧化作用

为研究连翘叶黄酮(Forsythia suspenseleaves flavonoids,FLF)的体外抗氧化作用,用水杨酸法研究FLF清除.OH的效果,并测定了FLF对连苯三酚自氧化体系的抑制作用。用硫代巴比妥酸(TBA)法测定了小鼠4种器官及肝线粒体、微粒体中的丙二醛(MDA)含量,用分光光度法测定了小鼠红细胞溶血和肝线粒体膨胀程度。结果表明,FLF可以清除.OH,抑制连苯三酚自氧化,并抑制.OH所致丙二醛的产生,减少红细胞溶血,减轻肝线粒体膨胀程度。说明FLF具有明显的抗氧化活性。     

Reaction conditions affecting the relationship between thiobarbituric acid reactivity and lipid peroxides in human plasma.

The thiobarbituric acid (TBA) reactivity of human plasma was studied to evaluate its adequacy in quantifying lipid peroxidation as an index of systemic oxidative stress. Two spectrophotometric TBA tests based on the use of either phosphoric acid (pH 2.0, method A) or trichloroacetic plus hydrochloric acid (pH 0.9, method B) were employed with and without sodium sulfate (SS) to inhibit sialic acid (SA) reactivity with TBA. To correct for background absorption, the absorbance values at 572 nm were subtracted from those at 532 nm, which represent the absorption maximum of the TBA:MDA adduct. Method B gave values of TBA-reactive substances (TBARS) 2-fold higher than those detected with method A. SS lowered TBARS by about 50% with both methods, indicating a significant involvement of SA in plasma TBA reactivity. Standard SA, at a physiologically relevant concentration of 1.5 mM, reacted with TBA, creating interference problems, which were substantially eliminated by SS plus correction for background absorbance. When method B was carried out in the lipid and protein fraction of plasma, SS inhibited by 65% TBARS formation only in the latter. Protein TBARS may be largely ascribed to SA-containing glycoproteins and, to a minor extent, protein-bound MDA. Indeed, EDTA did not affect protein TBARS assessed in the presence of SS. TBA reactivity of whole plasma and of its lipid fraction was instead inhibited by EDTA, suggesting that lipoperoxides (and possibly monofunctional lipoperoxidation aldehydes) are involved as MDA precursors in the TBA test. Pretreatment of plasma with KI, a specific reductant of hydroperoxides, decreased TBARS by about 27%. Moreover, aspirin administration to humans to inhibit prostaglandin endoperoxide generation reduced plasma TBARS by 40%. In conclusion, reaction conditions affect the relationship between TBA reactivity and lipid peroxidation in human plasma. After correction for the interfering effects of SA in the TBA test, 40% of plasma TBARS appears related to in vivo generated prostaglandin endoperoxides and only about 60% to lipoperoxidation products. Thus, the TBA test is not totally specific to oxidant-driven lipid peroxidation in human plasma.     

Lipid peroxidation and the thiobarbituric acid assay: standardization of the assay when using saturated and unsaturated fatty acids

Saturated fatty acids are less vulnerable to lipid peroxidation than their unsaturated counterparts. In this investigation, individual fatty acids of the C(16), C(18) and (20) families were subjected to the thiobarbituric (TBA) assay. These fatty acids were chosen based on their degree of saturation and configuration of double bonds. Interestingly, an assay threshold was reached where increasing the fatty acid concentration resulted in no additional decrease in the TBARS concentrations. Therefore, the linear range of TBARS inhibition was determined for fatty acids in the C(16) and C(20) families. The rate of TBARS inhibition was greater for the saturated than for unsaturated fatty acids, as measured from the slope of the linear range. These findings demonstrate the need to standardize the TBARS assay using multiple fatty acid concentrations when using this assay for measuring in vitro lipid peroxidation.     

A specific,accurate, and sensitive measure of total plasma malondialdehyde by HPLC

Malondialdehyde (MDA) is one of the most commonly reported biomarkers of lipid peroxidation in clinical studies. The reaction of thiobarbituric acid (TBA) with MDA to yield a pink chromogen attributable to an MDA-TBA₂ adduct is a common assay approach with products being quantified by ultraviolet-Vis assay as nonspecific TBA-reactive substances (TBARS) or chromatographically as MDA. The specificity of the TBARS assay was compared with both chromatographic assays for total plasma MDA. The levels of total plasma MDA were significantly lower than the plasma TBARS in each of the samples examined, and interestingly, the interindividual variation apparent in the level of plasma MDA was not evident in the plasma TBARS assay. Each of the four online chromatographic detectors yielded a precise, sensitive, and accurate determination of total plasma MDA, and selected-ion monitoring was the most-accurate assay (101.3%, n = 4). The online diode array detectors provided good assay specificity (peak purity index of 999), sensitivity, precision, and accuracy. This research demonstrates the inaccuracy that is inherent in plasma TBARS assays, which claim to quantify MDA, and it is proposed that the TBARS approach may limit the likelihood of detecting true differences in the level of lipid peroxidation in clinical studies.     

Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds

The occurrence of malondialdehyde (MDA), a secondary end product of the oxidation of polyunsaturated fatty acids, is considered a useful index of general lipid peroxidation. A common method for measuring MDA, referred to as the thiobarbituric acid-reactive-substances (TBARS) assay, is to react it with thiobarbituric acid (TBA) and record the absorbance at 532 nm. However, many plants contain interfering compounds that also absorb at 532 nm, leading to overestimation of MDA values. Extracts of plant tissues including purple eggplant (Solanum melongena L.) fruit, carrot (Daucuscarota L.) roots, and spinach (Spinacia oleracea L.) leaves were assessed for the presence of MDA and other non-MDA compounds absorbing at 532 nm. A method described herein corrects for these interferences by subtracting the absorbance at 532 nm of a solution containing plant extract incubated without TBA from an identical solution containing TBA. The reliability and efficiency of this spectrophotometric method was assessed by altering the relative ratios of exogenous MDA additions and/or extracts of red cabbage (Brassica oleracea L.) leaves containing interfering compounds and then measuring MDA recovery. Reliability was also validated through high-performance liquid chromatography and high-performance liquid chromatography-mass spectrometry techniques. Results indicated that over 90% of exogenously added MDA could be recovered through the improved protocol. If there were no corrections for interfering compounds, MDA equivalents were overestimated by up to 96.5%. Interfering compounds were not detected in vegetables such as lettuce (Lactuca sativa L.) and spinach which had low or negligible concentrations of anthocyanidin derivatives. Comparisons between the TBARS method presented here and two currently accepted protocols indicated that the new modified method exhibits greater accuracy for quantifying TBA-MDA levels in tissues containing anthocyanins and/or other interfering compounds. This modified protocol represents a facile and rapid method for assessment of lipid peroxidation in virtually all plant species that contain interfering compounds. Received: 28 August 1998 / Accepted: 29 September 1998     

UV-C irradiation-induced peroxidative degradation of microsomal fatty acids and proteins: protection by an extract of Ginkgo biloba (EGb 761).

After exposure of rat liver microsomes to UV-C irradiation, analysis of membrane fatty acids by gas chromatography confirmed that EGb 761, a drug containing a dosed and standardized extract of Ginkgo biloba, provides effective protection against free radical attack in vitro. This analysis, coupled with thiobarbituric acid (TBA) reaction, permitted qualitative and overall quantitative evaluation of radical-induced damage to polyunsaturated fatty acids (PUFA), as well as evidence of the antioxidant properties of the Ginkgo biloba extract. Assay of thiobarbituric acid reactive substances (TBARS) showed a correlation between TBARS concentration and the state of degradation of the polyunsaturated fatty acids. Mannitol (5.5 mM) did not prevent degradation of microsomal PUFA or malondialdehyde (MDA) production, nor did it prevent polymerization of membrane proteins. Low doses of EGb 761 were found to provide efficient protection of membrane PUFA regardless of individual susceptibility to peroxidation. This protection was accompanied by a decrease in the production of TBARS. EGb 761 also protected membrane proteins from the irreversible polymerization induced by these degradation products, but did not appear to prevent thiols oxidation into disulfide bonds.     

An improved malondialdehyde assay for estimation of thromboxane synthase activity in washed human blood platelets

After stimulation of the washed human blood platelets by arachidonic acid (AA), the concurrent evaluations for formed malondialdehyde (MDA) measured by the common photometrical thiobarbituric acid (TBA) method, and for thromboxane B2 (TXB2) measured by gas chromatography, revealed that the formed MDA exceeded the amount of TXB2 on a molar base. However, MDA and TXB2 originating from thromboxane synthase activity should be produced in approximately equimolar amounts. By treatment of the stimulated platelet samples with stannous chloride it is possible to reduce all peroxidized products of AA which generate MDA otherwise during the TBA reaction and to estimate MDA and TXB2 in a ratio of nearly 1:1. The stannous chloride treatment does not destroy the MDA and does not influence the TBA reaction with MDA. Therefore the simple and quick TBA method can be used after stannous chloride treatment for estimation of thromboxane synthase activity in AA stimulated washed human platelets.     

An improved malondialdehyde assay for estimation of thromboxane synthase activity in washed human blood platelets

After stimulation of the washed human blood platelets by arachidonic acid (AA), the concurrent evaluations for formed malondialdehyde (MDA) measured by the common photometrical thiobarbituric acid (TBA) method, and for thormboxane B₂ (TXB₂ measured by gas chromatography, revealed that the formed MDA exceeded the amount of TXB₂ on a molar base. However, MDA and TXB₂ originating from thromboxane synthesis activity should be produced in approximately equimolar amounts. By treatment of the stimulated platelet samples with stannous chloride it is possible to reduce all peroxidized products of AA which generate MDA otherwise during the TBA reaction and to estimate MDA and TXB₂ in a ratio of nearly 1:1. the stannous chloride treatment does not destroy the MDA and does not influence the TBA reaction with MDA. Therefore the simple and quick TBA method can be used after stannous chloride treatment for estimation of thromboxane synthase activity in AA stimulated washed human platelets.     

High-performance liquid chromatography analysis of the thiobarbituric acid adducts of malonaldehyde and trans,trans-muconaldehyde

A reversed-phase HPLC method is described for the separation and analysis of the thiobarbituric acid (TBA) adducts of the reactive aldehydes muconaldehyde (MUC) and malonaldehyde (MDA). The TBA adduct of malonaldehyde was synthesized, purified, and its structure elucidated, for use as standard in quantitative HPLC studies. A detection limit of 1 X 10(-14) mol was achieved for the MUC:TBA and MDA:TBA adducts using the double monochromator fluorometric detector, 7 X 10(-13) mol was the detection limit using a variable wavelength uv-visible detector. Direct on-line identification of the eluting aldehyde:TBA adducts was achieved by the use of a diode-array uv-visible detector. The chromatographic behavior of the adducts under different mobile phase conditions was also examined. This HPLC methodology was used for the identification of muconaldehyde as a product of benzene oxidation in a hydroxyl radical generating system.     

Interactions of Nitric Oxide with Lipid Peroxidation Products under Aerobic Conditions: Inhibitory Effects on the Formation of Malondialdehyde and Related Thiobarbituric Acid-Reactive Substances

Under aerobic conditions, exposure of peroxidized lipids to nitric oxide (NO) was found to result in a rapid decrease in the levels of thiobarbituric acid-reactive substances (TBARS). Addition of 10–100 μM NO to rat brain homogenates preincubated for 2 h at 37°C caused up to a 20% decrease in the levels of TBARS compared to controls. A similar inhibitory effect was observed on TBARS produced by Fe²⁺-induced decomposition of 15-hydroperoxyeicosatetraenoic acid (15-HPETE), due apparently to NO-induced decomposition of the hydroperoxide (ferrous oxidation/xylenol orange assay). Prostaglandin G₂ (PGG₂, 35 μM), as a model bicyclic endoperoxide, and malondialdehyde (MDA, 20 μM), the main component of TBARS, proved also susceptible to degradation by NO or NO donors (diethylamine NONOate, DEA/NO) at concentrations of 100 μM or higher in 0.05 M phosphate buffer, pH 7.4, and at 37°C, as indicated by the reduced response to the TBA assay. No significant effect on TBARS determination was caused by nitrite ions. These and other data indicate that NO can inhibit TBARS formation by decomposing primary lipid peroxidation products, chiefly 15-HPETE and related hydroperoxides, and, to a lesser extent, later stage TBARS precursors, including bicyclic endoperoxides and MDA, via nitrosation and other oxidative routes, without however affecting chromogenic reactions during the assay.     

Optimization of low-cost drying methods to minimize lipid peroxidation in <Emphasis Type="Italic">Spirulina platensis</Emphasis> grown in the Philippines

Three low-cost drying methods (sun, solar, and draft oven) were optimized to produce Spirulina powder of optimal quality. Optimization in the pre-dehydration stage included the use of two antioxidants, α-tocopherol and tertiary-butyl hydroquinone (TBHQ), and two blanching methods, microwave and water bath, to inactivate enzymes. The efficiency of the pre-dehydration treatments at minimizing lipid peroxidation were evaluated in terms of the product’s oxidative stability using the thiobarbituric acid (TBA) test. The sample with the lowest TBA reactive substance (TBARS) value was considered the most stable. TBHQ was found to be significantly better than α-tocopherol in minimizing lipid peroxidation in blanched samples while α-tocopherol was better than TBHQ in unblanched samples. Microwave blanching exerted a greater stabilizing effect than water bath blanching. The combined effect of TBHQ and microwave blanching was found to be the most effective pre-dehydration treatment for minimizing lipid peroxidation in drying Spirulina. Among the three low-cost optimized drying methods, sun-drying produced a dried product with the lowest TBARS value (0.472 mg malondialdehyde.kg⁻¹), which was closest to that of the spray-dried (control) sample (0.434 mg MDA.kg⁻¹). Draft oven and solar drying produced dried products with the same average TBARS value (0.56 mg MDA.kg⁻¹). Sun-drying, when optimized, produced a dried product that was almost as stable as the spray-dried product. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines