Fluorescent products and polyunsaturated fatty acids of human testes

Lipid soluble fluorescent pigments from human testis were fractionated by silicic acid column chromatography and silica gel thin layer chromatography. Fluorescence analyses revealed a family of at least 3 compounds with similar fluorescence properties, including excitation and emission maxima, reversible fluorescence quenching by alkaline pH, and fluorescence quenching by heavy metal chelation. These fluorescence characteristics strongly indicated the presence of the conjugated Schiff base fluorophore-N=C-C=C-N-. The chromatographic separations employed enabled a more definitive fluorescence characterization of the lipid soluble pigments known to accumulate late in tissues with age and as a result of lipid peroxidation. Total lipids and fatty acid composition of the total lipids were determined. Polyenoic acids constituted about 40% of the total fatty acids. Histological examination of the tissues revealed some degeneration and edema, but significant spermatogenesis and normal complement of Leydig cells.     

Studies on peroxidized lipids. V. Formation and characterization of 1,4-dihydropyridine-3,5-dicarbaldehydes as model of fluorescent components in lipofuscin

We investigated fluorescence properties of 1,4-dihydropyridine-3,5-dicarbaldehydes and their formation in mild reaction of primary amines and malonaldehyde, in order to clarify the role of malonaldehyde in the formation of fluorescent components of lipofuscin. The compounds exhibited fluorescence with excitation maxima at 375–405 nm and emission maxima at 435–465 nm, which was similar to those of lipofuscin and the fluorescent substances derived from the reaction of oxidized fatty acids with primary amines. Fluorescence of the compounds was greatly affected in acidic medium and little influenced in alkaline medium or by the metal chelator. The compounds lost fluorescence by treatment with sodium borohydride. They were inert to thiobarbituric acid reaction. Some of the fluorescence properties of the compounds were different from those of lipofuscin and the related fluorescent substances. Mild reaction of methylamine with pure malonaldehyde gave a single fluorescent compound, 1,4-dimethyl-1,4-dihydropyridine-3,5-dicarbaldehyde (Ia), and the reaction with the acid hydrolysate of tetramethoxypropane gave Ia and 1-methyl-4-(dimethoxyethyl)-1,4-dihydropyridine-3,5-dicarbaldehyde (IIa), the latter being produced from the impurity in the hydrolysate. These reactions produced a non-fluorescent Schiff base, a 1∶1-adduct of methylamine and malonaldehyde (IIIa), as a major product. It looks unlikely that malonaldehyde is the only product of lipid oxidation that produces fluorescent components in lipofuscin complex.     

Fluorescent products of lipid peroxidation of mitochondria and microsomes

Liver microsomes and mitochondria and heart sarcosomes from rats fed diets with varying α-tocopherol concentrations and lipid contents were peroxidized over a 6 hr time period. Lipid peroxidation was measured by absorption of oxygen, production of thiobarbituric acid (TBA) reactants and by development of fluorescence. The spectral characteristics of the fluorescent compounds were the same for all peroxidizing systems; the excitation maximum was 360 nm and the emission maximum was 430 nm. As time of peroxidation increased, uptake of oxygen and production of fluorescent compounds increased. These two parameters as well as production of TBA reactants were dependent upon dietary antioxidant and all three had an inverse relationship with the amount of dietary α-tocopherol. The relationship between absorption of oxygen and development of fluorescent compounds was also dependent upon dietary polyunsaturated fats (PUFA). Subcellular particles from animals fed higher levels of PUFA produced more fluorescent products per mole of oxygen absorbed than did those from animals on a diet with lower PUFA content. TBA reacting products increased with time during the initial phase of peroxidation: in the microsomal systems their production stabilized or decreased by 4–6 hr of peroxidation. Using the synthetic 1-amino-3-iminopropene derivative of glycine as standard for quantitation of fluorescence, the molar ratios of oxygen absorbed per fluorescent compound produced were calculated. This ratio for subcellular particles isolated from rats fed diets with PUFA ratios similar to those in the average American human diet was 393∶1. The fluorescent compounds had the same spectral characteristics as the lipofuscin pigment that accumulates in animal tissues as a function of age, oxidative stress or antioxidant deficiency. The fluorescent molecular damage represented by that accumulated in human heart age pigment by 50 years of age was calculated to have been caused by approximately 0.6 μmole of free radicals per gram of heart tissue.     

Fluorescent products from reaction of peroxidizing polyunsaturated fatty acids with phosphatidyl ethanolamine and phenylalanine

Fluorescent chromophores produced by reaction of peroxidizing arachidonic acid or methyl docosahexaenoate with synthetic dipalmityl phosphatidyl ethanolamine were lipid soluble, and those from reaction with phenylalanine were water soluble. In all reaction systems that contained polyunsaturated fatty acid and only one amine compound, the development of fluorescence was linearly related to oxygen absorption for 12–24 hr (p<0.001) and to the amount of thiobarbituric acid reacting materials until the rate of oxygen absorption decreased. The fluorochromes typically had maximum excitation at 360 nm and maximum emission at 430–440 nm, indicating that they were conjugated Schiff bases with the general structure R−N=C−C=C−N−R, where R represents the amino acid phenylalanine or the phospholipid phosphatidyl ethanolamine. The fluorochromes were similar to those extracted from isolated age pigments and tissues of animals that are aged, vitamin E-deficient, or stressed with highly unsaturated lipid diets.     

Fluorescent products of phospholipids during lipid peroxidation

During peroxidation, phosphatidyl ethanolamine and phosphatidyl serine formed fluorescent chromophores with maximum emission at 435 nm and maximum excitation at 365 nm. The development of fluorescence was related to formation of thiobarbituric acid reactive substance during lipid peroxidation. This relationship was studied by reacting dipalmityl phosphatidyl ethanolamine with the oxidation products of the methyl esters of arachidonic, linolenic and linoleic fatty acids. Reaction parameters affecting the development of lipid-extractable fluorescent chromophores are: the production of peroxidation products, especially malonaldehyde, from autoxidation of polyunsaturated fatty acids; the length of time these products react; and the availability of reactive amino groups on the phospholipids.     

Synthesis and characterization of some novel conjugated polyoxadiazoles with Schiff base structure

Some novel conjugated polyoxadiazoles with Schiff base structure were efficiently synthesized through polycondensation of 4,4′-(1,3,4-oxadiazole-2,5-diyl)dianiline with some dialdehydes. The polymers were characterized by IR, ¹H NMR, ultraviolet spectroscopy, fluorescence spectroscopy, gel permeation chromatography and thermal analyses. All polymers have high thermal stability with decomposition temperature higher than 260 °C. Some polymers have good solubility in common organic solvents. They also have strong absorptions at 294–345 nm, and strong emissions at 405–432 nm.     

Isolation and analysis of age-related fluorescent substances in rat testes

Fluorescent substances were extracted from rat testicular tissue with 2,2-dimethoxypropane (DMP) and analyzed by 2-dimensional thin layer chromatography (TLC). One substance that accumulated with increasing age of the animals was isolated and analyzed quantitatively by spectrophotofluorometry using quinine sulfate as a standard. This substance, which was designated as an age-related fluorescent substance (ARFS), exhibited an excitation maximum at 355 nm and an emission maximum at 490 nm. Its fluorescence was quenched by metal chelators and at alkaline pH, indicating it contained a conjugated Schiff base structure. Quantitative analysis of this substance in the testes of rats 1, 2, 11 and 20 months of age showed that it increased linearly with age. The relation of this substance to aging also was indicated by its detection in animals of different ages fed diets of both low and high unsaturation.     

Recent advances in assessment of marine lipid oxidation by using fluorescence

Lipid changes during food processing are important because of their impact on the final product quality. Lipid damage detection is limited because of the ability of lipid oxidation products (i.e., hydroperoxides and carbonyl compounds) to produce interaction compounds by reacting with nucleophilic food constituents. Fluorescence quantification at a single excitation/emission maximum of these interaction compounds has been employed in a qualitative way as a complementary tool for food quality assessment. The present work reviews recent research where simultaneous detection at different excitation/emission maxima was employed to assess lipid oxidation and quality changes during fish processing. A fluorescence shift towards a higher wavelength maxima was detected as a result of lipid damage; the shift was calculated as the ratio (δF) between two of the maxima tested (393/463 nm and 327/415 nm) and was investigated along different fish processes (freezing and frozen storage, refrigerated storage, cooking, and canning) and in complementary model systems where the influence of different factors (time and temperature of processing, amine and aldehyde composition and content, formaldehyde presence, and pH value of the medium) was checked. Determination of the δF value provided better results for quality change assessment in fish products than most of the lipid quality indices, in addition to being rapid and sensitive.     

Formation of fluorescent substances in reaction of aliphatic aldehydes and methylamine

Treatment of monofunctional aliphatic aldehydes (alkanals, 2-alkenals and 2,4-alkadienals) with methylamine at pH 7 produced fluorescence with excitation maxima at 340–360 nm and emission maxima at 410–470 nm. The reaction of 1-butanal and methylamine gave 2-ethyl-2-hexenal (ii), an aldol condensation product of 1-butanal, and 3,5-diethyl-2-propyl-1-methylpyridinium salt (i). The fluorescence was formed by the reaction of 1-butanal and/or ii with methylamine in the presence of molecular oxygen. Fluorescent products I, II and III formed by the reaction were partially purified. Fluorescence characteristics of I, II and III were close to those of the fluorophores derived from the reaction of oxidized fatty acids and primary amines, with respect to maximum wavelengths in excitation and emission, and the resistance to sodium borohydride reduction.     

Fluorescence kinetics of soybean flour oxidation

Changes in fluorescence and oxidation indices of soybean flour were studied during storage at 37, 50, and 60°C. The peroxide value and 2-thiobarbituric acid value did not increase in proportion to the extent of oxidation. The front-surface fluorescence of oxidized soybean flour had an excitation maximum around 350 nm and an emission maximum between 420 and 430 nm. The fluorescent compounds were soluble in the organic phase of chloroform/methanol (2∶1, vol/vol) after addition of water, and the solution showed excitation and emission maxima around 360 and 440 nm, respectively. The fluorescent compounds might have been produced from interactions of oxidizing soybean oil with other constituents of soybean flour. The fluorescence intensity in the organic phase increased steadily throughout the storage period. Fluorescence kinetics of soybean flour oxidation showed a zero-order increase, and the rate followed an Arrhenius relationship with an activation energy of 47.8 kJ/mol. These results suggest that analysis of fluorescence intensity in the organic phase is a useful indicator for determination of the oxidative deterioration of soybean flour.     

Preparation of schiff base adducts of phosphatidylcholine core aldehydes and aminophospholipids, amino acids, and myoglobin

We have prepared Schiff base adducts of the core aldehydes of phosphatidylcholine and aminophospholipids, free amino acids, and myoglobin. The Schiff bases of the ethanolamine and serine glycerophospholipids were obtained by reacting sn-1-palmitoyl(stearoyl)-2-[9-oxo]nonanoyl-glycerophosphocholine (PC-Ald) with a twofold excess of the aminophospholipid in chloroform/methanol 2∶1 (vol/vol) for 18 h at room temperature. The Schiff bases of the amino acids and myoglobin were obtained by reacting the aldehyde with an excess of isoleucine, valine, lysine, methyl ester lysine and myoglobin in aqueous methanol for 18 h at room temperature. Prior to isolation, the Schiff bases were reduced with sodium cyanoborohydride in methanol for 30 min at 4°C. The reaction products were characterized by normal-phase high-performance liquid chromatography and on-line mass spectrometry with electrospray ionization. The amino acids and aminophospholipids yielded single adducts. A double adduct was obtained for myoglobin, which theoretically could have accepted up to 23 PC-Ald groups. The yields of the products ranged from 12 to 44% for the aminophospholipids and from 15–57% for the amino acids, while the Schiff base of the myoglobin was estimated at 5% level. The new compounds are used as reference standards for the detection of high molecular weight Schiff bases in lipid extracts of natural products. Based on presentation at the AOCS Annual Meeting & Expo in Indianapolis, Indiana, April 28–May 1, 1996.     

Covalent binding of peroxidized phospholipid to protein: III. Reaction of individual phospholipids with different proteins

Various peroxidized phospholipids were reacted with proteins under N₂. In all cases, phospholipid is bound covalently to the proteins whose molecular size is increased. Both the amount of bound phospholipid and the increase in molecular size of the protein depends on the nature of the phospholipid. Ultraviolet (UV) absorption of the proteins is increased in qualitatively similar ways. Their difference spectra, which show a gradual increase in absorption from 400 nm toward shorter wavelength, differ from that of malonaldehyde-protein complexes. The various complexes of proteins and peroxidized phospholipids have similar fluorescence spectra showing two excitation maxima at 310–320 nm and at 340–350 nm, respectively, and emission maximum at ca. 400 nm. This is different from both fluorescence spectra of malonaldehyde-protein complexes and fluorescence spectra reported for proteins after reaction with peroxidized polyunsaturated fatty acids. Amino groups of the proteins are consumed in the reaction with peroxidized phospholipids. Blocking the amino groups decreases the binding of phospholipid considerably. Besides amino groups, other structures of the protein molecule react with the peroxidized phospholipids. The similar features of UV absorption, fluorescence, decrease of amino groups, and covalently bound phospholipid phosphorus of the various complexes suggest that they are formed by common type of reactions. The reactions seem to be different from those generally believed important between peroxidized lipid and protein. Important reacting species are compounds other than malonaldehyde. Preliminary report of this work was presented at the ISF/AOCS World Congress, New York, 1980.     

Effect of pH on a gossypol complex with phospholipids: Gossypol-PE

PE, one of the major phospholipids in oilseed soapstock, may react with gossypol to form Schiff bases. PE amounts to 20–30% of the phosphorus compounds in soap-stock. In this report, the dependence on pH of the Schiff base products between PE and gossypol was investigated using a spectrophotometer, an HPLC equipped with an ELSD, and an LC-MS system. We observed that at pH 7 the Schiff reaction product and reactants were clearly detected by ELSD, absorption, and LC-MS spectra. The absorption spectra displayed the characteristic peak for the Schiff bases around 430–440 nm. The absorption spectra also indicated that the reaction was pH dependent. The reaction temperatures were 60 and 90°C. The LC-MS spectra supported the formation of Schiff bases as well as methyl ether derivatives of gossypol in alcohol at the elevated temperatures. The implications of these experimental findings are presented in this paper.     

Non-Enzymatic Browning Reactions of Phospholipids

The formation and properties of the fluorescent compounds originated in the browning reactions of phospholipids have been investigated. The compounds exhibited fluorescence with excitation maximum 345–350 nm and emission maximum at 420–430 nm, which were similar to those of lipofuscin and fluorescent substances derived from the reaction of oxidized fatty acids with glutathione. The fluorescence of the compound was affected neither with the pH 2–11 nor by treatment with sodium borohydride. The fluorescent compounds formed in the browning reaction of peroxidized phosphatidylethanolamine are produced, mainly, by interaction of the amine group of phosphatidylethanolamine and saturated aldehydes produced through the decomposition of fatty acid hydroperoxides of phosphatidylethanolamine. Phospholipids do not interact per se with bovine serum albumin, and their interaction is similar to that of polyunsaturated fatty acid hydroperoxides: the phospholipids give secondary products (aldehydes) that subsequently react with protein.     

Synthesis and characterization of poly(p-phenylene vinylene) polymers containing the quinoxaline group

Summary Novel poly (p-phenylenevinylene) polymers containing the quinoxaline group were prepared by the Horner-Wadsworth-Emmons reaction of the various diphosphonic acid diethyl ester with a dialdehyde monomer. The spectral properties of products so obtained were characterized by UV-visible and fluorescence spectroscopy. The UV-visible absorbance of these polymers showed absorption bands at ca. 432∼440 nm, which corresponding to the π-π* transition of the conjugated system. Their maximum fluorescence appeared at ca. 488 ∼ 498 nm. The resulting polymers showed greenish blue emission in solution and an orange emission in solid state. Received: 13 December 2002/Revised version: 27 February 2003/ Accepted: 1 March 2003 Correspondence to Jae Yun Jaung     

Quantitative determination of organic solvent soluble lipofuscin pigments in tissues

Lipofuscin pigment determination in tissue extracts was quantitated by the use of its property of fluorescence. Chloroform: methanol tissue extracts were purified on Sephadex LH-20 columns before quantitative fluorescence measurements of the lipofuscin pigments. Interfering compounds separated by chromatography were retinol and a lower mol wt fluorescent compound. Irradiation of tissue extracts with ultraviolet light was not sufficient to eliminate the interference caused by retinol and the lower mol wt compound. Purified lipofuscin pigments from blood, lung, liver, spleen, brain, heart, and kidney tissues demonstrated distinct fluorescent emission maximum at 435 nm and excitation maxima between 345 and 350 nm. Paper No. 9370, Scientific Journal Series Article, Minnesota Agricultural Experiment Station, St. Paul.     

Optimization and validation of high-performance liquid chromatographic method for the determination of dowtherm ATM in edible oils and oleochemicals

A method using high-performance liquid chromatography and fluorescence detection is optimized and validated for the determination of Dowtherm A^TM in spiked oleochemicals and edible oils. The samples are directly injected into a reversed-phase C18 column, and Dowtherm A is detected using a fluorescence detector set at 247 nm excitation and 310 nm emission wavelengths. The simple isocratic mobile phase used is a mixture of methanol and water (90∶10, vol/vol) at a flow rate of 1 mL/min. The limits of quantitation are from 0.1 to 0.2 μg/g. Mean recoveries ranged from 93.0 to 116% with reproducibilities of 1.29–3.84%. The procedure provides a simple, reliable and sensitive method for determining Dowtherm A residue in oleochemicals and edible oils without prior sample cleanup or extraction.     

Preparation and fractionation of conjugated trienes from α-linolenic acid and their growth-inhibitory effects on human tumor cells and fibroblasts

Conjugated α-linolenic acid (ClnA) was prepared from α-linolenic acid (9,12,15–18∶3n−3, LnA) by alkaline treatment; we fractionated CLnA into three peaks by reversed-phase column-HPLC as evidenced by monitoring absorbance at 205, 235, and 268 nm. Peak I was a conjugated dienoic FA derived from LnA, whereas Peaks II and III were conjugated trienoic LnA. Proton NMR analysis showed that Peak III consisted of the all-trans isomer. The methylated Peak III was further divided into five peaks (Peaks IV–VIII) by silver ion column-HPLC. Peak V, a major constituent in the Peak III fraction, was identified as conjugated 10t,12t,14t-LnA by GC-EIMS and ¹H NMR analysis. Peaks III and V, which consisted of conjugated all-trans trienoic LnA, had stronger growth-inhibitory effects on human tumor cell lines than the other collected peaks and strongly induced lipid peroxidation as compared with Peaks I, II, and LnA. We propose that conjugated all-trans trienoic FA have the strongest growth-inhibitory effect among the conjugated trienoic acids and conjugated dienoic acids produced by alkaline treatment of α-LnA, and that this effect is mediated by lipid peroxidation.     

Assessment of quality changes in frozen sardine (Sardina pilchardus) by fluorescence detection

The formation of fluorescent compounds was tested as a quality assessment during the frozen storage of sardine at −18°C (up to 24 mon) and at −10°C (up to 120 d). The fluorescence ratio between two excitation/emission maxima (393/463 and 327/415 nm) was studied in the aqueous (δF _aq) and organic (δF _or) extracts after Bligh and Dyer extraction of the white muscle. Fluorescence results were compared to common quality indices [total volatile base-nitrogen (TVB-N); conjugated dienes; thiobarbituric acid index (TBA-i); and free fatty acids (FFA)]. δF _aq showed good correlations with storage time (r=0.80 and r=0.72 at −18 and −10°C, respectively) and TBA-i (r=0.92 and r=0.81). Principal-component analysis grouped δF _aq with quality indices that are sensitive for the assessment of fish damage during frozen storage at both temperatures (TBA-i and FFA at −18°C; BVT-N, TBA-i, and FFA at −10°C). According to these results, fluorescence detection of interaction compounds in the aqueous phase can provide an accurate method to assess quality differences during frozen storage of fish.     

基于苝酰亚胺席夫碱荧光探针的制备及其对镉离子的选择性识别研究

利用C=N异构机制,以苝酰亚胺为骨架设计合成了一种香草醛席夫碱荧光探针,通过红外光谱和质谱对其结构进行了表征。同时利用紫外-可见光谱仪和荧光光谱仪对其性能进行了研究,研究结果表明,当使用490 nm的激发波长对该化合物进行激发时,化合物对Cd^2+表现出良好的选择性,在530 nm处的荧光发射峰强度显著上升。干扰实验表明,Zn^2+、Cu^2+和Hg^2+对Cd^2+的识别存在干扰,大多数金属离子对识别过程影响不大。荧光实验数据分析结果表明两者之间形成了1∶1的键合关系,键合常数lgK为5.37。该荧光探针对Cd^2+的检出限为0.2 nmol/L(约22.8 ng/L),可以满足水体中镉离子的检测要求。