Increases in cholesterol 7-hydroperoxides in lipids of human skin by sunlight exposure.

Free and ester forms of cholesterol 7alpha- and 7beta-hydroperoxides (Ch 7-OOHs) in skin lipids of humans were separated and determined by high performance liquid chromatography with a chemiluminescence detector. We first demonstrated the presence of Ch 7-OOHs in lipids of human skin. The levels of Ch 7-OOHs found in skin lipids of healthy Japanese volunteers (n = 5) ranged from 2.78 to 25.2 pmol/cm2 skin, indicating large inter-individual differences. However, the intra-individual differences of Ch 7-OOHs levels in skin lipids between right and left arms were less than 25% (-16.4% to 24.0%). Inter-day differences of Ch 7-OOHs in 5 subjects at 1 week interval were also small (-36.7% to 47.7%). Additionally, we investigated effects of sunlight exposure on the levels of Ch 7-OOHs in skin lipids of healthy Japanese volunteers (n = 24). The levels of Ch 7-OOHs in skin lipids significantly increased from 10.0+/-6.7 to 38.9+/-38.0 pmol/cm2 skin by sunlight exposure (10-40 mJ/cm2/min) for 3 h. Therefore, natural sunlight exposure causes lipid peroxidation in skin lipids of humans. These results suggest that the level of Ch 7-OOHs is a good marker for lipid peroxidation in human skin.     

Lipid oxidation in the skin

Abstract

Skin is the largest organ of the body and exerts several physiological functions such as a protective barrier against moisture loss and noxious agents including ultraviolet irradiation. Oxidation of skin may impair such functions and induce skin disorders including photoaging and skin cancer. Skin surface lipids, a mixture of sebaceous and epidermal lipids, have unique species and fatty acid profile. The major unsaturated lipids are squalene, sebaleic aicd, linoleic acid, and cholesterol. Singlet oxygen and ozone as well as free radicals and enzymes are important oxidants for skin lipids. Squalene is the major target for singlet oxygen, giving rise to twelve regio-isomeric squalene hydroperoxides. Ultraviolet radiation activates lipoxygenase and cyclooxygenase, inducing specific enzymatic oxidation of lipids. Free radical mediated lipid peroxidation gives multiple oxidation products. Lipid oxidation products produced by these mechanisms are observed in human skin and induce various skin diseases, but in contrast to plasma and other tissues, identification and quantitative measurement of lipid oxidation products in skin are scarce and should be the subjects of future studies.     

LC-MS/MS analysis of carboxymethylated and carboxyethylated phosphatidylethanolamines in human erythrocytes and blood plasma

An amino group of phosphatidylethanolamine (PE) is considered as a target for nonenzymatic glycation, and the potential involvement of lipid glycation in the pathogenesis of diabetic complications has generated interest. However, unlike an early glycation product of PE (Amadori-PE), the occurrence and roles of advanced glycation end products of PE (AGE-PE) in vivo have been unclear. Here, we developed an LC-MS/MS method for the analysis of AGE-PE [carboxymethyl-PE (CM-PE) and carboxyethyl-PE (CE-PE)]. Collision-induced dissociation of CM-PE and CE-PE produced characteristic ions, permitting neutral loss scanning (NLS) and multiple reaction monitoring (MRM) of AGE-PE. By NLS analysis, a series of AGE-PE molecular species was detected in human erythrocytes and blood plasma. In LC-MS/MS analysis, MRM enabled the separation and determination of the predominant AGE-PE species. Between healthy subjects and diabetic patients, no significant differences were observed in AGE-PE concentrations in erythrocytes and plasma, whereas Amadori-PE concentrations were higher in diabetic patients. These results provide direct evidence for the presence of AGE-PE in human blood, and indicated that, compared with Amadori-PE, AGE-PE is less likely to be accumulated in diabetic blood. The presently developed LC-MS/MS method appears to be a powerful tool for understanding in vivo lipid glycation and its pathophysiological consequence.     

基于液质联用的莱茵衣藻极性甘油酯组定性定量分析

以模式藻株莱茵衣藻(Chlamydomonas reinhardtii)为材料, 基于液质联用技术对其极性甘油酯组进行定性定量分析。通过综合利用UPLC-ESI-Q-Trap/MS的一级质谱扫描(中性丢失或母离子扫描)及UPLC-ESI-Orbitrap/MS²的二级碎片信息扫描, 共鉴定出109种极性甘油酯分子; 再通过外标法利用UPLC-ESI-Q-Trap/MS在多级反应监测模式下对各分子进行靶向定量分析。结果表明, 莱茵衣藻的极性脂以糖脂MGDG、DGDG及甜菜碱脂DGTS为主, 所有极性脂的分子组成表明, DGDG、SQDG、DGTS及PI是C18脂肪酸的去饱和载体。该研究利用液质联用技术建立了莱茵衣藻极性甘油酯组的结构图谱及定量分析技术平台, 为微藻极性脂生物学功能及脂质代谢研究奠定了基础。     

An approach towards understanding the genesis of sunlight-induced skin cancer

The molecular basis of the sunlight-induced skin carcinogenesis has been elucidated. Of the two ultraviolet components of sunlight that reach the earth's surface the UV-B is known to be carcinogenic but the mode of action of UV-A, the predominant component of sunlight, is ill understood. Using the liposomes as a model system, it has been shown here that UV-A causes dose-dependent lipid peroxidation as estimated by measurements of conjugated dienes, lipid hydroperoxides, malondialdehydes and the fluorescent adducts (Schiff bases) produced by the reaction of MDA with glycine. Direct exposure to sunlight has also been shown to cause dose-dependent lipid peroxidation. The UV-A induced lipid peroxidation has also been shown to be dependent on dose rate. While the sodium formate, dimethyl sulphoxide, superoxide dismutase and EDTA do not have any significant effect, sodium azide, histidine, beta-carotene and dimethylfuran were shown to inhibit significantly the UV-A induced lipid peroxidation, thereby providing significant evidence of the involvement of singlet oxygen (1O2) as the initiating agent. The use of D2O in place of H2O as the liposome dispersing medium enhanced to great extent the UV-A induced lipid peroxidation, thereby lending additional support to the finding that singlet oxygen was the initiating agent. The possible mode of formation of 1O2 on exposure to UV-A was discussed. This study also highlighted the role of environmental factors on the sunlight-induced cutaneous damage. Finally, the relation between lipid peroxidation, DNA damage and carcinogenesis has been discussed in a way to suggest the possible link between sunlight exposure and causation of skin cancer.     

Ultraviolet-radiation induced skin inflammation: dissecting the role of bioactive lipids

Acute exposure of human skin to the ultraviolet radiation (UVR) in sunlight results in the sunburn response. This is mediated in part by pro-inflammatory eicosanoids and other bioactive lipids, which are in turn produced via mechanisms including UVR-induction of oxidative stress, cell signalling and gene expression. Sunburn is a self-limiting inflammation offering a convenient and accessible system for the study of human cutaneous lipid metabolism. Recent lipidomic applications have revealed that a wider diversity of eicosanoids may be involved in the sunburn response than previously appreciated. This article reviews the effects of UVR on cutaneous lipids and examines the contribution of bioactive lipid mediators in the development of sunburn. Since human skin is an active site of polyunsaturated fatty acid (PUFA) metabolism, and these macronutrients can influence the production of eicosanoids/bioactive lipids, as well as modulate cell signalling, gene expression and oxidative stress, the application of PUFA as potential photoprotective agents is also considered.     

High-temperature gas chromatography-mass spectrometry for skin surface lipids profiling

Skin surface lipids (SSLs) arising from both sebaceous glands and skin removal form a complex lipid mixture composed of free fatty acids and neutral lipids. High-temperature gas chromatography coupled with electron impact or chemical ionization mass spectrometry was used to achieve a simple analytical protocol, without prior separation in classes and without prior cleavage of lipid molecules, in order to obtain simultaneously i) a qualitative characterization of the individual SSLs and ii) a quantitative evaluation of lipid classes. The method was first optimized with SSLs collected from the forehead of a volunteer. More than 200 compounds were identified in the same run. These compounds have been classified in five lipid classes: free fatty acids, hydrocarbons, waxes, sterols, and glycerides. The advantage to this method was it provided structural information on intact compounds, which is new for cholesteryl esters and glycerides, and to obtain detailed fingerprints of the major SSLs. These fingerprints were used to compare the SSL compositions from different body areas. The squalene/cholesterol ratio was used to determine the balance between sebaceous secretion and skin removal. This method could be of general interest in fields where complex lipid mixtures are involved.     

An improved assay for platelet-activating factor using HPLC-tandem mass spectrometry

We describe an improved assay for platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) using HPLC-tandem mass spectrometry (LC-MS/MS). The present method can readily detect as little as 1 pg (1.9 fmol) of PAF, a significant improvement over previously described LC-MS/MS methods, and gives a linear response up to 1,000 pg of PAF. Our method also overcomes the artifacts from isobaric lipids that have limited the usefulness of certain existing LC-MS/MS assays for PAF. In the course of these studies, we detected three novel lipid species in human neutrophils. One of the novel lipids appears to be a new molecular species of PAF, and the other two have chromatographic and mass spectrometric properties consistent with stearoyl-formyl-glycerophosphocholine and oleoyl-formyl-glycerophosphocholine. These observations identify previously unknown potential interferences in the measurement of PAF by LC-MS/MS. Moreover, our data suggest that the previously described palmitoyl-formyl-glycerophosphocholine is not unique but rather is a member of a new and poorly understood family of formylated lipids.     

Involvement of lipid peroxidation and organic peroxides in UVA-induced matrix metalloproteinase-1 expression

Ultraviolet A (UVA) irradiation causes human skin aging and skin cancer at least partially through the activation of matrix metalloproteinases (MMPs). MMP-1, the interstitial collagenase, is responsible for the degradation of collagen and is involved in tumor progression in human skin. The present study uses human skin fibroblast cells (FEK4) to investigate the involvement of lipid peroxidation and the role of peroxides as possible mediators in MMP-1 activation by UVA. Preincubation with the antioxidants butylated hydroxytoluene and Trolox reduced UVA-dependent MMP-1 upregulation, suggesting that peroxidation of membrane lipids is involved. Blocking the iron-driven generation of lipid peroxides and hydroxyl radicals by different iron chelators led to a decrease in UVA-induced MMP-1 mRNA accumulation. Moreover, modulation of glutathione peroxidase activity by use of the specific inhibitor mercaptosuccinate (MS) or by the depletion of glutathione (using buthionine-S, R-sulfoximine, BSO), enhanced the UVA-dependent MMP-1 response. Finally, UVA irradiation generated a significant increase in intracellular peroxide levels which is augmented by pretreatment of the cells with BSO or MS. Our results demonstrate that lipid peroxidation and the production of peroxides are important events in the signalling pathway of MMP-1 activation by UVA.     

Application of the accurate mass and time tag approach in studies of the human blood lipidome

We report a preliminary demonstration of the accurate mass and time (AMT) tag approach for lipidomics. Initial data-dependent LC-MS/MS analyses of human plasma, erythrocyte, and lymphocyte lipids were performed in order to identify lipid molecular species in conjunction with complementary accurate mass and isotopic distribution information. Identified lipids were used to populate initial lipid AMT tag databases containing 250 and 45 entries for those species detected in positive and negative electrospray ionization (ESI) modes, respectively. The positive ESI database was then utilized to identify human plasma, erythrocyte, and lymphocyte lipids in high-throughput LC-MS analyses based on the AMT tag approach. We were able to define the lipid profiles of human plasma, erythrocytes, and lymphocytes based on qualitative and quantitative differences in lipid abundance.     

A systematic method for the sensitive and specific determination of hair lipids in combination with chromatography

A systematic method for the sensitive, precise and accurate determination of hair lipids, including trace amounts of intrinsic endogenous cholesterol (CH), ceramide/N-palmitoyl-DL-dihydrosphingosine (CER/PDS), cholesterol sulfate (CS) and chemically bound 18-methyl eicosanoic acid (18-MEA), has been developed in combination with TLC/FID (flame ionization detection), LC/MS and GC/MS. TLC/FID was used for the simultaneous determination of squalene (SQ), wax esters (WEs), triglycerides (TGs) and free fatty acids (FFAs). Optimal conditions for LC/MS to determine CS and 18-MEA were developed using selected ion monitoring (SIM) under the negative ion mode of electrospray ionization. An alternative procedure for the determination of 18-MEA was also established using commercially available heneicosanoic acid (HEA). In GC/MS, the optimal selection of ions for SIM of trimethylsilylated CH and CER/PDS, and the use of on-column injection has enabled their simultaneous detection. This newly developed method has been used to characterize the hair lipid composition from the proximal root end to the distal tip of chemically untreated hair fibers from two different females, and specific changes of hair lipids probably due to its origin and individuals have been demonstrated for the first time. This method may be useful for clarifying the important roles of intrinsic endogenous 18-MEA, CS, CH and CERs in the function of the cell membrane complex of hair fibers.     

Quantitative analysis of thymosin alpha1 in human serum by LC-MS/MS

A high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed to measure the thymosin alpha 1 (Talpha1) concentration in human serum. Tá1 in human serum was determined by solid phase extraction and reverse phase LC-MS/MS. The high-performance liquid chromatography (HPLC) system interfaced with the MS/MS system with a Turbo Ion spray interface. Positive ion detection and multiple reaction monitoring (MRM) mode were used for this human serum quantitation. Eight different concentration standards were used to establish the detection range. Six quality control (QC) and 2 matrix blanks were checked by calibration curves performed on the same day. The lower quantitation limit was 0.5 ng/mL Talpha1 in human serum. Calibration curves were established between 0.5 to 100 ng/mL by weighted linear regression. The correlation coefficients for different days were 0.9955 or greater. Quantitation of Talpha1 by the LC-MS/MS method is fast, accurate, and precise.     

The repair of DNA damages induced in normal human skin fibroblasts exposed to simulated sunlight

The induction and repair of DNA damages produced by exposure of normal human skin fibroblasts to the simulated sunlight produced by a solar simulator were examined. The photoproducts measured were pyrimidine dimers, E. coli endonuclease III-sensitive sites, 6-4 photoproducts, Dewar isomers, DNA-protein crosslinks, and DNA single-strand breaks. The results of these experiments serve to form a basis for the quantitation of damages induced by exposure to sunlight.     

Quantitative analysis of thymosin α1 in human serum by LC-MS/MS

1 high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed to measure the thymosin alpha 1 (Tα1) concentration in human serum. Tα1 in human serum was determined by solid phase extraction and reverse phase LC-MS/MS. The high-performance liquid chromatography (HPLC) system interfaced with the MS/MS system with a Turbo Ion spray interface. Positive ion detection and multiple reaction monitoring (MRM) mode were used for this human serum quantitation. Eight different concentration standards were used to establish the detection range. Six quality control (QC) and 2 matrix blanks were checked by calibration curves performed on the same day. The lower quantitation limit was 0.5 ng/mL T α1 in human serum. Calibration curves were established between 0.5 to 100 ng/mL by weighted linear regression. The correlation coefficients for different days were 0.9955 or greater. Quantitation of Tα1 by the LC-MS/MS method is fast accurate, and precise.     

Combined LC/MS-platform for analysis of all major stratum corneum lipids,and the profiling of skin substitutes

Ceramides (CERs), cholesterol, and free fatty acids (FFAs) are the main lipid classes in human stratum corneum (SC, outermost skin layer), but no studies report on the detailed analysis of these classes in a single platform. The primary aims of this study were to 1) develop an LC/MS method for (semi-)quantitative analysis of all main lipid classes present in human SC; and 2) use this method to study in detail the lipid profiles of human skin substitutes and compare them to human SC lipids. By applying two injections of 10 μl, the developed method detects all major SC lipids using RPLC and negative ion mode APCI-MS for detection of FFAs, and NPLC using positive ion mode APCI-MS to analyze CERs and cholesterol. Validation showed this lipid platform to be robust, reproducible, sensitive, and fast. The method was successfully applied on ex vivo human SC, human SC obtained from tape strips and human skin substitutes (porcine SC and human skin equivalents). In conjunction with FFA profiles, clear differences in CER profiles were observed between these different SC sources. Human skin equivalents more closely mimic the lipid composition of human stratum corneum than porcine skin does, although noticeable differences are still present. These differences gave biologically relevant information on some of the enzymes that are probably involved in SC lipid processing. For future research, this provides an excellent method for (semi-)quantitative, ‘high-throughput’ profiling of SC lipids and can be used to advance the understanding of skin lipids and the biological processes involved.     

Leukocytes utilize myeloperoxidase-generated nitrating intermediates as physiological catalysts for the generation of biologically active oxidized lipids and sterols in serum

The initiation of lipid peroxidation and the concomitant formation of biologically active oxidized lipids and sterols is believed to play a central role in the pathogenesis of inflammatory and vascular disorders. Here we explore the role of neutrophil- and myeloperoxidase (MPO)-generated nitrating intermediates as a physiological catalyst for the initiation of lipid peroxidation and the formation of biologically active oxidized lipids and sterols. Activation of human neutrophils in media containing physiologically relevant levels of nitrite (NO(2)(-)), a major end product of nitric oxide (nitrogen monoxide, NO) metabolism, generated an oxidant capable of initiating peroxidation of lipids. Formation of hydroxy- and hydroperoxyoctadecadienoic acids [H(P)ODEs], hydroxy- and hydroperoxyeicosatetraenoic acids [H(P)ETEs], F(2)-isoprostanes, and a variety of oxysterols was confirmed using on-line reverse phase HPLC tandem mass spectrometry (LC/MS/MS). Lipid oxidation by neutrophils required cell activation and NO(2)(-), occurred in the presence of metal chelators and superoxide dismutase, and was inhibited by catalase, heme poisons, and free radical scavengers. LC/MS/MS studies demonstrated formation of additional biologically active lipid and sterol oxidation products known to be enriched in vascular lesions, such as 1-hexadecanoyl-2-oxovalaryl-sn-glycero-3-phosphocholine, which induces upregulation of endothelial cell adhesion and chemoattractant proteins, and 5-cholesten-3beta-ol 7beta-hydroperoxide, a potent cytotoxic oxysterol. In contrast to the oxidant formed during free metal ion-catalyzed reactions, the oxidant formed during MPO-catalyzed oxidation of NO(2)(-) readily promoted lipid peroxidation in the presence of serum constituents. Collectively, these results suggest that phagocytes may employ MPO-generated reactive nitrogen intermediates as a physiological pathway for initiating lipid peroxidation and forming biologically active lipid and sterol oxidation products in vivo.     

Hydroxyl radical formation by UV-irradiated epidermal cells.

To elucidate the mechanism of sunlight-induced skin damage, guinea pigs were exposed to UV light (280-320 nm, UV B, 4 J/cm2) and a homogenate of the epidermis was examined by means of the thiobarbituric acid (TBA) test. Three hours after the exposure, TBA-malondialdehyde adducts had increased while glutathione reductase activity had decreased, indicating lipid peroxidation. To detect the initial species, spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was applied to a suspension of illuminated epidermal cells (0.5 J/cm2). An ESR signal obtained only with irradiation comprised a 1:2:2:1 quartet [a(N)= a(beta H) = 1.49 mT] attributable to a spin adduct of hydroxyl radicals. These results suggest that sunlight exposure of skin may lead to hydroxyl radical generation and simultaneous lipid peroxidation.     

Comprehensive analysis of the major lipid classes in sebum by rapid resolution high-performance liquid chromatography and electrospray mass spectrometry

Sebum is a complex lipid mixture that is synthesized in sebaceous glands and excreted on the skin surface. The purpose of this study was the comprehensive detection of the intact lipids that compose sebum. These lipids exist as a broad range of chemical structures and concentrations. Sebum was collected with SebuTape^TM from the foreheads of healthy donors, and then separated by HPLC on a C8 stationary phase with sub 2 µm particle size. This HPLC method provided high resolution and excellent reproducibility of retention times (RT). Compound mining was performed with time of flight (TOF) and triple quadrupole (QqQ) mass spectrometers (MS), which allowed for the classification of lipids according to their elemental composition, degree of unsaturation, and MS/MS fragmentation. The combination of the two MS systems detected 95 and 29 families of triacylglycerols (TAG) and diacylglycerols (DAG), respectively. Assignment was carried out regardless of positional isomerism. Among the wax esters (WE), 28 species were found to contain the 16:1 fatty acyl moiety. This method was suitable for the simultaneous detection of squalene and its oxygenated derivative. A total of 9 cholesterol esters (CE) were identified and more than 48 free fatty acids (FFA) were detected in normal sebum. The relative abundance of each individual lipid within its own chemical class was determined for 12 healthy donors. In summary, this method provided the first characterization of the features and distribution of intact components of the sebum lipidome.     

Simultaneous tissue profiling of eicosanoid and endocannabinoid lipid families in a rat model of osteoarthritis

We describe a novel LC method for the simultaneous and quantitative profiling of 43 oxylipins including eicosanoids, endocannabinoids, and structurally related bioactive lipids with modified acyl groups. The LC-MS/MS method uses switching at a defined time between negative and positive electrospray ionization modes to achieve optimal detection sensitivity for all the lipids. The validated method is linear over a range of 0.01–5 nmol/g (0.1–50 nmol/g for 2-arachidonoyl glycerol) with intra- and interday precision and accuracy between 1.38 and 26.76% and 85.22 and 114.3%, respectively. The method successfully quantified bioactive lipids in different tissue types in the rat, including spinal cord, dorsal root ganglia (DRGs), knee joint, brain, and plasma. Distinct regional differences in the pattern of lipid measured between tissue types were observed using principle component analysis. The method was applied to analyze tissue samples from an established preclinical rat model of osteoarthritis (OA) pain and showed that levels of 12-hydroxyeicosatetraenoic acid were significantly increased in the OA rat knee joint compared with controls, and that 15-hydroxyeicosatetraenoic acid was significantly increased in the DRGs in the model of OA compared with controls. The developed LC-MS/MS method has the potential to provide detailed pathway profiling in tissues and biofluids where the disruption of bioactive oxylipins may be involved in disease states.     

Lipid and lipid mediator profiling of human synovial fluid in rheumatoid arthritis patients by means of LC-MS/MS

Human synovial fluid (SF) provides nutrition and lubrication to the articular cartilage. Particularly in arthritic diseases, SF is extensively accumulating in the synovial junction. During the last decade lipids have attracted considerable attention as their role in the development and resolution of diseases became increasingly recognized. Here, we describe a capillary LC-MS/MS screening platform that was used for the untargeted screening of lipids present in human SF of rheumatoid arthritis (RA) patients. Using this platform we give a detailed overview of the lipids and lipid-derived mediators present in the SF of RA patients. Almost 70 different lipid components from distinct lipid classes were identified and quantification was achieved for the lysophosphatidylcholine and phosphatidylcholine species. In addition, we describe a targeted LC-MS/MS lipid mediator metabolomics strategy for the detection, identification and quantification of maresin 1, lipoxin A(4) and resolvin D5 in SF from RA patients. Additionally, we present the identification of 5S,12S-diHETE as a major marker of lipoxygenase pathway interactions in the investigated SF samples. These results are the first to provide a comprehensive approach to the identification and profiling of lipids and lipid mediators present in SF and to describe the presence of key anti-inflammatory and pro-resolving lipid mediators identified in SF from RA patients.