Noninvasive evaluation of the skin barrier in reconstructed human epidermis using speckle analysis: Correlation with Raman microspectroscopy.

BACKGROUND: Reconstructed epidermis models, obtained from 3D keratinocytes culture, have gained significant prominence as prototypes for safety and efficacy testing in skin research. To effectively evaluate these models, it is essential to perform molecular and functional characterization. The skin's barrier function is one of the essential aspects of the epidermis that needs to be assessed. A noninvasive method is thus required for the evaluation of the skin barrier in these models. With this perspective, the aim of this feasibility study is to apply the speckle technique for the assessment of the skin barrier in the Reconstructed Human Epidermis (RHE). MATERIALS AND METHODS: Speckle analysis as well as Raman microspectroscopy were performed on RHE samples at two maturation days, D17 and D20. RESULTS: Between D17 and D20, our study showed an increase in various Raman parameters, including stratum corneum percentage, lateral lipid packing, lipid-to-protein ratio, and protein secondary structure. Furthermore, the degree of light polarization and the speckle grain size also increased over this period. CONCLUSION: The speckle technique proved to be effective for evaluating the skin barrier in Reconstructed Human Epidermis (RHE) models. Comparison with Raman validates this approach and provides comprehensive molecular and functional characterization of reconstructive skin models.

Stratum corneum ceramide profiles in vitro, ex vivo, and in vivo: characterization of the α-hydroxy double esterified ceramides.

The presence of a new ceramide subclass, the 1-O-acyl omega-linoleoyloxy ceramides [1-O-E (EO) Cer], has been previously highlighted in reconstructed human epidermis (RHE). These ceramides are double esterified on two positions. The first is the 1-O position of the sphingoid base moiety with a long to very long chain of acyl residues (1-O-E), and the second is the position of the ω-hydroxyl group of the fatty acid moiety with linoleic acid (EO). Considering its chemical structure and hydrophobicity, this subclass can contribute to the skin barrier. Thus, it is important to determine whether this subclass is also present in native human stratum corneum (SC). This work compares ceramide structures of this novel subclass between RHE (in vitro) and two sources of human SC (in vivo and ex vivo) using normal-phase high-performance liquid chromatography coupled to high-resolution mass spectrometry (NP-HPLC/HR-MSn). The results confirm the presence of this double esterified ceramide subclass [1-O-E (EO) Cer] in human SC. The molecular profile obtained from the RHE was very close to that found in the human SC (in vivo and ex vivo). In addition, thanks to the targeted MS2/MS3 analysis, a new ceramide subclass was discovered and characterized in the three studied samples. We propose to name it [A-1-O-E (EO) Cer] because in these ceramides species, the fatty acid-esterified with the sphingoid base on the 1-O position-is hydroxylated on the α position. These results highlight the potential of both the analytical method and the characterization approach employed in this study.

Discriminative and quantitative analysis of norepinephrine and epinephrine by surface-enhanced Raman spectroscopy with gold nanoparticle suspensions.

Surface-enhanced Raman spectroscopy (SERS) is a powerful analytical technique capable of increasing the Raman signal of an analyte using specific nanostructures. The close contact between those nanostructures, usually a suspension of nanoparticles, and the molecule of interest produces an important exaltation of the intensity of the Raman signal. Even if the exaltation leads to an improvement of Raman spectroscopy sensitivity, the complexity of the SERS signal and the numbers of parameters to be controlled allow the use of SERS for detection rather than quantification. The aim of this study was to develop a robust discriminative and quantitative analysis in accordance with pharmaceutical standards. In this present work, we develop a discriminative and quantitative analysis based on the previous optimized parameters obtained by the design of experiments fixed for norepinephrine (NOR) and extended to epinephrine (EPI) which are two neurotransmitters with very similar structures. Studying the short evolution of the Raman signal intensity over time coupled with chemometric tools allowed the identification of outliers and their removal from the data set. The discriminant analysis showed an excellent separation of EPI and NOR. The comparative analysis of the data showed the superiority of the multivariate analysis after logarithmic transformation. The quantitative analysis allowed the development of robust quantification models from several gold nanoparticle batches with limits of quantification of 32 µg/mL for NOR and below 20 µg/mL for EPI even though no Raman signal is observable for such concentrations. This study improves SERS analysis over ultrasensitive detection for discrimination and quantification using a handheld Raman spectrometer.

Estimating the Analytical Performance of Raman Spectroscopy for Quantification of Active Ingredients in Human Stratum Corneum.

Confocal Raman microscopy (CRM) has become a versatile technique that can be applied routinely to monitor skin penetration of active molecules. In the present study, CRM coupled to multivariate analysis (namely PLSR-partial least squares regression) is used for the quantitative measurement of an active ingredient (AI) applied to isolated (ex vivo) human stratum corneum (SC), using systematically varied doses of resorcinol, as model compound, and the performance is quantified according to key figures of merit defined by regulatory bodies (ICH, FDA, and EMA). A methodology is thus demonstrated to establish the limit of detection (LOD), precision, accuracy, sensitivity (SEN), and selectivity (SEL) of the technique, and the performance according to these key figures of merit is compared to that of similar established methodologies, based on studies available in literature. First, principal components analysis (PCA) was used to examine the variability within the spectral data set collected. Second, ratios calculated from the area under the curve (AUC) of characteristic resorcinol and proteins/lipids bands (1400-1500 cm-1) were used to perform linear regression analysis of the Raman spectra. Third, cross-validated PLSR analysis was applied to perform quantitative analysis in the fingerprint region. The AUC results show clearly that the intensities of Raman features in the spectra collected are linearly correlated to resorcinol concentrations in the SC (R2 = 0.999) despite a heterogeneity in the distribution of the active molecule in the samples. The Root Mean Square Error of Cross-Validation (RMSECV) (0.017 mg resorcinol/mg SC), The Root Mean Square of Prediction (RMSEP) (0.015 mg resorcinol/mg SC), and R2 (0.971) demonstrate the reliability of the linear regression constructed, enabling accurate quantification of resorcinol. Furthermore, the results have enabled the determination, for the first time, of numerical criteria to estimate analytical performances of CRM, including LOD, precision using bias corrected mean square error prediction (BCMSEP), sensitivity, and selectivity, for quantification of the performance of the analytical technique. This is one step further towards demonstrating that Raman spectroscopy complies with international guidelines and to establishing the technique as a reference and approved tool for permeation studies.

Comprehensive characterization of the structure and properties of human stratum corneum relating to barrier function and skin hydration: modulation by a moisturizer formulation.

The stratum corneum (SC) is key in the maintenance of the biomechanical barrier and hydration of skin. Our previous investigations showed beneficial effects of a combination of emollients on water capture and retention and protein and lipid organization, all of which are linked to dryness and dry skin damage. Here, we show how a formulation containing an emollient combination ("Trio") and its basal formulation (placebo) impacted the descriptors of SC hydration in SC layers. Only the Trio formulation-not its placebo formulation-modified SC biomechanical drying stress behaviour and imparted a high capacity to protect it from dehydration. This was in accordance with findings at the molecular level using Raman analyses and at the structural level using cryo-scanning electron microscopy (SEM). After topical application, only the Trio formulation profoundly increased lateral packing of lipids and their compactness. Cryo-SEM showed that, unlike the placebo formulation, the Trio formulation prevented the water loss when applied before the dehydration process. In conclusion, these studies demonstrate that stresses in the SC due to dehydration can be alleviated using a formulation containing emollients that interact with the SC lipid components.

Biomolecular modifications during keratinocyte differentiation: Raman spectroscopy and chromatographic techniques.

From the basal layer until the stratum corneum, lipid and protein biomarkers associated with morphological changes denote keratinocyte differentiation and characterize each epidermis layer. Herein, we followed keratinocyte differentiation in the early stages using HaCaT cells over a period of two weeks by two complementary analytical techniques: Raman microspectroscopy and high-performance liquid chromatography coupled with high resolution mass spectrometry. A high concentration of calcium in the medium induced HaCaT cell differentiation in vitro. The results from both techniques underlined the keratinocyte passage from the granular layer (day 9) to the stratum corneum layer (day 13). After 13 days of differentiation, we observed a strong increase in the lipid content, decrease in proteins, decrease in DNA, and a decrease in glucosylceramides/ceramides and sphingomyelins/ceramides ratios.

Assessment of the skin barrier function in the reconstructed human epidermis using a multimodal approach at molecular, tissue and functional levels.

Reconstructed human epidermis models are used as epidermis alternatives in skin research studies. It is necessary to provide molecular and functional characterization in order to assess these models. Our aim is to establish a link between the barrier function and the structure and composition of the stratum corneum using several complementary techniques. The following three studies were performed on reconstructed human epidermis during the keratinocyte differentiation process: (i) caffeine percutaneous penetration kinetics, (ii) epidermis thickness measurement, stratum corneum formation and lipid organization by Raman microspectroscopy and (iii) lipid composition evolution by liquid chromatography coupled to high-resolution mass spectrometry. The results demonstrated that the caffeine penetration decreased along the differentiation process. Raman in-depth images demonstrated an increase in stratum corneum and RHE thickness accompanied by the evolution of lipid organization. Lipid analysis showed an increase of the ceramide amount and an inverse relationship between ceramide and its precursor levels during the differentiation process. Different behaviors between several ceramide subclasses are highlighted and they relied on the corresponding differentiation stages. The generation of the most important ceramides for the barrier function is closely followed. A period shift between lipid generation and their organization was found. Our analytical data allowed identifying the following 3 groups of maturation days: before day 15, between days 15 and 19, and after day 19. The chemical and physiological states of the barrier function for each group are described thanks to a multimodal approach.

Comprehensive characterization and simultaneous analysis of overall lipids in reconstructed human epidermis using NPLC/HR-MSn: 1-O-E (EO) Cer, a new ceramide subclass.

Stratum corneum lipids are responsible for the skin's barrier function. They are the final product of epidermis lipid biosynthesis. During this process, lipids evolve from simple to complex structures in three main levels respectively (stratum basal level, stratum granulosum level, and stratum corneum level). Our aim was to simultaneously analyze and characterize the structure of total epidermis lipids. A powerful analytical method (normal-phase liquid chromatography coupled with high-resolution mass spectrometry (NPLC/HR-MSn)) was developed in order to separate, in a single run, lipid classes with a wide polarity range. Chromatographic conditions were particularly designed to analyze lipids of intermediate polarity such as ceramides. Rich information was obtained about the molecular structure of keratinocyte differentiation biomarkers such as ceramides, glucosylceramides, and sphingomyelins and the microstructures of reconstructed human epidermis lipids using HR-MSn. A new subclass of ceramides, 1-O-Acyl Omega-linoleoyloxy ceramides [1-O-E (EO) Cer] has been highlighted. This class is double esterified on the 1-O-position of sphingoid base with long to very long chain acyl residues (1-O-E) and on the position of ω-hydroxyl group of fatty acid with the linolenic acid (EO). Considering its chemical structure and hydrophobicity, this subclass can contribute to the skin barrier. In addition, we detected a new epidermis sphingomyelins. Our lipidomic approach offers a direct access to epidermis biomarkers.

Measurement of the biomechanical function and structure of ex vivo drying skin using raman spectral analysis and its modulation with emollient mixtures.

An important aspect of the biomechanical behaviour of the stratum corneum (SC) is the drying stresses that develop with water loss. These stresses act as a driving force for damage in the form of chapping and cracking. Betasitosterol is a plant sterol with a structure similar to cholesterol, a key component in the intercellular lipids of the outermost layer of human skin, the SC. Cholesterol plays an important role in stabilizing the SC lipid structure, and altered levels of cholesterol have been linked with SC barrier abnormalities. Betasitosterol is currently applied topically to skin for treatment of wounds and burns. However, it is unknown what effect betasitosterol has on the biomechanical barrier function of skin. Here, by analysing the drying stress profile of SC generated during a kinetics of dehydration, we show that betasitosterol, in combination with two emollient molecules, isocetyl stearoyl stearate (ISS) and glyceryl tri-2-ethylhexanoate (GTEH), causes a significant modulation of the drying stress behaviour of the SC by reducing both the maximal peak stress height and average plateau of the drying stress profile. Raman spectra analyses demonstrate that the combination of betasitosterol with the two emollients, ISS and GTEH, allows a high water retention capacity within the SC, while the lipid conformational order by increasing the amount of trans conformers. Our study highlights the advantage of combining a biomechanical approach together with Raman spectroscopy in engineering a suitable combination of molecules for alleviating dryness and dry skin damage.

Clustering-based preprocessing method for lipidomic data analysis: application for the evolution of newborn skin surface lipids from birth until 6 months.

After life in utero and birth, the skin is submitted to an important process of adaptation to a relatively dry gaseous environment. Skin surface lipids (SSLs) contribute actively to the protection of the skin barrier. Within this context, our objective was to study the evolution of each lipid compound during the postnatal period. SSLs were collected from six newborns a few days after birth until the age of 6 months. Seventy samples were analyzed using high-temperature gas chromatography coupled to mass spectrometry (HT-GC/MS). The use of separative techniques coupled to mass spectrometry for the analysis of samples containing complex mixtures of lipids generates a large volume of data which renders the results interpretation very difficult. In this study, we propose a new approach to handle the raw data, a clustering-based preprocessing method (CB-PPM), in order to achieve (1) volume reduction of data provided by each chromatogram without loss of information, (2) alignment of time retention shift between different runs, (3) clustering of mass spectra of the same molecule in one qualitative group, (4) and integration of all data into a single matrix to be explored by chemometric tools. This approach allowed us to gather data variations in 256 qualitative groups and therefore enabled us to highlight the variation of compounds including those of low intensity. Moreover, the representation of all data gathered in one matrix rendered reading of the results rapid and efficient. Thus, using this approach, we have demonstrated an increase of cholesterol esterification with epidermal fatty acids (C20 to C25) with age. This epidermis participation in SSL production at a molecular level in the first period of life has not been previously shown. These data can be very interesting for the development and improvement of products destined for the protection of infant skin. Graphical abstract ᅟ.

In vivo Raman Microspectroscopy: Intra- and Intersubject Variability of Stratum Corneum Spectral Markers.

BACKGROUND: In vivo Raman spectroscopy is a powerful tool for real-time analysis and in situ evaluation of tissues such as the skin. The efficiency of this technique has been widely demonstrated as a label-free method for in vivo evaluation of the skin. The aim of this study is to gather information about inter- and intra-individual variations in the spectral descriptors of water content and structure, organization of the lipid barrier and structure of proteins in the stratum corneum (SC). METHODS: In vivo SC measurements were performed on 17 female volunteers aged 20-30 years (phototypes I and II). For intra-individual variability, spectral collection was performed on 5 successive days per volunteer. Shapiro-Wilk and Cochran tests were applied to check the normality and the homoscedasticity of variances. ANOVA was then applied to evaluate intra- and intergroup variability. RESULTS: ANOVA was performed on the spectral descriptors of water content and structure, organization of the lipid barrier and secondary structure of proteins in the SC. No significant intra- and interday variability was observed for all volunteers. Despite the low value of the total relative standard deviation, a highly significant variation was observed between volunteers. CONCLUSION: Interindividual variability for Raman measurements is significant for a set of volunteers with normal nondiseased SC and close phototypes. This variability should be taken into consideration as a threshold for significant variance when working in vivo.

Comparison of structure and organization of cutaneous lipids in a reconstructed skin model and human skin: spectroscopic imaging and chromatographic profiling.

The use of animals for scientific research is increasingly restricted by legislation, increasing the demand for human skin models. These constructs present comparable bulk lipid content to human skin. However, their permeability is significantly higher, limiting their applicability as models of barrier function, although the molecular origins of this reduced barrier function remain unclear. This study analyses the stratum corneum (SC) of one such commercially available reconstructed skin model (RSM) compared with human SC by spectroscopic imaging and chromatographic profiling. Total lipid composition was compared by chromatographic analysis (HPLC). Raman spectroscopy was used to evaluate the conformational order, lateral packing and distribution of lipids in the surface and skin/RSM sections. Although HPLC indicates that all SC lipid classes are present, significant differences are observed in ceramide profiles. Raman imaging demonstrated that the RSM lipids are distributed in a non-continuous matrix, providing a better understanding of the limited barrier function.

Effects of solar radiations on stratum corneum hydration: Part II, protective action of solar filters.

The skin surface lipids (SSLs) film, composed of sebum and keratinocyte membrane lipids, is crucial to the barrier function of the stratum corneum (SC). The first part of this study investigated the impact of solar radiation on the SC based on a novel hydration and dehydration approach using Raman spectroscopy. The SSLs were found to absorb solar light, and thus participate to the protection of the skin surface. However, the protective function of the SSLs may be limited and is dependent to the heterogenous distribution of SSLs over the body surface. To ensure comprehensive protection, synergistic measures such as the application of solar filters are necessary. In this second part of the study, we have evaluated the limits of the protection capacity of SSLs and explored the protective action of a solar filters on both SSLs composition and the water hydration and dehydration kinetics in the SC.

Effect of Th2 cytokines on ceramide subclasses based on a model of reconstructed human epidermis.

Atopic dermatitis (AD) is associated with chronic inflammation and an altered skin barrier. Lipids of the stratum corneum of AD patients are known to differ substantially in composition from those of healthy subjects. A reconstructed human epidermis (RHE) model has been developed in vitro in order to mimic the characteristics of AD. In this study, using this model, we compared lipid profile modifications between control RHE and RHE treated with Th2 cytokines in order to mimic AD. We focused particularly on the lipid profile of the ceramide subclasses: non-hydroxy sphingosine (NS) and esterified ω-hydroxy sphingosine (EOS), which have been reported to be clearly modified in atopic skin. RHE lipids were extracted and analysed using high-performance liquid chromatography coupled to high-resolution mass spectrometry. The following lipid profile changes were observed in Th2-cytokine-treated RHE: (i) an increase in ceramide NS composed of an unsaturated fatty acid chain; (ii) an increase in saturated ceramide NS with small total carbon content (≤40 carbon atoms), whereas NS with a higher total carbon content (≥42 carbon atoms) was decreased; and (iii) a decrease in ceramide EOS. These results are in accordance with reported lipid profiles of human atopic skin in vivo. Moreover, the in vitro model represents a useful tool to better understand the pathogenesis of AD which may be used for future screening of new effective treatments.

Hydration effects on the barrier function of stratum corneum lipids: Raman analysis of ceramides 2, III and 5.

The stratum corneum is the outermost layer of the skin; its barrier function is highly dependent on the composition and the structure as well as the organization of lipids in its extracellular matrix. Ceramides, free fatty acids and cholesterol represent the major lipid classes present in this matrix. They play an important role in maintaining the normal hydration levels required for the normal physiological function. Despite the advancement in the understanding of the structure, composition and the function of the stratum corneum (SC), the concern of "dry skin" remains important in dermatology and care research. Most studies focus on the quantification of water in the skin using different techniques including Raman spectroscopy, while the studies that investigate the effect of hydration on the quality of the barrier function of the skin are limited. Raman spectroscopy provides structural, conformational and organizational information that could help elucidate the effect of hydration on the barrier function of the skin. In order to assess the effect of relative humidity on the lipid barrier function; we used Raman spectroscopy to follow-up the evolution of the conformation and the organization of three synthetic ceramides (CER) differing from each other by the nature of their polar heads (sphingosine, phytosphingosine and α hydroxyl sphingosine), CER 2, III and 5 respectively. CER III and 5 showed a more compact and ordered organization with stronger polar interactions at intermediate relative humidity values, while CER 2 showed opposite tendencies to those observed with CER III and 5.

Effects of atmospheric relative humidity on Stratum Corneum structure at the molecular level: ex vivo Raman spectroscopy analysis.

Skin hydration plays an important role in the optimal physical properties and physiological functions of the skin. Despite the advancements in the last decade, dry skin remains the most common characteristic of human skin disorders. Thus, it is important to understand the effect of hydration on Stratum Corneum (SC) components. In this respect, our interest consists in correlating the variations of unbound and bound water content in the SC with structural and organizational changes in lipids and proteins using a non-invasive technique: Raman spectroscopy. Raman spectra were acquired on human SC at different relative humidity (RH) levels (4-75%). The content of different types of water, bound and free, was measured using the second derivative and curve fitting of the Raman bands in the range of 3100-3700 cm(-1). Changes in lipidic order were evaluated using νC-C and νC-H. To analyze the effect of RH on the protein structure, we examined in the Amide I region, the Fermi doublet of tyrosine, and the νasymCH3 vibration. The contributions of totally bound water were found not to vary with humidity, while partially bound water varied with three different rates. Unbound water increased greatly when all sites for bound water were saturated. Lipid organization as well as protein deployment was found to be optimal at intermediate RH values (around 60%), which correspond to the maximum of SC water binding capacity. This analysis highlights the relationship between bound water, the SC barrier state and the protein structure and elucidates the optimal conditions. Moreover, our results showed that increased content of unbound water in the SC induces disorder in the structures of lipids and proteins.

Photosensitizers incorporation in SOPC films at different hydration levels.

In the present work, two photosensitizing drugs, Temoporfin and Verteporfin have been studied. Both have regular approval in Europe, Temoporfin for the treatment of head and neck cancers and Verteporfin for the treatment of age-related macular degeneration (AMD). The treatment modality, known as "Photodynamic Therapy" (PDT), involves drug activation with visible light in the presence of oxygen and production of reactive oxygen species (ROS) to destroy the pathological tissues. Both drugs are inactive in the absence of light, presenting only few side effects. The incorporation of the two drugs into a SOPC bilayer -used as a model membrane- was studied by ATR-FTIR. An original approach was applied, involving lyotropic transitions and a very slow dehydration rate of the sample. In low water content and dry film, Temoporfin highly affects stretching vibrations of SOPC chains and polar groups, showing that Temoporfin is inserted into the bilayer in both apolar and polar regions. In fully hydrated layers, Temoporfin - SOPC interactions still take place but only impact Temoporfin vibration bands. Verteporfin shows smaller effect on both chain and polar groups' vibrations of SOPC, with the exception of choline group, suggesting that Verteporfin is inserted into the bilayer to a lesser extent and remains at the bilayer polar interface. These results can be used to better understand drugs behavior in biological media.

Glycerol and natural sugar-derived complex modulate differentially stratum corneum water-binding properties and structural parameters in an in vitro Raman-desorption model.

The epidermal protective functions are closely associated with skin hydration homeostasis. The understanding of different states of water binding is a rising concept in assessing topically applied formulations and their interaction within the stratum corneum (SC). In addition to global water content, primary bound water, partially bound water, and unbound water and barrier-related lipid lateral packing and protein secondary structure can be measured by Raman spectroscopy. This study aimed to establish an in vitro SC model to evaluate differences in the efficacy of a natural sugar-derived complex in combination with glycerol and a botanical extract in modulating SC water binding and structural proteins and barrier lipids. These compounds were selected due to their water-binding and soothing properties. The SC water profiles were assessed at the surface and in 8 µm SC depth. After a 12-hour hyperhydration and subsequent product incubation the measurements were performed during a 6 hours desiccation phase. The maximal water caption and the time until reaching a steady state are measured as well as water retention and resistance against water loss. Global water content, partially bound, and unbound water, as well as lipid and protein structures were assessed with confocal Raman microspectroscopy. Both the natural sugar-derived mixture and more pronounced, the same mixture with additional glycerol increased all three water-binding parameters at the surface and in 8 µm SC depth at the beginning and during the desiccation phase. Further addition of botanical extract did not result in an additional increase of the water-binding. All three formulations showed an increase in the lipid lateral packing values prevented the protein alteration as measured by ß-sheets signal compared to blank. The present model is suited for screening studies comparing the specific effects of different compounds on hydration states. The natural sugar-derived mixture Aquaxyl showed evidence for an improvement of all SC hydration states, lipid and protein structure which was further enhanced by the addition of glycerol 5%. This improvement was evidenced at the surface and within the SC for all hydration-related parameters, and the lipid as well the protein structures. The addition of botanical extract phytoessence blue daisy did not show further improvement.

Effects of solar radiations on stratum corneum hydration: Part I, protective role of skin surface lipids.

This study used Raman spectroscopy to develop a new approach to evaluate the effects of solar radiation on the stratum corneum (SC). The method measures the SC's hydration and dehydration kinetics by calculating the vOH/vCH ratio to monitor the relative water content during the drying process. The study also investigated the role of skin surface lipids (SSLs) in protecting the SC from solar radiation. The SSLs film is a complex mixture of free fatty acids, triglycerides, wax esters, squalene, free and esterified cholesterols, that play a crucial role in the skin's barrier function. The results showed that solar radiation alters the water content and balance within the SC, and SSLs provide protection by acting as an optical filter by absorbing some of the energy of the solar light. This is confirmed by high temperature gas chromatography coupled to mass spectrometry analyses by revealing a decrease in specific lipids after irradiating the SSLs .

Characterization of triglycerides photooxidation under solar radiations: A stepwise Raman study.

Triglycerides (TGs) are one of the main components of the glycerolipid family. Their main task in cells is to store excess fatty acids. TG energy storage is mainly concentrated in adipocytes. TGs and free fatty acids constitute the majority (57.5%) of the skin surface lipids (SSLs). TGs are essential for the formation of the skin water barrier. This work is the second part of a global study that aims to evaluate the effect of solar radiations on SSLs using vibrational spectroscopy. In the first part of this work, a stepwise characterization of free fatty acids was performed, and different spectral descriptors were used to follow the different structural modifications during the photo-oxidation process, that is hydrogen abstraction, formation of hydroperoxides and peroxyl radicals as primary oxidation products and the formation of aldehydes, ketones, alcohol as secondary products. In this second part, the photo-oxidation of TGs was evaluated using Raman spectroscopy. A decrease in the CH2/CH3 stretching bands ratio that confirmed the hydrogen abstraction, an increase in the 1165/1740 cm-1 ((δ(OH) and υ(C-O))/ν(C=O) (ester)) ratio indicated the formation of secondary oxidation products such as hydroperoxides. And finally, an increase in the 1725/1740 cm-1 (υ(C=O) (ald.)/υ(C=O) (ester)) ratio and the trans ν(C=C)/cis ν(C=C) ratio highlighted the formation of aldehydes, alcohols, ketone, trans secondary products and others.