Lipidomics‐based analysis of lipid differences between dry skin of women aged 22–28 years and 29–35 years

The skin condition of women is different at different ages, and skin surface lipids are also different. According to the “7–7 theory” of the Huangdi Neijing, the physiological condition of women changes significantly every 7 years, and women aged 22–28 are in the “4–7” stage as mentioned in the “7–7 theory” of the Huangdi Neijing. Women's skin is in different states at different ages and produces different lipids.

An in-depth study of SSL will help to understand the role of lipids and their specific regulatory functions.The skin condition of women is different at different ages, and SSL is also different.According to the "7-7 theory" of the Huangdi Neijing, the physiological condition of women changes significantly every 7 years, and women aged 22-28 are in the "4-7" stage as mentioned in the "7-7 theory" of the Huangdi Neijing.By studying the differences in facial lipids between the dry skin of women in these two age groups, we can find the key lipids and investigate the regulatory mechanisms.
In this paper, a comprehensive analysis of facial lipids in dry skin of women aged 22-28 years and 29-35 years was performed by ultra performance liquid chromatography coupled with quadrupole timeof-flight mass spectrometry (UPLC-Q-TOF-MS) using a lipidomics approach. 13Facial lipids were collected from dry-skinned women in two age groups and analyzed for SSL composition based on UPLC-Q-TOF-MS technique with the aim of providing guidance for the development of skin care products targeting dry-skinned women between these two age groups.

| Questionnaire development
The questionnaire was designed in four areas: basic information, life habits, work situation, and emotional stress.The survey of volunteers' basic information can be used to analyze the association between surface differential lipids and personal objective information (age, height, weight, BMI) in women with dry skin in the two subsequent age groups.Thirty-three volunteers with a mean age of 24.00 ± 1.80 years were recruited from women with dry (Group D1) skin aged 22-28 years, and 32 volunteers with a mean age of 32.63 ± 2.09 years from women with dry (Group D2) skin aged 29-35 years.The questionnaires were guided by professionals, and the results were statistically analyzed.

| Measurement of skin physiological parameters
After arriving at the laboratory in turn, the volunteers of D1 and D2 groups sat for 30 minutes in a constant temperature and humidity (25°C, 50 ± 10%) environment to start testing skin parameters, in turn testing moisture loss Tewameter TM300 (moisture loss test probe), moisture content Corneometer CM825 (moisture test probe), pH Skin-pH-Meter pH 905 (pH test probe), oil content, elasticity, a L b, and ITA value.

| Lipid collection and preservation
Prior to lipid collection, each volunteer was asked to clean their faces with water at 5 min intervals, and to register the time of washing their faces individually, and to sit for 30 min in a room with constant temperature and humidity (25°C; 50 ± 10% relative humidity).
The sebum sampling paper was then applied to the center of the volunteers' cheeks under contamination-free conditions, removed and curled in a centrifuge tube after 3 min, immediately labeled and stored on dry ice, and stored in a −80°C refrigerator to prevent correlation analysis, lipidomics, physiological parameters, questionnaire, skin surface lipids oxidation after all samples were taken on the same day (gloves were worn throughout the experiment to ensure that the samples were free of contamination).

| Lipid handling
The samples were removed from the refrigerator at −80°C, and the lipids were extracted using the modified Bligh and Dyer lipid extraction method, and the lipid extracts were blown dry under a nitrogen blower. 13

| Liquid phase and mass spectrometry conditions
The mobile phases were phase A (acetonitrile and water mixed at 2:3 v/v, 0.1% formic acid and 10 mmol/L ammonium formate) and phase B (acetonitrile and isopropanol mixed at 9:1 v/v, 0.1% formic acid and 10 mmol/L ammonium formate); the flow rate was 0.3 mL/ min.The column temperature was 50°C.The liquid chromatography gradient elution procedure was shown in Table 1.
An electrospray ionization source (ESI) was used, with positive ion mode acquisition and a mass scan range of 50 - 1200 m/z, using nitrogen in all gas paths.Leucine enkephalin (m/z = 554 277) was used as an external standard for accurate mass locking.The specific mass spectrometry parameters are shown in Table 2.

| Questionnaire results and analysis
The basic information of volunteers between D1 and D2 groups is shown in Table 3, except for age and BMI (p = 0.027), which were significantly different between the two groups, the rest of the basic information was not different.
The lifestyle habits of volunteers between D1 and D2 groups are shown in Table 4, which shows a significant difference in sleep time between the two age groups of female dry skin volunteers (p = 0.0133), with 32 dry skin women aged 29-35 years having a mean sleep time earlier than the 33 dry skin women aged 22-28 years.
The skin condition of the volunteers was closely related to their work situation and work environment, as shown in Table 5, there was no significant difference in the work situation between the two groups of volunteers in this study, and the difference in skin condition between them was not related to their work environment.
Emotional stress was also a key factor that had an effect on the skin, as shown in Table 6; there was no significant difference in emotional stress between the volunteers in D1 and D2 groups.
It indicates that the skin differences between these two groups of volunteers are not related to emotional stress.
The overall results of the questionnaire showed that women in the D2 group had a higher BMI than women in the D1 group and that women in the D1 group went to bed later than women in the D2 group, so there may be a relationship between the skin differences between the D1 and D2 groups. 14

| D1 and D2 physiological parameters results and analysis
The differences in moisture content, transcutaneous water loss dissipation value, oil content and pH between D1 and D2 groups are shown in Figure 1, where moisture content (p = 0.3305), TEWL value TA B L E 1 Liquid chromatography elution procedure.After 00:00 11 7 After 1:00 am 9 1 (Continues) (p = 0.9043), and oil content (p = 0.8633) were higher in D1 group than in D2 group, and pH value (p = 0.2815).
Was higher in D2 group than in D1 group, but there was no significant change.In addition, the above four physiological indicators were not statistically different in both D1 and D2 groups.
The skin elasticity and wrinkles at the corners of the eyes in the D1 and D2 groups are shown in Figure 2.There was no significant difference in the fine wrinkles at the corners of the eyes in both groups (p = 0.7359), but the skin elasticity in group D2 was significantly lower than that in group D1 (p = 0.000003).
The skin glossiness of group D1 and D2 was reflected by L value, a value, and b-value (as shown in Figure 3); there was no significant difference between D1 group and D2 group in terms of skin ruddyness (p = 0.4019); D1 group had significantly higher skin brightness (p = 0.0267) and ITA value (p = 0,001) than D2 group.D1 group had significantly lower skin dark yellowness (p = 0.0151) than D2 group.It means that the D1 group had fairer skin tone.

| OPLS-DA screening of facial differential lipids in dry skin women of two age groups
After entering the mass spectrometry data of the samples into EZinfo software, the OPLS-DA analysis mode was selected and the differences in lipid composition between the two groups were evaluated using Score plots, as shown in Figure 4, indicating that the SSL of the two groups are well separated and significantly different.

| D1, D2 eight types of lipid analysis
The 1453 lipids identified in the SSL of volunteers in groups D1 and D2 were divided into eight major categories: GP, GL, FA, SP, ST, SL, PR, and PK. Figure 5 shows the differences in the content of the total lipids and the eight major lipid classes were higher in the D2 group than in the D1 group, with glycerophospholipids significantly higher than in the D1 group, followed by glycerolipids, fatty acids.

| Correlation analysis of questionnaires and physiological parameters
From the results of the questionnaire study, the skin differences between the two age groups were related to BMI and sleep time.With age, metabolism slows down and the body becomes obese, and a high BMI is significantly associated with a decrease in skin capillary density. 15Capillary remodeling capacity and acetylcholine-mediated vasodilation are differentially impaired in obese women. 16This leads to problems such as reduced blood flow to the face and hyperpigmentation of the skin.People with high BMI also have high blood sugar content and are prone to non-enzymatic glycosylation (NEG), which produces Advanced Glycation End Products (AGEs). 17AGEs cross-link with collagen and elastin in the skin, reducing skin elasticity and connective tissue permeability, leading to skin aging and dull skin tone.with a content of up to 50%. 20Its structure is hydrophilic and can significantly promote the expression level of filament polymerization protein mRNA and protein. 21,22It is a natural moisturizing factor.
There are studies that prove. 23,24Ceramide can promote the proliferation of fibroblasts and increase collagen expression.It also inhibits matrix metalloproteinase (MMP-1) expression, so ceramide has some anti-aging effects and can increase skin elasticity.Melanin synthesis requires the involvement of protein kinase (ERK) and Akt/protein kinase B (PKB), and some studies have shown that Ceramide can delay the activation of ERK and PKB in melanocytes, thus inhibiting melanin production, so ceramide has whitening effect. 25Therefore, the content of Cer(d18:0/16:0) (LMSP02020001) is positively correlated with skin elasticity (R2) and skin brightness (L, a, b, ITA values).Among the GP-like lipids, LMSPGP03020013, LMSPGP 030 20014, and LMSP03020024 were negatively correlated with the elasticity value (R2), and they were differentially expressed in the D1 and D2 groups as shown in Figure 10.
[LMGP0302] are acetyl glycerol phosphates, which are biological macromolecules, and they all contain carbonyl groups in their structures, which are active metabolic intermediates with spontaneous reactive activity.When they form conjugates with adjacent unsaturated bonds or functional groups, they may cause cross-linking between biomolecules and form aggregated products that are difficult to remove, leading to skin aging.

| DISCUSS ION
Lipids on the human face are not only part of the skin barrier, but also play an important role in energy conversion, carbon reserves,

TA B L E 7
The most important differential lipids in D1 and D2 SSL.8][29] Therefore, the difference in BMI between the D1 and D2 groups, as well as the age difference, may account for the difference in skin brightness between the two age groups of women with dry skin.
The results of correlation analysis of differential lipids with differential physiological parameters showed that the expression content of Cer(d18:0/16:0) was significantly higher in group D1 than in group D2, and the elasticity value R2 was significantly lower in group D2 than in group D1.The correlation analysis of Cer(d18:0/16:0) and R2 between the two groups showed a significant positive correlation, that is, the difference of skin elasticity between the two groups was likely to be related to the content of Cer(d18:0/16:0) content.Cer(d18:0/16:0) is a neurosphingosine, and it has been shown that sphingosine extracted from wheat inhibits the activity of Human neutrophil elastase (HNE). 30gh expression of elastase disintegrates the elastic fiber network and reduces the skin's elasticity, resulting in wrinkles and skin relaxation.
In both D1 and D2 groups, three [LMGP0302] classes were all negatively correlated with elasticity values, and all were highly expressed in the D2 group, suggesting that the reduced skin elasticity in the D2 group may be related to the high expression of However, the present study still has some limitations, such as the influence of the intrinsic functional differences of the volunteers themselves on the reliability of this study, as well as the effects of manipulation and environment on the lipids during the experimental procedures.In addition to this, the functional studies of many lipids are still in the preliminary stage; therefore, in order to improve the reliability of lipidomic analysis, the increasing number of functionalities of skin lipids found should be investigated in addition to exploring the results through more experiments.

| 1457 BI
et al.only two ceramides, Cer(d18:0/16:0) and Cer(t18:0/19:0), were higher in the D1 group than in the D2 group.The two lipids with the greatest significant differences between the two groups were the FA class of erucamide (Erucamide) and the GP class of PS (O-18:0/17:0), both with the highest expression in the SSL of volunteers in the D2 group.

TA B L E 6
feel that difficult things are piling up and you cannot' Emotional stress of volunteers.F I G U R E 1 Moisture content (A), transcutaneous water loss dissipation value (B), oil content (C), and pH (D) of group D1 and D2.eight major categories of lipids as well as total lipids in the SSL of dry skin women aged 22-28 versus 29-35 years.As shown in the figure:

Subsequently, subclasses with
the same trend of change in lipid content were screened based on the relative trend of change in the eight major lipid classes to identify the subclass categories that led to significant changes in the eight major lipid classes mentioned above.The results are shown in Figure 6, and a total of 25 subclasses were identified as significantly different (p < 0.05), with trends in change consistent with the major major category to which they belonged.In the group of women aged 29-35 years, the subclasses of lipids that increased significantly were as follows: other fatty acyl [FA00], fatty acids and conjugates [FA01], arachidonic acid [FA03], et al.

F I G U R E 2 F I G U R E 3
Figure9A, and the content in group D2 was significantly higher (p < 0.001) than that in group D1, as shown by Figure3B, and the

F I G U R E 5 F I G U R E 6 F I G U R E 7
Relative mean content of total lipids and eight classes of lipids on the skin surface of women in group D1 compared with group D2 (***p < 0.001; **p < 0.01; *p < 0.05).| 1461BI et al. signal transduction and many other aspects.Differences in facial lipids vary between people of different ages and skin types, and this study investigated the differences in facial lipids between groups D1 and D2.Relative mean lipid content of lipid subclasses on the surface of dry skin of women aged 22-28 years (D1) and 29-35 years (D2) (***p < 0.001; **p < 0.01; * p < 0.05).Heat map of correlation between lipids and skin elasticity, b-value, L-value, ITA value.F I G U R E 8 LMSP02020001 in skin surface content in group D1 and group D2.(***p< 0.001; **p < 0.01; *p < 0.05).The results of the questionnaire showed that the BMI of group D1 was significantly higher than that of group D2.The results of skin physiological parameters showed that skin brightness was significantly higher in group D1 than in group D2.Studies26 have shown that people with a high BMI have elevated levels of pro-inflammatory factors (TNFα and IL-6), and a high BMI is also considered a chronic inflammation, which leads to impaired skin barrier function, enhanced tyrosinase activity, and increased melanin synthesis.In F I G U R E 1 0 Content and structure of LMGP03020013, LMGP03020014, and LMGP03020024 on the skin surface of group D1 and group D2.
the three [LMGP0302] classes.[LMGP0302] is a macromolecular acetylglycerophosphate class containing excess unsaturated carbonyl groups, and studies have shown that under physiological conditions, glucose reacts with amino acids, peptides, unsaturated fatty acids, and proteins to generate macromolecular products, and some long-chain macromolecular fatty acids in sebum can also cross-link with proteins, and protein cross-linking damage caused by these polymers is the main aging cause of aging.29,31In addition, lipids containing unsaturated structures are highly susceptible to oxidation, and the aldehydes and ketones produced by lipid peroxidation can act as signal transducers in the skin, transmitting signals through the sebaceous membrane and causing microinflammatory responses and oxidative stress in the skin.Oxidative stress causes the loss of expression activity of genes that maintain the basic physiological functions of cells by damaging DNA, which in turn leads to cellular senescence.32Some unsaturated macromolecular lipids peroxidise themselves to produce a series of peroxides that cause oxidative damage to cells, and their metabolite malondialdehyde (MDA) is a potent cross-linking agent, which can easily crosslink with proteins or nucleic acids to form lipofuscin that cannot be dissolved by lysosomes, and then accumulates in the skin tissue to form age spots.Therefore, unsaturated macromolecular lipids in the skin can lead to oxidative stress, microinflammation, and cross-linking reactions, affecting skin elasticity and accelerating skin aging.

Variables 22 ~ 28 years old Dryness (D1, 33) 29 ~ 35 years old Dryness (D2, 32) χ 2 p
A total of 1453 lipids were screened in the SSL of D1 and D2 groups, and 34 differential lipids were identified by screening differential lipids by p < 0.05, Fold Change >2, and VIP >1.The specific information is shown in Table7.Among the 34 differential lipids, 10 were FA, 14 SP, 6 GP, 2 GL, and 2 PK, of which 32 lipids were all high in the D2 group, and TA B L E 5 Volunteer work.