Evaluation of Serum Ceramides C16, C24, and Sphingosine-1-Phosphate (S1P) in Patients With Acne Vulgaris
Conditions: Acne Vulgaris
Sponsor: Sohag University

 
 
Official title :  
  
 Evaluation of Serum Ceramides C16, C24, and  
Sphingosine-1-Phosphate (S1P) in Patients  
with Acne Vulgaris  
  
  
NCT Number :  
Not yet assigned   
  
Date of the document :  
7\10\2025  
  
IRP Registration number :  
Soh-Med-25-10—11MS
 
 
 
 
 
 
 
 
Sohag University 
Faculty of Medicine 
Dermatology, Venereology and 
Andrology Department 

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Evaluation of Serum Ceramides C16, C24, 
and Sphingosine-1-Phosphate (S1P) in 
Patients with Acne Vulgaris 
 
A protocol of thesis submitted for partial fulfillment of Master degree in 
Dermatology,Venereology and Andrology 
 
by 
 
             Dalia Ahmed Helmy, MBBCh 
Resident of Dermatology, Venereology and Andrology , Elbalyana 
Central Hospital 
 
Supervised by 
 
Prof. Essam El Din Abdel Aziz Nada, MD 
Professor of Dermatology, Venereology and Andrology  
Faculty of Medicine 
Sohag University 
 
Dr. Doaa Gaber Abdelbaset, MD 
Lecturer of Dermatology, Venereology and Andrology 
 Faculty of Medicine  
Sohag University 
 
 
 
2025
 
 
 
 
 
 
 
 
Sohag University 
Faculty of Medicine 
Dermatology, Venereology and 
Andrology Department 

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Introduction 
Acne vulgaris (AV) is a chronic, inflammatory disease of the pilosebaceous 
unit that is globally prevalent and represents a great public health burden (Vasam et 
al., 2023). Acne affects approximately 9.4% of the world’s population, ranking the 
eighth most prevalent skin condition worldwide (Reynolds et al., 2024). It affects 
more than 85% of adolescents and frequently persists into adulthood (Guguluș et 
al., 2025).Clinically, acne vulgaris is characterized by a spectrum of lesions, 
including non-inflammatory comedones (open and closed) and inflammatory 
papules, pustules, nodules, and cysts, often leading to post-inflammatory 
hyperpigmentation and scarring (Vasam et al., 2023). 
         Lesions predominantly occur on regions rich in sebaceous glands such as the 
face, chest and upper back. The severity and chronicity of the condition, along with 
its visible disfigurement, often result in significant psychosocial distress, including 
anxiety, depression, and poor self-esteem which negatively impacting patients’ 
quality of life (Kazan, 2024). 
The pathogenesis of acne is multifactorial, involving a complex interplay 
between genetic predisposition, environmental exposures (e.g., humidity, pollution, 
comedogenic cosmetics), hormonal fluctuations, and immune dysregulation. The 
four classical pathogenic mechanisms include increased sebum production, 
abnormal follicular keratinization, colonization by Cutibacterium acnes and 
activation of innate and adaptive inflammatory responses (Alsaadoon et al., 2024). 
Although inflammation was once thought to occur secondary to microbial 
colonization, recent studies demonstrate that inflammatory processes are activated 
in the early stages of lesion development, even in clinically non-inflamed comedones 
(Tanghetti, 2013). 
Emerging evidence has highlighted the crucial role of lipids—particularly 
sphingolipids—in skin homeostasis, barrier function, and immune signaling. Among 
these lipids, ceramides (CERs) and sphingosine-1-phosphate (S1P) are of particular 

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interest due to their dual structural and signaling roles in the skin (Masuda-Kuroki 
et al., 2023). The outermost layer of the epidermis, the stratum corneum, consists of 
a lipid matrix comprising approximately equimolar concentrations of ceramides, free 
fatty acids, and cholesterol. This matrix is essential for maintaining skin barrier 
integrity and hydration, and any imbalance can contribute to dermatological 
disorders (Mijaljica et al., 2024). 
 
Ceramides are sphingolipid molecules composed of sphingosine and fatty acid 
chains of varying lengths. Long-chain ceramides (C16–C20) and very-long-chain 
ceramides (C22–C24) have been shown to exert differential effects on keratinocyte 
behavior and cutaneous inflammation. Specifically, recent data indicate that certain 
ceramide species play a role in modulating inflammation and cellular proliferation 
in the skin (Ho et al., 2022). For instance, C16 ceramide is often associated with 
pro-apoptotic and pro-inflammatory effects, whereas very-long-chain ceramides like 
C24 may contribute to anti-inflammatory functions and promote cell survival. 
Dysregulation in the balance of these ceramide species can thereby influence the 
severity and progression of inflammatory skin diseases, including acne vulgaris 
(Custodia et al., 2022; Thakkar et al., 2025).        Sphingomyelins (SMs), abundant 
in both circulation and cell membranes, serve as precursors to ceramides. These 
SMscan be hydrolyzed by sphingomyelinase (SMase) into ceramides, which in turn 
can be further metabolized into sphingosine and subsequently phosphorylated into 
Sphingosine-1-phosphate (S1P) by the action of sphingosine kinase . This 
conversion pathway is of great interest in inflammatory research (Thakkar et al., 
2025). 
Sphingosine-1-phosphate (S1P) is a potent bioactive lipid that plays diverse 
roles in immune regulation, angiogenesis, vascular integrity, and inflammatory 
signaling. In the skin, S1P regulates the behavior of keratinocytes, immune cells, 
and vascular endothelial cells. It promotes cellular proliferation and survival and can 
influence immune cell migration and cytokine production. Thus, alterations in serum 

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or tissue levels of S1P may reflect or contribute to the inflammatory milieu observed 
in acne vulgaris (Custodia et al., 2022; Su et al., 2024). 
 
Although the roles of sphingolipids have been studied in dermatologic disorders 
such as atopic dermatitis, psoriasis, and ichthyosis, data on their involvement in acne 
vulgaris remain limited. Most previous studies have focused on skin tissue analyses 
(Alizadeh et al., 2023; Delcheva et al., 2024), whereas investigations into serum 
sphingolipid profiles—especially ceramides C16, C24, and S1P—in acne patients 
are scarce. Understanding systemic alterations in these bioactive lipids may provide 
insights into the inflammatory and metabolic underpinnings of acne vulgaris, 
potentially identifying novel biomarkers or therapeutic targets. 
 
 
 
 
 
 
 
 
 
 

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Aim of the work 
1. To measure serum levels of ceramides C16 and C24 in patients with acne 
vulgaris. 
2. To determine the serum level of sphingosine-1-phosphate (S1P) in the same 
patient group. 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Patients and methods 
Study approval: The study will be approved by Research and Ethical committee 
at Faculty of Medicine, Sohag University. Informed written consent will be obtained 
from all participants after explanation of nature of the study. 
Study Design: This is a case-control, cross-sectional, analytical study  
Site of the Study: The study will be conducted at the Dermatology Outpatient 
Clinic in Sohag University Hospitals. 
Sample size calculation  
The sample size was determined to detect both group differences in serum lipid 
mediators and correlations with acne severity. Previous work in acne patients (Kaya 
et al., 2019) included only 30 cases and 20 controls but demonstrated significant 
lipid alterations. Using standardized effect sizes (α=0.05, power 80%), a moderate 
correlation (r≈0.40) requires ~47 subjects, while a moderate between-group 
difference (Cohen’s d≈0.6) requires ~44 per group. Accordingly, 40 patients and 40
 
matched controls (n=80) . 
 
Study Population: 
A. Inclusion Criteria:  
For Case Group  
This group will consist of (40) patients : 
• Both males and females aged 15–40 years. 
• Mild, Moderate, severe and very severe cases will be included. 
 

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For Control Group: 
• This group will consist of age- and sex-matched (40) healthy volunteers. 
 
 
 
B. Exclusion Criteria (for both groups): 
• Presence of any dermatological condition other than acne vulgaris (for cases). 
• Use of systemic or topical treatment such as isotretinoin or antibiotics within 
the last 3 months. 
• Pregnancy or lactation. 
• History of lipid-lowering therapy or supplements affecting ceramide or 
sphingolipid metabolism. 
All patients in this study will be subjected to: 
1. Clinical Evaluation  
A. History including: 
• Demographic data: Name, age, sex, residence and occupation. 
• History of present illness: 
o Age of onset of acne. 
o Duration of acne symptoms. 
• Medical History: 
o Presence of chronic diseases (e.g., diabetes, polycystic ovarian 
syndrome). 
o Menstrual and contraceptive history in females  
 
 

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B. Dermatological examination: 
 
o Lesion Assessment: Documentation of acne lesion types (comedones, 
papules, pustules, nodules, cysts) Presence of erythema,edema or 
redness 
o Distribution: Affected anatomical areas (face, chest, back, shoulders). 
o Scar Evaluation: Presence and severity of post-acne scarring 
(atrophic, hypertrophic). 
 
C. Acne Severity Assessment: 
Global Acne Grading System (GAGS): It is a system which evaluates acne 
severity by dividing the face, chest, and back into six regions, each assigned a 
factor according to its size. The most severe lesion type in each region is graded 
(0–4), multiplied by the regional factor, and summed to yield a global score (0–
44). Severity is then classified as mild, moderate, severe, or very severe. (Doshi 
A, 1997) 
The final global score is interpreted as follows: 
•       0 → No acne 
•       1–18 → Mild acne 
•       19–30 → Moderate acne 
•       31–38 → Severe acne 
•       39–44 → Very severe acne 
 

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2. Laboratory Investigation: 
Evaluation of Serum Ceramides (C16, C24) and Sphingosine-1-Phosphate 
(S1P). 
a. 
Sample Collection and Processing: 
• Timing: Morning samples will be collected after overnight fasting to reduce 
variability due to dietary lipid intake. 
• Procedure: 
o A volume of 5 mL of venous blood will be drawn using standard 
phlebotomy techniques. 
o Blood samples will be allowed to clot at room temperature and then 
centrifuged at 3000 rpm for 10 minutes. 
o The separated serum will be aliquoted into sterile cryovials and stored 
at −80°C until analysis to preserve lipid stability. 
b. 
Quantitative Measurement of Lipid Mediators: 
• Method of Analysis:  
• Quantitative determination of the target sphingolipids will be performed 
using liquid chromatography coupled with triple quadrupole mass 
spectrometry (LC-MS/MS) (Agilent 6460 Triple Quadrupole LC-MS/MS, 
USA). 
          Procedure: 
1. Add 100 µL serum into a glass vial. 
2. Add 400 µL methanol and 200 µL chloroform (containing internal 
standards). 
3. Vortex for 30 seconds. 
4. Add 200 µL water and 200 µL chloroform. 
5. Vortex and centrifuge at relative centrifugal force (1000_3000)g for 10 
minutes to separate layers. 

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6. Carefully collect the lower organic phase (contains lipids). 
7. Evaporate under nitrogen gas at room temperature or using a vacuum 
concentrator. 
          8. Reconstitute dried extract in 100 µL of methanol/water (9:1) with 0.1% 
(optimized according to instrument condition) formic acid for LC-MS/MS 
injection . 
 
 
 
 
Statistical Analysis 
Data will be statistically described in terms of mean_standard deviation (SD), 
median and range, or frequencies (number of cases) and percentages when 
appropriate. Comparison of numerical variables between the study groups will be 
done using Kruskal Wallis test. Within group comparison of numerical variables will 
be done using Wilcoxon signed rank test for paired (matched) samples. For 
comparing categorical data, Chi square (x2) test will be performed. Exact test will 
be used instead when the expected frequency is less than 5. Correlation between 
various variables will be done using Spearman rank correlation equation for non-
normal variables/non-linear monotonic relation. P values less than 0.05 will be 
considered statistically significant. All statistical calculations will be done using 
computer program SPSS  
 
 

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