A Clinical Approach to Validate the Biological Significance of LPC16:0 as a Discriminating and Pathogenic Biomarker of Fibromyalgia
Conditions: Fibromyalgia, Primary, Pain, Soreness, Muscle, Oxidative Stress, Metabolomics, Lipidomics
Sponsor: Kaohsiung Medical University Chung-Ho Memorial Hospital

Study protocol and statistical analysis of 
Bio-significance of LPC16:0 in Fibromyalgia 
 
Identifiers: 
NCT04832100    
Unique Protocol ID: KMUHIRB-G(I)-20170012 
 
Date: July 1, 2024 
 

This observational study is prospectively approved by the Institutional Review 
Board of Kaohsiung Medical University Hospital (KMUHIRB-(I)-20170012) and 
registered at ClinicalTrials.gov (identifier NCT04832100; date of registration: April 
5, 2021). Adult patients with FM are consecutively enrolled from the outpatient 
department since June 1, 2021. All participants are well informed and provided 
written informed consent. Potential participants are interviewed by neurologists, 
and those who met the 2011 American College of Rheumatology criteria for FM 
are included. To assess symptoms before pharmacotherapy and track the 
treatment responses, only patients with a new diagnosis of primary FM not 
receiving pharmacotherapy are included for analysis. None of the participants 
had received treatment with anti-epileptic agents, serotonin norepinephrine 
reuptake inhibitors (SNRIs), selective serotonin reuptake inhibitors (SSRIs) or 
tricyclic antidepressants within the 3 months preceding the study. Age- and sex-
matched healthy individuals are prospectively enrolled as healthy controls (HCs) 
from the outpatient department and public recruitment. Exclusion criteria for both 
patient and control groups included the presence of a systemic immune disorder, 
a known history of psychiatric or personality disorders, chronic pain disorders 
other than FM (e.g., diabetes neuropathic pain, neuralgia, etc.), malignancy, 
systemic use of corticosteroids, pregnancy, a major systemic disorder (e.g., 
kidney/liver dysfunction) and a chronic disease under poor control (e.g., diabetes 
mellitus or hypertension). We also excluded participants who might not be able to 
provide unbiased clinical information, such as those involved in ongoing litigation 
or filing compensation. 
 
Study design 
  The clinical data for patients and controls are obtained through questionnaires 
and interviews with the same pain specialists (KWL, YOF and CHH) throughout the 
study. Pain intensity of individuals is assessed with numeric rating scale for pain 
(NRS) and pain diffuseness by widespread pain index (WPI). The states of anxiety 
and depression are assessed using the Hospital Anxiety and Depression Scale 
(HADS). We considered the changes in FM severity as the outcomes of interest, 
which are assessed using the Revised Fibromyalgia Impact Questionnaire (FIQR) 

9. To record the initial therapeutic responses, all patients are under a follow-up 
procedure for at least one month after treatment. Cases with missing values or 
incomplete follow-up are excluded from analysis. All patients completed the 
questionnaires during the initial interview, and follow-up assessments are 
scheduled at 2 and 4 weeks to monitor their therapeutic responses. During the 
first visit, comprehensive interviews are performed. We initially educated patients 
about nonpharmacological therapies, such as exercise and relaxation techniques, 
and emphasize their importance throughout the treatment process. In this non-
trial observational study, all participants received adequate pharmacotherapy in 
accordance with existing therapeutic guidelines. All participants agreed to receive 
pharmacotherapy, and no placebo group is included. To target pain control and 
minimize interference with depressive symptoms, we used pregabalin (75 mg, 
twice a day) as initial pharmacotherapy, with or without a low dose of imipramine 
(25 mg, once a day). Participants who are intolerant to pregabalin or imipramine 
are excluded from analyses. No SNRIs, SSRIs, morphine or opioid-analogue drug 
is used for pain control during the initial 4 weeks of treatment. After 4 weeks, by 
clinical judgment, patients with sustained or deteriorated emotional symptoms 
are referred to a psychiatrist for further management, including cognitive 
behavioral therapy and antidepressants. 
 
Statistical analysis 
Data are expressed as mean ± standard deviation. Sample size calculations 
are performed using the statistical software G* Power (version 3.1.9.2), with a 0.05 
significance level (α = 0.05) and a power of 80% (β = 0.20) in all calculations. An 
effect size is determined according to previous literature. For a comparison of two 
groups, a sample size of 45 per group would have 80% power to detect an effect 
size of 0.6 using a t-test with a 5% two-sided significance level. For regression 
analysis, an optimal sample size is at least 26 for an effect size of 0.6 and four 
predictors (NRS, WPI, HADS-A and HADS-D scores). 
Between-group differences are assessed using the Student's t-test; Levene’s 
test is performed beforehand to confirm the homogeneity of variances between 
groups. The longitudinal analysis of changes after treatment are compared using 

repeated measures ANOVA. Pearson correlation is used to assess the correlation 
between parameters. Cluster analysis is performed to identify distinct groups 
based on anxiety and depression severity. The k-means clustering algorithm is 
used with the HADS-Anxiety (HADS-A) and HADS-depression (HADS-D) scores, 
and patients with similar manifestations are grouped into 2 subgroups. Repeated 
measures correlation (rmcorr) is used to determine the within-individual 
associations of clinical symptoms and disease severity, with repeated 
assessments across multiple individuals. Multiple linear regression (MLR) 
analysis is performed to determine the clinical factors associated with disease 
improvement after treatment. The linear mixed-effect regression model (LMM) is 
used to investigate the influences of symptomatic factors on FM severity during 
follow-up. Mediation analyses are conducted to assess the mediation effects of 
pain and mood symptoms on disease severity. Detailed information for 
correlation and mediation analyses is described in the Supporting Information. 
Two-tailed p < 0.05 is considered statistically significant. For the analyses of 
multiple tests, such as correlation analyses and ANOVA, the resulting p values are 
adjusted through Bonferroni correction, with p <0.0125 considered statistically 
significant. SPSS v20 (SPSS, USA) with the PROCESS macro used for analysis.