The Impact of Microbiota and Associated Blood Tryptophan Metabolites on Pain Perception in Patients Undergoing Lumbar Disc Herniation Surgery
Conditions: Microbial Substitution, Tryptophan Metabolism Alterations, Postoperative Pain, Acute
Sponsor: Dokuz Eylul University

The Impact of Microbiota and Associated Blood Tryptophan Metabolites on 
Pain Perception in Patients Undergoing Lumbar Disc Herniation Surgery  
 
Study Follow-Up Table 
Study starting from January 12, 2022, following ethical approval: 
1. Ethical approval from the DEU Non-Interventional Ethics Committee is estimated to 
be obtained in 2022. 
2. Commencement of patient data recording following the approval of the Ethics 
Committee. 
3. Regular maintenance of records in the SPSS computer program for the purpose of 
statistical analysis of the acquired data. 
4. Estimated completion of results by November 2022. 
5. Conducting statistical analysis after data collection. 
6. Evaluation of the correlation between tryptophan metabolites (picolinic acid, 3- 
Hydroxykynurenine, anthranilic acid, kynurenine, quinolinic acid, kynurenic acid, 
xanthurenic acid) routinely collected in blood samples after lumbar disc herniation 
surgeries and Visual Analog Scale (VAS) scores. 
 
 
Document Date: 30.03.2022 
Ethic committee decision dated 31.08.2022, numbered 7131-GO 
 
 
 
 
 
 
 
 
 
 
 
 
 

Study Protocol 
 
1. Introduction 
 
The human body is closely associated with a complex ecosystem known as the 
microbiota. The microbiota encompasses the total of microorganisms found in various 
regions of the body, from the digestive system to the skin and respiratory system. 
These microorganisms can have significant effects on health and disease, and recent 
research indicates that the gut microbiota, in particular, plays a decisive role in pain 
perception.[1] [2] 
 
In the past decade, research on the influence of gut microbiota on pain has been 
increasing.[3] The gut microbiota interacts not only with the digestive system but also 
with the nervous and immune systems.[4] These interactions may modulate pain 
mechanisms and contribute to chronic pain. Recent research suggests that gut 
microorganisms may regulate brain function and behaviour.[5] This has been 
considered an exciting development in the field of anesthesia, because the effect of 
intestinal microbiota on pain perception offers a new perspective in the development 
of anesthesia practices. 
 
Tryptophan can be converted into various metabolites by the gut microbiota, and 
the effects of these metabolites on pain are being investigated. [6] The effect of the gut 
microbiota on tryptophan metabolism is an important mechanism in regulating pain 
perception. [7], [8] 
An important feature of the microbiota is that the signaling molecules it produces 
through Tryptophan metabolism can affect pain mechanisms. Some bacteria, 
especially members of the microbiota such as Escherichia Coli, can produce 
tryptophan.[9] Furthermore, commensal bacterial species that produce AhR ligands 
play an important role in regulating AhR activity. [10], [11]. The ability of these bacteria 
to metabolize Tryptophan can lead to the production of the neurotransmitter 
tryptamine. [12].  
 

Pain is a subjective experience that cannot be directly observed or measured 
by biological tests. Therefore, the assessment of pain is largely based on the 
individual's own subjective interpretation. Although its adequacy in pain assessment is 
questioned, the method used is generally Visual Analogue Scale (VAS). 
In this study, we aimed to examine the effect of microbiota and tryptophan 
metabolites on pain perception in patients undergoing lumbar disc herniation surgery. 
We evaluated how the gut microbiota affects pain mechanisms by measuring levels of 
tryptophan metabolites and assessing pain severity in the pre- and postoperative 
periods. 
 
2. Materials and Methods 
 
In this study, we adhered to the STROBE (Strengthening the Reporting of 
Observational Studies in Epidemiology) guidelines to ensure the quality and 
transparency of the reporting of our observational study.  
Our study includes 37 lumbar disc herniation (LDH) surgeries performed in Dokuz 
Eylül University Faculty of Medicine Central Operating Rooms Brain and Nerve 
Surgery Hall. For the blood samples in which we evaluated tryptophan metabolites; 2 
mL of blood was used from the blood samples taken during routine surgery preparation 
for the patients and from the routine blood samples taken in the postoperative period, 
and no additional invasive intervention was performed. We elucidated the relationship 
between tryptophan metabolites (Picolinic Acid, 3-Hydroxykynurenine, Anthranilic Acid, 
Kynurenine, Quinolinic Acid, Kynurenic Acid, Xanthurenic Acid) and their variations due 
to microbiota alterations with VAS in acute pain. Preoperative, postoperative, 8th-hour, 
and 24th-hour blood samples were routinely collected from patients, with 2 ml of 
sample material used each time. Concurrently, pain information and vital signs were 
recorded. After the necessary centrifugation processes, the tryptophan metabolites 
were analyzed by the relevant laboratory team. 
In VAS assessment, patients are typically asked to choose a value between "0, no 
pain" and "10, the worst imaginable pain." VAS is an 11-point scale (ranging from 0 to 
10) that allows patients to numerically express the intensity of their pain.[13] 
 
2.1. 
Data Collection 
 

Blood samples were planned and collected from patients during the preoperative 
evaluation, at the 8th postoperative hour, and at the 24th postoperative hour, adhering 
to clinical routines for these procedures. Concurrently with each blood draw, the 
patient's VAS score, the nature of the pain, and vital signs were recorded. At the end 
of the surgery, procedures were performed in the recovery unit according to the 
protocols, and the same data along with vital signs were recorded. Blood samples 
collected from the patients were centrifuged at 4000 rpm for 10 minutes, after which 
the serum and plasma portions were transferred to Eppendorf tubes. The blood 
samples were stored at -80°C for preservation. For the same patients, 24-hour 
postoperative blood collection was performed, and similar data, including VAS scores, 
were recorded. 
2.2. 
Inclusion and Exclusion Criteria for Case Selection 
Inclusion Criteria: 
• 
Patients undergoing lumbar disc herniation surgery at Dokuz Eylül University 
Hospital. 
• 
Patients who have read and consented to the informed consent form for the 
study. 
• 
Age 18 years or older. 
• 
Surgery duration exceeding 2 hours. 
Exclusion Criteria: 
• 
Patients unwilling to participate in the study. 
• 
Surgery duration less than 2 hours. 
• 
Patients under the age of 18. 
• 
Patients with a history of abdominal surgery. 
• 
Patients who received antibiotic therapy for intestinal pathology within the last 3 
weeks. 
• 
Patients with known abdominal pathologies that are suspected to alter the 
microbial flora (e.g., Crohn's disease, ulcerative colitis, celiac disease). 
Patient records, clinical histories, examination findings, and relevant laboratory 
results were electronically recorded. All these data were documented on a prepared 
data collection form and transferred to an electronic database. 
 
2.3. 
Studying samples collected from patients 
 

Sample analyses were performed using an Ultivo tandem mass spectrometer 
(6465B, Agilent Technologies, Santa Clara, CA, USA) connected to an Agilent 
HPLC system. The analyses of kynurenine pathway metabolites were conducted 
using the CE-IVD certified and validated commercial Jasem Kynurenine Pathway 
Metabolites LC-MS/MS analysis kit (Sem Laboratory Devices Marketing Co., 
Istanbul, Turkey). The analytical process was completed using the mobile phases 
(mobile phase A and B), analytical column, calibrators, stable isotope-labeled 
internal standard (IS) mixture, and sample preparation reagents provided with the 
commercial kit. 
 
2.4. 
Sample Preparation for Kynurenine Pathway Metabolite Analysis 
 
Preparation of the calibrator set; 100 µl calibrator was taken into the HPLC vial, 50 
µl internal standard was added and diluted with 250 µl Reagent-1 and injected into the 
LC-MS/MS system. Sample Preparation; 100 µl serum/plasma/control sample was 
taken into Eppendorf, and 50 µl internal standard was added and vortexed for 5 
seconds after protein precipitation with 250 µl reagent-1. It was centrifuged at 4500rpm 
for 5 minutes. The resulting upper phase was taken into the HPLC vial and transferred 
to the LC-MS/MS system. 
 
2.5. 
Ethichs 
 
Ethics committee approval for this study was received locally from the University 
Izmir Dokuz Eylul University Hospital  (decision dated 31.08.2022, numbered 7131-
GO). The study was conducted following the principles of the Declaration of Helsinki. 
Since our study was planned retrospectively, approval was obtained from the ethics 
committee without obtaining informed consent from the patients. 
 
 
 
 
 
 
 
 
 

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1.1.