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ARTICLE
Preoperative and Postoperative Endogenous Melatonin and Anxiety Levels and Their Correlation in Living Liver Donors

Abstract

Objectives: High anxiety levels may lead to mental and physical changes that may affect quality of life. Melatonin has anxiolytic properties. It has been reported that administration of melatonin reduces anxiety. In this study, we examined the preoperative and postoperative anxiety levels of living liver donors and the correlation between anxiety levels and endogenous melatonin levels.
Materials and Methods: This prospective clinical study included 56 living liver donors who underwent right hepatectomy (39 women, 17 men; average age of 29 ± 7 years). The anxiety levels were evaluated by using the Spielberger State-Trait Anxiety Inventory Test with a form for this test used to measure the current state of anxiety score and another form used to measure the underlying anxiety score of the patient. These forms were applied preoperatively and postoperatively. Blood samples were taken simultaneously for melatonin levels. Melatonin levels were measured using high-pressure liquid chromatography. Our primary outcomes were to determine the preoperative and postoperative endogenous melatonin and anxiety levels of living liver donors and to investigate their correlations. Results: A statistically significant difference was observed between preoperative and postoperative state of anxiety scores. The preoperative and postoperative underlying anxiety scores were similar. A statistically significant difference was found between the preoperative endogenous melatonin level and postoperative endogenous melatonin level. A significant correlation was not observed between the preoperative and postoperative current and underlying anxiety levels or endogenous melatonin levels. Conclusions: Living liver donors had high anxiety levels during the preoperative and postoperative periods. A significant decrease was identified in the postope­rative hour 24 endogenous melatonin level. These results may lay the foundation for interventions that can identify emotional changes as well as control and improve the mental health of living liver donors.


Key words : Living donor hepatectomy

Introduction

Liver transplantation is the only treatment option for progressive, irreversible end-stage liver disease that is refractory to medical treatment.1,2 The failure to meet the increasing organ demand with deceased donors has increased interest in living donor liver transplant. It has been emphasized that the psychological and emotional state of living liver donors (LLDs) should be monitored, especially with regard to depression, and that clinical follow-up should be continued for up to 2 years after the surgical procedure.2

Anxiety is an important adaptation symptom in the presence of stressful conditions. High anxiety levels may lead to mental and physical changes that may affect quality of life.3 It has been reported that the quality-of-life scales for LLDs are either equal to or better than those for normal adults during the predonation period. However, it has been observed that the quality of life then decreases after donation compared with before donation and compared with control groups in 7% to 20% of LLDs.1,4

Glutamate, an excitatory neurotransmitter, has a role in the pathology of anxiety. Novel evidence has shown a strong correlation between the glutamate system and anxiety. Furthermore, melatonin was reported to be beneficial in ischemia-induced glutamatergic impairment. In addition, a correlation between melatonin release and glutamatergic inputs has been shown. Melatonin is a hormone that affects biorhythms through physiological mechanisms. Irregularities in melatonin secretion cause sleep disorders, psychosis in the intensive care unit, and many other behavioral disorders.5-7 Melatonin controls circadian rhythms, mood regulation, and anxiety. Disruption of circadian rhythms leads to helplessness and anxiety-like behavior in mice.8 Melatonin administration increases the level of gamma aminobutyric acid in the hypothalamus, cerebellum, and cerebral cortex.

Oral or sublingual administration of melatonin has been reported to reduce preoperative anxiety.9,10 Studies on the effects of oral melatonin administration on anxiety during the preoperative period have shown that exogenous melatonin administration reduced anxiety.7,9-14 However, no study has demonstrated a correlation between endogenous melatonin levels and anxiety in LLDs. Here, we hypothesized that a relationship between levels of endogenous melatonin and anxiety may exist.

In this study, we examined the preoperative and postoperative anxiety levels of LLDs as well as the correlation between anxiety levels and endogenous melatonin levels. Based on our literature review, the present study is the first trial on anxiety and endogenous melatonin levels in LLDs.

Materials and Methods

This prospective clinical study was carried out according to the American Society of Anesthesiologists classification of stages 1 to 2 and included 56 LLDs from 18 to 65 years old undergoing right hepatectomy (39 women, 17 men; average age of 29 ± 7 years). Approval was obtained from the Inonu University Faculty of Medicine Local Ethics Council, with protocols ensured to conform with the ethical guidelines of the 1975 Helsinki Declaration. Written informed consent was obtained from all participants. The trial was registered in the ClinicalTrial.gov database (NCT04230707). The study was performed at Inonu University Liver Transplant Institute, Malatya, Turkey, from July 2019 to July 2020.

In total, 60 patients were initially included in the study. Four patients were subsequently excluded because they did not want to complete the postoperative Spielberger State-Trait Anxiety Inventory (STAI) test. Therefore, 56 patients were analyzed. Donors were related to their recipients, and the relationship degree between the donor and the recipient was a first- or second-degree relationship. Patients were excluded if they had cardiovascular, neurologic, or psychiatric disorders; opioid tolerance; sleep disorders; hypnotic, neuroleptic, antidepressant, beta blocker, or steroid use; or allergy history.

The STAI 2019 test (STAI form TX-1) is a validated and commonly preferred test to measure anxiety. It is a questionnaire comprising two, 20-item scales used to measure state and trait anxiety. The anxiety levels of LLDs were evaluated by using the STAI, which also has a valid Turkish language version. The STAI-State (STAI-S) form was used to measure the current anxiety level. The STAI-Trait (STAI-T) form was used to measure the underlying (ongoing/personality) anxiety level of the patient. Each patient completed these forms preoperatively and postoperatively. The mean (total) score on the STAI has a minimum of 20 and a maximum of 80. The STAI scores are generally classified as no anxiety or low anxiety (score of 20-37), moderate anxiety (score of 38-44), and high anxiety (score of 45-80).15

Our primary outcomes in this study were to determine the preoperative and postoperative endogenous melatonin and anxiety levels of the LLDs and to investigate the correlation between them. No premedication was applied preoperatively. Electrocardiogram, arterial oxygen saturation, noninvasive blood pressure, and bispectral index measurements were monitored. All operations were started in the morning between 8:00 and 8:30 am. Vascular access was established prior to induction, and blood samples were drawn to determine the preoperative melatonin level.

Standard general anesthesia was applied. Anesthesia induction was performed with 2 to 3 mg/kg of propofol and 1 mg/kg of remifentanil, and muscle relaxation was induced by 0.6 mg/kg of rocuronium.

Anesthesia was maintained with 0.8 to 1 minimal alveolar concentration sevoflurane, a 40% oxygen-air mixture, and a 0.25 to 0.5 μg/kg/minute remifentanil infusion by keeping the bispectral index value between 40 and 60. The eyes of the patients were covered with a band throughout the surgery. The donors were extubated and moved to intensive care after the surgery. The patients were followed up in an isolated room in the intensive care unit for the first postoperative 24 hours. Lights were turned off from 11:00 pm to 06:00 am, thus maintaining an environment with optimal darkness to ensure that melatonin secretion was not affected.

The STAI scale was readministered 24 hours after the operation, and blood samples were drawn to determine the melatonin level. Melatonin can be measured by several methods, but chromatographic methods have been the most widely used separation techniques in this field in recent years.16,17 Changes in the plasma melatonin level were measured using the high-pressure liquid chromatography (HPLC) method (see Instrumentation for further explanation). Efficient analytical methods for the measurement of melatonin (N-acetyl-5-metoxytriptamine), together with opti­mized extraction protocols, should help confirm the presence of melatonin in various matrixes.

Sample pretreatments are required before chro­matographic measurement for the determination of melatonin, especially in plasma samples. Solid phase extraction (SPE) was used for the precon­centration and clean-up of melatonin from different samples prior to their instrumental analysis.17-19

Reagents and materials
A melatonin standard was purchased from Sigma-Aldrich. Samples of 1000 mg/L stock standard solutions were prepared by dissolving the accurately weighed pure compound in 10 mL of methanol and water (1:1 vol/vol). The melatonin calibration
curve was prepared from the stock standard solution with 1%:1% methanol-water (vol/vol) diluted to
5 concentrations between 0.2 and 200 pg/mL. Calibration curves of the standards were made daily by diluting stock standards in methanol and water (1:1 vol/vol). A calibration curve of the set of melatonin standards of known concentrations was used for quantitative evaluation of plasma melatonin levels.

Instrumentation
The chromatographic qualitative and quantitative measurements of melatonin were evaluated with a Shimadzu chromatography system that consisted of a scanning fluorescence detector. Analysis of melatonin was carried out using a Shimadzu HPLC, equipped with Shimadzu DGU-20 A5 model vacuum degasser and Shimadzu 20 ADXR solvent pump. Separations were performed using an ODS-C18
(150 mm × 4.6 mm, 5 μm) column operated at room temperature. The analysis was performed at an excitation wavelength of 275 nm and emission wavelength of 345 nm (Figure 1).17

Determination of melatonin was performed at the optimum separation condition by HPLC with isocratic elution at room temperature and a mobile phase consisting of 75 mM sodium acetate-acetonitrile (72:28, vol/vol) (pH 5). The flow rate was set at 1.0 mL/min, and the injection volume was 40 μL.

Sample preparation procedure with solid phase extraction
Plasma samples were mixed with 0.5 mL of sodium phosphate buffer (0.01 M, pH 7). Plasma samples were pre-extracted through an SPE cartridge (C-18, Supelco hydrophilic-lipophilic balance 30 mg/1 mL SPE tube) with dichloromethane in order to remove substances that could interfere with the subsequent melatonin analysis. The SPE cartridge was conditioned by successive washing with 1 mL of methanol and 1 mL of water. Samples were loaded and eluted with 1 mL of 90% ethanol-water, and the extract was evaporated to dryness with a nitrogen stream at 35 °C. The residue was reconstituted with 100 μL of acetonitrile-water (50:50, vol/vol) and injected into the HPLC system.18,19

Validation studies
Calibration curves, correlation factors (r) of the calibration curves, and linearity range were evaluated. Measurements of the limit of detection, limit of quantification, linearity, and recoveries were studied. Preoperative and postoperative plasma melatonin levels of an individual patient are shown by chromatogram in Figure 1A and Figure 1B, respectively. Figure 1C shows the HPLC chro­matogram of the standard solution containing 100 pg/mL of melatonin. The analysis was almost completed in 15 minutes.

Statistical analyses
The minimum sample size required was determined to be 19 to detect a statistically significant difference with a type I error (alpha) of 0.05, a test power
(1-beta) of 0.8, an effect size of 0.69, and a bidirectional alternative hypothesis.20

Data are summarized as means and standard deviations, minimums and maximums, or numbers and percentages. The Shapiro-Wilk test was used to examine whether the data were compatible with a normal distribution. The Wilcoxon paired 2-sample test was used to analyze the preoperative anxiety state of melatonin and continuity as well as for the postoperative anxiety state and continuity. Correlations between preoperative and postoperative anxiety states and preoperative and postoperative melatonin levels and the differences were calculated via the Spearman rho coefficient. P < .05 was accepted as statistically significant. Analyses were conducted using the IBM SPSS statistics 25.0 software package.

Results

The demographic data and laboratory results of
the patients are presented in Table 1. As shown in Table 2, a statistically significant difference was observed between the median (minimum to maximum) reoperative STAI-S score of 46 (27-61) and the median (minimum to maximum) postoperative STAI-S score of 41 (31-52) (P < .001). The median (minimum to maximum) preoperative STAI-T score of 46 (33-56) and the median (minimum to maximum) postoperative STAI-T score of 45 (34-69) were similar (P = .357).

The limit of detection, limit of quantification, and linear range for melatonin were found to be 0.01 pg/mL, 0.20 pg/mL, and 0.20 to 200 pg/mL, respectively. A statistically significant difference was found between the median (minimum to maximum) preoperative endogenous melatonin level of 47.39 pg/mL (5.38-524.26 pg/mL) and the median (minimum to maximum) postoperative endogenous melatonin level of 16.64 pg/mL (1.15-157 pg/mL) (P < .001) (Table 2).

A significant correlation was not observed between the preoperative and postoperative STAI-S and STAI-T scores or the preoperative and postoperative endogenous melatonin levels or their differences (Spearman rho correlation; P > .05). Details on this correlation are presented in Table 3 and Table 4.

Discussion

The present study indicated that LLDs had high preoperative STAI-S and STAI-T scores as well as high anxiety. The anxiety level decreased to moderate levels following the decrease in the STAI-S score 24 hours postoperatively, and the STAI-T score remained high as the anxiety level increased. In addition, the postoperative hour 24 endogenous melatonin level decreased significantly compared with the preoperative level. No correlation was seen between melatonin levels and anxiety.

The surgical procedure applied to LLDs puts not only a physical but also a psychological burden on many of these patients. Hence, volunteers selected for organ donation should be healthy both physically and emotionally.21 Given these burdens, it is obligatory to consider the psychological impacts of living donor liver transplant in a comprehensive manner and examine its long-term impacts. A better understanding of the psychological outcomes can be beneficial to solving the psychological problems that donors may experience. Moreover, identifying donors who may have a high risk for adverse psychological outcomes will enable the monitoring of donors and the development of the required treatments and beneficial interventions for transplant centers.1 Our finding that LLDs had high anxiety levels during both the preoperative and post­operative periods can be helpful in developing interventions for them.

In a study on the anxiety and stress levels of liver transplant candidates that measured anxiety via STAI,22 anxiety levels were found to be minimal in 1.92% of the patients, mild in 59.62%, moderate in 36.54%, and severe in 1.92%. There are many studies that have examined the psychological results of LLDs; however, these studies generally focus on a 1- to 2-year follow-up along with postoperative results and are mostly retrospective. In 2 such studies, 23.8% and 51.6% of donors had a mild level of anxiety 1 year and 2 years after the operation, respectively. Individual anxiety syndrome at a given point in time was reported by fewer than 5% of patients.1,2 In contrast, the prevalence and level of anxiety were high in our study group. This may be due to the use of the gold standard STAI test for identifying anxiety. Moreover, the preoperative anxiety measurement times for patients have not been clearly set forth in other studies. In our study, the STAI test was conducted in the surgery room immediately before surgery, which may be related to their high levels of anxiety. However, we could not find a study comparing the preoperative and postoperative anxiety levels of LLDs in the literature. Some pharmacological interventions or anesthetic medications, including benzodiazepines, lorazepam, oxazepam, diazepam, and midazolam, reduce anxiety levels. We did not use these medications to avoid bias.23

Melatonin synthesis is at a maximum level in a dark environment and during sleep.5 Compared with a placebo, oral or sublingual melatonin reduces preoperative anxiety 50 to 100 minutes after administration. Melatonin has an anxiolytic effect as effective as standard midazolam. In addition, it can reduce postoperative anxiety for up to 6 hours compared with a placebo.8,9,24 However, there are also reports indicating that the administration of melatonin to elderly surgical patients did not lead to a decrease in the level of anxiety.11

In animal studies, melatonin treatment ameliorated anxiety- and depression-like behaviors in mice. Melatonin treatment caused the differential expression of a number of proteins, including anxiety-associated proteins such as GSTP1, and was modulated in mouse hippocampus. Agomelatine (a melatonin agonist) is an antidepressant drug that acts through its effect on the MT1 and MT2 melatonergic receptors similar to melatonin. Melatonin creates an antidepressant effect by regulating the sleep-wake rhythm and central receptor activity (MT1 and MT2). Moreover, the anxiolytic effect of melatonin is possible through an interaction of the various effects of melatonin, including regulation of circadian rhythms and sedative, analgesic, anti-inflammatory, antioxidative, and oncostatic effects.8,24

In an examination of the effect of surgery on the endogenous melatonin level, there were significant decreases in plasma melatonin concentrations during the perioperative and postoperative periods.17 Similar results were also observed in our present study. The significant decrease in the melatonin level 24 hours after surgery may be explained by the shift in the sleep rhythm due to surgery-related stress and pain, lack of sleep, and inhibition of melatonin secretion by the increased cortisol caused by surgical stress.25

We observed no statistically significant correlation between endogenous melatonin levels and anxiety in the preoperative or postoperative period. However, 24-hour monitoring may be insufficient to detect a correlation between endogenous melatonin and anxiety. Evaluating the anxiety and mood of LLDs may be more beneficial over a wider time frame, including a period in which the stress related to surgery has disappeared.

A strength of the present study is that it was conducted in a homogeneous group of healthy persons, both physically and emotionally, undergoing the same surgical procedures.

Limitations of the study
The limitations of the study are as follows. First, we monitored the preoperative and postoperative anxiety and melatonin levels of the donors for a short time. Second, the physical environment, such as light and sound conditions, was not optimized and may have had an adverse impact on postoperative melatonin secretion. However, all donors were monitored in the same environment.

Conclusions

Living liver donors had high anxiety levels during the preoperative and postoperative periods. A statistically significant decrease was identified in the postoperative hour 24 endogenous melatonin level compared with the preoperative level. These results may lay the foundation for interventions that can identify emotional changes as well as control and improve the mental health of LLDs.


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DOI : 10.6002/ect.2021.0060


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From the 1Anaesthesiology and Reanimation, Inonu University Faculty of Medicine, the 2Analytical Chemistry, Inonu University Faculty of Pharmacy, and the 3Medical Pharmacology, Inonu University Faculty of Medicine, Malatya, Turkey
Acknowledgements: The authors have no conflicting relationships or anything to disclose. This study was supported by TUBITAK (Scientific and Technological Research Council of Turkey) with project number 217S871.
Corresponding author: Mehmet Ali Erdogan, Inonu University, School of Medicine, Department of Anaesthesiology and Reanimation, Malatya, Turkey
Phone: +90 341 06 60   
E-mail:drmalierdogan@gmail.com