Evaluation of pyruvate kinase and oxidative stressparametersindifferentiationbetween transudate and exudate in pleural liquids

Objectives: We aimed to investigate the usability of pleural pyruvate kinase (PK), total antioxidant status (TAS), and total oxidant status (TOS) as an alternative to Light’s criteria in exudate-transudate differentiation. Methods: This prospective study was conducted among 84 patients (42 transudates and 42 exudates) with pleural effusion. The levels of PK, TAS, and TOS were measured by using ELISA kits, and the ROC analysis was used to evaluate the diagnostic efficiency. Results: PK (p=0.001), TAS (p=0.027), and TOS (p=0.002) levels in pleural fluids were found to be significantly higher in the exudate group. The cut-off values for PK, TAS, and TOS were 10.64 U/L, 13.54 mmol trolox equivalent/L, and 13.88 μmol H2O2 equivalent/L, respectively. While the sensitivity values were 97.62 % for PK, 66.67 % for TAS, and 64.29 % for TOS, the specificity values were 80.95 % for PK, 52.38 % for TAS, and 57.14 % for TOS. Conclusions: PK levels in pleural effusion can be useful in suspected cases to differentiate between exudate and transudate in addition to Light’s criteria. However, pleural TOS and TAS parameters could not be as sensitive and specific as Light’s criteria.


Introduction
Pleural effusion (PE) is described as the accumulated fluid in the pleural space. PE is divided into two categories: transudates and exudates [1]. Exudates are liquids, cells, or other cellular materials that slowly leak from blood vessels from inflamed tissues in general. Transudates are fluids that pass through a membrane or squeeze through tissue or into the extracellular space of tissues. Transudates are thin, watery, and contain few cells or proteins [2].
Currently, the most commonly used method in distinguishing between transudate and exudate PE has been reported as Light's criteria [3,4]. The diagnosis of exudative PE is made in cases the fluid characteristics meet one of Light's criteria, which include a pleural/serum protein ratio >0.5, a pleural/serum lactate dehydrogenase (LDH) level >0.6, and a pleural LDH level over 2/3 of the reference value (or 200 IU) [5,6]. It has been reported that Light's criteria identify 98 % of pleural exudates, and they misclassify about 25 % of transudates as exudates [7]. Thus, there are various studies aimed to determine other parameters that can be used in the differentiation of transudates-exudates, especially with higher specificity for reducing the possibility of misclassification. It is crucial to distinguish between transudate and exudate for differential diagnosis and take appropriate action. For instance, the protein content of the pleural fluid is increased as a result of diuretic therapy, and it is misclassified as exudate in congestive heart failure patients with transudate pleural fluid [6,7]. Therefore, new parameters such as serum-pleural fluid albumin gradient, pleural fluid cholesterol level, pleural fluid/serum cholesterol measurement, and Costa criteria have been reported in the distinction of transudate-exudate as an alternative to Light's criteria [8].
Pyruvate kinase (PK) is a well-known enzyme that catalyzes the conversion of phosphoenolpyruvate and adenosine diphosphate (ADP) to pyruvate and adenosine triphosphate (ATP) in glycolysis and plays a role in the regulation of cell metabolism [9]. PK and oxidative stress parameters [total antioxidant status (TAS) and total oxidant status (TOS)] have been examined directly or indirectly in the diagnosis of various diseases [10,11]. However, to our knowledge, PK has not been tested in distinguishing between transudate and exudate in pleural liquids. Therefore, we have aimed to investigate the pleural PK and the oxidative stress parameters levels to differentiate between exudate and transudate as an alternative to Light's criteria.

Materials and methods
Patients who visited the Afyonkarahisar Health Sciences University Medical Faculty Hospital, Chest Surgery, and Chest Diseases Clinic between June 15, 2019, and December 1, 2020, were assessed. The study comprised 84 patients with PEs (42 transudates and 42 exudates as a result of distinction using Light's criteria).
The study included patients with pleural fluid aged 18 to 90. The study excluded patients who were younger than 18 years old, older than 90 years old, had bleeding diathesis and abnormal coagulation parameters, were unconscious, had hemothorax, chylothorax, empyema, pulmonary embolism, or had tuberculosis. It also excluded patients who were taking a diuretic, an antiplatelet, or an anticoagulant. After the physical examination, a chest X-ray and/or thorax computed tomography (CT) was taken. Thoracentesis was performed under sterile conditions, and two 20 mL fluid samples were taken into tubes separately from all patients with PE. Peripheral venous blood specimens were collected in serum vacuum tubes containing separator gel (volume of 8 mL) BD Vacutainer SST II advance (Becton Dickinson and Company Franklin Lakes, NJ, USA) simultaneously, and serum samples were obtained after centrifugation at 1,000 × g for 10 min. Afterward, two pleural fluid samples were centrifuged at 1,000 × g for 10 min, and only one pleura fluid sample for PK, TAS, and TOS was stored at −20°C until analysis. Another pleura fluid and serum samples were studied in the biochemistry laboratory for albumin, total protein, LDH, glucose, total cholesterol, triglyceride, and low-density lipoprotein cholesterol (LDL-C) on biochemistry autoanalyzer Cobas c501 (Roche Diagnostics, Mannheim, Germany) including the parameters of Light's criteria. Moreover, a smear, nonspecific culture, acid-fast bacillus (ARB) staining, mycobacteria culture, and fluid cytology were examined in pleural fluid. In addition, albumin and protein gradients (respectively, serum albumin level-pleural fluid albumin level, serum protein level-pleural fluid protein level) were calculated.
According to Light's criteria, fluids were evaluated as exudate if at least one of the following three criteria was present and as transudate if none was present [7,12].
-The ratio of pleural fluid protein to serum protein (protein P/S) is greater than 0.5 -The ratio of pleural fluid LDH to serum LDH (LDH P/S) is greater than 0.6 -Pleural fluid LDH and serum LDH greater than two-thirds of the upper limit of normal according to reference The conclusive diagnosis was made in patients whose pleural fluid samples revealed exudative fluid, whose etiological cause was unknown, and who were suspected of having malignancy or granulomatous pleuritis by performing pleural biopsy by closed pleural biopsy or Video-Assisted Thoracoscopic Surgery (VATS) technique.
Analysis of PK levels in pleural fluid samples PK levels of pleural fluid samples were measured by a microplate reader (BioTek, Epoch, Swindon-UK) using a specific ELISA kit (Bioassay Technology Laboratory, Shanghai, China). Linear detection ranges of the kit are 0.1-50 U/L for colorimetric assays and 0.01-2 U/L for fluorimetric assays run at 25°C for 30 min in a 96-well plate. The detection limit of the kit is 0.01 U/L.

Analysis of TOS levels in pleural fluid samples
TOS was determined using a novel automated measurement method developed by Erel [13] (Rel Assay Diagnostic, Gaziantep, Turkey). Oxidants in the sample oxidize the ferrous ion-O-dianisidine complex to a ferric ion. The oxidation reaction is enhanced by glycerol molecules, which are abundantly present in the reaction medium. The ferric ion makes a coloured complex with xylenol orange in an acidic medium. The colour intensity, which can be measured spectrophotometrically, is related to the total amount of oxidant molecules in the sample.

Statistical analysis
IBM (International Business Machines) SPSS (Statistical Package for the Social Sciences) v20 program was used for statistical analysis. G*POWER version 3.1.9.4 was used for power analysis. Power analysis was performed using the mean and standard deviation values obtained as a result of testing the quantitative data, and the power of the study was found to be 0.9511 under current conditions. As a result of the power analysis, 84 patients were included in the study. The conformity of quantitative data to the normal distribution was assessed using the Kolmogorov-Smirnov, Shapiro-Wilk, and graphical methods. Data were presented as mean ± standard deviation, number of subjects, and percentage. The Student's t-test was used if the two groups were normally distributed, and the Mann-Whitney U test was used if they did not fit the normal distribution. Receiver operating characteristic (ROC) curve analysis was performed to predict TAS, TOS, and PK cut-off values in transudate and exudate pleural fluid. The correlations among Light's parameters, PK, TAS, and TOS values were evaluated by the Pearson test. The Pearson chi-square or Fisher's exact test was used to compare qualitative data. A p value of <0.05 was considered statistically significant.

Results
The majority of the 84 patients with PEs had parapneumonic effusion (n=23) and lung cancer (n=32). Four cases of malignant melanoma, three cases of small cell lung cancer, and 14 adenocarcinomas were found among patients with lung cancer. Twenty-one (50 %) of the 42 patients with transudative fluid were male, and 21 (50 %) were female. Seventeen (40.48 %) and 25 (59.52 %) of the 42 patients with exudative fluid were men, respectively. Patients with exudate and transudate fluid had similar sex ratios (p=0.124).
The correlation values of TAS, TOS, and PK were determined as r=0.328, p=0.002; r=0.406, p<0.001, and r=0.657, p<0.001, respectively. In this study, the age range of 84 patients containing 38 females (45.24 %) and 46 males (54.76 %) were between 20 and 90 years, and their mean age was 69.07 ± 13.29 years. The mean age of females was 68.13 years, and the mean age of males was 69.78. The mean ages of males and females were similar (p=0.211).
The age range of patients with transudative fluid was 26-90 years, with a mean age of 71.83 ± 12.79 years. The age range for patients with exudative fluid was 20-87 years, with a mean age of 66.31 ± 13.51 years. Transudate/exudate and mean age did not differ statistically significantly (p=0.056). Demographic characteristics are shown in Table 1.
There was no statistical difference between the serum biochemical test (glucose, albumin, protein, LDH, total cholesterol, triglyceride, and LDL-C) values of the transudate and exudate groups. There was a statistically significant difference in the groups' pleural fluid biochemical values for glucose, albumin, protein, LDH, total cholesterol, triglycerides, and LDL-C (p values, respectively: p=0.001, p<0.001, p<0.001, p<0.001, p<0.001, p=0.004, p<0.001). Protein and albumin gradients were similar ( Table 2).
The two groups had differences in the mean values of TAS, TOS, and PK ( Table 3).
The cut-off values for TAS, TOS, and PK in transudateexudate separation were established as 13.54 mmol trolox equivalent/L, 13.88 μmol H 2 O 2 equivalent/L, and 10.64 U/L, respectively, in the ROC (Receiver Operating Characteristics) analysis. The sensitivity and specificity values for TAS, TOS, and PK are presented in Table 4 and Figure 1.
As a result of the cytological examination of the pleural fluid of the patients, 23 malignant (7 transudates, 16 exudates) and 61 benign (35 transudates, 26 exudates) results were obtained. It was statistically significant (p=0.028).
TAS, TOS, and PK were not statistically significant in the differentiation of benign/malignant (p>0.05) in Table 5.
A non-specific culture examination of the patient's pleural fluid revealed that there was no growth in 81 (96.43 %) of the cases. All were discovered in exudative pleural fluid during a non-specific culture examination; 1

Discussion
Pleural effusion is a common condition that clinicians have difficulty diagnosing and treating. Transudative and exudative PE must be distinguished first in patients with PE. Although Light's criteria are used internationally to distinguish transudate and exudate, the differentiation between transudate and exudate pleural fluid is still unclear in some cases [15]. In this study, we assessed the diagnostic utility of especially PK values and oxidative stress parameters in transudate-exudate differentiation in pleural liquids due to the necessity for alternative distinguishing indicators and/or biomarkers. We have demonstrated that the levels of PK, TAS, and TOS were statistically significantly higher in the exudative fluid. Additionally, in the differentiation of benign     and malignant pleural fluid, oxidative stress parameters and PK levels were not statistically significant. Free radicals and reactive oxygen species (ROS) that are generated in metabolic and physiological processes may be harmful to cellular components like nucleotides, proteins, and lipids [16,17]. ROS are removed via enzymatic and nonenzymatic antioxidative mechanisms. While oxidant molecules such as ROS increase in various pathological conditions, the effectiveness of antioxidant defense systems decreases. TOS and TAS are oxidative stress parameters that are widely used to demonstrate the oxidative balance in organisms [17][18][19][20]. It is known that oxidative stress increases in lung diseases [21,22]. A few studies have investigated the role of oxidative stress parameters in the differential diagnosis of transudative and exudative PE. They may be useful for the differentiation of transudate and exudate pleural fluid [23]. The study of Iclal et al. reported that pleural TAS value was significantly higher in exudate groups but not significant in TOS values according to the comparison of the transudate and exudate groups containing 14 and 47 people, respectively. Their ROC analysis showed that the cut-off value of pleural TAS was 0.87 (AUC: 0.794, sensitivity: 74 %, and specificity: 70 %) [24]. Similar to this study, we also found that TAS levels were significantly higher in the exudate group. However, we reported that TOS was significantly higher in exudative pleural fluid, contrary to the study of Iclal et al. In our study, the levels of TOS (AUC: 0.699, sensitivity: 64.29 % and specificity: 57.14 %) and TAS (AUC: 0.640, sensitivity: 66.67 % and specificity: 52.38 %) in the ROC analysis were not found as sensitive and specific as Light's criteria in the differentiation of transudate and exudate in the pleural fluids. Therefore, TAS and TOS analysis only points out the presence of oxidative stress and antioxidant capacity. Although this non-specific analysis can not be used as diagnostic markers in any pathological condition, they may be tools for observing the response to the therapy.
PK catalyzes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to adenosine diphosphate (ADP), yielding pyruvate and ATP. PK is one of the most critical enzymes for the control of metabolic pathways following glycolysis since, as an irreversible product of the reaction catalyzed by PK, pyruvate is used in various metabolic pathways [25,26]. Different isoenzymes of pyruvate kinase are expressed depending on the metabolic responsibilities of the various cells and tissues [9]. Pyruvate kinase L is a PK isoenzyme specifically found in tissues such as the liver and kidney, where gluconeogenesis reactions occur. Erythrocytes express pyruvate kinase R of PK isoenzymes. Pyruvate kinase M1 is located in tissues where large amounts of energy have to be rapidly provided, such as in muscles and the brain. Lung tissues and all cells with high rates of nucleic acid production, including all proliferating cells, including embryonic cells, adult stem cells, and particularly tumor cells, are characterized by the presence of pyruvate kinase M2 (PKM2) [27,28]. Zhang T. et al. reported that the differential cut-off value, sensitivity, and specificity were 28.67 U/mL (AUC: 0.856), 68.8 %, and 52.5 %, respectively, when they compared the diagnostic value of PKM2 in the benign and malignant PE groups with 85 and 125 subjects, respectively [29]. In another study with 34 lung cancer patients and 34 controls, Elia S. et al. reported that PKM2 was significantly higher in lung cancer with the cut-off value and sensitivity of PKM2 (32.9 U/mL and 85.7 % respectively), and they suggested that PKM2 could be used as a marker to distinguish between malignant and benign PE [30]. However, to our knowledge, PK has not been tested in distinguishing between transudate and exudate in Pleural Liquids. The total activities of PK enzymes were analyzed, but PKM2 of PK isoenzymes was not analyzed as a limitation of the study. Therefore, we concluded that the difference in the pleural fluid between benign and malignant was not statistically significant (p>0.05).
Although Light's criteria have been the accepted standard for distinguishing between transudates and exudates for several years, there are still some concerns with the misclassifications. Recently, some measurements have been made to differentiate between transudative PEs and exudative PEs. Hamm et al. reported significantly increased cholesterol levels in exudative pleural fluid [31]. Moreover, Garcia-Pachon investigated cholinesterase levels to differentiate between transudative and exudative pleural in pleural fluid [32]. In a study similar to our study concept in recent years, a significant increase in CRP level was found in the exudate group. The study demonstrated that the cutoff, sensitivity, and specificity values were determined as 3.31 mg/dL, 96.3 %, and 72.1 %, respectively, and they recommended that the level of CRP in the effusion fluid could help differentiate exudative from transudative PEs [33]. In our study, it was determined that the cut-off, sensitivity, and specificity values of PK were 10.64 U/L, 97.62 %, and 80.95 %. We have suggested that PK level can be useful in the distinction between transudate and exudate in addition to the Light's criteria.

Conclusions
This study established that there is no significant difference between malignant and benign PEs based on the usage of PK, TAS, and TOS. We believe that PK can be employed to distinguish between transudate and exudate in PE patients in addition to Light's criteria.