Energy metabolism, oxidative stress and immunological status in adult hip replacement patients and healthy individuals

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The study involved 16 HRP patients. Skeletal muscle and blood samples were taken during HR. Moreover, blood samples of 12 control subjects were used to isolate PBMCs. The activity of aerobic (malate dehydrogenase; MDH) and anaerobic (lactate dehydrogenase; LDH) metabolism enzymes and the level of carbonyl groups were measured in skeletal muscle homogenates and PBMC isolates. The indicators of inflammation and immunity were also assessed. The purity level of PBMCs isolates were determined using platelets to PBMC ratio. There was no relationship between MDH and LDH activities and carbonyl groups measured in skeletal muscle homogenate and PBMC isolate. Significantly higher inflammatory indexes (NLR and the number of neutrophils) and the level of protein carbonyl groups were also noted in the HRP group compared to the control group. Biological sciences/Immunology Biological sciences/Physiology Health sciences/Biomarkers Health sciences/Diseases Health sciences/Medical research PBMC LDH MDH oxidative stress NLR inflammation Figures Figure 1 Figure 2 Introduction The study of skeletal muscle energy metabolism in humans is an ongoing research topic, particularly with regard to identifying new clinical health markers in patients with cardiovascular diseases 1 , aging 2 , diabetes 3 , neuromuscular disorders 4 , hypokinesia 5 , and obesity 6 , among others. Changes in skeletal muscle metabolism are frequently observed in these patients, leading to a decline in their quality of life 7 . However, there is limited information available regarding the metabolism of the tensor fascia lata muscle in individuals scheduled for total hip replacement (THR). This muscle plays a crucial role in stabilizing the extended knee joint and the hip joint, as well as supporting the retention of the femur head in the acetabulum through its interaction with the iliotibial band. Additionally, it contributes to thigh flexion at the hip joint, thigh abduction, and inward rotation 8,9 . Changes in muscle metabolism can be indirectly measured by evaluating muscle function through clinical tests or directly analyzed by studying the activities of energy metabolism enzymes in biological material obtained from muscle biopsies. Muscle biopsies allow for a comprehensive analysis of various components of muscle metabolism, such as marker enzyme and metabolite activities 10 , transcripts 11 , and signaling pathways 12 . However, muscle biopsies are highly invasive and painful procedures. Therefore, finding an alternative biological material that is easily accessible and reflects metabolic changes in skeletal muscle is a significant challenge. One promising area of research is the use of peripheral blood mononuclear cells (PBMCs) in energy metabolism studies and assessing human health 13 . Due to their function, PBMCs play a critical role in the body's immune response. Studies have shown a connection between PBMCs and skeletal muscle in various diseases, such as major depression and cardiovascular diseases 14–16 . Decreased mitochondrial function in PBMCs has also been observed in prostate cancer patients 17 . These findings suggest that measuring energy metabolism and oxidative stress indices in PBMCs could potentially be used in the diagnosis and treatment of patients with different diseases. Inflammation may play a crucial role in the interaction between skeletal muscle and PBMCs. Consequently, several systemic inflammation parameters such as neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), lymphocyte to monocyte ratio (LMR), and systemic immune inflammation index (SII), have been identified as prognostic factors, reflecting inflammation in patients 18 . These indices have also been utilized to determine the immune status of adult patients undergoing THR surgery. To the best of our knowledge, there have been no previous studies investigating the relationship between the activity of PBMC energy metabolism enzymes and intraoperative muscle samples from patients with THR. Therefore, the purpose of this study was to compare the activity of energy metabolism marker enzymes and oxidative stress indicators in PBMC isolates with those measured in homogenates of the tensor fascia lata muscle in patients with THR. Additionally, the study aimed to compare oxidative energy and oxidative stress metabolism of PBMCs, as well as the and indicators of inflammation and immunity in HRP with healthy individuals. Results The obtained purity values of the present study are summarized in Table 2 . The average PLT/PMBC ratio in the isolates was 3.96 ± 1.4 and 4.85 ± 2.8, and the average degree of PBMC purity of isolates compared to the value obtained in whole blood was 3.39 ± 1.0 and 4.45 ± 2.1% in control and HRP group, respectively (Table 2 ). The results show that PBMC isolates are of good quality in both study groups, allowing further analyses to be performed. Nevertheless, the activity of energy metabolism enzymes in PBMC isolates did not correlate with respective enzymes in the tensor fascia lata muscle of the patients before HR (LDH, r = 0.066 and p = 0.808; MDH, r = -0.4809 and p = 0.059). Similarly, muscle carbonyl groups did not correlate with PBMC’s carbonyl groups (r = -0.1156 and p = 0.670). The mean value of LDH and MDH activities in skeletal muscle homogenates of HRP was 3101.2 ± 944,2 and 3572.0 ± 985.0 nmoles/min/mg of protein, respectively. The level of carbonyl groups in skeletal muscle homogenates of HRP was 0.86 ± 0.13 nmoles/mg of protein. Still, the comparison of energy metabolism in PBMC isolates indicated higher activity of MDH (p = 0.0002), as a representative of aerobic metabolism and tendency in LDH (p = 0.0898), as a representative of anaerobic metabolism in HRP group (Table 3 ). Modifications in energy metabolism were accompanied with changes in NLR index (Fig. 1 A) and the number of neutrophils (Fig. 1 B). The mean NLR values in the HRP group were significantly higher compared to the control group (p = 0.006) and amounted to 2.5 ± 0.6 and 1.8 ± 0.5, respectively (Fig. 1 A). The increase in the NLR value was mainly caused by a significantly higher content of neutrophils in the HRP group compared to the control group (p = 0.009; Fig. 1 B). NLR index and the number of neutrophils in the control and HRP groups. The average value of the NLR index and the number of neutrophils are shown in Fig. 1 A and 1 B. No differences between groups were observed in other inflammatory indices (Table 4 ). However, the level of carbonyl groups in HRP was significantly higher than in control group (p = 0.0301) and amounted to 0.57 ± 0.11 and 0.70 ± 0.16, respectively (Fig. 2 ). Discussion The presented PBMC isolation protocol is characterized by high isolate purity, and can be used in energy metabolism and oxidative stress studies. The previously applied protocol yielded a median value of 6.3, which was only 5.4% of the ratio observed in whole blood (median 117.2). Nevertheless, according to the authors, this contamination by PLTs can significantly contribute to the respiration of the PBMC fraction. Therefore, it has been suggested that the isolation method should be improved for better purity of the PBMC fraction 19 . However, we did not observe any correlation between the activities of energy metabolism enzymes measured in PBMC isolates and those measured in skeletal muscle homogenates. Additionally, there was no significant relationship between the levels of carbonyl groups in PBMC isolates and skeletal muscle homogenates. Nonetheless, this research demonstrated that patients before hip replacement surgery exhibited moderately increased inflammation and oxidative stress indicators. The use of PBMCs as a material to reflect changes in skeletal muscles in healthy individuals is controversial. Increased levels of proteins related to mitochondrial biogenesis and improved antioxidant capabilities of mitochondria have been observed in PBMCs of football players after 8-week training period 20 . By contrast, a 2-week high-intensity interval training protocol did not affect PBMC gene and protein markers of mitochondrial biogenesis. Moreover, mitochondrial respiration did not increase in PBMCs following training. Most importantly, PBMCs did not predict training effect on muscle mitochondrial respiration 21 . Similarly, in young women with varying body mass indices respiratory parameters were not correlated between permeabilized muscle fibers and intact PBMCs or platelets 1 . According to Sumbalova et al. (2020), the purity of PBMC isolates plays a significant role in measuring mitochondrial function 19 . However, previous studies evaluating the relationship between the respiratory activities of mitochondria in PBMC and skeletal muscle did not examine the purity of PBMC isolates 20,21 . Additionally, the correlations between platelets and permeabilized muscle in complex I leak and oxidative phosphorylation coupling efficiency were noted 1 . Due to the above, we assumed that using PBMC isolates as research material should first be verified by testing the quality of their isolation. Using a modified isolation procedure, the platelet contamination was lower than previously reported 19 . Since contamination by platelets can significantly contribute to the respiration of the PBMC fraction, the obtained purity values of the present study can be considered satisfactory and allows us to suggest the procedure for studying PMBC metabolism and oxidative stress. Muscle energy metabolism reflection in PBMC may occur, especially when the patients are subjected to inflammation and/or oxidative stress. In our study, we tested patients with osteoarthritis who qualified for primary THR without clinically significant comorbidities. Hip joint failure caused by osteoarthritis is the most common indication for THR, and other indications include but are not limited to, rheumatoid arthritis, avascular necrosis, traumatic arthritis, certain hip fractures, benign and malignant bone tumors, arthritis associated with Paget's disease, ankylosing spondylitis, and juvenile rheumatoid arthritis 22 . However, none of the activities of energy metabolism enzymes measured in PBMC isolates were correlated with those measured in skeletal muscle homogenates, despite high purity of PBMC. Our results, confirm previously reported data 1,2,21 indicating that PBMC isolates do not reflect the metabolism of the skeletal muscle in healthy individuals and in pathological conditions. Increased ROS accumulation and inflammation play a crucial role in etiology of various pathologies. The potential use of PBMC metabolism measurements in patients should not be excluded. We examined the markers of inflammation and oxidative stress in study patients with total hip replacement. Nevertheless, we recorded significantly higher average results of the NLR index and the number of neutrophils in the HRP group compared to the control group, indicating moderate inflammation 23 . However, it seems that the analysis of inflammatory markers should also be carried out individually because, in two patients, the value of the SII index exceeded the reference value and amounted to 1134.4 and 1339.5. This may be important in identifying biological markers of inflammation as a tool for predicting the risk of developing postoperative infectious complications at the preclinical stage. However, there are no studies documenting which indicators could be the most sensitive or showing the relationship between muscles and cells of the immune system, which is a possible direction for further research. As mentioned earlier, we did not observe a significant correlation between the levels of carbonyl groups in PBMC and skeletal muscle, although the concentration of carbonyl groups in PBMC isolates was significantly higher in the HRP group compared to the control group. Additionally, the measured levels of carbonyl groups in the muscle homogenates were at reference levels 24 , indicating the absence of oxidative stress in skeletal muscle. Conclusion Despite higher inflammatory and oxidative stress indexes in HRP than in controls, there was no correlation in metabolic indices between PBMC and skeletal muscle. However, the easy-to-use protocol for PBMC isolation and the high degree quality of these isolates give hope for using this biological material as the potential for diagnosing health and energy metabolism in metabolically ill individuals. Using indicators of immune status and oxidative stress measurable in good-quality PBMC isolates may help predict the risk of developing postoperative infectious complications at the preclinical stage. Materials and Methods Participants The experimental protocol was approved by the Ethics Committee of the Medical University of Gdansk (No. NKBBN/569/2021). All methods were performed in accordance with the relevant guidelines and regulations. Before starting the experimental procedure, all participants were informed about the procedure, risks, and expected outcomes and they provided their written informed consent for participation. This study recruited 20 consecutive patients undergoing total hip replacement (HRP group) who met the inclusion criteria (10 women and 10 men; age range: 24–68 years; mean age: 54.9 ± 10.0 years), and 12 control subjects were recruited for the experiment. The inclusion criteria were as follows: age ≥ 18 years of age and diagnosis of arthritis eligible for THR. The indication for THR in the whole study group was osteoarthritis. All the patients had radiographic evidence of joint damage and moderate-to-severe persistent pain or disability, or both, that was not substantially relieved by nonsurgical treatment, like analgetic and nonsteroidal anti-inflammatory drugs, physical therapy, or reduction in physical activities that provoke discomfort. According to The National Institute of Health, Bethesda, United States of America (NIH) consensus conference guidelines, discrepancies between the patient's expectations and the likely outcome were discussed in detail with the patient and family members before surgery 22 . Exclusion criteria were as follows: diagnosis of any cardiovascular disease or other chronic diseases known to lead to changes in skeletal muscle structure and function, taking medicines known to affect the immune system, and lack of written consent to participate in the study. The control group was similar in age and physical activity status to the HRP group, as well as in terms of parameters such as height or creatinine, glucose, hemoglobin and hematocrit concentrations in the blood. However, the subjects from the HRP group differed significantly from the control group in terms of body weight, BMI as well as systolic and diastolic blood pressure. All participants’ characteristics are presented in Table 1 . Muscle samples The tensor fascia lata muscle samples were collected under local or general anesthesia from patients undergoing THR at the Department of Orthopedics, Traumatology of the Locomotor System and Hand Surgery, University Hospital in Gdansk. Muscle samples were dissected free of visible fat and connective tissue, weighed, immediately frozen in liquid nitrogen, and stored at -80ºC until analysis. Muscle specimens were then minced and homogenized in a glass Teflon Potter-Elvehejm homogenizer in a 1:10 wt/vol dilution of buffer containing 50 mM potassium phosphate, 1 mM EDTA, and 1 mM dithiothreitol (pH 7.4). The resulting homogenates were stored at − 80 ºC until assayed. Blood samples Participants screened for the study arrived at the laboratory in the morning hours after an overnight fast. 8 mL of resting venous blood were drawn into each of 2 BD Vacutainer® CPT™ NC: 1 mL and Ficoll™: 2 mL (No 362782) tubes from which PBMCs were isolated. A detailed PBMC isolation protocol is provided below and in Scheme 1 . Additionally, blood was collected in 3KEDTA tubes for morphological analysis. White blood cell (WBC) count and differential leukocyte count were determined using an automated hematology analyzer (Sysmex XN-350, Global Medical Instrumentation, Inc (Mundelein, IL, USA)) in the whole blood. PBMC Isolation Protocol PBMCs were isolated from whole blood taken from the ulnar vein using density gradient centrifugation. In all cases, isolation of PBMCs was carried out within 1 h of collecting a whole blood sample. After mixing the tubes (x5 times), the blood was centrifuged at room temperature for 20 min at 1600x g. Spinning acceleration was set to 0, and braking was switched off (acceleration and brake settings were 0- min, 9- max- critical parameters for high-purity isolation of the PBMC). After centrifugation, the samples were mixed five times, and the plasma with PBMCs was decanted into a 50 mL falcon. The falcon was refilled up to 50 mL with Dulbecco's Phosphate Buffered Saline (DPBS). The tubes were then centrifuged at 4ºC for 10 min, 400x g (recommended centrifugation parameters for high-purity isolation of the PBMC). Spinning acceleration and braking were set to 9 and 6, respectively. The supernatant was then discarded, and the pellet consisting of PBMCs was dissolved in 15 mL of DPBS. After mixing, the tubes were centrifuged at 4ºC for 10 min at 120x g. Spinning acceleration and braking were set to 9 and 6, respectively. Then, the previous two procedures were repeated: the supernatant was discarded, and the pellet was dissolved in 15 mL of DPBS. After mixing, the tubes were centrifuged at 4ºC for 10 min at 120x g. Spinning acceleration and braking were set to 9 and 6, respectively. Finally, the supernatant was discarded, and the pellet was dissolved in 0.5 mL of MIRO5, consisting of 110 mM sucrose, 60 mM K-lactobionate, 20 mM HEPES, 20 mM taurine, 10 mM KH2PO4, 3 mM MgCl2, and 0.5 mM EGTA at pH 7.1 (about 30 million cells/mL) (Scheme 1 ). PBMCs prepared in this way were used to measure the activities of energy metabolism enzymes and the level of carbonyl groups. To determine the purity of the PBMC isolation, 50 µL of the isolate was taken and added to 450 µL of DPBS. Cells were counted on a Sysmex XN-350 hematology analyzer. PBMC Purity Determination The purity of PBMC isolation is measured by the ratio of the number of platelets (PLTs) to the amount of PBMC and expressed as the percent of PLT to PBMC ratio in whole blood. Maximal Enzyme Activities To characterize anaerobic and aerobic capacities, several pathways were examined. Specifically, the following enzyme activities were evaluated: (i) the anaerobic pathway was lactate dehydrogenase (LDH), and (ii) the aerobic pathway was malate dehydrogenase (MDH). For each assay, all samples were measured in duplicate, and the average activity over the linear portion of the absorbance–time relationship was used to represent enzyme activity. The following enzyme activities' maximum rates (V max ) were measured spectrophotometrically using a spectrophotometer (Cecil 9200 Super Aquarius; Cambridge, UK). Lactate Dehydrogenase Activity LDH activity was measured at 30°C, according to 25 . The concentration of pyruvate (PYR) was 2.1 mM. After 2 min of preincubation of tissue (10 µL of PBMC and 30 µL (1:10) homogenates) with buffer (50 mM KPi, 1 mM EDTA, pH 7.4) and PYR, NADH was added immediately before the measurement of the enzyme activity, and the reaction was initiated. The final volume in the cuvette was 1000 µL. The decrease in absorbance at 340 nm was measured for 2 min. The enzyme activities are expressed as nmol/min/mg of protein. Malate Dehydrogenase Activity MDH activity was measured at 30°C, according to 26 . Briefly, 30 µL of homogenate (1:10) or 10 µL of PMBC was incubated for 2 min in a buffer solution (50 mM Tris HCl, 5 mM EDTA, pH 7.6) supplemented with 10 µL freshly prepared oxaloacetic acid (20 mM). Next, 10 µL freshly prepared NADH (20 mM) was added to initiate the reaction. The final volume in the cuvette was 1000 µL. The decrease in absorbance at 340 nm was measured for 2 min. The enzyme activities are expressed as nmol/minute/mg of protein. Carbonyl protein groups assessement The carbonyl groups were measured in PBMC isolates and skeletal muscle homogenates from the HRP and control participants using the Biocell Protein Carbonyl Assay Kit (Biocell Corporation, New Zeland) according to the manufacturer's instructions. The values of the carbonyl groups are expressed as nmol per mg of protein. Indicators of Inflammation and Immunity Preoperative blood sampling was performed to measure neutrophil, lymphocyte, and platelet levels to calculate the NLR, PLR, LMR, and SII indices. NLR and PLR were defined as the total number of neutrophils or platelets divided by the total number of lymphocytes, respectively. LMR was defined as the total number of lymphocytes divided by the total number of monocytes. SII was calculated using the following formula: SII = (P × N)/L, where P, N, and L refer to peripheral platelet, neutrophil, and lymphocyte counts, respectively. International Physical Activity Questionnaire (IPAQ) – Polish Long Version. The level of physical activity of the patients and control subjects was assessed using the survey method 27,28 . The same interviewer conducted and analyzed the survey, and the test was performed on the same day as the blood collection. Based on the IPAQ questionnaire, physical activity was determined (in MET-min/week) in various domains of everyday life, such as during work, travel, housework, recreation, and sports, and sitting time was determined. Based on the results of the survey, respondents were classified into one of three categories of activity: low (below 600), moderate (600–1500 or 600–3000), or high (above 1500 or 3000 MET-min/week) (Table 1 ). Statistical Analysis Due to the availability of biological material the data of only 16 patients from the HRP group were used in the statistical analysis. Statistical analyses were performed using the software package Statistica v. 13.0 (StatSoft Inc., Tulsa, OK, USA). Shapiro-Wilk was used to test the normality of data distribution and t-Student or Mann-Whitney test was used to determine statistically significant differences between groups. A Pearson's/Spearman's product–moment correlation coefficient was computed to assess the correlations between enzyme activities in PBMC and skeletal muscle homogenates. The results were considered statistically significant when p < 0.05. Data are summarized as means ± SD. Declarations Funding This study was supported by grant from Medical University of Gdańsk, Research University, DB 71-01420 / 291-0005147. Conflict of interest All authors declare that they have no conflict of interest. Availability of data and material The data that support the findings of this study are available from the corresponding authors Author Contributions Conceptualization, BA, DF, LS, WZ and DS; Formal analysis, BA, DF, LS, RO, MZ, WZ and DS; Funding acquisition, DS; Investigation, BA, DF, LS, RO, MZ, AS, LK, TB, JA, WZ and DS; Supervision, WZ and DS; Validation, BA, DF, RO, AS, WZ and DS; Visualization, BA, DF, WZ and DS Writing – original draft, BA, DF, WZ and DS; Writing – review & editing, LS, RO, MZ, AS, LK, TB and JA. All authors read and approved the final manuscript. References Rose, S. et al. A comparative study of mitochondrial respiration in circulating blood cells and skeletal muscle fibers in women. American journal of physiology. Endocrinology and metabolism 317 , E503-E512, doi:10.1152/ajpendo.00084.2019 (2019). Maynard, S. et al. 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Characteristics Control group (n = 12) HRP group (n = 16) p-value Age (years) 49.75 ± 10.94 54.85 ± 9.90 0.0712 BMI (kg/m 2 ) 23.72 ± 3.26 28.9 ± 4.82* 0.0014 Systolic BP (mmHg) 114.58 ± 10.97 127.25 ± 12.38* 0.0020 Diastolic BP (mmHg) 71.25 ± 7.11 81.31 ± 10.27* 0.0076 Weight (kg) 70.17 ± 14.31 88.35 ± 16.51* 0.0030 Height (cm) 171.31 ± 0.10 174.75 ± 7.65 0.3598 Hematocrit (%) 43.71 ± 3.63 42.70 ± 3.05 0.4508 Hemoglobin (g/dL) 14.26 ± 1,43 14.69 ± 1.29 0.4861 Creatine (mg/dL) 0.84 ± 0.12 0.79 ± 0.12 0.1905 Glucose (mg/dL) 97.42 ± 14.69 96.62 ± 10.65 0.8698 Physical activity per week (MET-min/week) 3654.66 ± 256,49 3433.38 ± 866.94 0.6225 BMI - body mass index; BP - blood pressure; MET - metabolic equivalent per task. Values are expressed as mean ± SD. The * symbol denotes a statistically significant difference between the studied groups for p<0.05. Table 2. Analysis of PBMC components and the purity of isolates. Control group (n = 12) HRP group (n = 16) p -value Whole blood Monocytes (10 9 /L) 0.54 ± 0.1 0.56 ± 0.2 0.999 Lymphocytes (10 9 /L) 1.81 ± 0.4 1.83 ± 0.5 0.672 PBMC count (10 9 /L) 2.35 ± 0.4 2.39 ± 0.7 0.870 Platelets (10 9 /L) 269.50 ± 53 250 ± 61 0.386 platelets/PBMC ratio 118 ± 28 114 ± 44 0.773 PBMC isolates Monocytes (10 9 /L) 0.67 ± 0.2 0.75 ± 0.4 0.542 Lymphocytes (10 9 /L) 2.54 ± 0.9 1.50 ± 0.7 0.002 PBMC count (10 9 /L) 3.21 ± 1.1 2.25 ± 1.0 0.023 Platelets (10 9 /L) 12.7 ± 5.5 8.8 ± 4.7 0.054 platelets/PBMC ratio 3.96 ± 1.4 4.85 ± 2.8 0.507 PBMC purity isolates analysis PLT/PBMC ratio of isolates as % of whole blood PLT/PBMC ratio 3.39 ± 1.0 4.45 ± 2.2 0.132 Values are expressed as mean ± SD. The * denotes a statistically significant difference between the studied groups for p<0.05. Table 3. PBMC lactate and malate dehydrogenases activities. PBMC Con PBMC HRP p -value LDH activity (nmoles/min/mg protein) 1309 ± 158 1527 ± 426 0.0898 MDH activity (nmoles/min/mg protein) 888 ± 117 1360 ± 357* 0.0002 Values are expressed as mean ± SD. The * denotes a statistically significant difference between the studied groups for p<0.05. Table. 4 The values of the indicators of inflammation and immunity of patients. Control group HRP group p -value SII 505 ±194 613 ± 262 0.189 PLR 154 ± 39 149 ± 59 0.507 LMR 3.6 ± 1.3 3.4 ± 0.8 0.543 The table presents the mean values of indicators of inflammation and immunity of patients such as SII, PLR, and LMR for the control and HRP groups. Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Scheme1.png Scheme 1. Procedure of PBMC isolation Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3896983","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":273574886,"identity":"a5b85acc-e414-49b1-8eda-29e4952e8df0","order_by":0,"name":"Beata Andruszkiewicz","email":"","orcid":"","institution":"Medical University of Gdańsk","correspondingAuthor":false,"prefix":"","firstName":"Beata","middleName":"","lastName":"Andruszkiewicz","suffix":""},{"id":273574887,"identity":"e1bf81b1-71f2-4508-a16e-13fb87039c26","order_by":1,"name":"Damian Flis","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYDCCA2DEwM/HzMD4ACYiAaRkCGmRbGNmYDZA1sKDTwsDWAsDA5sEUVr4jp9OPPBzB4MEGzv7s8qvbdvk+A4wH7zNw3AHpxbJM7kbDvaeAWph5jG7Ldt221jyAFuyNQ/DM5xaDA7kbjjA28ZQB9TCdluy7XbihgM8ZtI8DIdxazn/dsPBv20gW9ifFQO11G84wP8Nv5YbuRsO84K1MJgxfmy7nWBwgIcNrxbJG283HJZtkwD5xVia4dxtw5mH2Ywt5xjg9gvf+dzNH9+22Ujw8x9/+PFH2W15vuPND2+8qbgjh0sLFEBihBlsMjMkWAjogALGHwg2kVpGwSgYBaNgJAAAbpBX3x4fJSYAAAAASUVORK5CYII=","orcid":"","institution":"Medical University of Gdańsk","correspondingAuthor":true,"prefix":"","firstName":"Damian","middleName":"","lastName":"Flis","suffix":""},{"id":273574888,"identity":"5195d06e-a48b-4836-8068-f56f450d5927","order_by":2,"name":"Lucjan Samson","email":"","orcid":"","institution":"Medical University of Gdańsk","correspondingAuthor":false,"prefix":"","firstName":"Lucjan","middleName":"","lastName":"Samson","suffix":""},{"id":273574889,"identity":"a8047511-5ed4-4801-a1a5-2b171739eb3b","order_by":3,"name":"Robert Olek","email":"","orcid":"","institution":"Poznan University of Physical Education","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"","lastName":"Olek","suffix":""},{"id":273574890,"identity":"308e71c7-3e3c-417b-ab4f-5e8ce098376a","order_by":4,"name":"Maciej Zrodowski","email":"","orcid":"","institution":"University Hospital in Gdańsk","correspondingAuthor":false,"prefix":"","firstName":"Maciej","middleName":"","lastName":"Zrodowski","suffix":""},{"id":273574891,"identity":"b6916c2d-6a92-413e-9e7a-79f59df764dd","order_by":5,"name":"Anna Siekierzycka","email":"","orcid":"","institution":"Medical University of Gdańsk","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Siekierzycka","suffix":""},{"id":273574892,"identity":"369a7f59-030f-4e4e-8500-522134fad854","order_by":6,"name":"Leszek Kalinowski","email":"","orcid":"","institution":"Medical University of Gdańsk","correspondingAuthor":false,"prefix":"","firstName":"Leszek","middleName":"","lastName":"Kalinowski","suffix":""},{"id":273574893,"identity":"dc157bfc-7980-4bad-907c-d25962fb7d8c","order_by":7,"name":"Tomasz Borkowski","email":"","orcid":"","institution":"Medical University of Gdańsk","correspondingAuthor":false,"prefix":"","firstName":"Tomasz","middleName":"","lastName":"Borkowski","suffix":""},{"id":273574894,"identity":"a7c10159-00fc-4c2a-9ef5-2d69fb8f15a2","order_by":8,"name":"Jędrzej Antosiewicz","email":"","orcid":"","institution":"Medical University of Gdańsk","correspondingAuthor":false,"prefix":"","firstName":"Jędrzej","middleName":"","lastName":"Antosiewicz","suffix":""},{"id":273574895,"identity":"2c1a0f07-e87b-4d2d-8df9-427626cf987c","order_by":9,"name":"Wiesław Ziółkowski","email":"","orcid":"","institution":"Medical University of Gdańsk","correspondingAuthor":false,"prefix":"","firstName":"Wiesław","middleName":"","lastName":"Ziółkowski","suffix":""},{"id":273574896,"identity":"2cc9ae24-a008-47f2-8c8c-2246d1f69050","order_by":10,"name":"Dominika Szalewska","email":"","orcid":"","institution":"Medical University of Gdańsk","correspondingAuthor":false,"prefix":"","firstName":"Dominika","middleName":"","lastName":"Szalewska","suffix":""}],"badges":[],"createdAt":"2024-01-25 11:29:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3896983/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3896983/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51330429,"identity":"94f85d37-6441-42c1-b73b-0c03660e1f9b","added_by":"auto","created_at":"2024-02-19 17:48:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":21027,"visible":true,"origin":"","legend":"\u003cp\u003eNLR index and the number of neutrophils in the control and HRP groups.\u003c/p\u003e\n\u003cp\u003eValues are expressed as mean ± SD.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3896983/v1/f46a46b4a95f4e40f93504ac.png"},{"id":51330431,"identity":"2f0f41e6-3f2b-464a-a17e-c737e0b163bf","added_by":"auto","created_at":"2024-02-19 17:48:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":13723,"visible":true,"origin":"","legend":"\u003cp\u003eThe concentration of carbonyl groups in the control and HRP groups.\u003c/p\u003e\n\u003cp\u003eValues are expressed as mean ± SD.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3896983/v1/ed5c3776bcbba83e67685b27.png"},{"id":58255525,"identity":"70c6e124-5709-4912-ac67-70729e4130d3","added_by":"auto","created_at":"2024-06-13 05:01:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":621062,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3896983/v1/ca103981-af34-4ea1-95f7-242dabbfeb9a.pdf"},{"id":51330428,"identity":"d1ef1e5e-4f9b-48fc-9420-2d2812bb77eb","added_by":"auto","created_at":"2024-02-19 17:48:11","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":200994,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 1. Procedure of PBMC isolation\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-3896983/v1/1fd0393cb4b755cc2c3d0fe2.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Energy metabolism, oxidative stress and immunological status in adult hip replacement patients and healthy individuals","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe study of skeletal muscle energy metabolism in humans is an ongoing research topic, particularly with regard to identifying new clinical health markers in patients with cardiovascular diseases \u003csup\u003e1\u003c/sup\u003e, aging \u003csup\u003e2\u003c/sup\u003e, diabetes \u003csup\u003e3\u003c/sup\u003e, neuromuscular disorders \u003csup\u003e4\u003c/sup\u003e, hypokinesia \u003csup\u003e5\u003c/sup\u003e, and obesity \u003csup\u003e6\u003c/sup\u003e, among others. Changes in skeletal muscle metabolism are frequently observed in these patients, leading to a decline in their quality of life \u003csup\u003e7\u003c/sup\u003e. However, there is limited information available regarding the metabolism of the tensor fascia lata muscle in individuals scheduled for total hip replacement (THR). This muscle plays a crucial role in stabilizing the extended knee joint and the hip joint, as well as supporting the retention of the femur head in the acetabulum through its interaction with the iliotibial band. Additionally, it contributes to thigh flexion at the hip joint, thigh abduction, and inward rotation \u003csup\u003e8,9\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eChanges in muscle metabolism can be indirectly measured by evaluating muscle function through clinical tests or directly analyzed by studying the activities of energy metabolism enzymes in biological material obtained from muscle biopsies. Muscle biopsies allow for a comprehensive analysis of various components of muscle metabolism, such as marker enzyme and metabolite activities \u003csup\u003e10\u003c/sup\u003e, transcripts \u003csup\u003e11\u003c/sup\u003e, and signaling pathways \u003csup\u003e12\u003c/sup\u003e. However, muscle biopsies are highly invasive and painful procedures. Therefore, finding an alternative biological material that is easily accessible and reflects metabolic changes in skeletal muscle is a significant challenge.\u003c/p\u003e \u003cp\u003eOne promising area of research is the use of peripheral blood mononuclear cells (PBMCs) in energy metabolism studies and assessing human health \u003csup\u003e13\u003c/sup\u003e. Due to their function, PBMCs play a critical role in the body's immune response. Studies have shown a connection between PBMCs and skeletal muscle in various diseases, such as major depression and cardiovascular diseases \u003csup\u003e14\u0026ndash;16\u003c/sup\u003e. Decreased mitochondrial function in PBMCs has also been observed in prostate cancer patients \u003csup\u003e17\u003c/sup\u003e. These findings suggest that measuring energy metabolism and oxidative stress indices in PBMCs could potentially be used in the diagnosis and treatment of patients with different diseases. Inflammation may play a crucial role in the interaction between skeletal muscle and PBMCs. Consequently, several systemic inflammation parameters such as neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), lymphocyte to monocyte ratio (LMR), and systemic immune inflammation index (SII), have been identified as prognostic factors, reflecting inflammation in patients \u003csup\u003e18\u003c/sup\u003e. These indices have also been utilized to determine the immune status of adult patients undergoing THR surgery.\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, there have been no previous studies investigating the relationship between the activity of PBMC energy metabolism enzymes and intraoperative muscle samples from patients with THR. Therefore, the purpose of this study was to compare the activity of energy metabolism marker enzymes and oxidative stress indicators in PBMC isolates with those measured in homogenates of the tensor fascia lata muscle in patients with THR. Additionally, the study aimed to compare oxidative energy and oxidative stress metabolism of PBMCs, as well as the and indicators of inflammation and immunity in HRP with healthy individuals.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe obtained purity values of the present study are summarized in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n\u003cp\u003eThe average PLT/PMBC ratio in the isolates was 3.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 and 4.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.8, and the average degree of PBMC purity of isolates compared to the value obtained in whole blood was 3.39\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 and 4.45\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1% in control and HRP group, respectively (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The results show that PBMC isolates are of good quality in both study groups, allowing further analyses to be performed.\u003c/p\u003e\n\u003cp\u003eNevertheless, the activity of energy metabolism enzymes in PBMC isolates did not correlate with respective enzymes in the tensor fascia lata muscle of the patients before HR (LDH, r\u0026thinsp;=\u0026thinsp;0.066 and p\u0026thinsp;=\u0026thinsp;0.808; MDH, r = -0.4809 and p\u0026thinsp;=\u0026thinsp;0.059). Similarly, muscle carbonyl groups did not correlate with PBMC\u0026rsquo;s carbonyl groups (r = -0.1156 and p\u0026thinsp;=\u0026thinsp;0.670). The mean value of LDH and MDH activities in skeletal muscle homogenates of HRP was 3101.2\u0026thinsp;\u0026plusmn;\u0026thinsp;944,2 and 3572.0\u0026thinsp;\u0026plusmn;\u0026thinsp;985.0 nmoles/min/mg of protein, respectively. The level of carbonyl groups in skeletal muscle homogenates of HRP was 0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 nmoles/mg of protein.\u003c/p\u003e\n\u003cp\u003eStill, the comparison of energy metabolism in PBMC isolates indicated higher activity of MDH (p\u0026thinsp;=\u0026thinsp;0.0002), as a representative of aerobic metabolism and tendency in LDH (p\u0026thinsp;=\u0026thinsp;0.0898), as a representative of anaerobic metabolism in HRP group (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eModifications in energy metabolism were accompanied with changes in NLR index (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA) and the number of neutrophils (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). The mean NLR values in the HRP group were significantly higher compared to the control group (p\u0026thinsp;=\u0026thinsp;0.006) and amounted to 2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 and 1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). The increase in the NLR value was mainly caused by a significantly higher content of neutrophils in the HRP group compared to the control group (p\u0026thinsp;=\u0026thinsp;0.009; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). NLR index and the number of neutrophils in the control and HRP groups. The average value of the NLR index and the number of neutrophils are shown in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB.\u003c/p\u003e\n\u003cp\u003eNo differences between groups were observed in other inflammatory indices (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eHowever, the level of carbonyl groups in HRP was significantly higher than in control group (p\u0026thinsp;=\u0026thinsp;0.0301) and amounted to 0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 and 0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe presented PBMC isolation protocol is characterized by high isolate purity, and can be used in energy metabolism and oxidative stress studies. The previously applied protocol yielded a median value of 6.3, which was only 5.4% of the ratio observed in whole blood (median 117.2). Nevertheless, according to the authors, this contamination by PLTs can significantly contribute to the respiration of the PBMC fraction. Therefore, it has been suggested that the isolation method should be improved for better purity of the PBMC fraction \u003csup\u003e19\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHowever, we did not observe any correlation between the activities of energy metabolism enzymes measured in PBMC isolates and those measured in skeletal muscle homogenates. Additionally, there was no significant relationship between the levels of carbonyl groups in PBMC isolates and skeletal muscle homogenates. Nonetheless, this research demonstrated that patients before hip replacement surgery exhibited moderately increased inflammation and oxidative stress indicators.\u003c/p\u003e \u003cp\u003eThe use of PBMCs as a material to reflect changes in skeletal muscles in healthy individuals is controversial. Increased levels of proteins related to mitochondrial biogenesis and improved antioxidant capabilities of mitochondria have been observed in PBMCs of football players after 8-week training period \u003csup\u003e20\u003c/sup\u003e. By contrast, a 2-week high-intensity interval training protocol did not affect PBMC gene and protein markers of mitochondrial biogenesis. Moreover, mitochondrial respiration did not increase in PBMCs following training. Most importantly, PBMCs did not predict training effect on muscle mitochondrial respiration \u003csup\u003e21\u003c/sup\u003e. Similarly, in young women with varying body mass indices respiratory parameters were not correlated between permeabilized muscle fibers and intact PBMCs or platelets \u003csup\u003e1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAccording to Sumbalova et al. (2020), the purity of PBMC isolates plays a significant role in measuring mitochondrial function \u003csup\u003e19\u003c/sup\u003e. However, previous studies evaluating the relationship between the respiratory activities of mitochondria in PBMC and skeletal muscle did not examine the purity of PBMC isolates \u003csup\u003e20,21\u003c/sup\u003e. Additionally, the correlations between platelets and permeabilized muscle in complex I leak and oxidative phosphorylation coupling efficiency were noted \u003csup\u003e1\u003c/sup\u003e. Due to the above, we assumed that using PBMC isolates as research material should first be verified by testing the quality of their isolation. Using a modified isolation procedure, the platelet contamination was lower than previously reported \u003csup\u003e19\u003c/sup\u003e. Since contamination by platelets can significantly contribute to the respiration of the PBMC fraction, the obtained purity values of the present study can be considered satisfactory and allows us to suggest the procedure for studying PMBC metabolism and oxidative stress.\u003c/p\u003e \u003cp\u003eMuscle energy metabolism reflection in PBMC may occur, especially when the patients are subjected to inflammation and/or oxidative stress. In our study, we tested patients with osteoarthritis who qualified for primary THR without clinically significant comorbidities. Hip joint failure caused by osteoarthritis is the most common indication for THR, and other indications include but are not limited to, rheumatoid arthritis, avascular necrosis, traumatic arthritis, certain hip fractures, benign and malignant bone tumors, arthritis associated with Paget's disease, ankylosing spondylitis, and juvenile rheumatoid arthritis \u003csup\u003e22\u003c/sup\u003e. However, none of the activities of energy metabolism enzymes measured in PBMC isolates were correlated with those measured in skeletal muscle homogenates, despite high purity of PBMC. Our results, confirm previously reported data \u003csup\u003e1,2,21\u003c/sup\u003e indicating that PBMC isolates do not reflect the metabolism of the skeletal muscle in healthy individuals and in pathological conditions.\u003c/p\u003e \u003cp\u003eIncreased ROS accumulation and inflammation play a crucial role in etiology of various pathologies. The potential use of PBMC metabolism measurements in patients should not be excluded. We examined the markers of inflammation and oxidative stress in study patients with total hip replacement. Nevertheless, we recorded significantly higher average results of the NLR index and the number of neutrophils in the HRP group compared to the control group, indicating moderate inflammation \u003csup\u003e23\u003c/sup\u003e. However, it seems that the analysis of inflammatory markers should also be carried out individually because, in two patients, the value of the SII index exceeded the reference value and amounted to 1134.4 and 1339.5. This may be important in identifying biological markers of inflammation as a tool for predicting the risk of developing postoperative infectious complications at the preclinical stage. However, there are no studies documenting which indicators could be the most sensitive or showing the relationship between muscles and cells of the immune system, which is a possible direction for further research. As mentioned earlier, we did not observe a significant correlation between the levels of carbonyl groups in PBMC and skeletal muscle, although the concentration of carbonyl groups in PBMC isolates was significantly higher in the HRP group compared to the control group. Additionally, the measured levels of carbonyl groups in the muscle homogenates were at reference levels \u003csup\u003e24\u003c/sup\u003e, indicating the absence of oxidative stress in skeletal muscle.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eDespite higher inflammatory and oxidative stress indexes in HRP than in controls, there was no correlation in metabolic indices between PBMC and skeletal muscle. However, the easy-to-use protocol for PBMC isolation and the high degree quality of these isolates give hope for using this biological material as the potential for diagnosing health and energy metabolism in metabolically ill individuals. Using indicators of immune status and oxidative stress measurable in good-quality PBMC isolates may help predict the risk of developing postoperative infectious complications at the preclinical stage.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003eParticipants\u003c/h2\u003e\n \u003cp\u003eThe experimental protocol was approved by the Ethics Committee of the Medical University of Gdansk (No. NKBBN/569/2021). All methods were performed in accordance with the relevant guidelines and regulations.\u003c/p\u003e\n \u003cp\u003eBefore starting the experimental procedure, all participants were informed about the procedure, risks, and expected outcomes and they provided their written informed consent for participation.\u003c/p\u003e\n \u003cp\u003eThis study recruited 20 consecutive patients undergoing total hip replacement (HRP group) who met the inclusion criteria (10 women and 10 men; age range: 24\u0026ndash;68 years; mean age: 54.9\u0026thinsp;\u0026plusmn;\u0026thinsp;10.0 years), and 12 control subjects were recruited for the experiment. The inclusion criteria were as follows: age\u0026thinsp;\u0026ge;\u0026thinsp;18 years of age and diagnosis of arthritis eligible for THR. The indication for THR in the whole study group was osteoarthritis. All the patients had radiographic evidence of joint damage and moderate-to-severe persistent pain or disability, or both, that was not substantially relieved by nonsurgical treatment, like analgetic and nonsteroidal anti-inflammatory drugs, physical therapy, or reduction in physical activities that provoke discomfort. According to The National Institute of Health, Bethesda, United States of America (NIH) consensus conference guidelines, discrepancies between the patient\u0026apos;s expectations and the likely outcome were discussed in detail with the patient and family members before surgery \u003csup\u003e22\u003c/sup\u003e. Exclusion criteria were as follows: diagnosis of any cardiovascular disease or other chronic diseases known to lead to changes in skeletal muscle structure and function, taking medicines known to affect the immune system, and lack of written consent to participate in the study. The control group was similar in age and physical activity status to the HRP group, as well as in terms of parameters such as height or creatinine, glucose, hemoglobin and hematocrit concentrations in the blood. However, the subjects from the HRP group differed significantly from the control group in terms of body weight, BMI as well as systolic and diastolic blood pressure. All participants\u0026rsquo; characteristics are presented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eMuscle samples\u003c/h3\u003e\n\u003cp\u003eThe tensor fascia lata muscle samples were collected under local or general anesthesia from patients undergoing THR at the Department of Orthopedics, Traumatology of the Locomotor System and Hand Surgery, University Hospital in Gdansk. Muscle samples were dissected free of visible fat and connective tissue, weighed, immediately frozen in liquid nitrogen, and stored at -80\u0026ordm;C until analysis. Muscle specimens were then minced and homogenized in a glass Teflon Potter-Elvehejm homogenizer in a 1:10 wt/vol dilution of buffer containing 50 mM potassium phosphate, 1 mM EDTA, and 1 mM dithiothreitol (pH 7.4). The resulting homogenates were stored at \u0026minus;\u0026thinsp;80 \u0026ordm;C until assayed.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003eBlood samples\u003c/h2\u003e\n \u003cp\u003eParticipants screened for the study arrived at the laboratory in the morning hours after an overnight fast. 8 mL of resting venous blood were drawn into each of 2 BD Vacutainer\u0026reg; CPT\u0026trade; NC: 1 mL and Ficoll\u0026trade;: 2 mL (No 362782) tubes from which PBMCs were isolated. A detailed PBMC isolation protocol is provided below and in Scheme \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Additionally, blood was collected in 3KEDTA tubes for morphological analysis. White blood cell (WBC) count and differential leukocyte count were determined using an automated hematology analyzer (Sysmex XN-350, Global Medical Instrumentation, Inc (Mundelein, IL, USA)) in the whole blood.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003ePBMC Isolation Protocol\u003c/h2\u003e\n \u003cp\u003ePBMCs were isolated from whole blood taken from the ulnar vein using density gradient centrifugation. In all cases, isolation of PBMCs was carried out within 1 h of collecting a whole blood sample. After mixing the tubes (x5 times), the blood was centrifuged at room temperature for 20 min at 1600x g. Spinning acceleration was set to 0, and braking was switched off (acceleration and brake settings were 0- min, 9- max- critical parameters for high-purity isolation of the PBMC). After centrifugation, the samples were mixed five times, and the plasma with PBMCs was decanted into a 50 mL falcon. The falcon was refilled up to 50 mL with Dulbecco\u0026apos;s Phosphate Buffered Saline (DPBS). The tubes were then centrifuged at 4\u0026ordm;C for 10 min, 400x g (recommended centrifugation parameters for high-purity isolation of the PBMC). Spinning acceleration and braking were set to 9 and 6, respectively. The supernatant was then discarded, and the pellet consisting of PBMCs was dissolved in 15 mL of DPBS. After mixing, the tubes were centrifuged at 4\u0026ordm;C for 10 min at 120x g. Spinning acceleration and braking were set to 9 and 6, respectively. Then, the previous two procedures were repeated: the supernatant was discarded, and the pellet was dissolved in 15 mL of DPBS. After mixing, the tubes were centrifuged at 4\u0026ordm;C for 10 min at 120x g. Spinning acceleration and braking were set to 9 and 6, respectively. Finally, the supernatant was discarded, and the pellet was dissolved in 0.5 mL of MIRO5, consisting of 110 mM sucrose, 60 mM K-lactobionate, 20 mM HEPES, 20 mM taurine, 10 mM KH2PO4, 3 mM MgCl2, and 0.5 mM EGTA at pH 7.1 (about 30 million cells/mL) (Scheme \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). PBMCs prepared in this way were used to measure the activities of energy metabolism enzymes and the level of carbonyl groups. To determine the purity of the PBMC isolation, 50 \u0026micro;L of the isolate was taken and added to 450 \u0026micro;L of DPBS. Cells were counted on a Sysmex XN-350 hematology analyzer.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003ePBMC Purity Determination\u003c/h2\u003e\n \u003cp\u003eThe purity of PBMC isolation is measured by the ratio of the number of platelets (PLTs) to the amount of PBMC and expressed as the percent of PLT to PBMC ratio in whole blood.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003eMaximal Enzyme Activities\u003c/h2\u003e\n \u003cp\u003eTo characterize anaerobic and aerobic capacities, several pathways were examined. Specifically, the following enzyme activities were evaluated: (i) the anaerobic pathway was lactate dehydrogenase (LDH), and (ii) the aerobic pathway was malate dehydrogenase (MDH). For each assay, all samples were measured in duplicate, and the average activity over the linear portion of the absorbance\u0026ndash;time relationship was used to represent enzyme activity. The following enzyme activities\u0026apos; maximum rates (V\u003csub\u003emax\u003c/sub\u003e) were measured spectrophotometrically using a spectrophotometer (Cecil 9200 Super Aquarius; Cambridge, UK).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eLactate Dehydrogenase Activity\u003c/h2\u003e\n \u003cp\u003eLDH activity was measured at 30\u0026deg;C, according to \u003csup\u003e25\u003c/sup\u003e. The concentration of pyruvate (PYR) was 2.1 mM. After 2 min of preincubation of tissue (10 \u0026micro;L of PBMC and 30 \u0026micro;L (1:10) homogenates) with buffer (50 mM KPi, 1 mM EDTA, pH 7.4) and PYR, NADH was added immediately before the measurement of the enzyme activity, and the reaction was initiated. The final volume in the cuvette was 1000 \u0026micro;L. The decrease in absorbance at 340 nm was measured for 2 min. The enzyme activities are expressed as nmol/min/mg of protein.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eMalate Dehydrogenase Activity\u003c/h2\u003e\n \u003cp\u003eMDH activity was measured at 30\u0026deg;C, according to \u003csup\u003e26\u003c/sup\u003e. Briefly, 30 \u0026micro;L of homogenate (1:10) or 10 \u0026micro;L of PMBC was incubated for 2 min in a buffer solution (50 mM Tris HCl, 5 mM EDTA, pH 7.6) supplemented with 10 \u0026micro;L freshly prepared oxaloacetic acid (20 mM). Next, 10 \u0026micro;L freshly prepared NADH (20 mM) was added to initiate the reaction. The final volume in the cuvette was 1000 \u0026micro;L. The decrease in absorbance at 340 nm was measured for 2 min. The enzyme activities are expressed as nmol/minute/mg of protein.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eCarbonyl protein groups assessement\u003c/h2\u003e\n \u003cp\u003eThe carbonyl groups were measured in PBMC isolates and skeletal muscle homogenates from the HRP and control participants using the Biocell Protein Carbonyl Assay Kit (Biocell Corporation, New Zeland) according to the manufacturer\u0026apos;s instructions. The values of the carbonyl groups are expressed as nmol per mg of protein.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eIndicators of Inflammation and Immunity\u003c/h2\u003e\n \u003cp\u003ePreoperative blood sampling was performed to measure neutrophil, lymphocyte, and platelet levels to calculate the NLR, PLR, LMR, and SII indices. NLR and PLR were defined as the total number of neutrophils or platelets divided by the total number of lymphocytes, respectively. LMR was defined as the total number of lymphocytes divided by the total number of monocytes. SII was calculated using the following formula: SII = (P \u0026times; N)/L, where P, N, and L refer to peripheral platelet, neutrophil, and lymphocyte counts, respectively.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eInternational Physical Activity Questionnaire (IPAQ) \u0026ndash; Polish Long Version.\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eThe level of physical activity of the patients and control subjects was assessed using the survey method \u003csup\u003e27,28\u003c/sup\u003e. The same interviewer conducted and analyzed the survey, and the test was performed on the same day as the blood collection. Based on the IPAQ questionnaire, physical activity was determined (in MET-min/week) in various domains of everyday life, such as during work, travel, housework, recreation, and sports, and sitting time was determined. Based on the results of the survey, respondents were classified into one of three categories of activity: low (below 600), moderate (600\u0026ndash;1500 or 600\u0026ndash;3000), or high (above 1500 or 3000 MET-min/week) (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eStatistical Analysis\u003c/h2\u003e\n \u003cp\u003eDue to the availability of biological material the data of only 16 patients from the HRP group were used in the statistical analysis. Statistical analyses were performed using the software package Statistica v. 13.0 (StatSoft Inc., Tulsa, OK, USA). Shapiro-Wilk was used to test the normality of data distribution and t-Student or Mann-Whitney test was used to determine statistically significant differences between groups. A Pearson\u0026apos;s/Spearman\u0026apos;s product\u0026ndash;moment correlation coefficient was computed to assess the correlations between enzyme activities in PBMC and skeletal muscle homogenates. The results were considered statistically significant when p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Data are summarized as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grant from Medical University of Gdańsk, Research University,\u0026nbsp;DB 71-01420 / 291-0005147.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no conflict of interest. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding authors\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, BA, DF, LS, WZ and DS; Formal analysis, BA, DF, LS, RO, MZ, WZ and DS; Funding acquisition, DS; Investigation, BA, DF, LS, RO, MZ, AS, LK, TB, JA, WZ and DS; Supervision, WZ and DS; Validation, BA, DF, RO, AS, WZ and DS; Visualization, BA, DF, WZ and DS Writing – original draft, BA, DF, WZ and DS; Writing – review \u0026amp; editing, \u0026nbsp;LS, RO, MZ, AS, LK, TB and JA. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eRose, S.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e A comparative study of mitochondrial respiration in circulating blood cells and skeletal muscle fibers in women. \u003cem\u003eAmerican journal of physiology. Endocrinology and metabolism\u003c/em\u003e \u003cstrong\u003e317\u003c/strong\u003e, E503-E512, doi:10.1152/ajpendo.00084.2019 (2019).\u003c/li\u003e\n \u003cli\u003eMaynard, S.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Relationships between human vitality and mitochondrial respiratory parameters, reactive oxygen species production and dNTP levels in peripheral blood mononuclear cells. \u003cem\u003eAging\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 850-864, doi:10.18632/aging.100618 (2013).\u003c/li\u003e\n \u003cli\u003eAnderson, E. 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Malate-aspartate and alpha-glycerophosphate shuttle enzyme levels in human skeletal muscle: methodological considerations and effect of endurance training. \u003cem\u003eActa physiologica Scandinavica\u003c/em\u003e \u003cstrong\u003e128\u003c/strong\u003e, 397-407, doi:10.1111/j.1748-1716.1986.tb07993.x (1986).\u003c/li\u003e\n \u003cli\u003eHallal, P. C. \u0026amp; Victora, C. G. Reliability and validity of the International Physical Activity Questionnaire (IPAQ). \u003cem\u003eMedicine and science in sports and exercise\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 556, doi:10.1249/01.mss.0000117161.66394.07 (2004).\u003c/li\u003e\n \u003cli\u003eBiernat, E., Stupnicki, R. \u0026amp; Gajewski, A. K. International Physical Activity Questionnaire (IPAQ)-Polish version. \u003cem\u003eWych. Fiz. Sport\u003c/em\u003e \u003cstrong\u003e51\u003c/strong\u003e, 47-54 (2007).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1. Participants characteristics.\u0026nbsp;\u003c/p\u003e\n\u003cdiv align=\"Left\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"527\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.97338403041825%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eCharacteristics\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.482889733840302%\" valign=\"top\"\u003e\n \u003cp\u003eControl group (n = 12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.38403041825095%\" valign=\"top\"\u003e\n \u003cp\u003eHRP group\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(n = 16)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.159695817490494%\" valign=\"top\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.97338403041825%\" valign=\"top\"\u003e\n \u003cp\u003eAge (years)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.482889733840302%\" valign=\"top\"\u003e\n \u003cp\u003e49.75 \u0026nbsp;\u0026plusmn; 10.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.38403041825095%\" valign=\"top\"\u003e\n \u003cp\u003e54.85 \u0026plusmn; 9.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.159695817490494%\" valign=\"top\"\u003e\n \u003cp\u003e0.0712\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.97338403041825%\" valign=\"top\"\u003e\n \u003cp\u003eBMI (kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.482889733840302%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e23.72 \u0026nbsp;\u0026plusmn; 3.26\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.38403041825095%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e28.9 \u0026plusmn; 4.82*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.159695817490494%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0014\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.97338403041825%\" valign=\"top\"\u003e\n \u003cp\u003eSystolic BP (mmHg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.482889733840302%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e114.58 \u0026plusmn; 10.97\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.38403041825095%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e127.25 \u0026plusmn; 12.38*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.159695817490494%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0020\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.97338403041825%\" valign=\"top\"\u003e\n \u003cp\u003eDiastolic BP (mmHg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.482889733840302%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e71.25 \u0026nbsp;\u0026plusmn; 7.11\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.38403041825095%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e81.31 \u0026plusmn; 10.27*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.159695817490494%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0076\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.97338403041825%\" valign=\"top\"\u003e\n \u003cp\u003eWeight (kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.482889733840302%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e70.17 \u0026nbsp;\u0026plusmn; 14.31\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.38403041825095%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e88.35 \u0026plusmn; 16.51*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.159695817490494%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0030\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.97338403041825%\" valign=\"top\"\u003e\n \u003cp\u003eHeight (cm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.482889733840302%\" valign=\"top\"\u003e\n \u003cp\u003e171.31 \u0026plusmn; 0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.38403041825095%\" valign=\"top\"\u003e\n \u003cp\u003e174.75 \u0026plusmn; 7.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.159695817490494%\" valign=\"top\"\u003e\n \u003cp\u003e0.3598\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.97338403041825%\" valign=\"top\"\u003e\n \u003cp\u003eHematocrit (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.482889733840302%\" valign=\"top\"\u003e\n \u003cp\u003e43.71 \u0026nbsp;\u0026plusmn; 3.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.38403041825095%\" valign=\"top\"\u003e\n \u003cp\u003e42.70 \u0026plusmn; 3.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.159695817490494%\" valign=\"top\"\u003e\n \u003cp\u003e0.4508\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.97338403041825%\" valign=\"top\"\u003e\n \u003cp\u003eHemoglobin (g/dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.482889733840302%\" valign=\"top\"\u003e\n \u003cp\u003e14.26 \u0026nbsp;\u0026plusmn; 1,43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.38403041825095%\" valign=\"top\"\u003e\n \u003cp\u003e14.69 \u0026plusmn; 1.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.159695817490494%\" valign=\"top\"\u003e\n \u003cp\u003e0.4861\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.97338403041825%\" valign=\"top\"\u003e\n \u003cp\u003eCreatine (mg/dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.482889733840302%\" valign=\"top\"\u003e\n \u003cp\u003e0.84 \u0026nbsp;\u0026plusmn; 0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.38403041825095%\" valign=\"top\"\u003e\n \u003cp\u003e0.79 \u0026nbsp;\u0026plusmn; 0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.159695817490494%\" valign=\"top\"\u003e\n \u003cp\u003e0.1905\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.97338403041825%\" valign=\"top\"\u003e\n \u003cp\u003eGlucose (mg/dL)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.482889733840302%\" valign=\"top\"\u003e\n \u003cp\u003e97.42 \u0026nbsp;\u0026plusmn; 14.69\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.38403041825095%\" valign=\"top\"\u003e\n \u003cp\u003e96.62 \u0026nbsp;\u0026plusmn; 10.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.159695817490494%\" valign=\"top\"\u003e\n \u003cp\u003e0.8698\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"38.97338403041825%\" valign=\"top\"\u003e\n \u003cp\u003ePhysical activity per week (MET-min/week)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.482889733840302%\" valign=\"top\"\u003e\n \u003cp\u003e3654.66 \u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026plusmn; 256,49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.38403041825095%\" valign=\"top\"\u003e\n \u003cp\u003e3433.38 \u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026plusmn; 866.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.159695817490494%\" valign=\"top\"\u003e\n \u003cp\u003e0.6225\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eBMI - body mass index; BP - blood pressure; MET - metabolic equivalent per task.\u003c/p\u003e\n\u003cp\u003eValues are expressed as mean \u0026plusmn; SD. The * symbol denotes a statistically significant difference between the studied groups for p\u0026lt;0.05.\u003c/p\u003e\n\u003cp\u003eTable 2. Analysis of PBMC components and the purity of isolates.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eControl group\u003c/p\u003e\n \u003cp\u003e(n = 12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eHRP group\u003c/p\u003e\n \u003cp\u003e(n = 16)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eWhole blood\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eMonocytes (10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.54 \u0026plusmn; 0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.56 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eLymphocytes (10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e1.81 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e1.83 \u0026plusmn; 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.672\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003ePBMC count (10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e2.35 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e2.39 \u0026plusmn; 0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.870\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003ePlatelets (10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e269.50 \u0026plusmn; 53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e250 \u0026plusmn; 61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.386\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eplatelets/PBMC ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e118 \u0026plusmn; 28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e114 \u0026plusmn; 44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.773\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003ePBMC isolates\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eMonocytes (10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.67 \u0026plusmn; 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.75 \u0026plusmn; 0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.542\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eLymphocytes (10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.54 \u0026plusmn; 0.9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.50 \u0026plusmn; 0.7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.002\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003ePBMC count (10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.21 \u0026plusmn; 1.1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.25 \u0026plusmn; 1.0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.023\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003ePlatelets (10\u003csup\u003e9\u003c/sup\u003e/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e12.7 \u0026plusmn; 5.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e8.8 \u0026plusmn; 4.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.054\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eplatelets/PBMC ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e3.96 \u0026plusmn; 1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e4.85 \u0026plusmn; 2.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.507\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003ePBMC purity isolates analysis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003ePLT/PBMC ratio of isolates as % of whole blood PLT/PBMC ratio\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e3.39 \u0026plusmn; 1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4.45 \u0026plusmn; 2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e0.132\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;Values are expressed as mean \u0026plusmn; SD. The * denotes a statistically significant difference between the studied groups for p\u0026lt;0.05.\u003c/p\u003e\n\u003cp\u003eTable 3. PBMC lactate \u0026nbsp;and malate dehydrogenases activities.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"587\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.25170068027211%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.68027210884354%\" valign=\"top\"\u003e\n \u003cp\u003ePBMC Con\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.14965986394558%\" valign=\"top\"\u003e\n \u003cp\u003ePBMC HRP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.918367346938776%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.25170068027211%\"\u003e\n \u003cp\u003eLDH activity (nmoles/min/mg protein)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.68027210884354%\"\u003e\n \u003cp\u003e1309 \u0026plusmn; 158\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.14965986394558%\"\u003e\n \u003cp\u003e1527 \u0026plusmn; 426\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.918367346938776%\"\u003e\n \u003cp\u003e0.0898\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"29.25170068027211%\"\u003e\n \u003cp\u003eMDH activity (nmoles/min/mg protein)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.68027210884354%\"\u003e\n \u003cp\u003e\u003cstrong\u003e888 \u0026plusmn; 117\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.14965986394558%\"\u003e\n \u003cp\u003e\u003cstrong\u003e1360 \u0026plusmn; 357*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.918367346938776%\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.0002\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eValues are expressed as mean \u0026plusmn; SD. The * denotes a statistically significant difference between the studied groups for p\u0026lt;0.05.\u003c/p\u003e\n\u003cp\u003eTable. 4 The values of the indicators of inflammation and immunity of patients.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eControl group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eHRP group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eSII\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e505 \u0026plusmn;194\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e613 \u0026plusmn; 262\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.189\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003ePLR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e154 \u0026plusmn; 39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e149 \u0026plusmn; 59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.507\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003eLMR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e3.6 \u0026plusmn; 1.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e3.4 \u0026plusmn; 0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25%\" valign=\"top\"\u003e\n \u003cp\u003e0.543\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eThe table presents the mean values of indicators of inflammation and immunity of patients such as SII, PLR, and LMR for the control and HRP groups.\u0026nbsp;\u003c/p\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"PBMC, LDH, MDH, oxidative stress, NLR, inflammation","lastPublishedDoi":"10.21203/rs.3.rs-3896983/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3896983/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe work aimed to compare (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) the activity of energy metabolism enzymes and oxidative stress indicators in homogenates of the tensor fascia lata muscle with peripheral blood mononuclear cell (PBMC) isolates in patients with hip replacement (HRP) and (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) PBMCs energy and stress metabolism, and indicators of inflammation and immunity of HRP and healthy individuals.\u003c/p\u003e \u003cp\u003eThe study involved 16 HRP patients. Skeletal muscle and blood samples were taken during HR. Moreover, blood samples of 12 control subjects were used to isolate PBMCs. The activity of aerobic (malate dehydrogenase; MDH) and anaerobic (lactate dehydrogenase; LDH) metabolism enzymes and the level of carbonyl groups were measured in skeletal muscle homogenates and PBMC isolates. The indicators of inflammation and immunity were also assessed. The purity level of PBMCs isolates were determined using platelets to PBMC ratio.\u003c/p\u003e \u003cp\u003eThere was no relationship between MDH and LDH activities and carbonyl groups measured in skeletal muscle homogenate and PBMC isolate. Significantly higher inflammatory indexes (NLR and the number of neutrophils) and the level of protein carbonyl groups were also noted in the HRP group compared to the control group.\u003c/p\u003e","manuscriptTitle":"Energy metabolism, oxidative stress and immunological status in adult hip replacement patients and healthy individuals","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-19 17:48:06","doi":"10.21203/rs.3.rs-3896983/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"10a6e34c-07d0-44b0-9a82-1cbb3e987a22","owner":[],"postedDate":"February 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":28827639,"name":"Biological sciences/Immunology"},{"id":28827640,"name":"Biological sciences/Physiology"},{"id":28827641,"name":"Health sciences/Biomarkers"},{"id":28827642,"name":"Health sciences/Diseases"},{"id":28827643,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2024-06-13T04:53:22+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-19 17:48:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3896983","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3896983","identity":"rs-3896983","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}