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The enzyme-linked immunosorbent assay (ELISA) detects antigen-antibody interactions by using enzyme-labelled conjugates and enzyme substrates that generate colour changes. This review aims to provide an overview of ELISA, its various types, and its applications in detecting metabolites in biological fluids. The article discusses the history of the assay, its underlying principles and procedures, common ELISA protocols, and the most accurate and reliable techniques for measuring peptide molecules in biological fluids. Additionally, we emphasize best laboratory practices to achieve consistent, high-quality results and outline the essential materials for setting up an ELISA laboratory, drawing from our over 30 years of experience in the field.

Background and Objectives: Obesity has become one of the most significant health problems nowadays, with its prevalence rapidly increasing. Approaches such as diet and exercise play an important role in the treatment of obesity. This study aimed to investigate the responses of uric acid, irisin, adiponutrin, adropin, and copeptin levels to exercise and metformin intervention in obesity. Materials and Methods: Thirty-six male Sprague–Dawley rats were randomly divided into seven groups: healthy control (HC), sham (S), obese control (OC), metformin (M), exercise (E), metformin + exercise (ME), and decapitation (D). After obesity was induced through a 12-week high-fat diet, obese rats underwent a 4-week aerobic exercise and metformin intervention. Results: Uric acid, irisin, adiponutrin, adropin, and copeptin levels were determined using an ELISA method. Copeptin levels significantly decreased in the ME group (p < 0.001). Irisin levels significantly increased in the E and ME groups (p < 0.001). The most notable increases in adropin levels occurred in the E and ME groups (p < 0.001). Uric acid levels were highest in the OC group but significantly lower in the E and M groups (p < 0.001). Adiponutrin levels did not change in response to exercise or metformin intervention in obesity (p > 0.05). Conclusions: These findings suggest that exercise and metformin intervention may play an effective role in obesity management.

Background. In this study, the effects of pomegranate-black carrot juice mixture on serum and erythrocytes of sedentary individuals who had exhaustion test were investigated. Methods. A total of 20 men voluntarily participated in the study. Blood samples were obtained from participants on three conditions. First, before the study, blood samples of participants were collected (baseline). Second, the same participants performed in the 20-meter shuttle run test for 1 week each day and were subjected to oxidative stress. Lastly, the same participants were given a mixture of pomegranate-black carrot juices (100 ml/100 ml) for a week, 45 minutes prior to the 20-meter shuttle run test, and the stress + supplement was performed. Blood samples were taken at the end of each process. Results. In the erythrocytes, while the oxidative stress condition malondialdehyde (MDA) level and carbonic anhydrase (CA) enzyme activity levels increased compared to the baseline, reduced glutathione (GSH) level, glutathione reductase (GR), and glutathione S-transferase (GST) enzyme activity levels decreased. In stress + supplement conditions, while GSH and GR levels increased according to oxidative stress conditions, CA and MDA levels decreased. While the lactate dehydrogenase (LDH) level of the oxidative stress condition increased compared to the baseline, the LDH level of the stress + supplement decreased compared to the oxidative stress condition. Conclusions. Our results showed that the level of oxidative stress in subjects exposed to the exhaustion test decreased with the mixture of pomegranate-black carrot juices.

Background Chromium histidinate (CrHis) and biotin are micronutrients commonly used to improve health by athletes and control glycaemia by patients with diabetes. This study investigates the effects of 8-week regular exercise training in rats together with dietary CrHis and biotin supplementation on glucose, lipids and transaminases levels, as well as protein expression levels of peroxisome proliferator-activated receptor gamma (PPAR-γ), insulin receptor substrate-1 (IRS-1) and nuclear transcription factor kappa B (NF-κB). Methods A total of 56 male Wistar rats were randomly divided into 8 groups of 7 animals each and treated as follows: Control, CrHis, Biotin, CrHis+Biotin, Exercise, CrHis+Exercise, Biotin+Exercise, and CrHis+Biotin+Exercise. The doses of CrHis and biotin were 400 μg/kg and 6 mg/kg of diet, respectively. The training program consisted of running at 30 m/min for 30 min/day at 0% grade level, 5 days per week, once a day for 6 weeks. Serum glucose, total cholesterol (TC), high-density lipoprotein cholesterol (HDL), triglycerides (TG), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured with an automatic biochemical analyzer. Muscle and liver PPAR-γ, IRS-1 and NF-κB expressions were detected with real-time polymerase chain reaction. Results Regular exercise significantly ( p  < 0.001) decreased glucose, TC and TG levels, but increased HDL cholesterol. Dietary CrHis and biotin supplementation exhibited a significant ( p  < 0.001) decrease in glucose (effect size = large; ƞ2 = 0.773) and TG (effect size = large; ƞ2 = 0.802) levels, and increase in HDL cholesterol compared with the exercise group. No significant change in AST and ALT (effect size = none) levels was recorded in all groups ( p  > 0.05). CrHis/biotin improves the proteins expression levels of IRS-1, PPAR-γ, and NF-κB (effect size: large for all) in the liver and muscle of sedentary and regular exercise-trained rats ( p  < 0.001). Conclusions CrHis/biotin supplementation improved serum glucose and lipid levels as well as proteins expression levels of PPAR-γ, IRS-1 and NF-κB in the liver and muscle of exercise-trained rats, with the highest efficiency when administered together. CrHis/biotin may represent an effective nutritional therapy to improve health.

Background and Objectives: Prediabetes (PD) is characterized by impaired glucose metabolism and is associated with an elevated risk of type 2 diabetes and cardiovascular diseases. This study aimed to investigate the effects of an 8-week core exercise intervention on glycemic control, lipid profiles, insulin sensitivity, body composition, and physical performance in prediabetic women. Materials and Methods: Eighteen prediabetic women aged 20–55 years were randomly allocated to either a core exercise group (n = 9) or a control group (n = 9). The intervention group completed 24 supervised core exercise sessions over 8 weeks, whereas the control group remained sedentary. Pre- and post-intervention evaluations included anthropometric measurements, flexibility and strength tests, fasting and postprandial glucose levels, HbA1c, insulin, HOMA-IR, lipid profiles, and serum iron levels. Non-parametric tests were used for statistical analysis, and a Principal Component Analysis (PCA) and hierarchical clustering were conducted to explore multidimensional metabolic changes. Results: Core exercise significantly improved the body weight, BMI, fat percentage, and circumferences (shoulder, chest, and hip), along with an enhanced flexibility and back-leg strength (p < 0.05). Glycemic indices (FBG, PBG, and HbA1c), insulin, and HOMA-IR levels were significantly reduced, while serum iron and HDL-C increased (p < 0.05). Lipid markers, including the TG, LDL-C, CHOL, and TG/HDL-C ratio, showed significant improvements. The PCA and cluster analyses identified three clusters reflecting metabolic risk, body composition, and protective factors. Conclusions: This study demonstrates that an 8-week structured core exercise program significantly improves glycemic control, lipid profiles, insulin sensitivity, and body composition in women with prediabetes. Multivariate analyses (PCA and hierarchical clustering) corroborate a metabolic shift towards a reduced insulin resistance and a more favorable cardiometabolic profile, supporting core training as a viable, evidence-based non-pharmacological intervention to mitigate metabolic risk.

Obesity, a major global health concern, is associated with systemic metabolic dysregulation. Spexin, a peptide implicated in appetite control and energy balance, may represent a biomarker and therapeutic target in obesity management. This study aimed to investigate tissue-specific modulation of spexin expression in obese male rats subjected to aerobic exercise and/or metformin treatment. Thirty-six Sprague–Dawley rats were randomly assigned to six groups (n = 6 per group): (i) control, (ii) obese control, (iii) exercise, (iv) metformin, (v) metformin + exercise, and (vi) a decapitation baseline group. Obesity was induced via a 12-week high-calorie diet. Subsequently, interventions were applied over 4 weeks: treadmill running (30 min/day, 5 days/week) and/or metformin (150 mg/kg/day). Post-intervention, body weight significantly decreased in intervention groups (p < 0.001) exercise (−13.7%), metformin (−14.6%), and metformin + exercise (−21.1%) compared to the obese control group. ELISA revealed tissue-specific effects on spexin expression. In skeletal muscle, spexin levels were highest in controls (628 ± 160.5 pg/mL), with a significant reduction in the metformin + exercise group (349 ± 84.7 pg/mL; p = 0.003, Cohen’s d = 2.17). In the liver, the control group showed the highest expression (443 ± 240.8 pg/mL), while metformin + exercise yielded the lowest (254 ± 20.4 pg/mL). In contrast, heart tissue maintained elevated spexin levels across all intervention groups, with the metformin + exercise group nearly matching control levels (617 ± 25.2 vs. 618 ± 53.2 pg/mL). Immunohistochemistry confirmed these patterns, with the highest cardiac histoscore in the metformin + exercise group (2.34 ± 0.09). Hierarchical clustering underscored distinct tissue-specific expression patterns, separating muscle from liver and heart. Collectively, these findings suggest that spexin is differentially regulated by exercise and metformin, with joint effects and complex, tissue-specific modulation. This highlights spexin’s potential as a biomarker and therapeutic target in precision obesity interventions.

Background and Objectives: In this study, the effects of an eight-week exercise and nutrition program on blood lipids, glucose, insulin, insulin resistance (HOMA-IR), leptin, ghrelin, irisin, malondialdehyde (MDA), and Growth Differentiation Factor 15 (GDF15) in overweight women were investigated. Materials and Methods: A total of 48 women volunteers participated in this study. The participants were randomly divided into four groups: control (C), exercise (E), nutrition (N), exercise + nutrition (E + N). While no intervention was applied to group C, the other groups participated in the predetermined programs for 8 weeks. At the beginning and end of this study, body composition was measured and blood samples were taken. Results: It was determined that the body composition components, lipid profile indicators, insulin, glucose, insulin resistance, leptin, ghrelin, irisin, and MDA parameters examined in this study showed positive changes in the intervention groups. Group E had a greater effect on body muscle percentage, MDA, and irisin levels, while group N had a greater effect on blood lipids and ghrelin levels. Conclusions: As a result, it is thought that lifestyle changes are important to improve cardiovascular health and combat obesity, and that maintaining a healthy diet together with exercise may be more effective.

Background and Objectives: In this study, the effects of a six-week training program and various diets on subfatin, asprosin, irisin, leptin, ghrelin and the lipid profile were investigated in overweight women. Materials and Methods: A total of 78 women voluntarily participated in the study. Groups: The study was divided into eight groups: Healthy Control, Obese Control, Obese + Vegetarian, Obese + Ketogenic, Obese + Intermittent Fasting, Obese + Exercise + Vegetarian, Obese + Exercise + Ketogenic and Obese + Exercise + Intermittent Fasting. While there was no intervention in the healthy and obese control groups, the other groups followed predetermined exercise and diet programs for 6 weeks. Blood samples were taken from the participants in the research group twice (before and after the interventions). An autoanalyzer was used to determine the lipid profile in the blood samples taken, and the ELISA method was used to analyze other parameters. Results: Overall, a significant difference was found in the values of weight, BMI, subfatin, ghrelin, leptin, cholesterol, triglyceride, HDL and LDL as a result of the exercise and diet interventions (p < 0.05). There was no significant difference in asprosin and irisin values (p > 0.05). Conclusions: In conclusion, regular exercise and dietary interventions in obese women can regulate lipid profile, ghrelin, leptin and asprosin levels, and increasing irisin with exercise can activate lipid metabolism and support positive changes in lean mass.

Background: Coffee is considered one of the most preferred and consumed beverage types in the world, and caffeine is known to increase physical performance due to its ergogenic properties. The aim of this study is to examine the effects of coffee consumption in different forms on cortisol, testosterone, lactic acid and anaerobic performance levels. Methods: A total of 15 licensed male football players participated in the research voluntarily. The research was implemented in a single-blind, counterbalanced, randomized and crossover study design. Participants were given caffeinated coffee (CK), decaffeinated coffee (placebo) (DK), powdered caffeine (in a gelatin capsule) (PC) and powdered placebo (maltodextrin in a capsule) (PM) on different days, and the Wingate test protocol was performed after the warm-up protocol. Blood samples were collected post-test. Cortisol, testosterone and lactic acid levels in the serum samples taken were determined by the ELISA method. Results: As a result, it was revealed that caffeinated coffee given to participants who exercise increased anaerobic power. However, it was observed that lactic acid levels were higher in placebo and decaffeinated coffee. The highest level of cortisol was found in caffeinated coffee and powdered caffeine compared to the placebo. Testosterone values were observed to be highest in caffeinated coffee and decaffeinated coffee compared to a placebo. Conclusions: The study suggests that the type of caffeine is a factor that affects absorption rate, which impacts performance and hormone levels.

Coenzyme Q10 (CoQ10) is a molecule that serves as a coenzyme for mitochondrial enzymes, playing a fundamental role in mitochondrial bioenergetics as an electron and proton carrier in the energy production process. This study aimed to examine the modulatory effects of moderate/high-intensity exercise and CoQ10 supplementation on tumstatin, lipid dynamics, and body mass in rats. This study used 42 male Wistar Albino rats in six groups: a control group (C), a moderate-intensity continuous training group (MICT), a high-intensity continuous training group (HICT), a coenzyme Q10 group (Q10), a moderate-intensity continuous training combined with Q10 group (MICTQ10), and a high-intensity continuous training combined with Q10 group (HICTQ10) to assess the effects of exercise and 5 mg/kg/daily CoQ10 supplementation. Rats underwent treadmill training, and tumstatin levels in plasma, cardiac, and skeletal muscle tissues were measured using ELISA and immunostaining techniques. In addition to the plasma, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), and total cholesterol (TC) levels were analyzed using enzymatic methods, with the LDL-C calculated using the Friedewald equation. The atherogenic index of plasma was determined by the TG/HDL-C ratio. As compared to group C, body mass was significantly affected by both exercise intensity and supplementation (p = 0.01, η2 = 0.37), with the MICTQ10 and HICTQ10 groups demonstrating the greatest reductions by day 50th (p = 0.0003, d = 4.02; p = 0.0001, d = 3.99). Lipid profiles varied significantly between groups. Compared to the C group, the MICTQ10 group exhibited the most substantial decreases in LDL-C (p = 0.03, d = 2.35) and TG levels (p = 0.03, d = 2.25), while the HICTQ10 group showed the most pronounced reduction in TC levels (p = 0.001, d = 6.41). Regarding tumstatin levels, skeletal muscle tumstatin levels were lowest in the HICTQ10 group (p = 0.01, d = 2.11). Moreover, cardiac muscle tumstatin levels were significantly lower in the MICTQ10, MICT, and HICTQ10 groups compared to in the C group (p = 0.004, d = 1.01). These findings suggest that both exercise intensity and CoQ10 supplementation exert notable physiological effects, particularly in modulating body mass, lipid metabolism, and tumstatin levels.