Effects of Cornelian Cherry Supplementation in Patients With Metabolic Syndrome

TRPV4 ion channel activity is a measure of the level and functioning of a membrane protein responsible for detecting mechanical, osmotic, and thermal stimuli

Serum alpha-crystallin A (CRYAA) concentration will be measured in stored serum samples using a quantitative enzyme-linked immunosorbent assay (ELISA). For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group. Results will be reported in ng/mL.

The aldehyde dehydrogenase 1A1 (ALDH1A1) test measures the level of an enzyme that helps remove toxic substances from cells.

The carboxymethyllysine (CML) assay is a test that assesses the level of advanced glycation end products. It serves as a marker of oxidative stress and vascular damage in diabetes, kidney disease and the ageing process

The determination of carboxyethyllysine (CEL) concentration is used to measure advanced glycation end products (AGEs) in serum or tissues. It helps to assess the extent of protein damage resulting from oxidative stress and ageing processes.

The sRAGE (soluble receptor for advanced glycation end-products) test measures the level of a protein in the blood that acts as a 'trap' for harmful sugar molecules and inflammatory substances.

The 4-hydroxynonenal (4-HNE) test is a laboratory analysis of a toxic lipid peroxidation product, used as an indicator of the severity of oxidative stress and cellular damage.

AOPP (advanced oxidation protein products) are advanced oxidation products of proteins, which indicate the extent of damage to plasma proteins caused by oxidative stress and inflammation.

An interleukin-6 (IL-6) test is a standard, widely available laboratory test used to quantify the concentration of this protein molecule (cytokine) in the blood. It helps with the rapid detection and monitoring of acute and chronic inflammatory conditions.

The sICAM-1 (soluble intercellular adhesion molecule-1) test measures the concentration of a protein that is released into the blood when the endothelium of blood vessels is damaged or stimulated, and by the immune system.

An analysis of the composition of the gut flora (microbiome) is a genetic or microbiological analysis of a stool sample, which involves a quantitative and qualitative assessment of microorganisms.

Fecal calprotectin concentration will be measured in stool samples as a marker of intestinal mucosal inflammation. Results will be reported as the concentration of calprotectin in feces, for example in µg/g of stool.

Fecal lactoferrin concentration will be measured in stool samples as a marker of neutrophil-associated intestinal inflammation. Results will be reported as the concentration of lactoferrin in feces, for example in µg/g of stool.

Serum calcium concentration will be measured in stored serum samples using a validated clinical chemistry method. For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group.

Fecal zonulin concentration will be measured as an exploratory marker associated with intestinal barrier function and intestinal permeability. Results will be reported as the concentration of zonulin in feces.

The concentration of bacterial lipopolysaccharide (LPS) will be measured in stool samples as an exploratory marker of the presence of Gram-negative bacterial components in the intestinal lumen. Results will be reported as the concentration of LPS in feces.

Lipopolysaccharide-binding protein (LBP) will be measured as a marker associated with host exposure and immune response to bacterial lipopolysaccharides. Results will be reported as the concentration of LBP in the analyzed biological material.

Soluble CD14 (sCD14) will be measured as a marker associated with innate immune activation and the host response to bacterial lipopolysaccharides. Results will be reported as the concentration of sCD14 in the analyzed biological material.

Fecal alpha-1-antitrypsin concentration will be measured as a marker associated with intestinal protein loss and intestinal mucosal barrier integrity. Results will be reported as the concentration of alpha-1-antitrypsin in feces.

Fecal pancreatic elastase concentration will be measured as a marker of exocrine pancreatic function. Results will be reported in µg/g of stool or another unit appropriate for the analytical method.

Fecal secretory immunoglobulin A (sIgA) will be measured as a marker of intestinal mucosal immune activity. Results will be reported as the concentration of secretory IgA in feces.

Selected beta-defensins will be measured in stool samples as markers of intestinal innate immune defense. Results will be reported as the concentration of individual beta-defensins in feces.

Fecal mucin concentration or selected mucin-associated markers will be measured to assess changes related to the intestinal mucus layer and mucosal protection. Results will be reported as the concentration of the analyzed mucin marker in feces.

The total concentration of short-chain fatty acids (SCFAs) will be measured in stool samples as a marker of intestinal microbial fermentation activity. Results will be reported as the total concentration of SCFAs, for example in µmol/g or mg/g of stool.

The relative proportions of acetate, propionate, butyrate and other selected short-chain fatty acids will be determined. Results will be expressed as the percentage contribution of each SCFA to the total measured SCFA pool.

The total concentration of bile acids will be measured in stool samples to assess changes in intestinal bile acid metabolism. Results will be reported as the total concentration of measured bile acids in feces.

The concentrations of selected primary, secondary and conjugated bile acids will be measured in stool samples. Results will be reported separately for each identified bile acid.

The ratio of primary to secondary bile acids in stool samples will be calculated from the concentrations of the identified bile acids. This measure will be used to assess changes in microbiota-associated bile acid transformation.

The concentrations of selected tryptophan metabolites will be measured in stool samples to assess changes in microbial and host-associated tryptophan metabolism. Results will be reported separately for each identified metabolite.

The concentrations of indole and selected indole derivatives will be measured in stool samples. Analytes may include indole, indole-3-acetic acid, indole-3-lactic acid, indole-3-propionic acid and other selected metabolites, depending on the analytical method.

The concentrations of selected phenolic acids and related microbial metabolites will be measured in stool samples. Results will be reported separately for each identified compound.

Selected fecal metabolites reflecting intestinal bacterial activity will be measured in stool samples. The analysis may include metabolites derived from the microbial fermentation of carbohydrates, proteins, amino acids and dietary polyphenols. Results will be reported separately for each selected marker.

Gut microbiome alpha diversity will be assessed using molecular analysis of stool samples, such as 16S ribosomal RNA gene sequencing or shotgun metagenomic sequencing. Alpha-diversity indices may include the Shannon index, Simpson index, Chao1 index and the number of observed taxa.

Gut microbiome beta diversity will be assessed using molecular analysis of stool samples, such as 16S ribosomal RNA gene sequencing or shotgun metagenomic sequencing. Differences in microbial community composition will be evaluated using appropriate distance measures, such as Bray-Curtis dissimilarity or UniFrac distance.

The relative abundance of selected intestinal bacterial taxa will be determined using 16S ribosomal RNA gene sequencing or shotgun metagenomic sequencing. Results will be expressed as the percentage or proportion of sequencing reads assigned to individual taxa at the phylum, family, genus or species level, as permitted by the analytical method.

Overall gut microbiome composition will be evaluated using molecular analysis of stool samples. Changes in the distribution of bacterial taxa and the structure of the microbial community will be assessed between study time points and study groups.

The ratio of the relative abundance of bacteria assigned to the phyla Firmicutes and Bacteroidota will be calculated from microbiome sequencing data. This parameter will be treated as an exploratory microbiome outcome.

The relative abundance of selected bacterial taxa considered potentially beneficial to intestinal health, such as Bifidobacterium, Lactobacillus, Akkermansia and selected butyrate-producing bacteria, will be assessed. The final list of taxa will depend on the resolution and quality of the sequencing data.

The relative abundance of selected potentially pro-inflammatory or opportunistic bacterial taxa will be assessed using microbiome sequencing data. The final list of taxa will be predefined in the statistical analysis plan or determined using an exploratory microbiome analysis.

When shotgun metagenomic sequencing is performed, the functional potential of the gut microbiome will be assessed based on the abundance of microbial genes, metabolic pathways or functional modules. Analyses may include pathways associated with short-chain fatty acid production, bile acid metabolism, tryptophan metabolism, intestinal barrier function and inflammatory processes.

Plasma asymmetric dimethylarginine (ADMA) concentration will be measured as an endogenous inhibitor of nitric oxide synthase and a marker associated with reduced nitric oxide bioavailability and endothelial dysfunction. Results will be reported as the concentration of ADMA in plasma.

Plasma symmetric dimethylarginine (SDMA) concentration will be measured as a marker associated with methylarginine metabolism, renal function and impaired L-arginine transport. Results will be reported as the concentration of SDMA in plasma.

Plasma dimethylamine (DMA) concentration will be measured as a metabolite associated with the degradation of asymmetric dimethylarginine by dimethylarginine dimethylaminohydrolase. Results will be reported as the concentration of DMA in plasma.

Plasma L-arginine concentration will be measured as the principal substrate for nitric oxide synthase. Results will be reported as the concentration of L-arginine in plasma.

Plasma citrulline concentration will be measured as a product of nitric oxide synthesis from L-arginine and as a marker associated with arginine-nitric oxide metabolism. Results will be reported as the concentration of citrulline in plasma.

The plasma L-arginine-to-ADMA ratio will be calculated as an exploratory indicator of nitric oxide synthase substrate availability relative to endogenous nitric oxide synthase inhibition.

The plasma ADMA-to-DMA ratio will be calculated as an exploratory marker associated with ADMA metabolism and dimethylarginine dimethylaminohydrolase activity.

Endothelial nitric oxide synthase (eNOS) concentration will be measured in stored serum samples using a quantitative enzyme-linked immunosorbent assay (ELISA). Change from baseline will be calculated separately at 3 and 6 months and summarized for each study group. Results will be expressed in ng/mL.

Inducible nitric oxide synthase (iNOS) concentration or activity will be measured as a marker associated with inflammation-related nitric oxide production. Results will be reported as iNOS concentration or enzymatic activity, depending on the analytical method.

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) concentration or activity will be measured as a marker of asymmetric dimethylarginine degradation and nitric oxide pathway regulation.

Dimethylarginine dimethylaminohydrolase 2 (DDAH2) concentration or activity will be measured as a marker associated with asymmetric dimethylarginine metabolism and vascular nitric oxide regulation.

Protein arginine methyltransferase 1 (PRMT1) concentration or activity will be measured as a marker associated with protein arginine methylation and the formation of methylated arginine derivatives, including ADMA.

Plasma 6-keto-prostaglandin F1 alpha concentration will be measured as a stable metabolite of prostacyclin and an indirect marker of prostacyclin production. Results will be reported as the concentration of 6-keto-prostaglandin F1 alpha in plasma.

Plasma thromboxane B2 (TXB2) concentration will be measured as a stable metabolite of thromboxane A2 and a marker associated with platelet activation and vasoconstrictive prostanoid activity.

Plasma prostaglandin F2 alpha (PGF2α) concentration will be measured as a marker of prostanoid pathway activity. Results will be reported as the concentration of PGF2α in plasma.

Plasma prostaglandin E2 (PGE2) concentration will be measured as a mediator associated with inflammation, vascular responses and immune regulation.

Plasma prostaglandin D2 (PGD2) concentration will be measured as a mediator involved in inflammatory, allergic and vascular processes.

Plasma 13,14-dihydro-prostaglandin E1 concentration will be measured as a metabolite associated with prostaglandin E1 metabolism. Results will be reported as the concentration of the analyte in plasma.

Plasma leukotriene B4 (LTB4) concentration will be measured as a pro-inflammatory lipid mediator associated with leukocyte recruitment and activation.

Plasma 15-deoxy-Δ12,14-prostaglandin J2 concentration will be measured as a cyclopentenone prostaglandin associated with the regulation and resolution of inflammatory responses.

The ratio of 6-keto-prostaglandin F1 alpha to thromboxane B2 will be calculated as an exploratory marker of the balance between prostacyclin-related vasodilatory activity and thromboxane-related platelet and vasoconstrictive activity.

The plasma eicosanoid profile will be assessed based on concentrations of selected prostaglandins, prostanoids and leukotrienes, including 6-keto-prostaglandin F1 alpha, TXB2, PGF2α, PGE2, PGD2, 13,14-dihydro-PGE1, LTB4 and 15-deoxy-Δ12,14-PGJ2.

Plasma anandamide, also known as arachidonoylethanolamide (AEA), concentration will be measured as an endogenous cannabinoid receptor ligand involved in the regulation of inflammation, metabolism, vascular function and pain signaling.

Plasma 2-arachidonoylglycerol (2-AG) concentration will be measured as a major endogenous cannabinoid receptor ligand involved in metabolic, immune and vascular regulation.

Plasma 1-arachidonoylglycerol (1-AG) concentration will be measured as an arachidonoylglycerol isomer associated with endocannabinoid metabolism. Results will be reported as the concentration of 1-AG in plasma.

Plasma palmitoylethanolamide (PEA) concentration will be measured as an endocannabinoid-related lipid mediator associated with anti-inflammatory and analgesic pathways.

Plasma docosatetraenoylethanolamide (DEA) concentration will be measured as an N-acylethanolamine associated with endocannabinoid-related lipid signaling. Results will be reported as the concentration of DEA in plasma.

The plasma endocannabinoid and endocannabinoid-related lipid profile will be assessed based on concentrations of AEA, 2-AG, 1-AG, PEA and DEA.

Serum high-sensitivity C-reactive protein (hsCRP) concentration will be measured as a marker of low-grade systemic inflammation. Results will be reported as the concentration of hsCRP in serum.

Serum procalcitonin concentration will be measured as a marker associated with systemic inflammatory responses and bacterial infection-related inflammation.

Serum amyloid A concentration will be measured as an acute-phase protein and a marker of systemic inflammatory activity.

Serum neopterin concentration will be measured as a marker of cellular immune activation, particularly activation of monocytes and macrophages.

Serum interleukin-10 (IL-10) concentration will be measured as a marker of anti-inflammatory and immunoregulatory activity.

Serum interleukin-15 (IL-15) concentration will be measured as a marker associated with immune-cell activation and inflammatory regulation.

Serum interleukin-1 alpha (IL-1α) concentration will be measured as a marker of pro-inflammatory signaling and tissue-associated immune activation.

Serum interleukin-8 (IL-8) concentration will be measured as a chemokine associated with neutrophil recruitment and systemic inflammatory activity.

Serum interleukin-18 (IL-18) concentration will be measured as a pro-inflammatory cytokine associated with innate and adaptive immune activation.

Serum tumor necrosis factor alpha (TNF-α) concentration will be measured as a marker of systemic pro-inflammatory activity.

The systemic pro-inflammatory cytokine profile will be assessed using selected cytokines, including IL-6, IL-15, IL-1α, IL-8, IL-18 and TNF-α.

The systemic anti-inflammatory cytokine profile will be assessed primarily using serum IL-10 and other predefined anti-inflammatory cytokines included in the laboratory panel.

Ratios between selected pro-inflammatory cytokines and IL-10 will be calculated as exploratory indicators of the balance between systemic pro-inflammatory and anti-inflammatory activity.

Selected circulating markers of vascular endothelial function will be measured in serum or plasma. The panel may include markers of endothelial activation, vascular inflammation, vascular adhesion, angiogenesis and endothelial injury.

Serum or plasma soluble intercellular adhesion molecule-1 (sICAM-1) concentration will be measured as a marker of endothelial activation and leukocyte adhesion.

Serum or plasma soluble vascular cell adhesion molecule-1 (sVCAM-1) concentration will be measured as a marker of endothelial activation and vascular inflammation.

Serum or plasma E-selectin concentration will be measured as a marker of endothelial-cell activation.

Plasma endothelin-1 concentration will be measured as a marker of endothelial vasoconstrictive activity.

Plasma von Willebrand factor concentration or activity will be measured as a marker associated with endothelial activation and vascular hemostatic function.

Reduced glutathione (GSH) concentration will be measured in blood or erythrocytes as a marker of intracellular antioxidant capacity.

Oxidized glutathione (GSSG) concentration will be measured in blood or erythrocytes as a marker of glutathione oxidation and oxidative stress.

The GSH-to-GSSG ratio will be calculated as an indicator of cellular redox balance. A lower ratio may indicate increased oxidative stress.

Glutathione peroxidase (GPx) activity will be measured in blood, erythrocytes or plasma as a marker of enzymatic antioxidant defense.

Superoxide dismutase (SOD) activity will be measured in blood, erythrocytes or plasma as a marker of enzymatic antioxidant defense against superoxide radicals.

Catalase activity will be measured in blood, erythrocytes or plasma as a marker of enzymatic antioxidant defense against hydrogen peroxide.

Total antioxidant status (TAS) will be measured in serum or plasma as an integrated marker of the overall antioxidant capacity of the biological sample.

Trolox equivalent antioxidant capacity (TEAC) will be measured in serum or plasma as an estimate of the total non-enzymatic antioxidant capacity. Results will be expressed as Trolox equivalents.

Protein carbonyl concentration will be measured in serum, plasma or other selected biological material as a marker of oxidative protein damage and protein carbonylation.

F2-isoprostane concentration will be measured in plasma, serum or urine as a marker of lipid peroxidation and systemic oxidative stress.

Selected markers of nitrosative stress will be measured in the predefined biological material. The analysis may include nitric oxide metabolites, nitrotyrosine or other validated indicators of reactive nitrogen species-mediated damage.

Serum or plasma 3-nitrotyrosine concentration will be measured as a marker of protein nitration and nitrosative stress.

Total nitrate and nitrite concentration will be measured in serum or plasma as an indirect marker of systemic nitric oxide production and metabolism.

Fasting plasma glucose concentration will be measured after an overnight fast as a marker of glucose metabolism. Results will be reported in mg/dL or mmol/L.

Fasting serum insulin concentration will be measured as a marker of pancreatic insulin secretion and insulin metabolism. Results will be reported in µIU/mL, mIU/L or another unit appropriate for the analytical method.

Plasma glucose concentration will be measured during an oral glucose tolerance test following administration of a standardized oral glucose load. Measurements will be performed at predefined time points, such as fasting and 30, 60, 90 or 120 minutes after glucose administration.

Serum insulin concentration will be measured at predefined time points during the oral glucose tolerance test to assess the insulin response to oral glucose administration.

The area under the plasma glucose concentration-time curve will be calculated from glucose measurements obtained during the oral glucose tolerance test.

The area under the serum insulin concentration-time curve will be calculated from insulin measurements obtained during the oral glucose tolerance test.

The Homeostatic Model Assessment of Insulin Resistance index will be calculated from fasting glucose and fasting insulin concentrations as an estimate of insulin resistance.

The Homeostatic Model Assessment of beta-cell function will be calculated from fasting glucose and fasting insulin concentrations as an estimate of pancreatic beta-cell function.

The Matsuda index will be calculated from glucose and insulin concentrations measured during the oral glucose tolerance test as an estimate of whole-body insulin sensitivity.

The insulinogenic index will be calculated from early glucose and insulin responses during the oral glucose tolerance test as an estimate of early-phase insulin secretion.

Serum total cholesterol concentration will be measured as a marker of lipid metabolism. Results will be reported in mg/dL or mmol/L.

Serum low-density lipoprotein cholesterol concentration will be measured or calculated as a marker of atherogenic lipoprotein metabolism.

Serum high-density lipoprotein cholesterol concentration will be measured as a marker of reverse cholesterol transport and cardiovascular risk.

Non-high-density lipoprotein cholesterol will be calculated as total cholesterol minus high-density lipoprotein cholesterol.

Serum triglyceride concentration will be measured as a marker of circulating lipid metabolism.

The ratio of total cholesterol to high-density lipoprotein cholesterol will be calculated as an exploratory marker of cardiovascular risk.

The ratio of low-density lipoprotein cholesterol to high-density lipoprotein cholesterol will be calculated as an exploratory marker of atherogenic lipid balance.

The atherogenic index of plasma will be calculated as the logarithm of the ratio of triglycerides to high-density lipoprotein cholesterol, using concentrations expressed in molar units.

Serum apolipoprotein A-I concentration will be measured as the principal protein component of high-density lipoproteins and a marker of reverse cholesterol transport.

Serum apolipoprotein B100 concentration will be measured as a marker of the number of circulating atherogenic lipoprotein particles.

The ratio of apolipoprotein B100 to apolipoprotein A-I will be calculated as an exploratory marker of the balance between atherogenic and anti-atherogenic lipoproteins.

Serum apolipoprotein E concentration will be measured as a marker associated with lipoprotein transport, lipid clearance and cholesterol metabolism.

Serum or plasma oxidized low-density lipoprotein concentration will be measured as a marker of oxidative modification of circulating lipoproteins.

Selected markers of lipid peroxidation will be measured in serum, plasma or urine. The analysis may include malondialdehyde, lipid hydroperoxides, thiobarbituric acid-reactive substances or F2-isoprostanes, depending on the predefined analytical protocol.

An integrated lipid metabolism profile will be assessed using total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, non-high-density lipoprotein cholesterol, triglycerides, apolipoprotein A-I, apolipoprotein B100, apolipoprotein E and selected markers of lipid oxidation.

Serum creatine kinase activity will be measured as a marker of skeletal muscle cell injury and muscle membrane disruption.

Serum creatine kinase-MB activity or concentration will be measured as an exploratory marker of muscle injury, depending on the analytical method and study population.

Blood lactate concentration will be measured as a marker of anaerobic metabolism and tissue metabolic activity.

Plasma free glycerol concentration will be measured as a marker of adipose tissue lipolysis and triglyceride breakdown.

Selected markers of skeletal muscle metabolism and injury will be measured in blood samples. The panel may include creatine kinase, creatine kinase isoenzymes, lactate and other predefined markers of muscle-cell metabolism.

Selected circulating markers of adipose tissue lipolysis will be assessed, including free glycerol and other predefined metabolites associated with triglyceride breakdown.

Serum or plasma non-esterified free fatty acid concentration will be measured as a marker of adipose tissue lipolysis and systemic lipid mobilization.

An integrated exploratory profile of adipose tissue metabolism will be assessed using free glycerol, free fatty acids, adipokines and other predefined markers of adipocyte function.

The concentration of 8-hydroxy-2'-deoxyguanosine will be measured in serum, plasma or urine as a marker of oxidative DNA damage.

Serum uric acid concentration will be measured as a marker of purine metabolism and as a compound associated with both antioxidant activity and cardiometabolic risk.

Serum osteocalcin concentration will be measured as a marker of osteoblast activity and bone formation.

Serum osteoprotegerin concentration will be measured as a regulatory marker of osteoclastogenesis and the receptor activator of nuclear factor kappa-B ligand pathway.

Serum Dickkopf-related protein 1 concentration will be measured as an inhibitor of Wnt signaling and a marker associated with bone formation regulation.

Serum C-terminal telopeptide of type I collagen concentration will be measured as a marker of bone resorption.

Serum receptor activator of nuclear factor kappa-B ligand concentration will be measured as a marker associated with osteoclast differentiation and bone resorption.

Serum receptor activator of nuclear factor kappa-B concentration or expression will be assessed as a marker of signaling involved in osteoclast development and activation.

The ratio of receptor activator of nuclear factor kappa-B ligand to osteoprotegerin will be calculated as an exploratory indicator of the balance between osteoclast activation and inhibition.

Serum procollagen type I N-terminal propeptide concentration will be measured as a marker of type I collagen synthesis and bone formation.

Serum bone-specific alkaline phosphatase activity or concentration will be measured as a marker of osteoblast activity and bone formation.

Serum tartrate-resistant acid phosphatase 5b concentration or activity will be measured as a marker of osteoclast number and bone resorption.

Urinary deoxypyridinoline concentration will be measured as a marker of collagen degradation and bone resorption. Results may be normalized to urinary creatinine concentration.

Serum fibroblast growth factor 23 concentration will be measured as a regulator of phosphate and vitamin D metabolism.

The bone formation marker profile will be assessed using osteocalcin, procollagen type I N-terminal propeptide, bone-specific alkaline phosphatase and other predefined markers of osteoblast activity.

The bone resorption marker profile will be assessed using C-terminal telopeptide of type I collagen, tartrate-resistant acid phosphatase 5b, urinary deoxypyridinoline and other predefined markers of osteoclast activity.

An integrated exploratory bone turnover profile will be assessed using markers of bone formation, bone resorption and RANK-RANKL-osteoprotegerin signaling.

Serum adiponectin concentration will be measured as an adipokine associated with insulin sensitivity, lipid metabolism and anti-inflammatory activity.

Serum leptin concentration will be measured as an adipokine associated with energy balance, appetite regulation and adipose tissue mass.

Serum soluble leptin receptor concentration will be measured as a marker of leptin-binding capacity and leptin signaling.

The free leptin index will be calculated from serum leptin and soluble leptin receptor concentrations as an exploratory indicator of biologically available leptin.

Serum resistin concentration will be measured as an adipokine associated with inflammation and insulin resistance.

Serum nicotinamide phosphoribosyltransferase, also known as visfatin, concentration will be measured as an adipokine and enzyme associated with energy metabolism and inflammation.

Serum omentin concentration will be measured as an adipokine associated with insulin sensitivity and vascular function.

Serum chemerin concentration will be measured as an adipokine associated with adipogenesis, inflammation and metabolic regulation.

Serum apelin concentration will be measured as an adipokine associated with cardiovascular regulation, glucose metabolism and energy balance.

Serum vaspin concentration will be measured as an adipokine associated with insulin sensitivity and metabolic regulation.

Serum retinol-binding protein 4 concentration will be measured as an adipokine associated with insulin resistance and retinol transport.

Serum fatty acid-binding protein 4 concentration will be measured as a marker of adipocyte function, lipid metabolism and cardiometabolic risk.

Serum interleukin-6 concentration will be measured as a marker of systemic and adipose tissue-associated inflammation.

Serum monocyte chemoattractant protein-1 concentration will be measured as a chemokine associated with monocyte recruitment and adipose tissue inflammation.

Plasma plasminogen activator inhibitor-1 concentration will be measured as a marker associated with impaired fibrinolysis, adipose tissue dysfunction and cardiometabolic risk.

Serum lipocalin-2, also known as neutrophil gelatinase-associated lipocalin, concentration will be measured as a marker associated with inflammation, metabolic dysfunction and renal injury.

Serum progranulin concentration will be measured as an adipokine and inflammatory mediator associated with obesity and insulin resistance.

Serum secreted frizzled-related protein 5 concentration will be measured as an adipokine associated with Wnt signaling, inflammation and metabolic homeostasis.

Serum angiopoietin-like protein 2 concentration will be measured as a marker associated with chronic inflammation, adipose tissue dysfunction and vascular disease.

Serum angiopoietin-like protein 4 concentration will be measured as a regulator of lipoprotein lipase activity, triglyceride metabolism and energy homeostasis.

Serum asprosin concentration will be measured as a fasting-induced glucogenic hormone associated with appetite, hepatic glucose production and insulin resistance.

An integrated exploratory adipokine profile will be assessed using selected markers of adipose tissue endocrine and inflammatory activity, including adiponectin, leptin, soluble leptin receptor, resistin, visfatin, omentin, chemerin, apelin, vaspin, retinol-binding protein 4, fatty acid-binding protein 4, tumor necrosis factor alpha, interleukin-6, monocyte chemoattractant protein-1, plasminogen activator inhibitor-1, lipocalin-2, progranulin, secreted frizzled-related protein 5, angiopoietin-like proteins 2 and 4, and asprosin.

Circulating caveolin-1 concentration will be measured as an exploratory marker associated with membrane signaling, lipid transport, endothelial function and metabolic regulation.

Total ghrelin concentration will be measured in plasma as a marker associated with appetite regulation, energy balance and glucose metabolism.

Acylated ghrelin concentration will be measured in appropriately stabilized plasma as the biologically active form of ghrelin.

Des-acyl ghrelin concentration will be measured in plasma as the major circulating non-acylated form of ghrelin.

The ratio of acylated ghrelin to des-acyl ghrelin will be calculated as an exploratory marker of ghrelin processing and biological activity.

The concentration or cellular expression of selected integrins will be assessed as markers of cell adhesion, extracellular matrix interactions, inflammation and vascular function. The specific integrin subunits will be predefined in the analytical protocol.

The concentration or cellular expression of selected cadherins will be assessed as markers of intercellular adhesion and tissue barrier integrity. The specific cadherin type will be predefined in the analytical protocol.

Serum or plasma soluble vascular endothelial cadherin concentration will be measured as a marker associated with endothelial junction integrity and vascular permeability.

E-cadherin concentration or cellular expression will be assessed as a marker of epithelial cell adhesion and tissue barrier integrity.

Chemokine-like receptor 1, also known as CMKLR1 or ChemR23, expression will be measured in the selected cell population as the principal functional receptor for chemerin.

G protein-coupled receptor 1 expression will be measured in the selected biological material as a receptor involved in chemerin binding and signaling.

C-C chemokine receptor-like 2 expression will be measured in the selected biological material as a non-classical chemerin-binding receptor involved in the regulation of local chemerin availability.

The expression profile of chemerin receptors will be assessed using CMKLR1, G protein-coupled receptor 1 and C-C chemokine receptor-like 2 in a predefined cell population or tissue-derived material.

Glycated hemoglobin will be measured in whole blood as an indicator of average blood glucose concentration during the preceding approximately 2 to 3 months. Results will be reported as a percentage or in mmol/mol.

Serum glycated albumin concentration or percentage will be measured as an indicator of average glycemic exposure during the preceding approximately 2 to 3 weeks.

Plasma methylglyoxal concentration will be measured as a reactive dicarbonyl compound associated with carbonyl stress and advanced glycation end-product formation.

Plasma 3-deoxyglucosone concentration will be measured as a reactive dicarbonyl intermediate involved in advanced glycation end-product formation.

Serum or plasma Nε-carboxymethyllysine concentration will be measured as a major advanced glycation end product associated with glycoxidative stress.

Serum or plasma Nε-carboxyethyllysine concentration will be measured as an advanced glycation end product associated with methylglyoxal exposure.

Serum, plasma or urinary pentosidine concentration will be measured as a fluorescent cross-linking advanced glycation end product.

Serum or plasma methylglyoxal-derived hydroimidazolone 1 concentration will be measured as a major methylglyoxal-derived advanced glycation end product.

Serum or plasma soluble receptor for advanced glycation end products concentration will be measured as a circulating decoy receptor associated with advanced glycation end-product signaling.

Total advanced glycation end-product concentration will be measured in serum or plasma using the predefined analytical method.

Skin autofluorescence may be measured non-invasively as an indirect marker of tissue accumulation of fluorescent advanced glycation end products.

An integrated exploratory advanced glycation end-product profile will be assessed using glycated hemoglobin, glycated albumin, methylglyoxal, 3-deoxyglucosone, carboxymethyllysine, carboxyethyllysine, pentosidine, methylglyoxal-derived hydroimidazolone 1, soluble receptor for advanced glycation end products and total advanced glycation end products.

The ratio of selected advanced glycation end products or total advanced glycation end products to soluble receptor for advanced glycation end products will be calculated as an exploratory indicator of the balance between glycation burden and circulating advanced glycation end-product-binding capacity.

An integrated exploratory carbonyl stress profile will be assessed using protein carbonyls, methylglyoxal, 3-deoxyglucosone and selected advanced glycation end products.

Serum alpha-crystallin B (CRYAB) concentration will be measured in stored serum samples using a quantitative enzyme-linked immunosorbent assay (ELISA). For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group. Results will be reported in ng/mL.

Serum inorganic phosphate concentration will be measured in stored serum samples using a validated clinical chemistry method. For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group.

Serum magnesium concentration will be measured in stored serum samples using a validated clinical chemistry method. For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group.

Serum thyroid-stimulating hormone (TSH) concentration will be measured in stored serum samples using a validated immunoassay. For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group.

Serum free thyroxine (FT4) concentration will be measured in stored serum samples using a validated immunoassay. For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group.

Serum free triiodothyronine (FT3) concentration will be measured in stored serum samples using a validated immunoassay. For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group.

Serum anti-thyroid peroxidase antibody (anti-TPO) concentration will be measured in stored serum samples using a validated immunoassay. For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group.

Serum anti-thyroglobulin antibody (anti-Tg) concentration will be measured in stored serum samples using a validated immunoassay. For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group.

Serum vascular endothelial growth factor A (VEGF-A) concentration will be measured in stored serum samples using a validated quantitative immunoassay. For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group.Serum vascular endothelial growth factor A (VEGF-A) concentration will be measured in stored serum samples using a validated quantitative immunoassay. For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group.

Serum vascular endothelial growth factor C (VEGF-C) concentration will be measured in stored serum samples using a validated quantitative immunoassay. For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group.

Plasma nitrate concentration will be measured in stored plasma samples using a validated colorimetric, fluorometric or chromatographic method as a marker of nitric oxide metabolism. For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group.

Plasma nitrite concentration will be measured in stored plasma samples using a validated colorimetric, fluorometric or chromatographic method as a marker of nitric oxide metabolism. For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group.

Plasma malondialdehyde (MDA) concentration will be measured in stored plasma samples using a validated chromatographic method as a marker of lipid peroxidation. For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group.

Serum malondialdehyde-modified low-density lipoprotein (MDA-LDL) will be measured in stored serum samples using a validated immunoassay as a marker of oxidative modification of low-density lipoproteins. For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group.

Fecal [acetate/propionate/butyrate/isobutyrate/valerate/isovalerate] concentration will be quantified in stored stool samples using a validated gas chromatographic method. For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group.

Fecal [acetate/propionate/butyrate/isobutyrate/valerate/isovalerate] concentration will be quantified in stored stool samples using a validated gas chromatographic method. For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group.

Fecal [acetate/propionate/butyrate/isobutyrate/valerate/isovalerate] concentration will be quantified in stored stool samples using a validated gas chromatographic method. For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group.

Fecal [acetate/propionate/butyrate/isobutyrate/valerate/isovalerate] concentration will be quantified in stored stool samples using a validated gas chromatographic method. For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group.

Fecal [acetate/propionate/butyrate/isobutyrate/valerate/isovalerate] concentration will be quantified in stored stool samples using a validated gas chromatographic method. For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group.

Fecal [acetate/propionate/butyrate/isobutyrate/valerate/isovalerate] concentration will be quantified in stored stool samples using a validated gas chromatographic method. For each participant, changes from baseline at 3 and 6 months will be calculated and summarized as the mean change for each study group.