Edited by: Pedro M. Baptista, Universidad de Zaragoza, Spain
Reviewed by: Nazri Mustaffa, Universiti Sains Malaysia (USM), Malaysia; Kunkai Su, Zhejiang University, China
This article was submitted to Gastroenterology, a section of the journal Frontiers in Medicine
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Chronic hepatitis C virus (HCV) infection is considered the leading cause of liver fibrosis development. Though fibrosis stays asymptomatic for many years, it progresses slowly to cirrhosis and end-stage liver disease and is the major cause of mortality and morbidity related to HCV (
Bohan was the first to describe the relationship between diabetes mellitus (DM) and liver cirrhosis (
Advanced glycation end products (AGEs) are defined as a heterogeneous group of irreversibly reactive derivatives. They are formed by non-enzymatic glycation, hence the name, and oxidation of lipids and proteins. They elicit and generate oxidative stress and subsequently induce inflammation (
It is observed in patients with cirrhosis that the onset of type 2 diabetes is linked to a decrease in insulin secretion rather than an increase though cirrhosis is found to be associated with increased IR leading to increased secretion (
To date, this problem has not been discussed properly in the research literature. The objective of this work, therefore, is to assess the possible correlation between AGE and the development of DM in patients with cirrhosis.
This is a cohort prospective pilot study. It was executed in the Department of Tropical Medicine (Mansoura University-Egypt), between October 2015 and August 2019. A total of 306 patients were enrolled consecutively. They had liver cirrhosis and were referred to our department. We included in our study only 165 patients who met the inclusion criteria. All the patients' clinical, hematological, demographic, and biochemical data were assessed and recorded at baseline and over the defined follow-up periods.
The inclusion criteria were patients (1) with liver cirrhosis and (2) 18 years or older. The exclusion criteria were (1) DM and/or HD; (2) impaired fasting glucose (IFG); (3) alcoholic liver disease; (4) liver cirrhosis due to hepatitis B virus, autoimmune hepatitis, NASH, and metabolic or cholestatic liver diseases; (5) collagen vascular diseases; (6) abdominal tuberculosis; (7) uncontrolled thyroid disorders; (8) kidney diseases and hematologic disorders; (9) peritoneal carcinomatosis; (10) pancreatitis; (11) bone marrow suppression; (12) cancers; (13) pregnancy and lactation; (14) osteoporosis; (15) smoking; (16) heart failure; (17) cerebrovascular accidents/Alzheimer's disease; (18) administration of immunosuppressive drugs or drugs likely to affect glucose metabolism; (19) administration of anticoagulant/antiplatelet treatment, hepatotoxic drugs, NSAIDs, and/or oral contraceptive drugs; and finally (20) patients with missing information.
Also, the control group included 72 healthy controls who were sex- and age-matched subjects (male/female = 50/22).
The baseline data were taken during the 1st week of the first visit, while the data for the end of study were taken during the last week of both 1st and 2nd year follow-up periods. The biochemical and radiological findings and clinical examination of our patients at the end of our study did not reveal any complications that would change the parameters in the subjects' health during the follow-up period that were not recorded at the start of the study.
We diagnosed liver cirrhosis by clinical assessment and biochemical tests, abdominal ultrasonography, liver biopsy for histopathological assessment, elastography, and endoscopic findings implying portal hypertension associated with stigmata of chronic liver disease (
All the cirrhotic complications during the follow-up period were treated based on standardized therapeutic measures (
Patients with cirrhosis classified as CTP-A and CTP-B, either naive or treatment-experienced patients, those who did not achieve sustained virologic response (SVR) after treatment with pegylated interferon (Peg IFN) and ribavirin (RBV) or with IFN/Sofosbuvir (SOF), or those who received SOF/RBV only were treated according to EASL guidelines (
Our protocol was designed according to the 2015 EASL recommendations for hepatitis C treatment (
Treatment-experienced patients can be re-treated with a SOF combined with DCV for 12 weeks with RBV or 24 weeks without it (
Hepatitis C viral RNA level was measured at baseline, through screening, at the end of treatment (either 12 or 24 weeks), and finally 12 weeks after that. We used Roche COBAS TaqMan HCV assay version 2.0 to measure the HCV RNA, which has a detection limit of 15 IU/ml. The primary virological outcome was to achieve SVR12, where the virus is undetected (below detection limit) for 12 or more weeks. On the contrary, virological failure is categorized as either non-response (HCV RNA is still detectable at the end of the treatment period) or relapse (HCV RNA becomes detectable again during follow-up after being undetectable at the end of the treatment period) (
AGE levels were measured at baseline and again after the completion of treatment by 12 weeks.
Safety was evaluated through laboratory tests, physical examinations, and reports of clinical adverse events at scheduled clinic visits and then treated according to the study schedule.
Patients were diagnosed with diabetes through clinical examination, history of antidiabetics administration, and/or fasting plasma glucose (FPG) of 126 mg/dl or more (≥7 mmol/l) (
Patients with HD usually show normal FPG, but abnormal response to an oral glucose tolerance test (OGTT), when at least two of the following three plasma glucose levels (measured during OGTT) are met or exceeded: fasting: 95 mg/dl (5.3 mmol/L), 1 h: 180 mg/dl (10.0 mmol/L), and 2 h: 155 mg/dl (8.6 mmol/L), which is mandatory for the diagnosis (
Hypersplenism in patients with liver cirrhosis has played a role in shortening erythrocyte life span and falsely lowering levels of HbA1c. Therefore, OGTT is required to identify DM or IGT in cirrhosis. Subjects with normal HbA1c (and FPG) and abnormal OGTT are likely to be those with HD; however, in most patients with increased FPG levels, diabetes is commonly type 2 DM.
Patients with fasting blood sugar of 110 mg/dl or more (≥6.1 mmol/L) but <126 mg/dl (<7 mmol/L) were considered prediabetic with IFG and were advised to have a glucose tolerance blood test (
We followed the García-Compeán et al. (
All our study data were collected by trained investigators. All patients were asked to complete a self-validated, standardized questionnaire that allowed us to collect information on their smoking habits, alcohol consumption, and medical and therapeutic history, especially malignancy, hypertension, and diabetes history if present. We used the formula
In our study, patients were considered vegetarian if they refrain from eating meat, but have two to three vegetable servings or five fruits servings during the day. One vegetable serving is equivalent to a cup of raw green leafy vegetables or ½ cup of other cooked or chopped raw vegetables while one fruit serving is equivalent to a medium-sized banana, orange, or apple, or ½ cup of canned/chopped fruit or juice (
We used the World Health Organization-developed Global Physical Activity Questionnaire (GPAQ), used in the STEPS questionnaire, to assess the physical activity. This questionnaire divides the physical activity into three distinct intensity levels: light, moderate, and vigorous according to three different behavioral actions: transport, work, and during leisure time (
Participants were considered sufficiently active when fulfilling or exceeding the minimum duration and intensity of physical activity every week per WHO recommendations, which are moderate-intensity for 150 min, vigorous-intensity for 75 min of or an equivalent combination of both to reach at least 600 MET min per week with each activity performed in at least 10-min long sessions (
Six milliliters of fresh venous blood was aspirated from all participants following overnight fasting and divided into 4 ml with separating gel for serum samples and 2 ml on EDTA for full blood count (FBC). The serum samples were centrifuged for 10 min at 1,000–3,000 RPM, and then the serum was collected and divided into aliquots and kept in a −20°C freezer until later analysis.
We used the Dimension Xpand Plus chemistry analyzer (Siemens Technology, Princeton, New Jersey) to assess serum creatinine and complete liver function tests, the CELL-DYN Emerald 22 Hematology Analyzer (Abbott, Wiesbaden, Germany) to assess complete blood count (CBC), the Spinreact kits [Sant Esteve De Bas (GI), Spain] to measure serum triglycerides (TG) and cholesterol, and enzyme-linked immunosorbent assay (ELISA) kits to measure the fasting serum insulin (FSI) levels (Calbiotech, Spring Valley, California), plasma AGEs (MyBioSource, San Diego, CA 92195-3308, USA; Cat No. MBS267540), and serum C-peptide [DiaMetra, Garibaldi, 18 20090 SEGRATE (MI) Italy; Cat No. DKO077]. As for measuring the hemoglobin A1c (HbA1c), we used ion exchange resin chromatography kits (StanBio, 1261 North Main street, Boerne, Texas USA) procedure No (0350). The eGFR is calculated by the abbreviated Modification of Diet in Renal Disease (MDRD) study equation:
All participants underwent an OGTT at baseline and at the end of the 1st and 2nd year follow-up periods. Patients developing diabetes were confirmed with OGTT for proper diagnosis during the follow-up period.
We used the following HOMA formulas to calculate the basal insulin secretion and sensitivity indices (
β-cell function (HOMA-β) =
Insulin resistance (HOMA-IR) =
Hepatitis C viral RNA was extracted from patients' sera using QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). First, we amplified the core region by RT-PCR in the Biometra thermal cycler (Analytik Jena Company, Germany) using specific primers common to all genotypes (
All procedures and the study protocols were evaluated and accepted by the Mansoura Institutional Review Board. In addition, informed consents were obtained from all participants. The study was carried out in agreement with the Helsinki Declaration's recommendations.
Our results were plotted and analyzed by the Social Package of Statistical Science (SPSS) software version 20 (SPSS Inc., Chicago, IL, USA). Quantitative data are described as mean ± SD. We used the Kolmogorov–Smirnov test to determine the compatibility of normally distributed data. We also used Mann–Whitney
Only 144 patients out of the enrolled 165 who fulfilled the inclusion criteria have completed the study where 7 were dropped due to non-compliance along the follow-up period and 14 died from complications of cirrhosis, e.g., hepatorenal syndrome (
Flowchart of the patients included in this study. IGT, Impaired glucose tolerance; DM, diabetes mellitus; CHB, chronic hepatitis B; AIH, autoimmune hepatitis; GIT, Gastrointestinal tract.
Baseline clinical, demographic, and biochemical characteristics of enrolled participants.
Age (years) | 57.6 ± 5.5 | 56.2 ± 4.9 | 0.07 |
Sex (male/female) | 93/51 | 50/22 | 0.82 |
Diet | |||
Vegetarian | 58 (40.3) | 32 (44.4) | 0.57 |
Non-vegetarian | 86 (59.7) | 40 (55.6) | 0.56 |
Body mass index (kg/m2) | 26 ± 1.1 | 25.8 ± 0.4 | 0.14 |
Physical activity | |||
≥600 MET min/week | 32 (22.2) | 18 (25) | 0.64 |
<600 MET min/week | 112 (77.8) | 54 (75) | 0.65 |
Hypertension | |||
Present | 6 (4.2) | – | – |
Absent | 138 (95.8) | 72 (100) | 0.08 |
Family history of diabetes | |||
Absent | 130 (90.3) | 66 (91.7) | 0.74 |
Present | 14 (9.7) | 6 (8.3) | 0.73 |
Patients receiving DAA ( |
|||
SVR | 90 (79) | – | – |
Non-responder | 24 (21) | – | – |
Patients not receiving DAA | 30 | – | – |
Hemoglobin (gm/dl) | 9.2 ± 0.7 | 12 ± 0.4 | <0.001 |
WBCs (×103/cm2) | 3.5 ± 0.4 | 6.2 ± 1.6 | <0.001 |
Platelet count (×103/cm2) | 60.6 ± 24.8 | 196.6 ± 47.5 | <0.001 |
Total cholesterol (mg/dl) | 178.1 ± 13.9 | 175.4 ± 10.2 | 0.15 |
Triglyceride (mg/dl) | 122.8 ± 16 | 125.8 ± 9.1 | 0.14 |
AST (U/L) | 42.2 ± 14.6 | 28 ± 5.4 | <0.001 |
ALT (U/L) | 39.4 ± 13.9 | 28.3 ± 4.7 | <0.001 |
GGT (U/L) | 38.5 ± 11 | 21.6 ± 5.1 | <0.001 |
ALP (IU/ml) | 102.4 ± 15.2 | 53.8 ± 11.3 | <0.001 |
Bilirubin (mg/dl) | 2.5 ± 1.3 | 0.9 ± 0.1 | <0.001 |
Albumin (g/dl) | 3 ± 0.4 | 4.2 ± 0.2 | <0.001 |
INR | 1.5 ± 0.3 | 0.9 ± 0.1 | <0.001 |
Creatinine (mg/dl) | 1.2 ± 0.3 | 0.9 ± 0.2 | <0.001 |
eGFR | 77.3 ± 9.5 | 95.7 ± 6.1 | <0.001 |
Child-Pugh score | 7.7 ± 2.6 | – | – |
MELD score | 16.8 ± 4 | – | – |
FPG (mg/dl) | 88.3 ± 7.7 | 87.3 ± 4.8 | 0.31 |
HbA1c | 4.52 ± 0.56 | 4.41 ± 0.56 | 0.18 |
C-peptide (ng/ml) | 4.28 ± 0.68 | 2.8 ± 0.32 | <0.001 |
Insulin (mIU/ml) | 16.1 ± 2.3 | 8.7 ± 1.5 | <0.001 |
HOMA-IR | 3.8 ± 0.5 | 1.9 ± 0.2 | <0.001 |
HOMA-β | 135.7 ± 11.6 | 138.4 ± 8.4 | 0.08 |
AGEs (ng/ml) | 77.8 ± 54.3 | 14.3 ± 1.5 | <0.001 |
We followed up the participants by hematological and biochemical blood tests every year for 2 years after the initial baseline assessment.
Fourteen patients (10%; CTP-A = 3, CTP-B = 6, and CTP-C = 5) out of all the patients examined during the follow-up period (
AGE levels in all patients throughout the study period. AGEs, Advanced glycation end products; DM, diabetes mellitus.
In the control group, there were no statistically significant changes in AGE levels between the baseline and 1 year follow up, as well as at the end of study (15.3 ± 2.2 vs. 15.8 ± 2.25;
Spearman correlation analysis showed that there were significant positive correlations between AGEs and age (
Clinical, demographic, and biochemical characteristics of patients with and without DM during the follow-up period.
Age (years) | 55.8 ± 5.3 | 58.4 ± 5.5 | 0.09 |
Sex (male/female) | 9/5 | 84/46 | 0.99 |
Diet | |||
Vegetarian | 6 (42.9) | 52(40) | 0.83 |
Non-vegetarian | 8 (57.1) | 78 (60) | 0.84 |
Body mass index (kg/m2) | 26.6 ± 1.2 | 26 ± 1.1 | 0.07 |
Physical activity | |||
≥600 MET min/week | 3 (21.4) | 29 (22.3) | 0.94 |
<600 MET min/week | 11 (78.6) | 101 (77.7) | 0.93 |
Hypertension | |||
Present | 1 (7.1) | 5 (3.8) | 0.55 |
Absent | 13 (92.9) | 125 (96.2) | 0.6 |
Family history of diabetes | |||
Absent | 12 (86) | 118 (91) | 0.55 |
Present | 2 (14) | 12 (9) | 0.56 |
Patients receiving DAA | |||
SVR | 9 (64.3) | 81 (62.3) | 0.88 |
Non-responder | 2 (14.3) | 22 (16.9) | 0.8 |
Patients not receiving DAA | 3 (21.4) | 27 (20.8) | 0.96 |
Hemoglobin (gm/dl) | 9.1 ± 0.7 | 9.2 ± 0.7 | 0.61 |
WBCs (×103/cm2) | 3.5 ± 0.3 | 3.5 ± 0.4 | 0.99 |
Platelet count (×103/cm2) | 87 ± 36 | 57.5 ± 21.8 | <0.001 |
Total cholesterol (mg/dl) | 180.4 ± 10.9 | 177.8 ± 14.2 | 0.51 |
Triglyceride (mg/dl) | 128.6 ± 11.2 | 122.1 ± 16.3 | 0.15 |
AST (U/L) | 48.3 ± 21.6 | 41.6 ± 13.6 | 0.1 |
ALT (U/L) | 45.9 ± 21.4 | 38.7 ± 12.8 | 0.07 |
GGT (U/L) | 36.3 ± 7.6 | 38.8 ± 11.2 | 0.42 |
ALP (IU/ml) | 112.8 ± 17.4 | 101.3 ± 14.5 | 0.007 |
Bilirubin (mg/dl) | 2.7 ± 1.6 | 2.5 ± 1.3 | 0.6 |
Albumin (g/dl) | 3.4 ± 0.5 | 3 ± 0.4 | <0.001 |
INR | 1.5 ± 0.3 | 1.6 ± 0.3 | 0.23 |
Creatinine (mg/dl) | 1.2 ± 0.3 | 1.3 ± 0.3 | 0.24 |
eGFR | 76.6 ± 9.3 | 79.4 ± 10 | 0.32 |
Child-Pugh score | 7.6 ± 2.4 | 7.7 ± 2.6 | 0.89 |
MELD score | 16.4 ± 4.8 | 16.8 ± 3.8 | 0.72 |
FPG (mg/dl) | 95.6 ± 3.9 | 87.5 ± 7.6 | <0.001 |
HbA1c | 4.68 ± 0.58 | 4.45 ± 0.53 | 0.16 |
C-peptide (ng/ml) | 3.95 ± 0.88 | 3.29 ± 1.9 | 0.2 |
Insulin (mIU/ml) | 17.7 ± 1.9 | 16.9 ± 1.7 | 0.1 |
HOMA-IR | 4.2 ± 0.4 | 3.8 ± 0.5 | 0.004 |
HOMA-β | 140 ± 12.3 | 135.3 ± 11.5 | 0.15 |
AGE (ng/ml) | 129.1 ± 17.8 | 72.2 ± 54.1 | <0.001 |
There is no significant difference in sex, age, diet, BMI, physical activity, family history of diabetes, hypertension, patients receiving DAA, hemoglobin, WBCs, total cholesterol, serum TG, AST, ALT, GGT, serum bilirubin, INR, serum creatinine, eGFR, serum TG, HbA1c, C-peptide, serum insulin, CTP and MELD classifications, or HOMA-β between the two groups (all
Univariate and multivariable Cox regression analysis models in the studied patients to predict DM development.
AGE | 1.004 | 1.0014 to 1.0073 | 0.004 | 1.005 | 1.0014 to 1.0083 | 0.006 |
HOMA-IR | 3.412 | 1.2437 to 9.3617 | 0.018 | 3.355 | 1.1635 to 9.6719 | 0.026 |
HOMA-β | 1.036 | 0.9872 to 1.0874 | 0.015 | – | – | – |
Age | 0.9301 | 0.8481 to 1.0202 | 0.012 | 0.901 | 0.8177 to 0.9916 | 0.034 |
We re-evaluated the multivariable Cox regression analysis model after adjusting multiple confounders using the formerly described parameters at baseline related to the development of DM during the 2 year follow-up period. This revealed that the only factors independently associated with DM development are AGEs, HOMA-IR, and age (
Using the ROC curve, at a cutoff value of more than 82.4 ng/ml, AGEs had 99.23% specificity, 100% sensitivity, 0.992 AUC, 93.33% positive predictive value (PPV), 100% negative predictive value (NPV), and
Receiver operating characteristic curve of AGEs in detecting DM in patients with cirrhosis. AGEs, advanced glycation end products; DM, diabetes mellitus.
Patients with CTP-A (
There were no significant differences in AGEs at baseline and end of therapy regarding SVR (
Regarding the development of DM, there was no statistically significant difference between patients who received therapy and those who did not (11/114
Two patients (CPT-A) were treated with oral hypoglycemic agents (Glimepiride 1 mg) while 12 patients (CPT-B and CPT-C) were treated with insulin.
The most common adverse events were fatigue (8 and 28.8%), anemia (14.5 and 26.9%), headache (6.5 and 15.4%), RBV dose reductions (4.8 and 9.6%), hyperbilirubinemia (11.3 and 23.1%), and pruritus (6.5 and 11.5%) in CTP-A and CTP-B patients, respectively.
We saw more serious side effects in CTP-B patients than in CTP-A; 6.5 and 9.6% developed ascites, 1.6 and 5.8% had hepatic encephalopathy, 3.2 and 7.7% had GIT bleeding, 0.0 and 3.8% had renal impairment, and 6.5 and 9.6% had hepatocellular carcinoma (HCC) in CTP-A and CTP-B, respectively.
No adverse effects related to the treatment of DM were reported during the follow-up period regarding the drugs' safety and tolerability.
AGEs were recently proven to play a role in the fibrosis and/or cirrhosis of CHC patients through autophagy induction and hepatic stellate cells (HSC) activation (
The liver is the main site for AGE catabolism and clearance from the circulation; in one study, it successfully removed more than 90% of AGEs intravenously injected in rats through endocytosis (
The most clinically significant finding was the increased levels of AGEs as an independent factor for the prediction of DM development in these patients.
As mentioned above, AGEs activate multiple intracellular signaling pathways controlling different cellular functions, which in turn lead to pathophysiological effects (
AGEs contribute to both IR and β-cell damage and death leading to impaired insulin function. Many studies investigated the cytotoxic potential of AGEs on pancreatic β-cells. Shu et al. (
Hepatitis C viral protein favors IR, which plays a crucial role in accelerating hepatic fibrosis and increasing its severity in infected patients (
AGEs alter insulin signaling through many mechanisms. They modify the insulin directly, which in turn alters its action resulting in impaired glucose uptake, reduced insulin clearance, or further increased insulin secretion. They increase RAGE expression and decrease NAD-dependent deacetylase sirtuin-1 (SIRT1) expression, which alters its signaling and induces inflammation. They stimulate PKC β and upregulate TNF-α (
If HCV caused the increase in AGE levels (
Contrary to expectations, this study did not find a significant correlation between HCV therapy with DAAs and DM development in patients with cirrhosis, while in non-diabetic lean patients, therapy of HCV enhances peripheral (but not hepatic) insulin sensitivity in CHC without significant fibrosis (
The principal theoretical implication of this study is that several other aspects besides HCV infection must be kept in mind when interpreting variables of glucose homeostasis in such patients, e.g., AGEs, IR, and the improvement in perceived well-being throughout therapy, which led to the adoption of a more dynamic lifestyle by these patients (
The AGE–RAGE axis is an important player in HSC activation and resulting liver fibrosis. Reducing the AGE–RAGE signaling, by controlling high glucose, sensitizing insulin function, avoiding overcooked foods, and oxidant supplement digestion (
The most striking result to emerge from the data is that age may be considered as an independent factor for the prediction of DM development in this study. This is probably because insulin sensitivity decreases with age and compensation of β-cell function becomes insufficient in the face of increasing IR (
This study, as far as we know, is the first to add to this expanding research field by uncovering the impact of AGEs in such patients. These findings have significant implications in the understanding of the involvement of AGEs in the development of DM.
There are certain limitations to generalize these results: first, small sample size; second, single-center study; third, the cause of liver cirrhosis is solely HCV; fourth, the effect of liver transplantation on the levels of AGEs and the development of DM in such patients (
Despite these limitations, HOMA-β and HOMA-IR are the most broadly utilized markers for β-cell function and IR in epidemiological studies and are widely used for comparing insulin secretion and IR among various population-based studies (
In conclusion, AGEs were evidently related to increased incidence of DM, especially in patients with liver cirrhosis. This may be a simple yet effective method to lower the risk of DM and liver fibrosis in these patients.
The data that support the findings of this study have restrictions and so are not publicly available. Data are however available from the authors upon reasonable request.
The studies involving human participants were reviewed and approved by Mansoura Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.
AA-R, NM, and SZ contributed to the concept and/or design of the study and performed the statistical analysis. MosA, ME, and MohA contributed to the acquisition of the data, interpreted data critically, and revised the manuscript. AH drafted the manuscript. RasE critically revised the manuscript and recruited and followed up with patients. RanE acquired, analyzed and interpreted data, and revised the manuscript. WE and NE-W contributed to the analysis and interpretation of data and performed the statistical analysis. All authors approved the final version of the article, including the authorship list.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors thank the patients and staff of the Tropical Medicine Department and the lab specialists for their significant aid.
advanced glycation end products
diabetes mellitus
hepatogenous diabetes
Basal metabolic index
Receptor for Advanced Glycation Endproducts
estimated glomerular filtration rate
Homeostatic Model Assessment-Insulin Resistance
hepatitis C virus
Sofosbuvir
daclatasvir
impaired fasting glucose
Child-Turcotte Pugh
Model for End-Stage Liver Disease
nonalcoholic steatohepatitis
complete blood count
triglycerides
Global Physical Activity Questionnaire
protein kinase C
nuclear factor kappa-B
reactive oxygen species.