|Year : 2021 | Volume
| Issue : 6 | Page : 1671-1678
|Study Protocol for a Randomized Controlled Trial Evaluating the Effectof Chromium Picolinate Supplementation on Gene Expression of TumorNecrosis Factor-α and DNA Damage in Metabolic Syndrome Patients
Hassan Mozaffari-Khosravi1, Sara Jambarsan2, Fazad Karimpour3, Seyyed Ebrahim Hosseini4, Behrooz Ebrahimzadeh Kour1
1 Nutrition and Food Security Research Center; Department of Nutrition, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
2 Department of Biostatistics and Epidemiology, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
3 Social Determinants of Health Research Center, Yasuj University of Medical Science, Yasuj, Iran
4 Cellular and Molecular Research Center, Yasuj University of Medical Sciences, Yasuj, Iran
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|Date of Web Publication||27-Jul-2022|
| Abstract|| |
Metabolic syndrome (MS) is caused by environmental factors as well as genetic. Human studies of efficacy of chromium for glucose and lipid metabolism and insulin function is not still definitive. Furthermore, the effect of chromium supplementation on the expression of inflammatory genes in patients with MS has not been studied. We will assess effects of chromium picolinate supplementation on gene expression of tumor necrosis factor-α (TNF-α) and DNA damage in MS patients. In this triple-blind randomized placebo-controlled clinical trial, 48 MS patients will be randomly assigned into two groups to receive daily 400 μg chromium picolinate supplement or placebo for 12 weeks. The outcome measures include of change in fasting blood sugar, glycosylated hemoglobin A1C, inflammatory biomarkers, lipid profile, blood pressure, gene expression of TNF-α, and 8-hydroxy-deoxyguanosine concentration as DNA damage biomarker, will be quantified at baseline and end of intervention. This protocol was approved by Institutional Research Ethics Committee School of Public Health Shahid Sadoughi University of Medical Sciences (Approval ID: IR.SSU.SPH.REC.1399.141).
|How to cite this article:|
Mozaffari-Khosravi H, Jambarsan S, Karimpour F, Hosseini SE, Kour BE. Study Protocol for a Randomized Controlled Trial Evaluating the Effectof Chromium Picolinate Supplementation on Gene Expression of TumorNecrosis Factor-α and DNA Damage in Metabolic Syndrome Patients. Saudi J Kidney Dis Transpl 2021;32:1671-8
|How to cite this URL:|
Mozaffari-Khosravi H, Jambarsan S, Karimpour F, Hosseini SE, Kour BE. Study Protocol for a Randomized Controlled Trial Evaluating the Effectof Chromium Picolinate Supplementation on Gene Expression of TumorNecrosis Factor-α and DNA Damage in Metabolic Syndrome Patients. Saudi J Kidney Dis Transpl [serial online] 2021 [cited 2022 Sep 25];32:1671-8. Available from: https://www.sjkdt.org/text.asp?2021/32/6/1671/352428
| Introduction|| |
Metabolic syndrome (MS), a prevalent metabolic disorder, is defined as the existence of at least three factors of abdominal obesity, hyperglycemia, hypertension, decreased high-density lipoprotein (HDL), and hypertriglyceridemia. Etiologically, obesity and resulting accumulation of fat leads to the releasing the inflammatory cytokines and ultimately insulin resistance. Impaired clearance of lipid profiles from blood into tissues from insulin resistance eventually causes cardiovascular complications. In addition, oxidative stress from chronic hyperglycemia can cause defective antioxidant system and exacerbation of inflammation, which leads to an increase in inflammatory biomarkers such as interleukins (ILs) and tumor necrosis factor-α (TNF-α) in blood. These mechanisms leading to imbalance between antioxidant defense system and free radicals production in the body can even damage the DNA or genotoxicity, and increased the expression of inflammatory genes. This syndrome increases the risk of chronic diseases and even mortality.,,,,, The prevalence of this syndrome has increased sharply over the past decades and has been estimated 10%–84% in different age groups in various studies.,, In Iran, its prevalence was estimated to be more than 30%–50% in adults., In today’s machinery life, unhealthy diet (high energy intake, especially simple sugars such as fructose), sedentary lifestyle, alcohol and tobacco intake, and even lack of enough daily sleep along with genetic factors have been found to be effective in this pathological condition; thus, the role of nutritional interventions in prevention and reduction of its complications is important.
Chromium as an essential mineral is a cofactor for many metabolic pathways of body and is effective in improving metabolic disorders, including insulin resistance and impaired glucose metabolism. Trivalent chromium is found in fruits, vegetables, meat, fish, and brewer’s yeast. Due to its low biological availability, daily requirement of chromium is not met through the usual diet.
Although mechanisms of the effect of chromium on insulin resistance, have been shown, human studies still show uncertainty and contradiction. The effect of chromium supplementation on the inflammatory mediators such as IL-6 and TNF-α, although in cell and animal model studies, have been shown, but in a few human studies, it has been confirmed, and the exact mechanism of this effect is still unknown.
To the best of our knowledge, the effect of chromium supplementation on gene expression of inflammatory biomarkers in MS has not been studied as clinical trial; therefore, the aim of this study will be to investigate the effect of trivalent chromium picolinate supplementation on gene expression of TNF-α, DNA degradation, glycemic indexes, and lipid profiles in MS patients.
| Patients and Methods|| |
This study is a two-arm, triple-blind randomized placebo-controlled clinical trial. Its protocol was approved by Institutional Research Ethics Committee of University of Medical Sciences.
Considering type, I error of 5% (a = 0.05), type II error of 20% (b = 0.20; power = 80%) and based on the following formula for calculating sample size and amount of gene expression of TNF-α as the main variable (S1, S2 = 0.25), the initial number of sample in each group will be 20 persons and considering the possible loss of samples during the study, the final sample size for each group will be 24 persons.
Patients with the following characteristics will be assigned to the study (having at least three of the five conditions):
• Waist circumference ≥88 cm for women and ≥102 cm for men
• Systolic blood pressure and diastolic greater than or equal to 130 and 85 mm Hg respectively or use of blood pressurelowering medication
• Fasting blood sugar (FBS) ≥100 mg/dL or use of hypoglycemic drugs
• Blood triglycerides (TG) ≥150 mg/dL or the use of lipid-lowering drugs
• HDL <50 mg /dL in women and less than 40 mg/dL in men
• The patients will be 20–70 years old.
The following will be excluded from the study: patients who are taking any multivitamins or dietary supplements or medications, pregnant and lactating women, those who have uncontrolled blood pressure, uncontrolled blood sugar, heart arrhythmias and chronic obstructive pulmonary disease, stroke, or any other disease that requires medical treatment.
In the first stage, from registered information of people living in Dena county (SisakhatAdult Cohort Center), all eligible persons will be listed and numbered. Then, 48 patients will be randomly selected using Random Numbers Generator software. In the next step, using the above-mentioned software as a random sequencing tool, participants will be divided into two groups A (intervention) and B (placebo). Participants, laboratory technician, statistical analyst, and researcher will not be aware from patient grouping until the end of the study, and only a trusted health expert who has no involvement in the process of study will be informed. The tablets received by both groups will be the same in terms of color, smell, and appearance. The label of the boxes containing the tablets will be removed and only will be marked with a special code.
First group (intervention) will receive 400 μg of trivalent chromium picolinate tablets (Eurho Vital company, Germany), and other groups (placebo) will receive equal amounts of placebo tablets for 12 weeks. The tablets received by both groups will be the same in terms of color, smell, and appearance. The dose of the drug was selected on the basis that despite the fact that so far no cases of poisoning and side effects of this supplement have been reported and from 100 μg to 600 μg have been prescribed in various studies,, but a dose of 400 μg will be used. The duration of different studies had been from eight to 24 weeks,, so to observe the relative effectiveness and prevent excessive sample loss during the study, intervention duration will be 12 weeks. An overview of the study is shown in [Figure 1]. Standard Protocol Items: Recommendations for Interventional Trials diagram including the time points of study are presented in [Table 1].
|Table 1. Template of recommended content for the schedule of enrolment, interventions, and assessments.|
*Weight, waist circumstance, FBS, HbA1C, blood pressure, lipid profile, IL-1, IL-6, TNF-α, 8-HDG, Gene expression of TNF-α. FBS: Fasting blood sugar, HbA1C: Hemoglobin A1C, IL: Interleukins, TNF-α: Tumor necrosis factor-α, 8-HDG: 8-hydroxy-deoxyguanosine.
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The primary outcomes will be gene expression related to inflammatory cytokine TNFa, Serum concentration of IL-1, 6 and TNF-α, FBS, glycosylated hemoglobin A1C (HbA1C), TG, low-density lipoprotein (LDL), HDL and total cholesterol; and the secondary outcomes: blood pressure, weigh, and waist circumference of participants.
Anthropometric variables will be measured both beginning and after the intervention. Participant’s weight will be quantified without shoes and with the least possible clothes with a standard scale made by Mizan Company, with an accuracy of 0.1 kg. The scales will be tested at the beginning and at time intervals when working with standard 5 kg weights. Patient’s height will be measured in standing position and without shoes, using the vertical plane of the height gauge with an accuracy of 0.1 cm. Waist circumference is also measured as the minimum value obtained between the ends of the ribs and the umbilical cord using nonstretching tape. Body mass index (BMI) will be calculated by dividing the weight [kg by the height (m2)]. Patients should be fast when measuring weight and waist circumference.
Biochemical Measurement and Gene Expression Assay
At baseline and endpoint of intervention, after 12–14 overnight fasting and before taking medication, 5 mL venous blood will be taken from each patient by a laboratory technician. Blood samples are immediately centrifuged to separate serum.
Serum concentration of FBS, glycosylated HbA1C, TG, LDL, HDL, and total cholesterol will be measured using enzymatic colorimetric method, automatic analyzer. Concentration of ILS 1, 6, and TNF-α as inflammatory markers and 8-hydroxy-deoxyguanosine (8-OH-dG) as indicator of DNA damage will be measured using enzyme-linked immunosorbent assay.
Isolation of lymphocyte cells, RNA extraction, and cDNA synthesis
From fasting blood samples, extraction of lymphocyte cells will be done using 50% precool solution (Sigma-Aldrich, Dorset, UK) gradient by centrifugation for 20 min and 3000 rpm at 4°C. Total RNA extraction will be based on acid guanidinium-phenol-chloroform procedure using RNX™-plus reagent (Cinnacolon, Tehran, Iran) according to the manufacturer’s instructions. RNAs will be treated with DNAase I (Fermentas, Lithuania) for the elimination of any genomic DNA contamination. The isolated RNA will be reverse transcribed to cDNA library using Moloney murine leukemia virus reverse transcriptase.
Gene expression assay
To quantify the gene expression of TNF-α, mRNAs will be extracted using reverse transcription-polymerase chain reaction method, and primers will be designed using the Glyceraldehyde-3-phosphate dehydrogenase primers as appropriate internal standard. Graphs of gene expression will be drawn Using Light Cycler technology in the relevant software. The relative expression of the gene in the two groups will be compared with the Pffafih method or the same 2-ΔΔct method.
| Statistical Analysis|| |
Data were analyzed using the IBM SPSS Statistics version 23.0 (IBM Corp., Armonk, NY, USA). Continuous variables will be reported as mean ± standard deviation and categorical data as number or percentage. The normality of the distribution of variables will be checked using Kolmogorov–Smirnov test. Analyzing the differences in parametric continuous and asymmetric variables between the two groups will be done by independent samples t-test and the Mann-Whitney U test, respectively. Differences in qualitative variables between study groups will be assessed by Chi-square test. The effect of the intervention in each group will be analyzed by Paired t-test. For outcome variables with no normally distribution log transformation or two related sample tests (Wilcoxon) and Kruskal–Wallis tests will be used. For adjusting the baseline factors and individuals’ characteristics (BMI, gender, age), ANCOVA test will be applied. Statistically significant level for all tests will be considered the value P <0.05.
| Ethical consideration|| |
This study protocol was registered in the Ethics Committee of University of Medical Sciences and University of Medical Sciences. The main goals and method of the study, its duration, the importance of no change in their usual habits and diet and physical activity will be described for all patients. Participation in the study will be voluntary, and leave the study during the project will be possible. Written consent will be signed by all patients. The information obtained from each subject will be kept completely confidential, and identity information of patients will be remaining secreted.
| Discussion|| |
It is hypothesized that chromium supplementation as an essential element can be useful in the management of many metabolic disorders including MS, by improving blood glucose metabolism and lipid profiles, and also by reducing inflammatory mediators can improve the immune system. However, the effect of different compounds of chromium in human studies has not shown definitive results, and evidences are conflicting and may be not clinical significant, and even without any beneficial effect; so this study will be conducted to investigate the effects of chromium picolinate supplementation on glycemic index, lipid profile, blood concentration of TNF-α, and its gene expression in MS patients. The structure and design of controlled randomized selection and measurement of inflammatory gene expression along with other biochemical factors are the strengths of the study. Of course, it should be noted that although statistical tests can be used to adjust the effect of different values of diet and physical activity, because these values are based on patients’ self-report, they can be with error. It is hoped that this study can have significant effects on improving the indicators of MS.
Trial registration: Iranian Clinical Trial Registry: (IRCT20200919048757N1, at 2020- 10-18.http://www.irct.ir).
Conflict of interest: None declared.
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Behrooz Ebrahimzadeh Kour
Nutrition and Food Security Research Center; Department of Nutrition, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd
Source of Support: None, Conflict of Interest: None
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