DOI: https://doi.org/10.21203/rs.3.rs-258673/v1
This study aimed to examine the synergistic effect of Vitamin D (VD) supplement and mindfulness on insulin resistance and different aspects of cognitive function in VD deficient type 2 diabetic women.
In this randomized-controlled trial, totally 225 women with type 2 diabetes mellitus (T2DM) and VD deficiency were randomly allocated to five groups: (1) mindfulness, (2) mindfulness and VD, (3) mindfulness and placebo, (4) VD, and (5) placebo. Mindfulness includes 12 sessions and VD patients received a daily 4000 IU oral dosage (two capsules) with 28,000 IU vitamin D weekly for 12 weeks. Sun exposure, anthropometric indices, dietary intake, physical activity, energy levels, and laboratory analyses were measured at the pre-test and after 12-week supplementation. Dependent variables include cognitive function (Risky decision making, IQ and Trail Making Test) and insulin resistance.
In baseline, measures were not different among the groups. At the end of the treatment, cognitive function results showed improvement for all groups except the “placebo” group. About other groups, there was not any differences between VD and mindfulness groups (or combinations with placebo). However, in “VITAMIN + MINDFULNESS” group, a greater improvement compared to other groups was observed (P < 0.05). This result exactly repeated for insulin resistance. Furthermore, there were not any significant difference among groups for IQ (as one aspect of cognitive function) (P > 0.05). Moreover, there was not any differences among groups for sunlight exposure and BMI (P > 0.05). Only in combined group (VD + Mindfulness) the fasting blood sugar (FBS) significantly reduced that shows the synergistic effect of VD supplementation and Mindfulness (P < 0.05).
Combining VD Supplementation and mindfulness can reduce insulin resistance and improve different aspects of cognitive function in VD Deficient patients.
Currently type 2 diabetes mellitus (T2DM) is recognized as a global epidemic and estimated to increase exponentially by more than 500 million patients worldwide in 2035(1). Diabetic patients are at greater risk for long-term difficulties concerning the feet, eyes, heart, kidneys, brain, and nervous system (1). In patients with T2DM, the capacity to generate insulin is not lost, even extreme in some diabetic patients, but the result of insulin is weak, so insulin is approximately lacking in these patients. Recent studies showed that the onset of T2DM is linked with insulin resistance (IR) caused by reduced insulin sensitivity(2, 3).The effects of IR on body include (1) feeling hungry even after a meal, (2) frequent infections, (3) increased or frequent urination, (4) feeling more tired than usual, etc. (3, 4). Therefore, special attention should be paid to improving IR in patient’s withT2DM.
On the other hand, diabetes heightens the risk of disabilities on cognitive function. Based on recent researches, the frequency of dementia in diabetic patients is two times higher than the general population. Cognitive dysfunctions also significantly affect the ability of self-management in diabetic patients(5).
In recent researches, results showed that in T2DM patients, by promoting the inflammation, vitamin D deficiency enhances the IR. In other words, vitamin D insufficiency is linked to IR related diseases, especially T2DM(2). Therefore, based on new researches vitamin D supplementation can improve IR(6). Moreover, this supplement could improve cognitive functioning in patients with T2DM(5). However, because of some limitations such as small sample size and unclear inclusion criteria the results are contradictory(5, 7).
Another treatment that demonstrated promising results in T2DM is mindfulness. In accordance with previous researches, mindfulness can improve underlying physiological and behavioral determinants that influence insulin resistance such as stress arousal, physical activity, sleep quality, and eating unhealthy habits(8). This treatment also was accompanied with improvement in immediate memory, thinking, and attention could enhance cognitive function. It has also enhance cognitive function with effects on dorsolateral prefrontal cortex, amygdala, and the limbic system (9).
Treatment for type T2DM involves a complex combination of behavioral-cognitive and biological factors that require multifaceted efforts to manage the disease and reduce the health burden. However, despite the bio-psycho-social nature of diabetes, there is not any study that has examined this combination of treatments. Moreover, when vitamin D and mindfulness used as monotherapy, although they are effective, but they have on average, a small effect size. There has accordingly been interesting in consolidating pharmacological and psychotherapeutic treatments in a trial to improve effectiveness (7–10).In addition, behavioral intervention by improving the empathy and therapeutic alliance can enhance the drug compliance. Therefore, regarding with this combination, we can directly and indirectly modify problems (11, 12).
Accordingly, this is the first randomized clinical trial to examine the effect of combined vitamin D supplementation with mindfulness on insulin resistance and cognitive function in T2DM patients.
Study Design and Participants
This study was a randomized single-blind placebo-controlled clinical trial. In the present study, participants include all T2DM patients with vitamin D insufficiency or deficiency were recruited from hospitals in Tehran, Iran. This sampling was conducted during 1 September 2019 to 25 October 2019.T2DM was diagnosed by a physician with regard to WHO guidelines(13).From 26 October 2019, to January18, 2020, interventions have been implemented. The presented research is a piece of a larger cohort project concerning examining various treatment for long-term improvement in metabolic syndrome. Because in Iran, the rate of diabetes in women is much higher than men, all participants are women (14).
Inclusion criteria include (1) vitamin D insufficiency or deficiency [between 10 (ng/mL) to 30 (ng/mL) serum vitamin D], (2) age of 18 to 60 years, and (3) willingness to participation. Exclusion criteria include (1)had psychiatric or neurological disorders, (2) took vitamin D or multivitamin supplements during the last three months, (3) using any illicit substance and alcohol, (4) being in pregnant period, (5)two or more absent in mindfulness groups, (6)using vitamin D supplements over the last 4 months and, (7) Pregnancy or breastfeeding.
Randomization and blinding
Participants were randomly allocated into either intervention or placebo groups (with PC version of random table). All participants blinded to receive vitamin D or placebo. In order to hide the allocation of individuals to the groups, closed envelopes containing the code of the assigned group of each person were used along with the number of the person on the envelope.
Sample size
Regarding 0.05 as type 1 error and 0.2 as type 2 error (with 80%power) based on prior research, 35 patients were required in each group. For covering possible dropouts, sample size 25% increased and reach 45 subjects for each group (15).
Grouping
The subjects are randomly allocated into the following groups:
Interventions
Supplement: In vitamin D groups, patients received a daily 4000 IU oral dosage (two capsules) with 28,000 IU vitamin D weekly for 12 weeks (26 October 2019 until 18 January 2020). This supplement includes fourteen oily drops weekly (sobhan darou, Iranian pharmaceutical company).Placebo (Subhan darou) groups receive totally same drops (without any vitamin D content) and time.
Mindfulness: mindfulness consisted of 12 sessions (meeting once a week for 90 minutes) with one psychotherapist and his co-therapist. The intervention protocol was an adaptation of mindfulness to medicine context (MBSR) that introduced by L McCracken(16).Sessions consist of activity-based, interactive, motivational content. This treatment focuses on relaxation (and yoga) exercises and techniques. Some sessions also concentrate on cognitive restructuring, self-compassionate and receiving social support. Other elements of mindfulness based on stress reduction include: implement mindfulness while breathing, exercise meditation while lying, Tarrying to be in the present moment (here and now), ticking off balmy thoughts, recognizing and accepting unpleasant feeling, emotions and thoughts, moving from the intrapersonal life to the interpersonal world, recognizing steady life and planning for more healthy lifestyle, organizing for individual care, seeking the right refuge for diabetic problems, planning for solving diabetic problems, and how to moderating life-style in diabetes and vitamin D deficiency. For comparing the adherence, audios of sessions were recorded with the permission of all the members. Then, a mindfulness psychotherapist checked the content of secessions. The sessions were divided into 30-minute modules that were taken for adherence check, randomly. The treatment position and the occurrence process were evaluated. Based on the intervention manual, the modules assessed the adherence level as either enough or not sufficient. The majority of content (93%) was judged as implemented adequately.
Measures
Laboratory analyses: ten milliliters of fasting blood sample from every patient was obtained after nine hours of fasting at the start and at the completion of the study. The blood samples stored at −80°C (with ten minutes centrifuging at 3000 rpm) until further analysis with centrifuged to isolate serums. Fasting blood glucose (FBS), measure was performed by spectrophotometry utilizing Pars Azmun kit (Iran) in auto-analyzer equipment (BT3000, Italy).
Insulin resistance: The formula for measurement of insulin resistance was homeostatic model assessment for insulin resistance (HOMA-IR) that was calculated as: (HOMA IR= (Fasting insulin (μU/ml) ×Fasting glucose ((mmol/l) ÷22.5). Patients were demanded be fasting for nine hours prior to come in for blood measuring. To increase the validity of the insulin and glucose outcomes, ere the blood sample, patients filled out a fasting questionnaire that required particular subjects discussing the last time they had consumed any food or liquids . In fact, for assurance that the patients were obeying the fasting contract we used a questionnaire. Questions in this scale include common and less commonly considered foods like breath mints, tea, supplements and etc.
Sun exposure time: Before implementing intervention and in the post-test by a validated questionnaire the sun exposure rate was assessed. The span of sun exposure was measured by total hours exposing to the sun in the last two week and divided into two.(17).
Dietary intakes: For assessing the nutrients intake and energy levels, the fourth version of Nutritionist software modified for Iranian foods was used by a trained dietitian(18) (three-day food records include one weekend day and two weekdays were collected).
Anthropometric assessments: Height was measured while the subjects were standing, not wear shoes and shoulders were in a normal position by a wall stadiometer to the nearest 0.1 cm (Seca, Germany). Weight was measured by digital scale with an accuracy of 0.1 kg (Seca 808, Germany) with light clothing and no shoes. Calculation of the body mass index (BMI, kg/m2) was done as below formula:
Weight (kg)/Height (m2)
To decrease subjective errors, all measurements were performed by the same technician.
Physical activity: The brief version of the International Physical Activity Questionnaire (IPAQ) was applied for determining the level of physical activity participants(19).
Cognitive function tasks
Three examinations were utilized for evaluation of different aspects of cognitive function:
Statistical analysis
For assessing data SPSS version 26 for windows (SPSS Inc., Chicago, IL) was employed and we used paired sample t-test, the one-way ANOVA, and post-hoc test (Scheffe). P-values <0.05 were considered statistically significant.
Two hundred twenty-five (225) TDM2 were included in our research. Finally, 201participants completed the examination [Figure 1].In the administration of vitamin D and placebo for diabetic patients, no side effects were recorded.
Demographic variables were measured in pre-test and end of invention. In pre-test stage participants were not statistically different among groups. (P > 0.05). Yet, in post-intervention there are statistically differentiation for FBS, physical activity, and energy intake (P < 0.05) [Table 1].
Characteristic | placebo | Placebo + mindfulness | mindfulness | Vitamin | Vitamin + Mindfulness | P value |
|---|---|---|---|---|---|---|
Age (years)a | 46.4(8.9) | 46.3(8.2) | 45.9(10.6) | 45.1(9.6) | 47.6(10.2) | 0.8 |
DURATION OF DIABETES (YEARS | 14.3(3.6) | 14.02(4.2) | 14.8(4.1) | 14.3(3.8) | 14.3(3.7) | 0.9 |
base line Vitamin D level (ng/mL) | 24.6 ± 5.5 | 25.4 ± 5.4 | 25.3 ± 5.2 | 27.4 ± 5.07 | 26.2 ± 5.7 | 0.1 |
FBS (pre-test) | 152.7(24.8) | 156.1(25.8) | 161.5(22.1) | 163.3(19.5) | 157.6(23.02) | 0.25 |
FBS (post-test) | 155.5(23.4) | 150.6(23.01) | 148.2(25.6) | 159.87(2.8) | 128.09(26.2) | 0.00* |
FBS duringtimeb | P > 0.05 | P > 0.05 | P > 0.05 | P > 0.05 | P = 0.00* | ---- |
sun (pre-test) | 1.9(1.4) | 1.4(1.3) | 1.9(1.2) | 1.9(1.5) | 1.8(1.4) | 0.37 |
sun (post-test) | 1.8(1.3) | 2.3(1.3) | 1.8(1.3) | 1.7(1.3) | 2(1.4) | 0.23 |
BMI(pre) | 29.4(3.36) | 30.41(3.5) | 30(3.2) | 30.8(3.34) | 29.7(3.36) | 0.36 |
BMI(post) | 29.8(3.01) | 30.4(2.09) | 30.17(3.6) | 30.4(2.9) | 29.7(3.30) | 0.7 |
BMI during time | P > 0.05 | P > 0.05 | P > 0.05 | P > 0.05 | P > 0.05 | ---- |
Energy (pre) | 2206.22(305.09) | 2242.27(351.24) | 2141.51(346.4) | 2174.6(310.2) | 2238.02(308.71) | 0.5 |
Energy (post) | 2244.7(308.68) | 1970.58(281.72) | 1954.97(269.6) | 1939.9(355.91) | 1730.25(277.24) | 0.00* |
energyDURING-TIMEB | P > 0.05 | P < 0.05* | P < 0.05* | P < 0.05* | P < 0.05* | |
PHY (pre) | 12.5(5.02) | 12.57(5) | 12.33(5.8) | 12.7(5.9) | 13.67(5.6) | 0.98 |
PHY (post) | 12.12(4.3) | 15.38(5.1) | 15.05(4.2) | 12.8(4.4) | 15.2(4.6) | 0.002 |
| a = one way anova, b = paired sample t-test, FBS = Fasting blood sugar (mg/dL), Sun: Sunlight exposure, Energy: Energy (pre-test) (kCal/day), PHY: Physical activity (MET/hours*week). VD = Vitamin D intake (mcg/day) | ||||||
Based on these results, in all groups except for “placebo”, we found a reduction in FBS. But only in “Vitamin + Mindfulness” group we had a statistically reduction in post-treatment rather than pre-test (P < 0.05). For energy intake, all groups except for “placebo” showed a statistically reduction in post-intervention (p < 0.05). In fact, both vitamin D and mindfulness exercises can improve energy intake. A comparison among other groups which received vitamin D or mindfulness as a component of their intervention package, done with post-hoc (Scheffe) analysis. Table 2 demonstrated that, “Vitamin + Mindfulness” group significantly reduced in energy intake rather than other groups. Also there was not any significant difference between “mindfulness” and “Vitamin D” in energy intake (P < 0.05).For physical activity, results showed that only in groups with “mindfulness”, a significant reduction in physical activity was observed (P < 0.05). Yet, among groups with mindfulness component there were no significant differentiation. Additionally, there was not any differences between placebo and vitamin D groups [Table 2].
BMI | |||||||
|---|---|---|---|---|---|---|---|
Group I | Group J | Mean difference | P value | Group I | Group J | Mean difference | P value |
placebo | Placebo + mindfulness | 274.11 | 0.003* | Placebo + mindfulness | mindfulness | 15.61 | 0.9* |
mindfulness | 289.72 | 0.001* | Vitamin | 30.68 | 0.99 | ||
Vitamin | 304.8 | 0.001* | Vitamin + Mindfulness | 240.33 | 0.01* | ||
Vitamin + Mindfulness | 514.44 | 0.001* | |||||
mindfulness | Vitamin | 15.7 | 0.9 | ||||
Vitamin + Mindfulness | 224.71 | 0.02* | Vitamin | Vitamin + Mindfulness | 209.64 | 0.04* | |
Physical activity | |||||||
Group I | Group J | Mean difference | P value | Group I | Group J | Mean difference | P value |
placebo | Placebo + mindfulness | -3.16 | 0.001* | Placebo + mindfulness | mindfulness | 0.38 | 0.69 |
mindfulness | -2.77 | 0.004* | Vitamin | 2.61 | 0.007* | ||
Vitamin | -0.55 | 0.56 | Vitamin + Mindfulness | 0.15 | 0.8 | ||
Vitamin + Mindfulness | -3 | 0.002* | |||||
mindfulness | Vitamin | 2.22 | 0.02* | ||||
Vitamin + Mindfulness | -0.2 | 0.81 | Vitamin | Vitamin + Mindfulness | -2.45 | 0.01* |
For Cognitive function tasks, results demonstrated that in pre-test there were no difference among groups (P > 0.05) [Table3]. But, at the end-of-treatment, for risky decision-making result showed improvement for all groups except “placebo” group. About others groups, although other groups demonstrated significant improvement, but “VITAMIN + MINDFULNESS” has a greater improvement rather than other groups (P < 0.05). This result exactly repeated for insulin resistance. (P < 0.05). Yet. For SIGT results showed there is not any significantly difference among group before and after of implementation of intervention (P > 0.05). However, in Vitamin + Mindfulness group, we faced with somewhat improving, But, this improvement is not statically significant (P > 0.05).
Characteristic | placebo | Placebo + mindfulness | mindfulness | Vitamin | Vitamin + Mindfulness | P value |
|---|---|---|---|---|---|---|
IOWA (pre-test) | 0.92(5.9) | -0.38(5.2) | 0.51(5.4) | 0.72(5.1) | -0.13(5.7) | 0.76 |
IOWA (post-test) | 2.30(5.1) | 6.28(5.6) | 6.3(4.6) | 5.97(4.7) | 9.89(3.8) | 0.001* |
IOWA duringtimeb | P > 0.05 | P < 0.05* | P < 0.05* | P < 0.05* | P < 0.05* | |
SGIT (pre) | 32.5(3.8) | 32.9(3.2) | 32.4(4.05) | 31.8(3.6) | 31.6(3.3) | 0.45 |
SGIT (post) | 32.2(3.9) | 33.1(4.1) | 31.6(3.5) | 31.6(4.2) | 33(3.7) | 0.25 |
SGITduring time | P > 0.05 | P > 0.05 | P > 0.05 | P > 0.05 | P = 0.07 | --- |
TMT (pre) | 92.8(13.3) | 94.3(14.1) | 94.1(17.4) | 93.6(14.6) | 93.7(14.9) | 0.9 |
TMT (post) | 97.9(17.4) | 86.4(14.1) | 84.4(16.7) | 81.01(13.5) | 68.6(15.9) | 0.001* |
TMT DURING-TIMEB | P > 0.05 | P < 0.05* | P < 0.05* | P < 0.05* | P < 0.05* | |
INSULINE (pre) | 5.8(1.7) | 5.5(1.4) | 5.5(1.3) | 5.1(1.4) | 5.7(1.6) | 0.39 |
INSULINE (post) | 5.7(1.2) | 4.4(1.5) | 4.1(1.08) | 4.4(1.2) | 3.1(1.2) | 0.001* |
during time | P > 0.05 | P < 0.05* | P < 0.05* | P < 0.05* | P < 0.05* | |
| a = one way anova, b = paired sample t-test, Ruler:Ruler drop method, TMT:Trial making test; | ||||||
IOWA | |||||||
|---|---|---|---|---|---|---|---|
Group I | Group J | Mean difference | P value | Group I | Group J | Mean difference | P value |
placebo | Placebo + mindfulness | -3.98 | 0.01* | Placebo + mindfulness | mindfulness | -0.02 | 0.9 |
mindfulness | -4.01 | 0.011* | Vitamin | 0.33 | 0.89 | ||
Vitamin | -3.65 | 0.024* | Vitamin + Mindfulness | -3.55 | 0.01* | ||
Vitamin + Mindfulness | -7.53 | 0.001* | |||||
mindfulness | Vitamin | 0.35 | 0.89 | ||||
Vitamin + Mindfulness | -3.52 | 0.03* | Vitamin | Vitamin + Mindfulness | -3.88 | 0.011* | |
TMT | |||||||
Group I | Group J | Mean difference | P value | Group I | Group J | Mean difference | P value |
placebo | Placebo + mindfulness | 11.3 | 0.03* | Placebo + mindfulness | mindfulness | 2.1 | 0.98 |
mindfulness | 13.4 | 0.006* | Vitamin | 5.5 | 0.65 | ||
Vitamin | 16.8 | 0.001* | Vitamin + Mindfulness | 17.8 | 0.001* | ||
Vitamin + Mindfulness | 29.2 | 0.001* | |||||
mindfulness | Vitamin | 3.38 | 0.9 | ||||
Vitamin + Mindfulness | 15.7 | 0.001* | Vitamin | Vitamin + Mindfulness | 12.3 | 0.01* | |
INSULINE | |||||||
Group I | Group J | Mean difference | P value | Group I | Group J | Mean difference | P value |
placebo | Placebo + mindfulness | 1.30 | 0.001* | Placebo + mindfulness | mindfulness | 0.34 | 0.8 |
mindfulness | 1.65 | 0.001* | Vitamin | 0.02 | 0.9 | ||
Vitamin | 1.33 | 0.001* | Vitamin + Mindfulness | 1.2 | 0.001* | ||
Vitamin + Mindfulness | 2.59 | 0.001* | |||||
mindfulness | Vitamin | -0.31 | 0.8 | ||||
Vitamin + Mindfulness | 0.97 | 0.014* | Vitamin | Vitamin + Mindfulness | 1.26 | 0.001* | |
| DSP: Digit Span Backwards, CPT: continuous performance test, INSULINE: Insulin Resistance | |||||||
Our research revealed that twelve weeks supplementation with 28,000 IU vitamin D per week combined with mindfulness decreased insulin resistance and improved cognitive functions among vitamin D deficient Type 2 Diabetic women. This is the first study in combination of vitamin D with 3rd wave behavior therapies and demonstrated high feasibility and efficacy. Regarding the results, vitamin D and mindfulness training (Mindfulness based on stress reduction protocol) in separated groups could improve insulin resistance and cognitive function. Furthermore, when they were combined in a package, they could significantly better than separated groups treating these variables. These results are in line of previous studies.
For example, in Shomaker research, mindfulness training reduced insulin resistance in diabetic adolescents with one-year follow-up(8).Also various meta-analysis showed that vitamin D can reduce insulin resistance in different physical conditions(23, 24). About cognitive function Klainin-Yobas et.al showed that mindfulness can improve cognitive function and emotional state in elderly individuals with moderate cognitive impairment (25). Moreover, in a randomized control trial, Byrn et.al reported that vitamin D supplementation in a variety of dosages can improve cognition in patients with T2DM(26).
About efficacy of vitamin D and mindfulness on IR above researches showed similarity of our data with previous research literature .Mindfulness with developing of basic physiological and/or behavioral factors that influence IR such as physical activity, eating habits, sleep behaviors, and stress arousal, reduce insulin resistance. Also, in type 2 diabetes patients, vitamin D deficiency lead to promote inflammation and enhance proportion of p-p65/RelB. RelB and p65 protein and in results enhances insulin resistance. Generally, animal investigations revealed that vitamin D insufficiency may trigger insulin resistance in T2DM rats by increasing inflammation through the NF-kB pathway. Therefore, taking vitamin D can break this pathway and reduce inflammation (2, 8, 27). All in all, as diabetes is a bio-psycho-social problem our combined package with targeting biological and behavioral aspects of IR, so we faced with higher improvement rather than separated pathway to reduce the IR. For cognitive function, low levels of 25(OH)D is associated with increased risk of cognitive impairments. Furthermore, vitamin D has a vital role in healthy brain function and deficiency of vitamin D may lead to cognitive impairment(28) and vitamin D supplements improved cognitive function in various medical populations(29, 30).
Mindfulness by affecting on dlpfc, amygdala, PFC and limbic system, has revealed improvements in cognitive functions (i.e., executive function, working memory and attention) in younger and older patients (25). Therefore, combination of vitamin D and mindfulness revealed synergistic effects on cognitive functions. However, there is not any difference among groups for IQ. In fact, IQ is somewhat permanent ability and changing IQ is difficult. Furthermore, the IQ score of patients were in normal range and there was not any impairment that needs to be improved. So all improvements happened in other cognitive functions that were impaired.
About demographic variables, there were any difference among groups for sunlight exposure and BMI. As, these variable highly associated with life-styles for changing them, patients need to do variety changes in their lifestyles and this action needs more times(7). For FBS, only in combined group (VD + Mindfulness) the FBS significantly reduced that demonstrated the synergistic effects. For other groups we needed more times for examining their results. In fact, in a 16-weeks vitamin D supplementation, a reduction in FBS in vitamin D group was observed (7), but there is not any similar research for the effect of mindfulness on FBS. More examinations for mindfulness effects on metabolic profiles are needed.
Although various strengths of this study, we have some limitations first, we were unable to explore the seasonal changes that could impact the result. Also, we need monitoring follow-up but as there is the first pilot these limitations must be eliminated to future studies. Future studies must reduce these limitations.
Combining vitamin D and mindfulness training could improve cognitive functions and insulin resistance in vitamin D deficient type 2 diabetic.
Ethics approval and consent to participate: Written informed consent (about participation in the study) was received from all patients before the beginning of the study. The scales used in this research were all filled anonymously and a numeric code was used. This project was assessed and certified by the ethics committee of Kermanshah University of medical science (Ir.kums.rce.1399.0492). Moreover, this study is registered in the Thailand Registry of Clinical Trials (TCTR20200629004).
Consent for publication: during sampling individual session was held. We received consent for publication results from each participant.
Availability of data and material: Data of participants who consented to the public sharing of data are accessible from the corresponding author upon reasonable demands.
Competing interests: The authors certify that they have no competing interests.
Funding: This study was not funded.
Authors' contributions: The first author and corresponding author conceived of the presented idea. The first author with second author developed the protocols and performed the sampling. Second author and fourth. The author verified the analytical methods. The corresponding author encouraged the first author to investigate this matter and supervised the results of the findings. All authors discussed the results and contributed to the final manuscript.
Acknowledgements: We would like to express our gratitude to the respected staff of the hospitals affiliated with Kermanshah University of Medical Sciences who have cooperated in the implementation of this study.