F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.126650.1 Research Article Articles Impact of serum vitamin D level on selected bone-related markers in obese- type 2 diabetes patients

[version 1; peer review: awaiting peer review]

 Faris Raheem Mohanad Conceptualization Data Curation Formal Analysis Investigation Methodology Software Validation Writing – Original Draft Preparation 1 H Ali Shatha Investigation Methodology Project Administration Software Supervision Validation Visualization Writing – Review & Editing 1 M. A. AL-Nuaimi Ali Conceptualization Methodology Supervision Validation Visualization Writing – Review & Editing 2 G. Shareef Laith Formal Analysis Software Supervision Visualization Writing – Review & Editing https://orcid.org/0000-0001-7773-8474 a 3 Department of Clinical Laboratory Science, College of Pharmacy, University of Baghdad, Baghdad, 10011, Iraq Department of Pharmacy, Gilgamesh Ahliya University, Baghdad, 10022, Iraq Department of Pharmacy, Al-Rasheed University College, Baghdad, 10011, Iraq Laithalkunani@yahoo.com

No competing interests were disclosed.

 13 1 2023 2023 12 56 17 10 2022 Copyright: © 2023 Faris Raheem M et al. 2023 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: Type 2 diabetes mellitus (T2DM) is accompanied by an increased risk for skeletal fractures. The causes are probably a mix of factors, including poor glycemic control, a higher risk of falling due to hypoglycemia, osteopenia, bone quality deterioration, and drug side effects related to bone fragility. Undercarboxylated osteocalcin (ucOC) is a protein generated by osteoblasts that affects insulin secretion and sensitivity.

Methods: A total number of 47 obese (BMI ˃ 30) patients with confirmed type 2 diabetes were selected depending on the American Diabetes Association ADA criteria. The control participants were similar to the patients in age and gender, with a total number of 43 obese (BMI ˃ 30) and healthy subjects chosen from the general population. The selected subjects were grouped according to their serum vitamin D levels. Their blood specimen was used for assaying ucOC, parathyroid hormone (PTH), and vitamin D3 levels by specific ELISA kits, and to estimate calcium (Ca 2+) levels and inorganic phosphate (PO 4 3 −) via enzymatic colorimetric methods.

Results: Serum parathyroid hormone and inorganic phosphate median (IQR) values were markedly increased in patients with T2DM when compared to healthy controls, whereas serum calcium and ucOC levels were lowered significantly in diabetic patients when compared to healthy controls. This was irrespective of serum vitamin D levels.

Conclusions: Elevated serum levels of PTH and PO 4 3 −values in obese type 2 diabetic patients compared to obese non-diabetic controls were accompanied by a significant decrease in ucOC and Ca 2+ levels, irrespective of serum vitamin D levels. Hence, serum vitamin D3 levels had no significant impact on levels of ucOC, PTH, Ca 2+, and PO 4 3 − in obese patients with type 2 diabetes.

 Type 2 Diabetes Mellitus Bone Metabolism Undercarboxylated Osteocalcin Parathyroid Hormone The author(s) declared that no grants were involved in supporting this work. Expression of Concern

Expression of Concern (20 th October 2023): We, the Publisher and Editor of F1000Research, are issuing an Expression of Concern for the following article:

Faris Raheem M, H Ali S, M. A. AL-Nuaimi A and G. Shareef L. Impact of serum vitamin D level on selected bone-related markers in obese- type 2 diabetes patients [version 1; peer review: awaiting peer review]. F1000Research 2023, 12:56 ( https://doi.org/10.12688/f1000research.126650.1).

After publication of this article, questions about the integrity of the ethical approval and methodology were brought to the attention of the F1000 Editorial Team. We have reached out to the authors requesting that they respond to the concerns raised and provide additional information to support the integrity of the content. However, despite multiple attempts to contact the authors, and their institution, they have not responded to our queries within the requested timeframe. Therefore, as we continue to work through the issues raised, we advise readers to interpret the information presented in the article with due caution. The authors have been sent notification about this Expression of Concern.

 Editorial note

Editorial Note (13 th July 2023): Since publication, concerns have been raised to the Editorial Team regarding the ethical approval for this study, as well as overlap in the sample and methods to other papers by these authors. The Editorial Team requested explanations regarding the similarities on 19 th May and 6 th June 2023, and the institution was contacted on 21 st March, 6 th April and 25 th April 2023 to verify the ethical approval. Neither the author nor institution have provided responses to our requests. The Editorial Team will update this Editorial Note as the situation progresses. Peer review activity has been suspended for this article until we receive an explanation from the authors/institution.

 Introduction

Diabetes mellitus (DM) is a metabolic disorder described by a chronic increase in blood glucose and varying levels of impairment in the metabolism of carbohydrates, lipids, and proteins. 1 – 3 However, type 2 diabetes (T2DM) is companied by an altered bone mineral density (BMD), depending on the type of antidiabetic treatment, a paradoxically elevated hazard for skeletal fractures. 4 The causes are probably a mix of factors, including poor glycemic control, a higher risk of falling due to hypoglycemia, osteopenia, bone quality deterioration, and drug side effects, which may increase the risk of fractures and bone fragility. 5 , 6 On the other hand, undercarboxylated osteocalcin (ucOC) is a protein released by osteoblasts that affects insulin release and sensitivity. 7 UcOC regulates insulin secretion and sensitivity by acting on pancreatic beta-cells and adipocytes. So, the bone has been given a new role as an endocrine organ with extra-skeletal functions. 8 Insulin signaling increases osteocalcin production and osteoblastic differentiation by inhibiting Twist2, a Runx2 inhibitor, and FoxO1, resulting in carboxylated osteocalcin accumulation in the bone matrix. Adiponectin also increases osteocalcin expression and osteoblast development through the AMP-activated protein kinase (AMPK) signaling pathway. Insulin and adiponectin increase the RANKL/OPG ratio (receptor activator for nuclear factor κB ligand), which activates osteoclasts and speeds up bone turnover. Activated osteoclasts decarboxylate bone matrix-embedded osteocalcin, which is released into the bloodstream as ucOC. Meanwhile, ucOC elevates insulin production in the pancreas and adiponectin production in adipose tissue. 9 The aim of this research was to estimate the impact of vitamin D3 levels on bone metabolism presented by estimating undercarboxylated osteocalcin, calcium, inorganic phosphate, and parathyroid hormone (PTH) levels in type 2 diabetics with obesity in comparison to healthy people with equivalent levels of vitamin D.

 Methods Ethical consideration

The research protocol received formal clearance from the University of Baghdad's ethics committee on April 10, 2021 (ethics board approval code: 193220). In addition, each research subject signed a written informed consent form.

 Study design

In an observational, case-control study, adult diabetes subjects were compared to healthy subjects.

 Setting

A case-controlled study was conducted as a multicenter study from April 2022 to July 2022, comprising the diabetes centers at The Medical City Complex and Al-Kadhimiya Teaching Hospital.

 Variables

The primary aim of this study was to estimate parathyroid hormone, undercarboxylated osteocalcin, calcium, inorganic phosphate, and vitamin D in study groups.

 Sample size

Subjects' number was determined by the tool G*Power (RRID: SCR 013726) version 3.1.9.7. The sample size was ensured to be not less than 90 participants, with a 95% confidence interval, 90% power, a two-tailed alpha of 0.05, and an effect size of 0.80 (f). This study comprised of 47 diabetic patients and 45 healthy subjects as controls.

 Selection criteria

Adults of both genders were included, subjects diagnosed with type 2 DM depending on American Diabetes Association diagnostic criteria and not starting a treatment or on sulfonylureas only, and obese participants (BMI ≥ 30).

 Exclusion criteria

Patients with a history of hepatic disease, chronic kidney disease (CKD) patients, malignant, any history of using drugs like insulin, metformin, vitamin D, and pregnant or breastfeeding women.

 Study groups

A total number of 90 obese (BMI ˃ 30) subjects were included. The selected subjects were grouped according to their serum vitamin D levels as follows:

Group-1: Involved 22 patients (4 males, 18 females) diagnosed with type 2 DM and having normal vitamin D levels in serum ≥ 22.5 ng/mL.

Group-2: Involved 25 patients (5 males, 20 females) diagnosed with type 2 DM and have low vitamin D levels in serum < 22.5 ng/mL.

Group-3: Involved 20 healthy participants as control (4 males, 16 females), having normal vitamin D levels in serum ≥ 22.5 ng/mL.

Group-4: Involved 23 healthy participants as control (5 males, 18 females), having low vitamin D levels in serum < 22.5 ng/mL.

 Study procedure

Venous blood samples (5 mL) were gained from each participant after fasting for not less than 12 hours and collected in a gel tube (with no anticoagulant). The samples were left for 15—30 minutes at room temperature to allow them to clot and then centrifuged for 10—15 minutes at 4400 round per minute (rpm) to get serum. The resulting serum was then kept frozen (−20°C) until their analysis for ucOC, 10 (PTH), 10 and vitamin D3 11 using sandwich ELISA-specific kits. A detailed study of the procedures is listed below:

The standard was placed in the plate's wells from zero concentration in the first well to the sixth well. Then the serum was poured into the remaining wells and sealed with the kit's sealer.

The plate was placed in the incubator for one hour. It was then washed with inert phosphate buffer, which was made by dilution of the phosphate buffer in distilled water in an amount that was equal to multiplying the concentration of the phosphate in the vial by the volume of the vial solution, in order to remove all the extra unbound materials that were in the serum of participants (for ucOC plate washing in this step was not done).

The conjugate solution in all the kits was prepared by first adding 0.1 mL of Biotin-labeled antibody working solution, waiting 30 minutes, and then adding the enzyme HRP with avidin solution. After another 30 minutes of incubation, both biotin and avidin bound together, allowing conjugation of antibodies with enzyme HRP. The plate was then sealed and incubated for one hour at 37 °C. (The enzyme HRP is used to convert the colorless contact between conjugated antibodies and antigen into a blue interaction.) After the incubation period, the cover was removed and the plate was rinsed three times with an inert phosphate buffer.

Being light and sensitive, a solution of tetramethyl benzidine (TMB) was added to each well, and the plate was covered and kept in the dark for 15 to 30 minutes. The antigens we were looking for are indicated by the color blue, and the intensity of the color indicates the amount of antigen present in the serum.

When a stop solution (acid solution) was administered to the wells, the color changed from blue to yellow. The plate was then put in the ELISA device reader to read the data at 450 nm, which is the best wavelength for reading the yellow color.

In addition, the remaining serum part was used for analyzing calcium by a method based on specific binding of cresolftalein complexone (OCC), a metallochromic indicator, and calcium at alkaline pH with the resulting shift in the absorption wavelength of the complex. The intensity of the cromophore formed was proportional to the concentration of total calcium in the sample absorbance read at a wavelength of 570 nm 12 and inorganic phosphate, 13 where inorganic phosphate reacted with molybdic acid forming a phosphomolybdic complex. Its subsequent reduction in alkaline medium gave a blue molybdenum color, with an intensity proportional to the amount of phosphorus present in the sample, the absorbance was read at 740 nm against the reagent blank.

 Materials and instruments

The analysis used for the chemicals was as pure as it could be. The chemical kits utilized in the current study are summarized in Table 1.

Chemical kits used in this study.
Chemicals Provider Cat. No. REF No. Web Link
Vitamin D3 MyBioSource (American) MBS264661 NA https://www.mybiosource.com/human-elisa-kits/vitamin-d3-vd3/264661
Undercaroxylated osteocalcin Elabscience (USA) E-EL-H6118 NA https://www.elabscience.com/p-human_ucoc_undercarboxylated_osteocalcin_elisa_kit-356241.html
Parathyroid hormone MyBioSource (American) MBS263675 NA https://www.mybiosource.com/human-elisa-kits/parathyroid-hormone-pth/7722808
Calcium LINEAR chemicals (Spain) NA 1115000 https://www.linear.es/wp-content/uploads/2018/03/1115000I-Rev.-03.pdf
Inorganic phosphate LINEAR chemicals (Spain) NA 1148010 https://www.linear.es/en/portfolio_page/phosphorus/
Statistical analysis

IBM SPSS Statistics (RRID: SCR_016479) version 27 software for Microsoft Windows was used throughout the statistical analysis. First, the normality of data distribution was tested using the Shapiro-Wilk test. A non-parametric test was employed for the study since the data is not normally distributed, and descriptive statistics were presented as (median (interquartile range)) (IQR). Next, the four studied groups were compared using the Kruskal-Wallis test to assess their significance level. A p-value of < 0.05 was treated to be significant.

 Results

The studied groups demonstrated non-significant differences in median (IQR) of age, height, weight, and BMI among the four studied groups, as shown in Table 2.

Anthropometric measures among studied groups.
Variables D.M. with Vit. D > 22.5 ng/dl (N=22) D.M. with Vit. D < 22.5 ng/dl (N=25) Control with Vit. D > 22.5 ng/dl (N=20) Control with Vit. D < 22.5 ng/dl (N=23) P-value
Age (years) 50.50 (17.75) 52.0 (13.0) 45.5 (11.75) 51.0 (20) 0.588
Height (cm) 160.5 (9.0) 163 (13.5) 161 (8.0) 162 (14.0) 0.328
Weight (kg) 91 (12) 93 (12.5) 89 (8.5) 91 (8.0) 0.496
BMI 34.56 (3.27) 34.45 (2.79) 33.895 (3.25) 34.54 (3.57) 0.740

N=number of subjects, BMI= body mass index, (Kruskal) insignificant at a level of P>0.05.

No significant difference in PTH and PO 4 3− serum levels between the two groups with diabetes nor between the two healthy control groups were shown. At the same time, the two diabetes patient groups showed significantly higher PTH and PO 4 3− values than controls. Also, no significant difference in ucOC and Ca 2+ serum levels between the diabetic and the control groups was found. However, the two control groups had higher ucOC and Ca 2+ values than the patient groups, as shown in Table 3.

Bone-related markers (PTH, Ca <sup>2+</sup>, PO <sub>4</sub>=, and undercarboxylated osteocalcin) levels among studied groups.
Variables D.M. with vit. D > 22.5 ng/dl (N=22) D.M. with vit. D < 22.5 ng/dl (N=25) Control with vit. D > 22.5 ng/dl (N=20) Control with vit. D < 22.5 ng/dl (N=23)
PTH (Pg/ml) 40.3587 (28.75) a 42.150 (28.75) a 24.1211 (23.55) b 26.4133 (17.15) b
Ca 2+ (mg/dl) 8.702 (1.31) b 8.05 (0.91) b 9.39 (0.68) a 9.1 (0.86) a
PO 4= (mg/dl) 4.376 (1.19) a 4.4 (1.23) a 3.74 (0.75) b 3.656 (1.08) b
ucoC (Pg/ml) 37.5345 (5.890) b 39.475 (11.075) b 122.65 (72.711) a 104.156 (31.71) a

N=number of subjects, ucOC=undercarboxylated osteocalcin, PTH= parathyroid hormone, PO 4 3− Inorganic Phosphate, Ca 2+=calcium, Superscripts (a,b,c,d) refer to significant differences among groups, such that (a) the level is significantly higher than the level in (b) and the last is significantly higher than the level in (c) and so on.

 Discussion

Vitamin D levels are lowered in obese individuals in comparison to non-obese; this may be explained by the diminished degrees of exercise, exposure to sunlight, and reserving 25 hydroxy-vitamin D in adipose tissues. 14 , 15 Since older people are less effective than younger people in producing vitamin D from sunlight and older people's kidneys have a lower ability to transform vitamin D into its active form, age could be predicted to correlate with vitamin D levels adversely. However, for this study, there were no significant differences in confounder (BMI and age) among the studied groups (as shown in Table 2) to decrease the confusion in results. Together with PTH, vitamin D mediates bone mineralization and maintains calcium homeostasis in the circulation. In addition, vitamin D modulates calcium absorption from the small gut. According to studies, low vitamin D levels have been linked to several ailments; this is possible because vitamin D has anti-inflammatory and immune-modulating characteristics. 16 Most research shows that people with insulin-dependent diabetes have a lower bone mineral density (BMD). 17 Still, with type 2 diabetes, some studies reported a decrease, 18 others reported an increased, 19 and many others reported no change. 20 This study found no significant difference in serum PTH, ucOC, Ca 2+, and PO 4 3− levels between the two groups of patients with T2DM nor between the two groups of healthy control subjects.

In contrast, the patients' groups had higher significant PTH and PO 4 3− values than the control group. Both patient groups showed a considerable decrease in ucOC and Ca 2+ in their serum levels compared to control, so no correlation was found between vitamin D and PTH, ucOC, Ca 2+, PO 4 3− in this study, while a significant correlation between diabetic and these bone markers was observed. Studies conducted in vivo and in vitro have shown that the primary factor in reducing blood sugar, boosting insulin production, and reducing insulin resistance was likely the ucOC component. 21 A case-control study of 153 subjects diagnosed newly with type 2 diabetes and 306 controls showed an association of ucOC with risk of diabetes. 22 However, many other studies didn't show a strong correlation between ucOC and indices of glycemia. 23 , 24 A survey by Kramer CK et al. involving 494 women who had a serial metabolic characterization reported that vitamin D exerts its indirect action by controlling calcium flow via the cell membrane and intracellular calcium, while secondary hyperparathyroidism is caused by secondary vitamin D deficiency. Diabetes is also linked to elevated PTH levels. Cell dysfunction, insulin resistance, and hyperglycemia were all independently predicted by hypo vitamin D levels with high PTH levels. 25 In T2DM subjects, the serum calcium level was markedly lower than the serum level in the control group. The serum levels of inorganic phosphate in the T2DM patients' group were also lower than in the control group. These results agreed with others 26 , 27 ; Numerous factors including a decrease in insulin levels, which inhibit bone formation by stimulating osteoblast proliferation, a disturbance of calcium homeostasis, and hyperglycemia, which increases calcium excretion in the urine, are most likely to held responsible for the drop in serum calcium levels. 28 Due to more significant amounts of the complexed ion, 20 lowering serum-ionized calcium should encourage PTH secretion in these individuals. As opposed to this, other investigations 29 found no differences in PTH readings between diabetics and non-diabetics. Further research found that patients with T2DM had lower serum levels of PTH compared to the control group, 27 , 30 , 31 in addition to two large-scale U.S. studies among community adults. 32

Limitations

Low patient numbers limited this study. So, we suggest elevating the number of participants in future research.

 Conclusion

Increased PTH and PO 4 3− values in obese diabetes patients compared to obese non-diabetic controls significantly decreased ucOC and Ca 2+ levels. Furthermore, serum vitamin D3 levels did not substantially impact serum levels of ucOC, PTH, Ca 2+, and PO 4 3− in obese type 2 diabetic patients.

Recommendations

In light of the present study, there is a need to include a larger sample size to investigate the relationship between glycemic measure control and serum levels of vitamin D3.

 Author contributions

Conceptualization: Mohanad Faris Raheem. Data curation: Mohanad Faris Raheem. Formal analysis: Shatha H Ali. Investigation: Mohanad Faris Raheem. Methodology: Ali M. A. Al-Nuaimi. Software: Laith G. Shareef. Validation: Shatha H Ali. Visualization: Ali M. A. Al-Nuaimi - original draft: Mohanad Faris Raheem. Writing - review & editing: Laith G. Shareef, Shatha H Ali.

 ORCID iDs

Mohanad Faris Raheem https://orcid.org/0000-0003-2988-6071

Shatha H. Ali https://orcid.org/0000-0002-3682-2386

Ali M. A. Al-Nuaimi https://orcid.org/0000-0001-5185-1049

Laith G. Shareef https://orcid.org/0000-0001-7773-8474

Data availability Underlying data

Zenodo: Demographic data along with bone-related biomarkers. https://doi.org/10.5281/zenodo.6969297. 33

The underlying information for this project is as follows:

Article’s data.xlsx (Demographic data along with bone-related biomarkers)

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

 Acknowledgments

The authors would like to thank the participating subjects and the team of diabetes centers for their help during sample collection, especially M.B.Ch.B Harith Abass Hadi.

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