|Year : 2021 | Volume
| Issue : 1 | Page : 138-143
Study of vitamin D supplementation on selected hematological and inflammatory parameters in type 2 diabetes mellitus with vitamin D deficiency
Deepali Sadashiv Jankar1, Kanchan Chandrashekhar Wingkar1, Sujata V Kanetkar2, Chintamani Dilip Bodhe3
1 Department of Physiology, KIMS, Karad, Maharashtra, India
2 Department of Pathology, KIMS, Karad, Maharashtra, India
3 Department of Physiology, GMC, Miraj, Maharashtra, India
|Date of Submission||05-Jun-2020|
|Date of Decision||18-Oct-2020|
|Date of Acceptance||22-Nov-2020|
|Date of Web Publication||29-Jul-2021|
Dr. Chintamani Dilip Bodhe
Department of Physiology, GMC, Miraj - 416 410, Maharashtra
Source of Support: None, Conflict of Interest: None
Introduction: Diabetes mellitus (DM) results from decreased insulin secretion and/or increased insulin resistance. Vitamin D deficiency (VDD) is linked with decreased insulin secretion, increased insulin resistance, inflammation, and complications of DM. Few studies have reported association of VDD with anemia or decreased hemoglobin level in diabetic patients. Animal studies have reported some improvement in various blood parameters such as hemoglobin (Hb), hematocrit, red blood cell (RBC) and white blood cell count, and mononuclear cell count after Vitamin D supplementation. Neutrophil–lymphocyte ratio (NLR) and platelet–lymphocyte ratio (PLR) are cheap, easy, reproducible, and dynamic parameters of inflammation with high predictive value. NLR can be associated with DM and its complications. PLR is reported to be associated with cardiovascular disease, diabetic complications, end-stage renal disease, and malignancies. Both have prognostic value. Materials and Methods: Sixty-three Type 2 DM (T2DM) patients on oral hypoglycemic agents aged 30–60 years with VDD (Vitamin D level <20 ng/ml) participated in this comparative and interventional study. Vitamin D, Hb, total leukocyte count (TLC), mean corpuscular volume (MCV), platelet, red cell distribution width (RDW), mean corpuscular hemoglobin (MCH), MCH concentration (MCHC), packed cell volume (PCV), RBC count, NLR, and PLR were determined at baseline. Participants received Vitamin D 2000 IU daily orally for 12 weeks. All the parameters were estimated again after 12 weeks. Results: We found no correlation of Vitamin D with Hb, TLC, MCV, platelet, RDW, MCH, MCHC, PCV, RBC count, NLR, and PLR at baseline. Extremely significant rise in Vitamin D; nonsignificant fall in Hb, TLC, RDW, MCH, NLR, and PLR; and nonsignificant rise in MCV, MCHC, RBC count, and PCV were reported. Conclusion: There was no association of Vitamin D with hematological and inflammatory parameters. Nonsignificant improvement in the parameters was seen with Vitamin D supplementation.
Keywords: Blood, endocrine, inflammation, metabolism
|How to cite this article:|
Jankar DS, Wingkar KC, Kanetkar SV, Bodhe CD. Study of vitamin D supplementation on selected hematological and inflammatory parameters in type 2 diabetes mellitus with vitamin D deficiency. J Datta Meghe Inst Med Sci Univ 2021;16:138-43
|How to cite this URL:|
Jankar DS, Wingkar KC, Kanetkar SV, Bodhe CD. Study of vitamin D supplementation on selected hematological and inflammatory parameters in type 2 diabetes mellitus with vitamin D deficiency. J Datta Meghe Inst Med Sci Univ [serial online] 2021 [cited 2021 Sep 23];16:138-43. Available from: http://www.journaldmims.com/text.asp?2021/16/1/138/322612
| Introduction|| |
Diabetes mellitus (DM) results from decreased insulin secretion and/or increased insulin resistance. Worldwide, 463 million adults were estimated to be suffering from DM in 2019 by the International Diabetes Federation. In India, 74 million adults were reported to have DM and it is estimated to rise to 101 million by 2030. Long-standing DM leads to several microvascular and macrovascular complications. These affect the cardiovascular system, renal function, brain, eyes, and extremities. Apart from suffering to the patient, this puts the family into emotional and economic stress.,
The drugs employed in the treatment of DM, mainly the hypoglycemic agents, aim to maintain the target blood sugar level. However, frequently, there are fluctuations in blood sugar level and with eventual failure of beta cells glycemic control deteriorates. This necessitates early identification and correction of the risk factors associated with DM. Nutritional deficiencies, sedentary lifestyle, stress, and obesity are such risk factors associated with DM.
Literature has reported association of Vitamin D deficiency (VDD) with DM. VDD is linked with decreased insulin secretion, increased insulin resistance, inflammation, and complications of DM., Although Vitamin D can be formed under skin in sunlight, lifestyle modifications result in decreased Vitamin D formation and VDD. Hence, it is advocated that Vitamin D supplementation may restrict the inflammatory damage to beta cells, improve insulin secretion and response to insulin, and thus improve glycemic control. Thus, the complications of DM can be prevented or delayed.
Few studies have reported association of VDD with anemia or decreased hemoglobin level (Hb)., However, none has studied the effects of Vitamin D supplementation on these parameters. Animal studies have shown some improvement in various blood parameters such as Hb, hematocrit, red blood cell (RBC) and white blood cell (WBC) count, and mononuclear cell count after Vitamin D supplementation. Altered levels of these blood parameters are considered to be due to the oxidative stress induced by hyperglycemia. However, other study showed no change in the blood parameters.
WBC count and individual leukocytes counts are common markers of inflammation. However, it is difficult to establish the diagnosis using WBC count and neutrophil or lymphocyte count. Again, they do not provide any predictive information. Other inflammatory markers such as interleukin–1 (IL) 1, IL-6, IL-8, tumor growth factor, and tumor necrosis factor are associated with diabetic complications. However, their assays are expensive, need standardization, and are not routinely available.
Neutrophil–lymphocyte ratio (NLR) and platelet–lymphocyte ratio (PLR) are cheap, easy, reproducible, and dynamic parameters of inflammation with a high predictive value. NLR is associated with inflammatory diseases such as DM, metabolic syndrome, hypertension, cardiovascular disease (CVD), end-stage renal disease (ESRD), and malignancies. Thus, NLR can be associated with DM and its complications., PLR is reported to be associated with CVD, diabetic complications, ESRD, and malignancies. Both being markers of inflammation have prognostic value.
Few studies are available stating association of NLR and/or PLR with DM, diabetic complication with inconsistent findings.,, However, there is no study on relation of Vitamin D with NLR and PLR in diabetics.
Hence, we proposed the present study to determine the relation of Vitamin D with various hematological and inflammatory parameters in Type 2 DM (T2DM) and study the effect of Vitamin D supplementation on these parameters.
Aim and Objectives
To study the effects of Vitamin D supplementation on hematological and inflammatory parameters in T2DM patients with VDD.
- To study the selected hematological and inflammatory parameters in T2DM patients with VDD
- To find the association of Vitamin D with the selected hematological and inflammatory parameters
- To study the selected hematological and inflammatory parameters after 12 weeks of Vitamin D supplementation.
| Materials and Methods|| |
This is a comparative and interventional study. Sixty-three T2DM patients on oral hypoglycemic agents aged 30–60 years with VDD (Vitamin D level <20 ng/ml) and willing to participate in the study were included. Patients with T1DM, cardiovascular, renal, hepatic endocrine, and neural complication and gestational DM and patients on insulin treatment were excluded. Participants received Vitamin D 2000 IU daily orally for 12 weeks. Packet of HI-D containing 30 tablets of 2000 IU of Vitamin D3 (calcitriol) was given to each patient. After 1 month, the patient was asked to submit empty packet and then the next packet for 30 days was given. In this way, compliance for the intervention was assessed. Total three packets for 12 weeks were given to each patient. All the parameters were estimated at baseline and 12 weeks after.
Estimation of Vitamin D was done by enzyme immunoassay
25-OH Vitamin D is a better indicator of status of Vitamin D in the body though biologically active form is 1, 25-OH Vitamin D. The ST AIA-PACK 25-OH Vitamin D is a one-step delayed competitive enzyme immunoassay done on TOSOH autoanalyzer.
Test cups contain lyophilized twelve magnetic beads coated with anti-25-OH Vitamin D sheep monoclonal antibody with sodium azide as a preservative. Vitamin D conjugate contains vials with liquid 25-OH Vitamin D conjugated to bovine alkaline phosphatase. Before actual determination of Vitamin D, first serum was pretreated with the pretreatment reagent containing sodium hydroxide which dissociates 25-OH Vitamin D from its binding proteins. During first incubation, 25-OH Vitamin D from the pretreated sample is bound to 25-OH Vitamin D-specific monoclonal antibody immobilized on magnetic beads. In this test enzyme labelled 25-OH vitamin D was added to the reaction mixture, which competes with the 25-OH Vitamin D for binding to antibody on magnetic beads in the reaction mixture. The magnetic beads are washed to remove unbound enzyme-labeled 25-OH Vitamin D during second incubation and then incubated with 4MUP, which is a fluorogenic substrate. 25-OH Vitamin D in the test sample is inversely related to the amount of enzyme-labeled 25-OH Vitamin D that binds to the beads. The machine constructs a standard curve and unknown Vitamin D concentration was calculated. The values obtained have units in ng/ml.
Ethylenediaminetetraacetic acid bulb was used for the hematological investigations. Blood Hb level and hematological parameters, which include total leukocyte count, differential count, platelets, red cell distribution width (RDW), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), MCH concentration (MCHC), RBC count, and packed cell volume (PCV), were estimated on NIHON KOHDEN Automated Hematology Analyzer. The analyzer measures various parameters by electrical resistance detection method. Volumetric impedance method for cell counting is used in this machine. NLR and PLR were calculated using standard formula.
Instat 3 software was used for statistical analysis. Baseline correlation of Vitamin D with the hematological parameters was done by Pearson's correlation. Before and after comparison of the parameters was done by paired t-test.
This study was approved by the Institutional Ethics Committee of Krishna Institute of Medical Sciences, Deemed University, Karad with ref no KIMSU/IEC/01/2015 dated : 5th March 2015.
| Results|| |
- Number of participants: 63
- Age group: 50.4 ± 6.20
- Sex: Male:female, 34:29.
[Table 1] shows extremely significant rise in Vitamin D; nonsignificant fall in Hb, total leukocyte count (TLC), RDW, MCH, NLR, and PLR; and nonsignificant rise in MCV, MCHC, RBC count, and PCV.
|Table 1: Comparison of hematological parameters before and after Vitamin D supplementation (paired t-test)|
Click here to view
[Table 2] shows no correlation of Vitamin D with Hb, TLC, MCV, platelet, RDW, MCH, MCHC, PCV, RBC count, NLR, and PLR at baseline.
| Discussion|| |
We found no correlation of Vitamin D with Hb, TLC, MCV, platelet, RDW, MCH, MCHC, PCV, RBC count, NLR, and PLR at baseline. There was extremely significant rise in Vitamin D; nonsignificant fall in Hb, TLC, RDW, MCH, NLR, and PLR; and nonsignificant rise in MCV, MCHC, RBC count, and PCV after 12 weeks of Vitamin D supplementation.
For the sake of discussion, we have divided the parameters into two groups: hematological parameters and inflammatory markers.
Vitamin D and hematological parameters (hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, packed cell volume, and red blood cell count)
The coexistence of anemia in T2DM patients is on an increase. The cause of anemia may be traditional like nutritional deficiencies or DM itself in these patients. Anemia in diabetics increases the risk for coronary heart disease and CVD. Thus, it becomes necessary to screen the hematological parameters and find modifiable risk factors that are common to both DM and the hematological abnormality.
The number of studies on hematological parameters in T2DM with VDD and Vitamin D supplementation is quite limited. Sharif et al. reported high prevalence of anemia in diabetic males. Significant association of VDD with Hb level was reported by Yu et al., Nardin et al., and Meguro et al. Bella et al. reported lower RBC, Hb, and Hct in diabetic mice than control. However, Vitamin D supplementation had significant effect on these parameters in their study. Some studies have reported decreased average life span of RBCs in diabetics.
We found only one study that too in rats in whom first diabetes was induced and then the effect of Vitamin D and calcium supplement was studied. Anyakudo et al. reported no significant difference at baseline in Hb, PCV, MCV, MCH, MCHC, TLC, and DLC in diabetic rats and controls. There was no significant difference in these parameters 6 weeks after Vitamin D and calcium supplement.
The possible mechanisms that result in decreased RBC count, Hb, and hematocrit in DM could be:
- DM leads to autonomic neuropathy that results in decreased erythropoietin secretion and thus contributes to decreased RBC production and anemia
- There may be simultaneous nutritional deficiencies
- Vitamin D may have favorable effect on erythropoiesis and deficiency of Vitamin D may decrease erythropoiesis
- DM-associated hyperglycemia leads to decreased deformability, increased adhesion, and increased osmotic fragility of RBCs. Vitamin D by having favorable effect on glucose metabolism may decrease the adverse effects of hyperglycemia on RBCs.
However, our subset of patients was not having any complications. It is quite likely that in patients with prolonged and uncontrolled DM, these effects may be more obvious. Hence, a detailed study with inclusion of patients with uncontrolled DM or DM with complications is suggested for further research.
Vitamin D and inflammatory markers (total leukocyte count, neutrophil–lymphocyte ratio, platelet–lymphocyte ratio, and red cell distribution width)
T2DM is associated with chronic low-grade inflammation. Pro-inflammatory cytokine release by macrophages inhibits insulin secretion. Hyperglycemic state also affects certain WBCs. Increased TLC counts are usually associated with diabetic complications. Increased TLC count is conventional marker of inflammation. However, it does not give any idea about the cause of DM. Very limited studies are available that determine the effect of Vitamin D supplementation on TLC count in DM.
Lower WBC counts in diabetic mice were reported by Bella et al. They also reported increase in WBC count on Vitamin D supplementation. They suggested increased WBC migration to blood from tissues as the underlying mechanism. Atile et al. reported significantly high NLR in VDD group. Magri et al. reported strong association of RDW with diabetic nephropathy.
NLR and PLR are cheap, easy, reproducible, and dynamic parameters of inflammation with high predictive value. NLR is associated with inflammatory diseases such as DM, metabolic syndrome, hypertension, CVD, ESRD, and malignancies. Thus, NLR can be associated with DM and its complications., PLR is reported to be associated with CVD, diabetic complications, ESRD, and malignancies. Both being markers of inflammation have prognostic value.
In literature, no study was available on relation of Vitamin D with NLR and PLR in diabetics. However, some other studies are available. Chittawar et al. and Liu et al. have reported few studies with increased NLR in diabetic nephropathy and diabetic retinopathy; and increased PLR in diabetic retinopathy. They also have mentioned few studies with no association of NLR and PLR with diabetic complications.
NLR and PLR are found to be markers of chronic inflammation – DM, hypertension, and diabetic complication. Our study has reported a nonsignificant decline in NLR and PLR with Vitamin D supplementation. This suggests that Vitamin D may have favorable effect on NLR and PLR by decreasing inflammation. The anti-inflammatory and anti-oxidant role of Vitamin D helps decrease inflammation.
RDW indicates anisocytosis, i.e., variability in RBC size.,, RDW is a routine blood investigation, but mainly used in anemia. However, high RDW is increasingly found to be associated with increased cardiovascular morbidity and mortality in general population.
DM can affect RBC size in following manner - hyperglycemia, leads to glycation of Hb, decreased deformability of RBCs and decreasing life span of RBCs, inflammation associated with DM decreases RBC production and maturation.
Mathen et al. reported strong association of RDW with diabetic nephropathy.
VDR are expressed on the surface of neutrophils, monocytes and natural killer cells. Vitamin D can influence the activity of all types of T cells and can regulate cytokines as well. Vitamin D modulates the antimicrobial response of these cell types. Simultaneously, it decreases the production of pro-inflammatory cytokines and proliferation of natural killer cells and lymphocytes, thus giving anti-inflammatory effect.
VDD can contribute to inflammatory response by increased production of pro-inflammatory cytokines. Vitamin D by immune modulator action prevents the effects of pro-inflammatory cytokines on erythroid precursor cells and stimulates their proliferation.
However, we could not find any study on association of Vitamin D with RDW in T2DM nor could we find any study on effect of Vitamin D supplement on RDW in T2DM.
The reasons why we could not get significant change in RDW, NLR, and PLR could be:
- We excluded patients with diabetic complications. As high NLR and PLR values are mostly associated with complications of diabetes, it is possible that these values were not abnormally elevated in our study group. Thus, there was limited scope for anti-inflammatory action of Vitamin D to be evident. Hence, there was slight decline in NLR and PLR
- Ethnic differences in response to Vitamin D
- Relatively small sample size
- Relatively small dose and/or duration of Vitamin D supplementation.
General measures like fortification of food with Vitamin D, screening of high risk population for Vitamin D deficiency should be done regularly and corrective measures should be taken.
The strengths of the study are inclusion of parameters that were not done by previous studies, satisfactory patient compliance, and no change in patient medication or lifestyle.
The limitations of the study include relatively small sample size and duration and we measured the total level of Vitamin D; instead, blood levels of bioavailable or active Vitamin D should have been measured.
| Conclusion|| |
There was no association of Vitamin D with hematological and inflammatory parameters. Nonsignificant improvement in the parameters was seen with Vitamin D supplementation.
Suggestions for further studies:
- Studies with Vitamin D supplement with high dose and for longer duration in T2DM patients with diabetic complications are needed
- Bioavailability of Vitamin D and concentration of free or active Vitamin D should be given due consideration.
We are grateful to all participants in this study.
Financial support and sponsorship
The study was funded by Krishna Institute of Medical Sciences Deemed to be University, Karad.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Mitchell DM, Leder BZ, Cagliero E, Mendoza N, Henao MP, Hayden DL, et al
. Insulin secretion and sensitivity in healthy adults with low Vitamin D are not affected by high-dose ergocalciferol administration: A randomized controlled trial. Am J Clin Nutr 2015;102:385-92.
Abdullah O, Omran J, Enezate T, Mahmud E, Shammas N, Mustapha J, et al
. Percutaneous angioplasty versus atherectomy for treatment of symptomatic infra-popliteal arterial disease. Cardiovasc Revasc Med 2018;19:423-8.
Fowler MJ. Diabetes treatment Part 2: Oral agents for glycemic management. Clin Diabetes 2007:25:131-4.
Shab-Bidar S, Neyestani TR, Djazayery A. Vitamin D receptor Cdx-2-dependent response of central obesity to Vitamin D intake in the subjects with type 2 diabetes: A randomised clinical trial. Br J Nutr 2015;114:1375-84.
Ryu OH, Lee S, Yu J, Choi MG, Yoo HJ, Mantero F. A prospective randomized controlled trial of the effects of Vitamin D supplementation on long-term glycemic control in type 2 diabetes mellitus of Korea. Endocr J 2014;61:167-76.
Yu JR, Lee SA, Lee JG, Seong GM, Ko SJ, Koh G, et al
. Serum Vitamin D status and its relationship to metabolic parameters in patients with type 2 diabetes mellitus. Chonnam Med J 2012;48:108-15.
Nardin M, Verdoia M, Schaffer A, Barbieri L, Marino P, De Luca G, et al
. Vitamin D status, diabetes mellitus and coronary artery disease in patients undergoing coronary angiography. Atherosclerosis 2016;250:114-21.
Palacios C, Gonzalez L. Is Vitamin D deficiency a major global public health problem? J Steroid Biochem Mol Biol 2014;144PA: 138-45.
Chittawar S, Dutta D, Qureshi Z, Surana V, Khandare S, Dubey TN. Neutrophil-lymphocyte ratio is a novel reliable predictor of nephropathy, retinopathy, and coronary artery disease in Indians with type-2 diabetes. Indian J Endocrinol Metab 2017;21:864-70.
Akbas EM, Gungor A, Ozcicek A, Akbas N, Askin S, Polat M. Vitamin D and inflammation: Evaluation with neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio. Arch Med Sci 2016;12:721-7.
Liu J, Liu X, Li Y, Quan J, Wei S, An S, et al
. The association of neutrophil to lymphocyte ratio, mean platelet volume, and platelet distribution width with diabetic retinopathy and nephropathy: A meta-analysis. Biosci Rep 2018;38:BSR20180172.
Manual of NIHON KOHDEN Automated Hematology Analyzer.
Meguro S, Tomita M, Katsuki T, Kato K, Oh H, Ainai A, et al
. Plasma 25-hydroxyvitamin D is independently associated with hemoglobin concentration in malesubjects with type 2 diabetes mellitus. Int J Endocrinol 2011;2011:362981.
Bella LM, Fieri I, Tessaro FH, Nolasco EL, Nunes FP, Ferreira SS, et al
. Vitamin D modulates hematological parameters and cell migration into peritoneal and pulmonary cavities in alloxan-diabetic mice. Biomed Res Int 2017;2017:7651815.
Engström G, Smith JG, Persson M, Nilsson PM, Melander O, Hedblad B. Red cell distribution width, haemoglobin A1c and incidence of diabetes mellitus. J Intern Med 2014;276:174-83.
Anyakudo MM, Adebukola A. Effects of calcium and Vitamin D-fortified diet on glycemic profile, biochemical parameters and selected haemostatic and haematological indices in diabetic rats. Curr Res Nutr Food Sci 2015;3. doi: http://dx.doi.org/10.12944/CRNFSJ.3.1.02
Guadarrama-López AL, Valdés-Ramos R, Martínez-Carrillo BE. Type 2 diabetes, PUFAs, and Vitamin D: Their relation to inflammation. J Immunol Res 2014;2014:860703.
Atile NS, Yavaş R, Bilir BE, Bilir B. Association of neutrophil to lymphocyte ratio with Vitamin D levels in type 2 diabetes mellitus. Endocrine Abstracts 2016;41:OC3.4
Magri CJ, Fava S. Red blood cell distribution width and diabetes-associated complications. Diabetes Metab Syndr 2014;8:13-7.
Raghavan PR. Metadichol® and red cell distribution width (RDW) in CKD patients. J Stem Cell Res Ther 2017;7:392.
Malandrino N, Wu WC, Taveira TH, Whitlatch HB, Smith RJ. Association between red blood cell distribution width and macrovascular and microvascular complications in diabetes. Diabetologia 2012;55:226-35.
Mathen PG, Thabah MM, Zachariah B, Das AK. Decreased bone mineral density at the femoral neck and lumbar spine in south Indian patients with type 2 diabetes. J Clin Diagn Res 2015;9:OC08-12.
Payne JF, Ray R, Watson DG, Delille C, Rimler E, Cleveland J, et al
. Vitamin D insufficiency in diabetic retinopathy. Endocr Pract 2012;18:185-93.
Ryu OH, Chung W, Lee S, Hong KS, Choi MG, Yoo HJ. The effect of high-dose Vitamin D supplementation on insulin resistance and arterial stiffness in patients with type 2 diabetes. Korean J Intern Med 2014;29:620-9.
[Table 1], [Table 2]