TITLE OF THE TOPIC

“ASSOCIATION OF VITAMIN D WITH BLOOD SUGAR AND

HBA1C IN TYPE 2 DIABETES MELLITUS”

SYNOPSIS

BRIEF RESUME OF THE INTENDED WORK:

6.1 Need for the study:

Diabetes Mellitus is one of the fastest growing global health

emergencies of the 21st century. In 2021, it was estimated that 537
million people have diabetes, and this number was projected to reach
643 million by 2030 and 783 million by 2045. It is also predicted that
6.7 million people in the age group of 20-79 years will die with
diabetes related causes in 2021. In 2019, Indian scenario showed that
77 million individuals had diabetes and is expected to reach 134
million by 2045.1 Therefore, effective preventive measures are
needed and modifiable risk factors should be identified and
explored.

Diabetes mellitus is a group of metabolic diseases characterized

by hyperglycemia resulting from defects in insulin secretion, insulin
action, or both. The chronic hyperglycemia of diabetes is associated
with long-term damage, dysfunction, and failure of various organs,
especially the eyes, kidneys, nerves, heart, and blood vessels.2

Diabetes mellitus is classified on the basis of the pathogenic

process leading to hyperglycemia. There are two broad categories of
Diabetes mellitus, designated as either Type 1 Diabetes Mellitus or
Type 2 Diabetes mellitus. Type 1 Diabetes Mellitus develops as a
result of autoimmunity against insulin-producing beta cells, resulting
in complete or near-total insulin deficiency in the age group of less
than 30 years. Type 2 Diabetes Mellitus is a heterogeneous group of
disorders characterized by variable degrees of insulin resistance,
impaired insulin secretion, and increased hepatic glucose production
is commonly seen in above 40 years of age.3

Diabetes Mellitus can affect multiple organ systems and is

responsible for the morbidity and mortality associated with the
disease. Diabetes related complications can be divided into vascular
and nonvascular complications. The vascular complications of
Diabetes Mellitus are further subdivided into microvascular
complications like retinopathy, nephropathy and neuropathy, and
macro vascular complications like coronary artery disease,
peripheral arterial disease, and cerebrovascular disease.
Nonvascular complications include infections, skin changes, and
hearing loss.3

Vitamin D is a fat-soluble steroid hormone. In response to

ultraviolet radiation of the skin, a photochemical cleavage results in
the formation of Vitamin D from 7-dehydrocholesterol. Vitamin D from
plant sources is in the form of vitamin D2 (ergocalciferol), whereas
that from animal sources is vitamin D3 (cholecalciferol). These two
forms have equivalent biologic activity and are activated equally well
by the vitamin D hydroxylases in humans.4

There are two successive hydroxylations that happen to vitamin

D precursors (vit D2 and D3), regardless of their source. Initially, in
the liver, vitamins D2 and D3 are converted to 25(OH)D2 and
25(OH)D3, respectively, by the action of 25-hydroxylase. These are
converted to the biologically active form of vitamin D (1,25(OH)2D) in
the kidneys by the action of 1-α-hydroxylases. Vitamin D, like other
steroid hormones, is transported through the bloodstream linked to
vitamin D-binding protein (DBP), a specific protein that belongs to the
albuminoid family and has binding sites for all vitamin D metabolites
and a high affinity for 25OHD and 1,25(OH)2D, thereby creating a
large pool of circulating 25OHD, which prevents rapid vitamin D
deficiency.5 Recent studies have shown a relationship between
Vitamin D and Type 2 Diabetes Mellitus.

Vitamin D plays an important role in the regulation of pancreatic

β cells by calcitriol interacting with calcium flux regulating receptors
on the β cells. On the other hand, Vitamin D could influence the
regulation of insulin secretion by opening and closing of calcium
channels and improve insulin sensitivity by stimulating the
expression of insulin receptors and activating peroxisome
proliferator activated receptor delta (PPAR- δ).6

Vitamin D insufficiency has been linked to lower insulin release

and insulin resistance in Type 2 diabetes. Low vitamin D is associated
with Type 2 Diabetes, which attributed this to two main causes; First,
vitamin D stimulates insulin secretion by pancreatic cells; thus,
vitamin D deficiency is associated with insulin resistance. Second,
vitamin D deficiency causes inflammation and increases inflammatory
markers.7

Vitamin D deficiency might play a role in the development of

diabetes and its complications by decreasing pancreatic insulin
secretion, peripheral insulin resistance and down-regulation of
insulin receptor gene.8 Vitamin D deficiency may also reduce ability
of β cells to convert pro insulin to insulin.6

Glycated haemoglobin (HbA1c ) reflects average blood glucose

levels over the previous 3 months and serves as a crucial marker for
long-term glycaemic control in Type 2 Diabetes Mellitus.9 The meta
analysis study observed that Vitamin D supplementation interrupted
the increase in plasma HbA1c levels and delayed the development of
diabetic complications.10

6.2 Review of Literature

Salih YA et al., conducted a study on the level of Vitamin D and

relationship between Vitamin D and glycemic control in Type 2
Diabetes mellitus. A case control study was done on 310 participants;
155 were type 2 diabetes mellitus patients and rest 155 were normal
controls. Subjects with any other complications were excluded from
the study. The serum vitamin D had shown significantly lower level in
patients with poor glycemic control compared to good glycemic
control. A significant difference in the level of vitamin D was noticed
in Type 2 diabetic patients between more than 5 years and less than 5
years duration of diabetes mellitus. It also observed an inverse
association between Vitamin D and HbA1c level in Type 2 Diabetic
patients. Fasting blood sugar levels were lower in diabetic patients
with sufficient Vitamin D when compared to Vitamin D insufficient and
deficient.11

Mishra A et al., conducted a study on the effect of

Hypovitaminosis D on glycemic control in Type 2 Diabetes Mellitus
patients. A case control study was done on 250 Type 2 diabetic
patients, among these 125 were Type 2 diabetic patients with
Hypovitaminosis D and remaining 125 Type 2 diabetic patients with
sufficient vitamin D as control. Patients without any complications
were alone included in the study. The study observed that Fasting
blood sugar was significantly elevated in Hypovitaminosis D subjects
compared to sufficient Vitamin D diabetic subjects. It also identified
that significantly elevated levels of HbA1c in Hypovitaminosis D
diabetic subjects compared to diabetic subjects with sufficient
Vitamin D. The study suggested that hypoglycaemic medication alone
is not effective, in addition to that sufficient Vitamin D also essential to
maintain normal glycemic control in Type 2 diabetic patients and
avoid related comorbidities.12

Bhattacharya S et al., conducted a study to know the relationship

between Vitamin D status and HbA1c levels. A case control study was
done on 102 known type 2 diabetes mellitus patients and 330 controls
in the age group of 20-80 years. The T2DM patients were divided into
two groups–those with controlled sugars (HbA1c ≤7) and those with
uncontrolled sugars (HbA1c >7) and vitamin D levels were studied in
them. Subjects with other complications and other diseases were
excluded from the study. The study had identified that Vitamin D
levels are lower in uncontrolled diabetic subjects who have more
than 7% of HbA1c when compared to well controlled diabetic
subjects with less than 7% of HbA1c and healthy subjects. The study
suggested that Vitamin D is mandatory to improve glycemic control
and reduced comorbidities in Type 2 diabetic subjects.13

Kusumadevi MS et al., conducted a study to examine the

association between Vitamin D and HbA1c levels in patients with
Type 2 diabetes mellitus. A cross sectional study was conducted on 50
type 2 diabetic patients. Patients with other complications were
excluded from the study. The study revealed that there was no
statistically significant correlation between HbA1c and Vitamin D
levels in Type 2 diabetic patients.14

Varma M et al., conducted a study to understand the

relationship between Vitamin D deficiency and Type 2 diabetes
mellitus. The study population comprised of 500 participants which
were divided into two groups: Vitamin D deficient (Vitamin D level <
20 ng/mL) and Non-Vitamin D deficient (Vitamin D level > 20 ng/mL).
The study participants without any complications were included in the
study. The study revealed that there was no statistically significant
association between Vitamin D status and HbA1c levels.15

Panah AD et al., conducted a study to examine HbA1c and

Vitamin D levels in Diabetic patients. A cross sectional study was
conducted on 102 diabetic patients. Diabetic patients with
complications and other diseases are excluded from the study. The
study revealed that there was no significant relationship between
Vitamin D and HbA1c and also no significant relationship between
Vitamin D and Fasting blood sugar.16

Since previous studies had reported inconsistent results on

relationship of Vitamin D with Fasting blood sugar and HbA1c. Hence,
the study was designed to assess the association between Vitamin D,
Fasting blood sugar and HbA1c.

6.3 Objectives of the Study:

 To estimate the level of Vitamin D, Fasting blood sugar and

HbA1c in Type 2 Diabetes Mellitus patients.

 To evaluate the association of Vitamin D with Fasting blood

sugar and HbA1c in Type 2 Diabetes mellitus patients.

7. MATERIALS AND METHODS

7.1 Source of data :

 The study will be carried out on 40 clinically diagnosed cases of
Type 2 Diabetes Mellitus patients and 40 age and gender matched
controls.

Group – I – Healthy controls

Group – II – Type 2 Diabetic Patients

 Study design: Cross Sectional Case Control study.

 Study period: This study will be conducted over a period of one

year from April 2024 to March 2025.

 Place of study: Central Diagnostic laboratory

Inclusion criteria:

• All Adult males and females in the age group of 30 to 70 years

diagnosed with Type 2 Diabetes Mellitus

• All Healthy individuals in the age group of 30 to 70 years

• Patients with HbA1c >6.5%

Exclusion criteria:

• Patients with Type 1 Diabetes Mellitus

• Pregnant women with gestational diabetes mellitus

• Patients on treatment with statin, insulin ,Vitamin D, OCP’s ,

Steroids

• Individuals with habit of alcoholism and smoking

• Known case of hypo/hyperthyroidism and

hypo/hyperparathyroidism

• Patients with liver and kidney diseases

• Patients with malignancy

• Patients with hypertension

• Patients with arthritis, cardiac disease

7.2 Method of Collection of Data (Including Sampling Procedures if
any)

Patients of both genders in the age group of 30 to 70 years

who present as clinically diagnosed cases of Type 2 Diabetes Mellitus
and their age and gender matched controls will be considered.
Detailed history will be taken (Age, gender, height, weight, education
, H/O of present illness , family history, duration of disease, frequency
of outdoor activity, duration of sun exposure, occupation and
complication if any). For the study, 5ml of fasting venous blood will be
collected under sterile conditions from the anticubital vein.

• 2 ml of blood will be collected in EDTA tube for HbA1c

estimation

• The remaining volume of blood will be collected in plain serum

tube allowed to clot and serum will be separated after centrifugation
at 3000 rpm for 10 minutes and aliquoted and stored at -20°C for
vitamin D estimation and Fasting blood sugar estimation.

a) Estimation of glycated haemoglobin (HbA1c ) by High Performance

Liquid Chromatography:

Principle: HbA1c, the glycated form of haemoglobin A, arises from

non-enzymatic glucose attachment to the β-chain of the haemoglobin
molecule. The principle of HPLC for HbA1c measurement relies on
charge differences between this molecule and other haemoglobin
fractions like HbA0 (non-glycated). A cation-exchange resin acts as
the stationary phase in the column. This resin holds negatively
charged groups that attract positively charged molecules like
haemoglobin fractions. The mobile phase, typically a phosphate
buffer, flows through the column with varying pH and ionic strength,
influencing the interaction between haemoglobin and the resin. The
extracted haemoglobin sample containing HbA1c and other fractions
is injected onto the column. Haemoglobin fractions interact with the
resin based on their net charge. HbA1c , having a slightly more
negative charge due to the attached glucose, binds less tightly to the
resin, allowing for earlier elution. The mobile phase carries fractions
at different rates depending on their affinity for the resin. HbA1c
elutes slightly earlier than HbA0, achieving separation. Fractions pass
through a detector, commonly using UV absorbance at 415 nm. Each
fraction absorbs light differently, enabling quantification. The
chromatogram displays peaks for each eluted fraction based on their
absorbance signal. The area under each peak represents the relative
amount of that fraction. The percentage of HbA1c is calculated by
dividing the HbA1c peak area by the total area of all haemoglobin
peaks and multiplying by 100.17,18,19

b) Estimation of Vitamin D by Beckman Coulter's Access 25(OH)

Vitamin D Total Assay:

Principle: The Access 25(OH) Vitamin D Total Assay is a paramagnetic

particle chemiluminescent immunoassay (CMIA) designed for the
quantitative determination of total 25-hydroxyvitamin D (25(OH)D) in
human serum and plasma. It utilizes a competitive binding approach
to achieve accurate measurement. A dedicated releasing agent
liberates 25(OH)D from its transport protein, vitamin D binding
protein (DBP), in the sample. The freed 25(OH)D then competes with
a chemiluminescently labeled 25(OH)D analogue for binding sites on
specific anti-25(OH)D antibodies attached to paramagnetic particles.
Unbound labeled analogue is removed through washing steps,
leaving only the bound molecules associated with the particles. A
25(OH)D analogue-alkaline phosphatase conjugate is introduced,
further competing for binding sites with the labeled analogue and the
sample's 25(OH)D. After another washing step, a chemiluminescent
substrate is added, reacting with the bound phosphatase conjugate to
produce light. The intensity of the emitted light is inversely
proportional to the amount of bound 25(OH)D (more sample means
less bound analogue, leading to less light). A luminometer measures
the light intensity, which is then used to calculate the 25(OH)D
concentration in the sample based on a pre-established calibration
curve.20

c) Estimation of Fasting blood Sugar by Glucose oxidase peroxidase

(GOD-POD) method:
Principle: Glucose oxidase enzyme (GOD) oxidizes the specific
substrate beta-D-glucose to gluconic acid and hydrogen peroxide is
liberated. Peroxidase enzyme acts on hydrogen peroxide to liberate
nascent oxygen (O). Nascent oxygen then couples with 4-amino-
antipyrine and phenol to form red quinoneimine dye. The intensity of
colour is directly proportional to concentration of glucose in plasma.
The intensity of colour is measured colorimetrically at 530 nm and
compared with that of a standard treated similarly.21

Statistical analysis:

The data collected will be entered into the excel sheet and will
be analysed using SPSS version 25.0. Qualitative variables as mean
+/- SD. Chi square test or Fisher test will be applied for qualitative
variables. Comparison of the parameters among groups will be done
by analysis of variance (ANOVA). Pearson’s correlation will be used
to correlate between parameters done in this study.

7.3 Does the study require any investigations or interventions to be

conducted on patients or other humans or animals?

Yes

The present study requires Vitamin D, HbA1c , Fasting blood

sugar values of the patient.

7.4 Has ethical clearance been obtained from your institution in case
of 7.3a?

Will be obtained

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