Vitamin D3, methods of measurement and it's correlation with diabetes mellitus

BY Assistant lecturer: Shaymaa Mahdi
Shaymaa.m@uokerbala.edu.iq

Type 2 diabetes mellitus (T2DM)
Diabetes mellitus is a serious metabolic disorder that has become
increasingly prevalent. The number of people with diabetes is
expected to increase from 285 million to 435 million people
worldwide by 2030.
T2DM manifests as a result of insulin resistance, increased
hepatic glucose production and b-cell failure.

Vitamin D3
Vitamin D, although traditionally classified as a vitamin, actually
performs functions that are analogous to those of a hormone. It
plays an integral role in maintaining the health of your bone,
teeth and joints and assisting the immune system to function
properly.

Vitamin D synthesis pathway
Exposure to UVB sunlight activates 7-dehydrocholesterol (7-
DHC), a pre-cursor structure made from cholesterol and found
predominantly within the epidermis, to form pre-vitamin D3. Pre-
vitamin D3 then isomerizes to vitamin D3 which is then carried to
the liver where it is hydroxylated by 25-hydroxylase and then the
kidney by 25(OH)D3-1α-hydroxylase to form the biologically active
1,25-dihydroxyvitamin-D3 (1,25(OH)2D3). 1,25(OH)2D3 is now
free to exert its effects by binding to vitamin D receptor (VDR) in
the target tissues.

1,25 OH Vitamin D Effects
* Increase calcium absorption by the intestinal mucosa.
* Decreased renal calcium and phosphorus excretion.
•
* increasing bone calcification.
*it plays a role in immune function and more recently is thought to
play a role in T2DM.

Non-classical Actions of Vitamin D
Suppress cell growth/proliferation and Regulate apoptosis.
Modulate immune responses
Susceptibility to infections
Susceptibility to autoimmune disorders for example T1DM
Suppress renin-angiotensin system
Stimulate insulin secretion so it has a role in T2DM

Vitamin D deficiency
The signs of Vitamin D deficiency can range from bone pain and
muscle weakness to depression and weakened immune system,
while long-term deficiency can result in obesity, Hypertention,
osteoporosis, Alzheimer’s disease, type 2 diabetes and many other
diseases.

Causes of Vitamin D Deficiency
Vitamin D deficiency can occur for a number of reasons:
Don't consuming adequate amount of the vitamin D over time: eg: Strict vegan
diet.
Limited exposure to sunlight : if you are homebounded, wear long robes or head
coverings for religious reasons, or have a job that prevents sun exposure, you will
be at risk for vitamin D deficiency.

Renal failure
Cause inability to convert vitamin D to its active form.
Malabsorption
Cause intestinal tract cannot adequately absorb vitamin D.
Obesity: Vitamin D is extracted from the blood by fat cells, altering its release
into the blood circulation. People with a BMI ≥ 30 often have low blood levels of
vitamin D.
Dark skin. The pigment of melanin reduces the skin's ability to make vitamin D
in response to sunlight exposure.
Reduction of skin synthesis
-Sunscreen use (SPF 15 reduces vitamin D3 synthesis by 99%)
-
Aging (reduction of vitamin D3 synthesis by 75% in elderly since 7-
dehydrocholesterol is reduced in a people of 70 year old).
-

Vitamin D deficiency…
In brief:
If you don’t expose to the sunlight adequately, follow a vegetarian diet or suffer
from milk allergies, you may be at risk for vitamin D deficiency.

Tests for Vitamin D Deficiency
The most effective way of measuring vitamin D status is to
measure serum concentration of 25(OH)D3, not 1,25(OH)2D3; this
is due to the rapid clearance rate of the Latter.
A level of 20-50 ng/mL is considered adequate for healthy
people, with anything below 20 ng/ml considered deficient.
However, it is now known that raising the amount of vitamin D in
your body to around 60-80 ng/ml can help keep blood glucose levels
under control, which is vital for people with diabetes.

Who Should Be Screened for Vitamin D
Deficiency
Elderly and Obese people.
Home bounded or institutionalized patients
Patients with malabsorption problems.
Patients with osteoporosis or osteopenia, Chronic kidney or chronic
liver disease
Patients with nonspecific musculoskeletal pain
Patients who take medications that induce cytochrome P-450
enzymatic activity.

Vitamin D Deficiency….

Solutions
Exposing to the sunlight for 15-20 minutes each day
Alternatively, you can get the daily requirement of vitamin D
through dietary supplements and foods for example: nuts, oily fish,
eggs, powdered milk and some fortified cereals.

Treatment for Vitamin D Deficiency
-concentration of less than 20 ng/ml is generally considered
inadequate, requiring treatment.
-Treatment involves getting more vitamin D through diet and
supplements.
-
Guidelines from the Institute of Medicine increased the RDA of
vitamin D to 600 IU for everyone ages 1-70, and raised it to 800 IU
for adults older than age 70 to optimize bone health. The safe upper
limit was also raised to 4,000 IU.
-

Vitamin D and Diabetes
Vitamin D deficiency and diabetes have one major trait in
common: both are pandemic. The International Diabetes Federation
estimates the number of people with diabetes worldwide to be nearly
285 million, or 7% of the world's population. This number is
expected to exceed 435 million by 2030. In the United States, an
estimated 79 million people have pre-diabetes.

Mechanisms of action
There is an Accumulating evidence indicates that insufficient
concentrations of circulating 1,25(OH)2D3 are associated with
glucose intolerance, insulin resistance, metabolic syndrome and
increased risk for development of both type 1 and type 2 diabetes.
First, the β-cell of the pancreas that secretes insulin has been shown
to contain VDRs as well as the 1 alpha hydroxylase enzyme
Second evidence indicates that vitamin D treatment improves
glucose tolerance and insulin resistance ( stimulate insulin receptor
expression).

Mechanisms of action
In addition, many animal studies showed that 1,25 –dihydroxyvitamin D3
(1,25(OH)2D3) stimulates the pancreatic β-cell to secrete insulin by Vitamin D3
has an indirect effect on insulin secretion through the Calcium where vitamin D
contributes to normalization of extracellular calcium, ensuring normal calcium
flux through cell membranes since Ca stimulate insulin release to the blood;
therefore, low vitamin D may diminish calcium's ability to increase insulin
secretion.
The relationship between vitamin D deficiency and insulin resistance could
develop through inflammation, as vitamin D deficiency is associated with
increased inflammatory markers. In addition, genetic polymorphisms of vitamin D
–related genes may predispose to impaired glycemic control and type 2 diabetes.

Aid weight loss
good vitamin D status → reduce (PTH) levels which in the long-term → weight loss and
reduce risk of obesity, which is a major risk factor for type 2 diabetes.
Regulate appetite
vitamin D ↑hormone leptin, which controls body fat storage and triggers the sensation of
satiety and lowering hunger levels.
Reduce belly fat
↑ vitamin D → ↓cortisol, higher and more prolonged levels of this hormone in the blood
can lead to ↑ abdominal (or visceral) fat, which is linked to various health problems
including T2DM.

Take Home Points
Vitamin D deficiency is common
-At least 800 IU of vitamin D3 /day is needed to maintain vitamin D sufficiency
-Sensible sun exposure.

Summary and Conclusion
Although the role of vitamin D in helping to regulate blood glucose remains
poorly understood, vitamin D status appears to play a role in the development and
treatment of diabetes.
According to Danescu et al. “both animal and human studies support the notion
that adequate vitamin D supplementation may decrease the incidence of type 1 and
possibly type 2 diabetes mellitus and may improve the metabolic control in the
diabetes state. However, the exact mechanisms are not clear and need further
investigation.”

Methods of Vitamin D measurement

Clinical significance of Vitamin D
measurement
Historically, vitamin D has been known for its role in the mineralization of teeth
and bones through regulation of calcium and phosphorus homeostasis. More
recently, there is emerging evidence of the role of vitamin D in protection against
risk for malignant neoplasms, cardiovascular disease, and diabetes, along with
osteoporosis and other bone disorders.
Redefining what is considered to be a sufficient plasma level of 25-hydroxy
vitamin D would potentially reclassify more people as vitamin D insufficient and
trigger the need for treatment and monitoring of vitamin D levels.

Clinical significance of Vitamin D
measurement
The relationship of blood vitamin D levels to these disorders is not entirely clear,
so standardization of laboratory methods for vitamin D analysis and redefining
the reference range that indicate health and disease are of most importance.
In 2010, new guidelines from The Institute of Medicine (IOM) defined sufficient
levels of total vitamin D as 20 to 50 ng/mL and toxicity as greater than 50
ng/mL. Evidence shows that total 25-hydroxy vitamin D is the most clinically
significant form. The methods used in routine laboratory testing should be able
to measure total 25-hydroxy vitamin D and to convey that information clearly to
clinicians.

Analytical Methods for Quantification of
Vitamin D
Historically, vitamin D was measured by competitive binding methods, high-
performance liquid chromatography (HPLC), and radioimmunoassay (RIA).This
method has been used to establish reference ranges during the past decade.
The DiaSorin 25-hydroxyl vitamin D assay is a 2-step procedure that involves a
rapid extraction of 25-hydroxy vitamin D and other hydroxylated metabolites
from serum or plasma, followed by a competitive RIA procedure using an
antibody with specificity for 25-hydroxy vitamin D
‫ز‬

Analytical Methods for Quantification
of Vitamin D
Newer chromatographic methods have been developed to improve sensitivity,
to simplify steps, and to measure all forms of vitamin D.
One example is a liquid chromatography–mass spectrometry (LC-MS/MS) method
that was developed to analyze all forms and metabolites of vitamin D
simultaneously, including D2, D3, and 25-hydroxy vitamin D in serum. The process
uses an ionization detector technique known as atmosphericpressure photo
ionization (APPI) to provide additional sensitivity for analysis. The method is less
difficult compared with other LC methods because it doesn’t require pre-
concentration steps.

Analytical Methods for Quantification
of Vitamin D
High-performance liquid chromatography methods quantitate 25-hydroxy vitamin
D2 and D3. HPLC methods are available in kit form (Hitachi High-Technologies
Corporation Tokyo, Japan, and Thermo Fisher Scientific, Sunnyvale, CA) in an
effort to standardize test quality and to make the assays more cost effective and
less labor intensive. The Hitachi method uses a reverse phase column and diode
array detection, which allow for highly sensitive simultaneous analysis at optimal
wavelengths.

Immunoassay Methods
Several United States Food and Drug Administration (FDA)–approved immunoassay
methods are available, including:
1- quantitative chemiluminescent immunoassay (CLIA) methods.
The method measures total 25-hydroxy vitamin D and other hydroxylase vitamin
D metabolites in human serum.
During the first step, 25-hydroxy vitamin D is dissociated from its binding protein
and binds to the specific solid phase antibody, followed by the addition of
vitamin D-isoluminol tracer; unbound material is removed with a wash cycle. In
the next step, the reagents are added to initiate the chemiluminescent reaction.
The light signal is detected by a photomultiplier as relative light units; this
measurement is inversely proportional to the concentration of 25-hydroxy
vitamin D.

Immunoassay Methods
2-Enzyme Linked immunosorbent Assay (ELISA):
Serum 25-hydroxy vitamin D3 (25(OH)D3) is determined quantitatively in human
serum or plasma sample by using ELISA technique.
This assay is based on the sandwich ELISA principle where each well of the
supplied microliter plate has been pre-coated with a target specific capture
antibody. Standards or samples are added to the wells and the target antigen
binds to the capture antibody. A Horseradish Peroxidase (HRP)-conjugated
detection antibody is then added which binds to the captured antigen. Unbound
antigen and detection antibody is washed away. A tetra methyl benzidine (TMB)
substrate is then added which reacts with the HRP enzyme resulting in color
development. A sulfuric acid stop solution is added to terminate color
development reaction and then the optical density (OD) of the well is measured
at a wavelength of 450 nm.

Immunoassay Methods
The sandwich ELISA principle for vitamin D measurement."

Vitamin D3, methods of measurement and it's correlation with diabetes mellitus

Vitamin D3, methods of measurement and it's correlation with diabetes mellitus

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