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Vitamin D and its clinical applications
Dr. Rohini C Sane
Vitamin D (CHOLECALCIFEROL )-HISTORY1919- M C Collumn ( experimental Rachitis )
Healthy Human
deprived sunlight
Rachitis ------ Twist
ANTIRACHITIS FACTOR (CORD LIVER OIL)
Rachitis cured
Rachitis
Vitamin D (CHOLECALCIFEROL )
1931- Augus and co-workers –isolation of Vitamin D and named as Calciferol.
Oto Diels and Kurt Alder – elucidation of structural aspects of Vitamin D
( Noble price 1950 )
Dietary Sources of vitamin D
Milk
Cheese
Curd
Egg Yolk
Fish
Cord Liver Oil
Fortified dairy products
ESSENTIAL STRUCTURAL CHARACTERISTICS OF PROVITAMIN D
1. OH Group at C3 of Cyclopentanoperhydrophenanthrene Ring
2. Two Conjugated DOUBLE BONDS between C5---C6 and C7---C8
3. Hydrocarbon ring at C17
ACTIVATION OF VITAMIN D BY UV LIGHT
( plants ) Ergosterol Provitamins 7 Dehydrocholesterol (Animals)
UV LIGHT PHOTOLYSIS
Ergocalciferol (D2) Cholecalciferol (D3)
No absorption in human body Absorbed by intestine epithelium
No Nutritional Value ANTIRACHITIS ACTIVITY
Cholecalciferol (D3)
CH3+ONE DOUBLE BOND+
ERGOCALCIFEROL
Exposure to sunlight – induces synthesis of vitamin D ( before 9am and after 5pm )
ACTIVATION OF VITAMIN D (plant source ) BY UV LIGHT
Ergosterol -Provitamin (plant source )
Intra Molecular Rearrangements
Ergocalciferol (Vitamin D2)-Active Vitamin D
ACTIVATION OF VITAMIN D (Animal source ) BY UV LIGHT
cholesterol (animal source)
7 dehydrocholecalciferol ( ProvitaminD3 )
Intra Molecular Rearrangements
Secosterol (cis configuration )
Cholecalciferol (Vitamin D3)(trans configuration )
UV light
ACTIVATION OF VITAMIN D BY UV LIGHT
ACTIVATION OF VITAMIN D BY UV LIGHT
cholesterol (animal source)
7 dehydro cholecalciferol ( ProvitaminD3 )
UV light
Intra Molecular Rearrangements
Secosterol (cis configuration )
Cholecalciferol (Vitamin D3)(trans configuration )
Synthesis of VITAMIN D
Dietary requirement of vitamin D
Category Dietary requirement of vitamin D
Children 10 micrograms ( 400 IU )/DAY
Adults 5- 10 micrograms (200- 400 IU )/DAY
Pregnancy/lactation / recovery of bone
fractures
10 micrograms ( 400 IU )/DAY
Above age of 58yrs /menopause 15 micrograms ( 600 IU )/DAY
Sometimes upto 100O IU)
Obesity /overweight associated with
hypothyrodism
800 IU ( Supplementation of D3 than D2 as
D3 has longer half life )
VITAMIN D IS A STEROID HORMONE
Similarities between VITAMIN D and a STEROID HORMONE:
(1) CYCLO PENTANO PER HYDRO PHENANTHRENE
(2) Synthesis under skin by UV light (7
DEHYDROCHOLESTEROLCHOLECALCIFEROL )
Target organs : bone ,kidney ,intestine ( away from site of synthesis )
(4) Cytosolic Receptors
(5) Self regulated synthesis ( feed back inhibition )
(6) Works in association with other hormones like PTH,CALCITONIN,CALCITRIOL
(7) USE OF ACTINOMYCIN
DNA ( cal binding protein ) RNA affected ( transcription /translation inhibited )
SYNTHESIS and ACTIVITY OF CALCITRIOL DECREASED
Mechanism of action of VITAMIN D and a Steroid Hormone
Comparison between Calcitriol and Calcitonin
Calcitriol Calcitonin
Class of hormone Steroid hormone
( active form of Vitamin D )
Peptide hormone released
by a thyroid gland
Function in human
body
Increases serum Calcium
levels
Decreases serum Calcium
levels
Absorption & Transport of Vitamin D
SITE OF SYNTHESIS OF CALCITRIOL
VITAMIN D ( 7 DEHYDROCHOLESTEROL)/prohormone /provitamin
BILE SALTS
----------------------------------------------------------------------------------------------- ---------------------------7 DEHYDROCHOLESTROL DUODENUM & JEJUNUM
CHYLOMICRONS ,Alpha 2-Globulins ,LIPOPROTEINS(Vitamin D binding protein) BLOOD ,LYMPH
7 DEHYDROCHOLESTEROL
UV LIGHT
CHOLE CALCIFEROL SKIN
-----------------------------------------------------------------------------------------------------------------------------------
CHOLE CALCIFEROL LIVER
25 HYDROXYLASE* , O2 ,NADPH ,BILE SALTS
25 HYDROXY CHOLECALCIFEROL (25 OH CHOLECALCIFEROL -25 DHCC – Major storage form )
------------------------------------------------------------------------------------------------------------------------------------------
25 HYDROXY CHOLECALCIFEROL KIDNEY
1 HYDROXYLASE ◊, NADPH ,Mg 2+ O2 ( Molecular )
1,25 DIHYDROXY CHOLECALCIFEROL ( Vitamin D3 - MOST POTENT FORM with three hydroxyl groups 1,3,25)
Characteristics of 25 HYDROXYLASE*
Characteristics of 25 HYDROXYLASE* are
1. A microsomal enzyme monooxygenase
2. Product of a gene CYP27 A1
3. Coenzymes required by 25 HYDROXYLASE* : NADPH and Cytochrome
P450
4.
Characteristics of 1 HYDROXYLASE◊
Characteristics of 1 HYDROXYLASE ◊ are
1. Located in mitochondria of proximal convoluted tubules of kidney
2. mono-oxygenase enzyme
3. Product of a gene CYP27 B1
4. Coenzymes required by 1 HYDROXYLASE ◊ : NADPH ,Ferrodoxin and
Cytochrome P450
5.
Comparison of 25 HYDROXYLASE* and 1 HYDROXYLASE◊
Characteristic of
enzyme
25 HYDROXYLASE* 1 HYDROXYLASE◊
SITE OF ACTIVITY LIVER proximal convoluted
tubules of KIDNEY
SUBCELLULAR
COMPARTMENT FOR
ACTIVITY
A microsomal enzyme
monooxygenase
mitochondrial mono-
oxygenase enzyme
Product of gene CYP27 A1 CYP27 B1
Coenzymes
required
NADPH and Cytochrome P450 NADPH ,Ferrodoxin and
Cytochrome P450
Product of activity 25 dihydro cholecalciferol
(25 DHCC –Storage form )
1,25 dihydro
cholecalciferol (calcitriol-
most active form )
Metabolism and effects of vitamin D
24,25 dihydroxy cholecalciferol
1. 24,25 dihydroxy cholecalciferol is formed by Hydroxylation at 24th position .
2. It is an inactive form.
3. Adequate Vitamin D and high Calcium level favor 24-hydroxylation
4. 24,25 dihydroxy cholecalciferol is isomerized to 1, 25 dihydroxy cholecalcitriol
on the need of body .
Target organs of Vitamin D –Bone, Intestine , Parathyroid glands
Molecular Mechanism of vitamin D3
BONE,PLACENTA ,KIDNEY ,LIVER, INTESTINE
1,25 DI HYDROXY CHOLE CALCIFEROL OR CALCITRIOL
Transcription of DNA followed by Translation of mRNA ( Calcium binding protein)
Synthesis of Calcium binding protein
Activated hormone receptor complex
Increase concentration calcium binding protein
Absorption of Calcium by intestinal epithelial cells (from DIET )
Absorption of Calcium by KIDNEY (from RENAL FILTRATE)
Absorption of Calcium by BONES( leading to MINERALIZATION of bones )
Synthesis of Calcium binding protein by Vitamin D
Calbindin( Calcium binding protein)
BONE,PLACENTA ,KIDNEY ,LIVER, INTESTINE
Specific nuclear receptor
Dimer
with
RXR
Functions of Vitamin D in Intestine
I -Action of Vitamin D in Target organ – Intestinal Villous cells
------------------------------------------------------------------------------
(a) Increase synthesis of calcium binding protein (Calbindin) increase absorption
of calcium by epithelial cells of intestine( passive absorption )
Intestinal cell blood ( active transport –Sodium-Calcium exchange mechanism or
by pumping Calcium –Calbindin complex )
(b) increase activity of Alkaline Phosphatase
(c) increase activity of Phytase increase hydrolysis of phytic acid
(d) lower p H to facilitate absorption of Calcium and Phosphorous
Functions of Vitamin D
II Action of vitamin D in target organ – Bone osteoblasts
(1) Increased Calcitriol Synthesis Calcification Of Bones Development Of
Bones by increasing activity of osteoblast Growth
(2) Increase Citrate levels increase absorption of Calcium
(3) Increase production of bone matrix proteins (Collagen ,Osteocalcein and
Osteopontin )increase bone density
(4 )Calcitriol acts on all three types of cells –osteoblast,osteoclast and osteocytes
Action of vitamin D in target organ – Bone osteoblasts
(bone mineralization ) Biochemical changes induced by Vitamin D during bone mineralization
25 hydroxy D3 Calcitriol by osteoblast
Mineralization and differentiation of osteoblast
Secretion of Nuclear factor Kappa B ligand ( RANKEL-a cytokine )
Osteoblastogenesis by from multinucleated precursors is induced
Osteoblastic bone resorption and increase in alkaline phosphatase activity to provide adequate Calcium and phosphorous
Mineralization of bone promoted
Hypercalcemia induces secretion of fibroblastic growth factor 23 (FGF23)from osteocytes
Regulation of 25 hydroxylase activity ( FGF 23 upregulates this enzyme )
Complete negative feedback loop on PTH secretion
PTH increased production of bone matrix proteins Collagen and osteocalceinbone growth
Biochemical changes induced by Vitamin D during bone mineralization
25 hydroxy D3 Calcitriol by osteoblast
Mineralization and differentiation of osteoblast
Secretion of Nuclear factor Kappa B ligand ( RANKEL-a cytokine )
Osteoblastogenesis by from multinucleated precursors is induced
Osteoblastic bone resorption and increase in alkaline phosphatase activity to provide adequate Calcium and phosphorous
Mineralization of bone promoted
Hypercalcemia induces secretion of fibroblastic growth factor 23 (FGF23)from osteocytes
Regulation of 25 hydroxylase activity ( FGF 23 upregulates this enzyme )
PTH increased production of bone matrix proteins Collagen and osteocalceinbone growth
Action of vitamin D in target organ –Kidney(distal renal tubular cells)
III Action of vitamin D in target organ –Kidney(distal tubular
renal cells)
(1)Increase in the reabsorption of Calcium and Phosphorous
(2) PTH conserves only Calcium and increase excretion of Phosphorous by kidney
(3) therefore PTH lower Serum Phosphorous levels
(4) 1,25 DHCC level directly regulate PTH secretion
REGULATION OF CALCITRIOL FORMATION
Hormonal levels of Calcitriol is controlled by
1. Serum Calcium levels
2. Serum Phosphorous levels
3. PTH
4. Regulation of Calcitriol formation by feed back control- Calcitriol itself
HORMONAL REGULATION OF SERUM CALCIUM LEVELS(by Calcitriol )-1A, 3
LOW DIATARY CALCIUM / LOW SERUM CALCIUM
SERUM PTH INCREASES †
SERUM CALCITRIOL INCREASES
BONE CALCIUM MOBALIZATION INCREASES INTESTINAL CALCIUM ABSORPTION INCREASES RENAL TUBULAR CALCIUM
ABSORPTION INCREASES
INCREASE IN SERUM CALCIUM LEVELS(homeostasis )
† Stimulatory effect of hypocalcemia on 1-alpha-hydroxylase is through PTH
II REGULATION OF CALCITRIOL SYNTHESIS
REGULATION OF CALCITRIOL SYNTHESIS by SERUM CALCIUM LEVELS
DECREASE SERUM CALCIUM LEVELS ( HYPOCALCEMIA )(NORMAL SERUM CALCIUM 9-11mg /dl)
INCREASE IN PTH
INCREASE IN ACTIVIITY OF 1 ALPHA HYDROXYLASE
INCREASE SERUM CALCITRIOL
INCREASE ABSORPTION CALCIUM FROM INTESTINE , KIDNEY
PTH DEMINERALIZATION OF BONES INCREASE SERUM CALCIUM LEVELS
INCREASE IN SERUM CALCIUM LEVELS
ACTION OF CALCIUM ON CALCITRIOL IS INDIRECT( THROUGH PTH )
ACTION OF CALCITONIN AND PLASMA CALCIUM LEVELS -1 B
LOW SERUM CALCIUM LEVELS
DECREASE IN SERUM CALCITONIN
INCREASE IN INCREASE IN INTESTINAL INCREASE IN
CALCIUM MOBALIZATION CALCIUM ABSORPTION RENAL ABSORPTION OF CALCIUM
FROM BONES
INCREASE IN SERUM CALCIUM LEVELS(homeostasis )
2. Regulation of calcitriol synthesis- serum inorganic phosphorous levels
DECREASE IN SERUM INORGANIC PHOSPHOROUS LEVELS
INCREASE IN 1ALPHA HYDROXYLASE ACTIVITY
INCREASE IN CALCITRIOL SYNTHESIS
INCREASE IN SERUM INORGANIC PHOSPHOROUS LEVELS (HOMEOSTASIS )
3. REGULATION OF CALCITRIOL SYNTHESIS –BY PTH
Regulation of Calcitriol synthesis by serum Calcium levels and PTH
DECREASE SERUM CALCIUM LEVELS ( HYPOCALCEMIA )(NORMAL SERUM CALCIUM 9-11mg /dl)
INCREASE IN PTH
INCREASE ACTIVITY OF 1 ALPHA HYDROXYLASE
INCREASE IN SERUM CALCITRIOL
INCREASE IN ABSORPTION CALCIUM FROM INTESTINE , KIDNEY
PTH DEMINERALIZATION OF BONES INCREASE SERUM CALCIUM LEVELS
INCREASE IN SERUM CALCIUM LEVELS (HOMOESTASIS)
ACTION OF CALCIUM ON CALTRIOL IS INDIRECT( THROUGH PTH )
III REGULATION OF CALCITRIOL SYNTHESIS by PTH
PTH and Calcitriol
Regulation of Calcitriol synthesis- by feedback mechanism
4. CONCENTRATION OF CALCITRIOL BY FEEDBACK MECHANISM
INCREASE CONCENTRATION OF CALCITRIOL
DECREASE 1 HYDROXYLASE and 25 HYDROXYLASE ACTIVITY
DECREASE SYNTHESIS OF CALCITRIOL
INCREASE OF 24 DIHYROXYLASE ACTIVITY
SYNTHESIS OF 24,25 DIHYDROXY CHOLECALCIFEROL
❖ STORAGE FORMS OF VITAMIN D
a) 24 ,25 DIHYOXYCHOLE CALCIFEROL (MOST POTENT STORAGE FORM )
b) 23,26 DIHYOXYCHOLE CALCIFEROL
c) 24,26 DIHYOXYCHOLE CALCIFEROL
Causes of deficiency manifestations of Vitamin D
Causes of deficiency manifestations of Vitamin D are
A. Dietary Vitamin D Insufficiency
B. Lack of sunlight exposure ( cover the body with purdah /bedridden
/children restricted in house /inhabitants of Northern latitude )
C. Liver diseases ( obstructive jaundice /steatorrhea ) decrease
availability bile salts malabsorption of fat soluble Vitamins )
D. Kidney diseases ( excessive loss of calcium in urine )
E. Liver /renal diseases adversely affect hydroxylation reactions involved
in activation of Vitamin D .
F. Prolonged treatment of anti-convulsant drugs
G. Genetic mutations in a gene coding for 1 hydroxylase enzyme or /and
25 hydroxylase enzyme abnormality in vitamin D activation
RICKETS
RICKETS IS A DEFICIENCY OF VITAMIN D IN CHILDREN
CAUSES OF RICKETS :
(1) DIETARY DEFICIECNCY VITAMIN D
(2) LACK OF UV EXPOSURE
(3) DISEASES OF LIVER / KIDNEY/ INTESTINE
(4) ABNORMALITY IN BINDING SITE (Nuclear binding site - for hormone receptor complex )
(5) GENETIC CAUSES
GENETIC CAUSES OF RICKETS
TYPE I ----1 HYDROXYLASE * DEFICIENCY /MUTATION
25 HYDROXY CHOLE CALCIFEROL
1 HYDROXYLASE *
1,25 DIHYDROXY CHOLE CALCIFEROL
TYPE II ----25 HYDROXYLASE DEFICIENCY/ /MUTATION
TYPE III ----MUTATION IN DNA FOR VITAMIN D RECEPTORS (REPLACEMENT OF SINGLE AMINO ACIDS OF RECEPTOR FROM DNA BINDING SITES )
CLINICAL FEATURES OF RICKETS
1. Insufficient mineralization of bones bones become soft and pliable
bone growth is affected
2. Bone deformities : Weight bearing bone bent
3. Bow legs ,knock-knee, bossing frontal bones and pigeon chest
4. rickety rosary: an enlargement of the epiphysis at lower end of ribs and
osteochondral junction beading of ribs
5. Harrison’s sulcus : a transverse depression passing outwards from the
coastal cartilage of axilla .This is due to the indentation of lower ribs at
the site of attachment of diaphragm.
CLINICAL FEATURES OF RICKETS
(1) REPEATED FRACTURES/BONE
DEFORMITIES:
DEFICIECY OF VITAMIN D
DEFECTIVE OSTEOBLAST FORMATION
NO VASCULARIZATION &
MINERALIZATION OF BONES
BONES BECOME SOFT
(2) HOT CROSS BUNS :FONTENELLES
DOSENOT CLOSE (IRREGULARITY IN
CALCIFICATION )
(3) SWELLING IN ANKLE ,KNEE,
ELBOW:
CARTILAGE DOSENOT DEGENERATE
EPIPHYSAL CARTILAGE
RICKETS
LABORATORY DIAGNOSIS OF RICKETS :
(1) DECREASE IN SERUM VITAMIN D LEVELS
(2) INCREASE IN SERUM ALKALINE PHOSPHATASE LEVELS
Methods to measure CONCENTRATION OF 1,25 HYDROXY D3:
a) RIA
b) HPLC
c) CLIA
d) LC Tandem mass spectroscopy
MANAGEMENT OF RICKETS
(1) SUPPLEMENTATION OF DIETARY MILK PRODUCTS ,EGG,FISH LIVER OIL,
(2) SUNLIGHT EXPOSURE
(3) RDA OF VITAMIN D =400 IU (IU .025 MICROGRAMS OF D3)
(4 ) RENAL RICKETS : DECREASED SYNTHESIS OF CALCITRIOL IN KIDNEY
(TREATMENT CALCITRIOL,)
OSTEOMALACIA ---ELDERLY RICKETS
ELDERLY RICKETS (OSTEOMALACIA )
ELDERLY RICKETS (OSTEOMALACIA ): Greek word OSTEON : bone ,and
MALAKIA :softness
OSTEOMALACIA :Bones are softened due insufficient mineralization and
increased osteoporosis prone to get repeated fractures
Diagnosis of Osteomalacia by biochemical Tests
a) Low Serum Calcium levels
b) Low Serum inorganic Phosphorous levels
c) Low Serum Vitamin D levels
d) High Serum Alkaline Phosphatase (bone iso-enzyme ) levels
ELDERLY RICKETS (OSTEO MALACIA )
Causes of ELDERLY RICKETS (OSTEO MALACIA )
(1)CHANGES IN LIFE STYLE - Lack of exposure to sunlight ( Purdah/
USE OF SUNSCREEN )
(2)Pregnancy( To fulfil requirement of two individuals )
(3)Lactation
(4)Obesity ( very common as fat soluble vitamin is stored in adipose
tissue not released for utilization )
(5)Advance in age
(6)presence of high concentration of Melanin decrease formation of
Vitamin under skin.
OPTIMAL CONCENTRATION OF 25 HYDROXY D3= MORE THAN 30 ng/ml ( SEVERE DEFICIENCY
10ng/ml )
Different Types of Rickets and their management Types of Rickets management of Rickets
1 Rickets (Classical Vitamin D deficiency) Dietary supplementation of Vitamin D
2 Hypophoshatemic Rickets ( defective tubular
reabsorption of phosphate )
Dietary supplementation of Vitamin D
along with Phosphate
3 Fanconi’s syndrome (( defective tubular reabsorption of
phosphate ,bicarbonate ,glucose and amino acids )
Gene /stem cell therapy ?
4 Renal Rickets ( Vitamin D not synthesized due to kidney
disease )
Administration of Vitamin D
5 End organ responsiveness to 1,25 DHCC ( decrease in
number of cytosolic receptors / structurally abnormal
receptors )
Gene /stem cell therapy ?
HYPERVITAMINOSIS D
HYPERVITAMINOSIS D( Serum concentration more than150 ng/ml )
Cause : EXCESSIVE CONSUMPTION OF VITAMIN D (more than 10-100 TIMES RDA )
SYMPTOMS ( DUE TO HIGH SERUM CALCIUM LEVELS )
1. DEMINERALIZATION OF BONE ,TEETH
2. INCREASE CALCIUM LEVELS CalcificationRENAL CALCULI/ DEPOSITION IN BONES
ARTHRITIS
3. Loss of appetite
4. Nausea,
5. Vomiting
6. Loss of wieght
7. Increased thirst
8. Hypertension
9. Polyuria
Manifestation of Vitamin D Toxicity
HYPERVITAMINOSIS D
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