courtesy all mentioned sources ;

1. Vitamin D
Dr.Ghulam Murtaza
Resident Chemical Pathology
DIMC

2. Introduction
Fat soluble / steroid hormone
Major forms are Vitamin D2 (ergocalciferol) and D3 (cholecalciferol)
(differ chemically only in their side-chain structures)
Both forms are well absorbed in the small intestine. Absorption occurs by simple passive diffusion and by a
mechanism that involves intestinal membrane carrier proteins
Neither aging nor obesity alters vitamin D absorption from the gut [NIH].
Latitude ,Season, sunscreen used
& skin pigmentation influence the production of vitamin D

3. Bioactivation
Vitamin D obtained from sun exposure, foods, and supplements is biologically inert
Must undergo two hydroxylation in the body for activation
The first hydroxylation, which occurs in the liver, converts vitamin D to 25-
hydroxyvitamin D [25(OH)D], also known as “calcidiol.
The second hydroxylation occurs primarily in the kidney and forms the
physiologically active 1,25-dihydroxyvitamin D [1,25(OH)2D], also known as
“calcitriol”

5. 25Hydroxyvitamin D
• The concentration of 25(OH)D in
plasma is Appro: 10-65ng/ml
• The half life of 25(OH)D is 2 to 3
weeks
• At 25(OH)D concentration near
30ng/ml dietary absorption of
calcium is maximum
• Test that determined the vitamin
D blood level
1,25 Dihydroxy vitamin D
• Norma circulating concentration
are approx. are 15 to 60 pg /ml
(about 1/1000 0f 25(OH)D
• The plasma half life is 4 to 6 hours
• Its biological active hormones
• Circulating Concentration of 1,25
(OH)2D are tightly regulated by
PTH, Phosphate, Calcium ,
• Only 0.4 % is free

6. In circulation :
• In circulation vitamin D 25(OH)D & 1,25(OH)2D are bound to D-
Binding Protein (DBP) ,a specific high affinity transport protein .
• DBP belongs to the albumin & alpha fetoprotein gene family
• DBP is constitutively synthesized by liver & circulates in great excess
(at about 400mg/L)
• DBP concentration are increased in pregnancy & with estrogen
therapy & are decreased in nephrotic syndrome

7. • In contrast to 25(OH)D, circulating 1,25(OH)2D is generally not a good
indicator of vitamin D status because it has a short half-life measured in
hours, and serum levels are tightly regulated by parathyroid hormone,
calcium, and phosphate .Levels of 1,25(OH)2D do not typically decrease until
vitamin D deficiency is severe [1 NIH vitamin D ].

8. BIOLOGICAL ACTION OF 1,25
DIHYDROXYVITAMIN D

9. Clinical Significance :
• Vitamin D nutritional status is best determined through the
measurement of 25 (OH)D than vitamin D because 25 (OH)D is the
main circulating form of vitamin D .
• Plasma 25 (OH)D is les affected by day to day variation ,exposure to
sunlight or food intake .
• Measurement of 25 (OH)D is relatively easy ,to measure
• Breast milk infants ,strict vegetarian ,darker skin pigmentation &
elderly

10. Abnormal circulating Concentration of
25(OH)D
DECREASED
25(OH)D :
Inadequate
exposure to
sunlight
Inadequate
dietary vitamin
D
Vitamin D
malabsorption
Severe
hepatocellular
carcinoma
Loss of 25-
hydroxylase
activity
Increased
catabolism
( drugs like
phenytoin
phenobarbital )

12. Abnormal circulating Concentration of
1,25(OH)D
Decrease 1,25(OH)2D
Renal Failure
Hyperphosphatemia
Hypomagnesemia
Pseudohypoparathyroidism
Hypercalcemia of malignancy
Rickets (VDD)
Increased 1,25(OH)2D
Granulomatous disease
Primary hyperparathyroidism
Lymphoma
1,25(OH)2D intoxication
24,25- Hydroxylase deficiency

13. Rickets
• Disease of growing
bone.
• Refer to changes at the
growth plate caused by
deficient mineralization
of bone.
• Occurs before the
growth plates fuse
deficient
mineralization
at the growth
plate
Osteomalacia
softening of the bones
impaired mineralization of the bone matrix
after the growth plates have fused
The softened bones of children and young adults
lead to bowing during growth
especially in weight-bearing bones of the legs

16. Measurement of Vitamin D
• All assays should measures D2 & D3 metabolites equally because
both D2& D3 are metabolized to produce biological active
1,25(OH)2D .
• The University of Ghent has developed an ID-LC/MS/MS
25(OH)vitamin D Reference Measurement Procedure (RMP) for
Vitamin D in human serum that is traceable to NIST Standard.
• Different methods like extraction & depolarization ,column
chromatography ,HPLC has been used to measure vitamin D

18. Limitations:
• Do not use samples that contain fluorescein. Fluorescein levels > 0.10
µg/mL can produce falsely elevated results in this assay .
• patients undergoing retinal fluorescein angiography can retain
amounts of fluorescein in the body for up to 72 hours post-treatment.
• Do not use hemolyzed samples. Hemoglobin at concentrations > 155
mg/dL will cause falsely depressed values.
• Patient samples may contain heterophilic antibodies that could react
in immunoassays to give falsely elevated or depressed results

19. Recommended dose :
• One of the problems with vitamin D terminology is the archaic
method used to express dose, international units or IU. One thousand
IU of vitamin D sounds like a lot; in fact, it is only .025 mg or 25
micrograms; i.e., one mcg is 40 IU
• Second, the amount of vitamin D needed varies with body weight,
body fat, age, skin color, season, latitude, and sunning habits.
• Supplementation with 1,000 IU per day will usually result in about a
10-ng/mL elevation of serum 25(OH)D when given over 3-4 months.

20. • In the absence of significant UVB exposure, input from diet and
supplements of approximately 1,000 IU (25 mcg) per day for every 15
kg of body weight may be needed; i.e., an obese 150-kg adult may
require up to 10,000 IU per day to achieve a 25(OH)D level of 50
ng/mL.
• However daily recommended dose allowance is 40 IU (10ug) & > 60
years is 800 IU .

21. Reference ranges

22. SUMMARY

23. Q.1: A 30 year old female complains of backache and generalized
weakness. Her biochemical profile was as following :
• Calcium: 7.5 mg/dl
• Phosphorous: 2.2 mg/dl
• Alkaline phosphatase : 107 U/l
She is most likely suffering from:
A- Hypomagnesaemia B- Hypoparathyroidism
C Osteitis fibrosa cystica D- Paget’s Disease
E- Vitamin D deficiency

24. References
• Use of vitamin D in clinical practice Article in Alternative Medicine
Review: a Journal of Clinical Therapeutic · April 2008
• Invited critical review 25-Hydroxyvitamin D: Analysis and clinical
application Zengliu Su, Satya Nandana Narla, Yusheng Zhu ⁎
Department of Pathology and Laboratory Medicine, Medical
University of South Carolina, Charleston, SC, USA
• Tietz text book of clinical chemistry volume 3 sixth edition .