INTRODUCTION

1. 2. 3. Approaches to evaluate vitamin status: Biochemical methods Clinical evaluation Dietary methods

Why Biochemical assessment? Clinical evaluation effective in diagnosis of latestage vitamin defficiencies- involving physiologic dysfunction or morphological changes. Dietary evaluation- diets and food habits likely to provide insufficient amounts of vitamin intake Biochemical assessment- Detection of early-stage vitamin defficiencies.

BIOCHEMICAL ASSESSMENTDEFINITION
Biochemical assessment is the analysis of specific enzymes / substances that are produced as a result of chemical reactions in the body.

REQUIREMENTS OF USEFUL BIOCHEMICAL METHODS

Correlate with the rate of vitamin intake and respond to deprivation of the vitamin Relate to a meaningful period of time Relate to normal physiologic function Be measurable in an accessible specimen Be technically feasible, reproducible and affordable Have an available base of normative data.

BIOCHEMICAL ASSESSMENT OF VITAMIN D

SERUM ALKALINE PHOSPHATASE SERUM 25- HYDROXY VITAMIN D SERUM VITAMIN D3 SERUM 1,25-DIHYDROXY VITAMIN D

BIOCHEMICAL ASSESSMENT OF VITAMIN D

Over 40 metabolites of vitamin D Very short half life in the circulation Vitamin D half life of almost 24 hours 25 hydroxy vitamin D: half life: 21-30 days 1,25 dihydroxy vitamin D: half life: 4-15 hrs Measurement of 25 hydroxy Vitamin D is a better indicator of Vitamin D stores - obtained from sunlight - obtained from dietary sources.

Why 25-hydroxy vitamin D?

25 OH-D circulates in nmol/L concentrations while 25dihydroxy Vitamin D circulates in pmol/L concentrations.

 1. 2. 3. 4.

MAIN METHODS TO ESTIMATE 25OHD are Immunoassay HPLC attached to fluoroscence Mass spectrometry detection Tandem MS

IMMUNOASSAYS
Immunoassay techniques are especially suited for the analysis of compounds at low concentration and in samples with little or no preparation, since their detection limits are usually within nanogram to picogram range they are analytical methods based on the specific immuno-reaction between an antibody(Ab) and an antigen(Ag), for the determination of the amount of either reactant in the solution.

 IMMUNOASSAYS: automated and incorporated into large commercial analyser systems- excellent functionality and ability to measure large numbers of samples routinely Weakness: issues with calibration, standardisation, inability to quantify vitamin D2 and D3 separatelythey give an estimation of total 25-OHD

HPLC
High-performance liquid chromatography, HPLC, is a chromatographic technique used to separate the components in a mixture, to identify each component, and to quantify each component.

MASS SPECTROMETRY
Mass Spectrometry is an analytical laboratory technique to separate the components of a sample by their mass.

 TANDEM MS ASSAYS Simulataneous estimation of 25OH Vitamin D2 and D3 More sensitive than immunoassays More labour intensive Require greater level of technical expertise

CONCLUSION
Measurement of serum 25OHD is the best way of estimating Vitamin D status Assay used should be able to recognise all forms of 25OHD [D2 and D3]- Tandem MS more preferable.

REFERENCES
Biochemical assessments of vitamins and trace minerals Robert.A.Jacob and David. E.Milne Vitamins : Fundamental Aspects in Nutrition and Health (3rd Edition) Combs, Gerald F. Nutrition and Diet Research Progress : Vitamin D Deficiency Lerner, Vladimir Miodownik, Chanoch Vitamin D and Bone Health: A Practical Clinical Guideline for Patient Management :National Osteoporosis Society, UK Nutritional Biochemistry of Vitamins: 2nd edition: David.A.Bender.