Bioassay of Vitamin D

Bioassay of vitamin D by curative method

1. BIOASSAY
OF
VITAMIN-D
KOPPALA RVS CHAITANYA
ASSISTANT PROFESSOR
DEPARTMENT OF PHARMACOLOG Y

2. The activity of a preparation of anti-rachitic vitamin (vitamin D) is determined by
comparing the anti-rachitic activity with that of standard preparation of vitamin D by suitable
method; when so determined it is expressed in units per gram.
STANDARD PREPARATION AND UNIT:
➢ The standard preparation is a preparation of crystalline vitamin D3.
➢ The unit is the anti-rachitic activity contained in 0.025mg of the standard preparation and is
same as the international unit.
SUGGESTED DETAILS OF METHOD:
CURATIVE METHOD: Not less than 40 young rats of either sex, shortly after being weaned are
used for the test. About 10 litters of 4 or 8 rats are selected, the heaviest in any litter not exceeding
the lightest by more than 10g. They are fed for about three weeks on a rachitogenic diet which may
consists of
Content in % Content in %
Ground yellow maize 33 Ground yellow maize 76
Whole wheat 33 Wheat gluten 20
Wheat gluten 15 OR Calcium carbonate 03
Gelatin 15 Sodium chloride 01
Calcium carbonate 03
Sodium chloride 01
➢ The development of the necessary degree of rickets may be determined in each rat under
light ether anesthesia by taking X-ray photographs of the proximal ends of the tibia or the
distal ends of ulna and radius.
➢ The rats are divided into 4 groups, 1 rat from each litter set of 4 or 2 rats from each litter
set of 8 being assigned to each group.
➢ The rats in 2 groups receive doses of x and nx units (where n is suitable value such as 2 or
3) respectively of the standard preparation, while the rats of the other two groups receive,

3. respectively doses of the preparation being tested in the same ratio, 1 to 2 or 1 to 3 as the
doses of standard preparation. Suitable doses of the standard preparation may vary from 2
to 8 units for lower dose and a corresponding value for the higher dose. Each rat may
receive the whole of its dose at once or the dose may be divided into 8 daily doses.
➢ The same procedure should be adopted for all the rats of any one assay.
➢ Ten to fourteen days after receiving the doses of standard preparation or of the preparation
being tested if given in one amount or ten to fourteen days after receiving the first fraction
of dose, the rats are killed and the extent which the rickets has been cured is estimated by
x-ray photographs or by examination of bones after staining in either instance by reference
for standard scale (In order to stain the bones, the distal ends of Ulna and Radii may be
removed, immersed 24 hours in 4%w/v of formaldehyde cut longitudinal section immersed
in a 1.5%w/v silver nitrate for few minutes and exposed to light for few minutes and
transferred to water)
➢ The amount of healing produced by a given dose of Vitamin-D varies in different rats;
therefore, this amount for the rats for each group is averaged and the average amount of
healing receiving the preparation being tested are compared with the amounts of healing in
the rats receiving the standard preparation.
➢ Limits of error: The limits of error of a test are determined from the data of the test if the
estimate of the slope of the curve relating of response to the log of dose not exceeds eight
times it’s St. Error fiducial limits should be calculated.