Lecture 14 - Vitamins Flashcards
definition of a vitamin
low molecular weight compounds which humans/organisms depend on organic matter as a source of nutrients
require small quantities for normal metabolism
classifications of vitamins?
based on solubility
- fat soluble (D E A K)
- water soluble (B group and vit C)
units of vitamins
mg (10^-3) or Mg (10^-6)
OR
international units (IU)
US pharmacopeia (USP)
% Daily value
what is IU?
international units
a unit of measurement for the amount of a substance based on measured biological activity or effect
used for vitamins, hormones, vaccines
the precise definition of 1 IU differs from substance to substance
what treatments are used in the extraction of vitamins?
heat acid alkali solvents enzymes
how do you prepare the sample for vitamin analysis?
extraction of vitamins from its biological matrix
each extraction procedure is specific for each vitamin and are designed to stabilize the vitamin
what is the typical vitamin extraction procedure for ascorbic acid?
cold extraction with metaphosphoric acid/acetic acid
what is the typical vitamin extraction procedure for vit B1 and B2
boiling or autoclaving in acid plus enzyme treatment
what is the typical vitamin extraction procedure for niacin
autoclaving in acid (noncereal products) or alkali (Cereal products)
what is the typical vitamin extraction procedure for for folate
enzyme extraction with alpha-amylase, protease and gamma-glutamyl hydrolase
what is the typical vitamin extraction procedure for vit A, E or D
organic solvent extraction, saponification and re-extraction with organic solvents
why is it challenging to extract vit D in milk?
therefore, ____ is important for vit D determination.
- milk has relatively low concentrations of vit D
- poor stability to heat, light, oxidation
- possible interference from milk components (eg. fat, AA, emulsifiers)
therefore, sample prep is important
what are 3 classifications of vitamin assays?
- bioassays: involves animals
- microbiological assays: uses of protozoan organisms, bacteria and yeast
- physiochemical assays: methods includes
- spectrophotometric
- fluorometric
- chromatographic
- enzymatic
- immunological
- radiometric
what vitamins does the bioassay method analyze?
- used for vit B12 and D analysis
describe the line test for vit D (bioassay method)
- vit D assay uses rat models
- AOAC method 936.14 is known as the line test
- the line test for vit D is based on the cure of experimentally induced rickets in rats, measured by bone calcification
describe the experimentally induced bone calcification in rats for vit D analysis?
- bone sections (longitudinal) are stained with silver for observing calcification
- compare the extent of line formation with the reference group receiving the defined amount of vit D
what are microbiolocal methods/assays limited to?
- limited to analysis of water soluble vitamins
- methods are sensitive and specific for each vitamin
what is the principle of microbiolocal methods/assays?
- the growth of a microorganism is proportional to their requirement for a specific vitamin
- the growth of a microorganism in an extract of a vitamin containing sample is compared against the growth of the same organism in the presence of known quantities of that vitamin
what are used as test organisms in microbiolocal methods/assays?
bacteria
yeast
protozoans
how is growth measured in microbiolocal methods/assays?
in terms of turbidity, acid production, gravimetric, respiration
turbidity (in bactiera and yeast)
what is the procedure of the microbiolocal method of niacin analysis?
- weigh sample
- add H2SO4
- autoclave 30 mins at 121-123 degC
- filtrate mixture
- add difco niacin assay medium
- autoclave 10 mins
- add lactobacillus plantarum
- incubate
- measure optical density and compare with the bacteria grown in a medium of known niacin amount
what is the procedure of the microbiolocal method of folate analysis?
- weigh sample
- add buffer
- add alpha amylase, protease, conjugate
- deactivate enzymes by boiling mixture
- filtrate
- dilute with bacterial growth medium
- autoclave
- add lactobacillus casei
- incubate
10 measure optical densitry and compare with that of bacteria grown in a medium containing a known amount of folate
what is Vit A sensitive to?
UV light air prooxidants high temp moisture
what is the only acceptable method to measure vit A activity?
HPLC: high performance liquid chromatogrpahic method
what is the principle of HPLC for vit A measurement?
- type of HPLC analysis (physicochemical method)
- test sample is saponified with ethanolic KOH
- vit A is extracted into organic solvents and concentrated
- vit A isomers (trans and cis retinol) levels are determined by HPLC on a silica column
steps of HPLC for vit A measurement?
- transfer sample to digestion flask
- add ethanolic pyrogallol solution and ethanolic KOH
- stirr at room temp for 18 hr
- extract vit A with hexan diethyl ether
- analyze organic layer
what is vit C susceptible to?
oxidative deterioration, enhanced by high pH and ferrir and cupric ions
in vit C analysis, if there is significant amounts of ferric Fe and cupric Cu ions present in the sample, what should you do?
include a chelating agent (eg EDTA)
what reaction occurs in vit C analysis?
what signals the endpoint?
L-ascorbic acid is oxidized to L-dehydroascorbic acid by 2,6-dichloroindophenol (redox indicator dye)
endpoint: excess unreduced dye appears rosepink in acid solution
in vit C analysis, what color is:
- L-ascorbic acid?
- 2,6 dichloroindophenol?
- reduced 2,6 dichloroindophenol?
- L-dehydroascorbic acid?
- l-ascorbic acid is colorless
- 2,6-dichloroindophenol is red
- reduced 2,6 dichloroindophenol is colorless
- L-dehydroascorbic acid is brown
describe the procedure of thiamine analysis
- extract with dilute acid
- enzymatic hydrolysis of thiamine’s phosphate-esters (using diesterase)
- chromatographic cleanup (purification)
- oxidation to thiochrome
- measurement using fluorescence intensity
