CC2 (LIPID AND LIPOPROTEIN ANALYSES) Flashcards
SAMPLE for cholesterol measurement
plasma or serum
PATIENT PREP for cholesterol measurement
usual diet 2 weeks prior testing
May be tested from nonfasting spx (little effect on TC)
May be tested from nonfasting spx
CHOLESTEROL
PRECAUTIONS for cholesterol measurement
- Avoid hemolyzed sample (free Hgb – false ↑)
- Avoid icteric sample
- No water contamination (dehydration is included in chole measurement)
- Precise and accurate timing for color development
COLOR DEVELOPER MIXTURE FOR CHOLE:
- Glacial acetic acid
- Acetic anhydride
- Conc. sulfuric acid
These makes it possible for the cholesterol to be carried out in colorimetric assay
Double bonds and hydroxyl group in the sterol structure
CDC Reference method for CHOLESTEROL
ABELL-LEVY-BRODIE-KENDALL METHOD
Used for cholesterol extraction
Hexane
Used for cholesterol hydrolysis
Alc. KOH
General steps for cholesterol method
Cholesteryl esters: hydrolyzed with alc. KOH
Unesterified cholesterol: extracted with hexane
Measured using: LIEBERMANN – BURCHARD reaction or SALKOWSKI reaction
NON-ENZYMATIC / CHEMICAL METHOD for CHOLESTEROL
ABELL KANDALL
Principle: Cholesterol dehydration & oxidation to form a colored compound (measured colorimetrically)
NON-ENZYMATIC / CHEMICAL METHODS (ABELL KANDALL)
Involves colorimetry only
One-step method
Involves extraction, colorimetry
Two-step method
Involves saponification, extraction, colorimetry
Three-step method
Involves precipitation, saponification, extraction, colorimetry
Four-step method
Chemical reaction after hydrolysis and hexane extraction of cholesterol
LIEBERMANN – BURCHARD reaction or SALKOWSKI reaction
Cholesterol + Sulfuric acid + Acetic anhydride –>
LIEBERMANN – BURCHARD: Green soln. (Cholestadienyl monosulfonic acid)
SALKOWSKI: Red soln. (Cholestadienyl disulfonic acid)
Product and solution color in LIEBERMANN – BURCHARD reaction for cholesterol
Cholestadienyl monosulfonic acid
Green soln.
Product and solution color in SALKOWSKI reaction for cholesterol
Cholestadienyl disulfonic acid
Red soln.
T/F
Color intensity absorbance = Cholesterol concentration
T
ENZYMATIC METHOD for CHOLESTEROL
CHOLESTEROL OXIDASE
Chemical equation for ENZYMATIC METHOD (CHOLESTEROL OXIDASE)
TC (cholesteryl ester and cholesterol) + H2O ▬Cholesteryl esterase▬► Cholesterol + FA
Cholesterol oxidase
Cholesterol + O2 ▬Cholesterol oxidase▬► 4-cholestenone + H2O2
H2O2 + Dye ▬Peroxidase▬► Color (Quinoneimine dye – violet/pink)
End product & color of enzymatic method (Cholesterol oxidase)
Quinoneimine dye – violet/pink
SAMPLE for TAG
plasma or serum
PATIENT PREP for TAG
Strict fasting (12 hours)
If + FBS: 10 hours
Transfer of phosphate group
Kinase
CDC Reference method for TAG
MODIFIED VAN HANDEL AND ZILVERSMITH
Chemical equation for NON-ENZYMATIC/CHEMICAL METHOD of TAG
HYDROLYSIS: TAG ▬Alcoholic KOH▬► Glycerol + FA
Glycerol + Periodic acid ▬▬► Formaldehyde
Formaldehyde + Chromotropic acid → Blue soln (Van Handel and Zilversmith)
or
Formaldehyde + Diacetyl acetone + NH3 → Yellow soln (Hantzch Condensation)
2 methods included in NON-ENZYMATIC/CHEMICAL METHOD for TAG
Van Handel and Zilversmith (Colorimetric)
Hantzch Condensation (Fluorometric)
Equation of Van Handel and Zilversmith (Colorimetric)
Formaldehyde + Chromotropic acid → Blue soln
Equation of Hantzch Condensation (Fluorometric)
Formaldehyde + Diacetyl acetone + NH3 → Yellow soln
T/F
Color intensity absorbance = TAG concentration
T
Part of TAG structure utilized in analysis
Glycerol
Colorimetric method for TAG
Color of solution
Van Handel and Zilversmith
Blue