LIPIDS LAB Flashcards
preferred sample
serum collected in SST
preferred for LPP assays
edta plasma
fasting hours required
12-14 hour
Turbidity of the chylomicrons can interfere with the
spectrophotometric methods of lipid profile by?
falsely increased result
specimen type for total cholesterol and HDL
non-fasting specimen
specimen type for TAG and LDL
fasting specimens
- can lead to decreased plasma volume in relation to volume of pack RBC
- can interfere with the
result
hemoconcentration
long period storage of
serum/plasma
-70C or lower
short-term storage of serum/plasma (up to a month or two)
-20C
cholesterol measurement methods
can be chemical or enzymatic
fasting requirement for cholesterol measurement?
8-12hours
specimen type for cholesterol measurement
fasting or non-fasting specimen
chemical methods for measuring cholesterol
- Libermann-Burchardt Rxn
- Salkowski Rxn
end product of Libermann-Burchardt Rxn
Cholestadienyl Monosulfonic Acid
end color of Libermann-Burchardt Rxn
green
color developer mixture of Libermann-Burchardt Rxn
a. glacial acetic acid
b. acetic anhydride
c. concentrated sulfuric acid
end product of salkowski rxn
Cholestadienyl Disulfonic Acid
end color of salkowski rxn
red
general methods for measuring cholesterol
- one-step method
- two-step method
- three-step method
- four-step method
one-step method
colorimetry (Pearson, Stern, and Mac Gavack)
two-step method
extraction + colorimetry (Bloors)
three-step method
saponification + extraction + colorimetry (Abell-Kendal)
four-step method
saponification + extraction + colorimetry + precipitation (Schoenheimer, Sperry, Parekh, and Jung)
what are the reagents for step methods?
- Liebermann-Burchardt (colorimetry)
- Petroleum ether (saponification)
- Alcoholic potassium hydroxide
- Digitonin (precipitation)
CDC REFERENCE METHOD FOR CHOLESTEROL
MEASUREMENT
Gas Chromatography Mass Spectrophotometry (GC-MS)
TRADITIONAL REFERENCE METHOD FOR
CHOLESTEROL
Abell, Levy and Brodie Method
enzymatic methods for measuring cholesterol
cholesterol oxidase rxn
interference for enzymatic method of measuring total cholesterol
- ascorbic acid, bilirubin, hgb
- ascorbic acid: ↑ascorbic acid ↓ TC
- bilirubin: oxidized by H2O2
- hemoglobin: has pseudo-peroxidase activity that diminish H2O2 produced in the cholesterol oxidase rxn
- Uses hexane extraction / petroleum ether after
hydrolysis with alcoholic KOH followed by reaction with
Liebermann-Burchardt color reagent - EC: green
Abell, Levy and Brodie Method
gold standard method for cholesterol
ISOTOPE DILUTION MASS SPECTROMETRY
fasting hours requirement for TAG measurement
10-12 hours
specimen for TAG measurement
either plasma or serum
CDC Reference Method for TAG Measurement
- Saponification: alcoholic KOH
- Extraction: choloroform
- Treatment w/ silicilic acid: to remove phospholipids
- EP: pink chromophore
interference for TAG measurement
HAB
- hemolysis
- ascorbic acid
- bilirubin
methods for measuring TAG
- chemical method
- enzyme method
chemical methods for measuring TAG
- colorimetric method - Van Handel and Zilversmith
- fluorometric method - Hantzsch Condensation
Van Handel and Zilversmith
(colorimetric)
- TAG + KOH → Glycerol + Fatty Acid
- Glycerol + Periodate → Formic Acid + Formaldehyde
- Formaldehyde + Chromotropic Acid → Chromogen
Hantzsch Condensation
(fluorometric)
- TAG + KOH → Fatty Acids + Glycerol
- Glycerol + Periodate → Formic acid + Formaldehyde
- HCHO + Diethyl acetone + NH3 → Diacetyl Lutidine Compound
Involves liberation of fatty acids from cholesterol, from
triglycerides and KOH
Hantzsch Condensation
enzymatic methods for measuring TAG
- Glycerol Kinase Method
Reaction A for Glycerol Kinase Method
- TAG + Lipase → Glycerol + Fatty acid
- Glycerol + ATP + Glycerol Kinase → Glycerolphosphate +ADP
- ADP + Phosphoenol pyruvate + Pyruvate Kinase → ATP + Pyruvate
- Pyruvate + NADH + H ion + Lactate dehydrogenase → Lactate + NAD
Reaction B for Glycerol KInase Method
- TAG + Lipase → Glycerol + Fatty Acid
- Glycerol + ATP + Glycerol Kinase → GlycerolPO4 + ADP
- Glycerol PO4 + NAD + GlycerolPO4dehydrogenase → DihydroxyacetonePO4 + NADH
- NADH + Tetrazolium dye + Diaphorase → Formasan + NAD+
Conditions where there is increased TAG
- Hypothyroidism
- Alcoholism
- Nephrotic Syndrome
- Hyperlipoproteinemia tyeps I, IIb, III, IV, V
- Pancreatitis
Conditions where there is decreased TAG
HMM… B-aba ng tag
- Hyperthyroidism
- Malabsorption syndrome
- Malnutrition burns
- Brain infection
