LIPIDS AND LIPOPROTEIN MEASUREMENT Flashcards
can be measured in non-fasting individuals
TOTAL CHOLE
HDL
Requires fasting individuals
TAG
LDL
Ideal time in fasting
12 hours before venipuncture
women have lower level than men except in
childhood and after the early fifties
Sex
NCEP abbreviation
National Cholesterol Education Program
- at least ___- hours before
blood specimens are taken for lipid and lipoprotein
analysis
Adult treatment Panel 3
Total cholate , HDL, LDL, TAG can be measured in fasting patients only
TRUE or FALSE
FALSE
What are the lipid profile
total cholesterol, HDL, LDL, TAG
POSTURE
When standing patient reclines =
↓10% TC, LDL, HDL
Position of the patient must be standardized for
venipuncture
sitting position
NCEP recommend:
patient be seated for __________ before sampling to prevent _____________
5 minutes / hemoconcentration
This is an accommodation for patients who may
be unable or unwilling to fast for 12 hours
Adult treatment panel 3
Plasma: Ultracentrifugation & electrophoresis (LPP
analysis)
o The samples can be cooled to ______ immediately
to retard changes that can occur in the
lipoproteins at room temperature
4°C
Plasma should _______ remain in contact with the _________
NOT - cells overnight
Protein aggregation occurs___________ using
what sample??
less frequently - SERUM
When applying _________ where LDL is
calculated (Plasma/Serum)
Friedewald equation
The two different calculations in VLDL
TAG/ 2.175 mmol/l - TAG/5 = mg/dL
Large osmotic effects
(falsely low result)
cyan (CITRATE)
- Because of its relatively
high molecular weight, it
has little effect on plasma
volume - But can alter
electrophoretic mobility
Green (HEPARIN
*Preferred because it
retards certain kinds of
oxidative and enzymatic
alterations that occur in
the lipoproteins during
storage.
Pink (EDTA)
TC, TAG, HDL, Apolipoproteins – can be analyzed
with ______ samples
FROZEN
STORAGE
Long-term (>_ mos) at -____C
* Short-term (__ mos.) at - ___C
> 2 MOS AT 70 C
1-2 MOS AT 20C
__________ are NOT appropriate for
ultracentrifugal analysis because the triglyceriderich lipoproteins do NOT withstand freezing
Frozen samples
Medical disorders that lead to secondary
dyslipoproteinemia include ______, ______, ______
Thyroid, hepatic and kidney disease
Even in the presence of the anticoagulant,
protein aggregation can occur in plasma that is
stored in the refrigerator for a few days or frozen
for longer periods
serum
what is the color of liebermanns burchard test ________which indicates ________
Green color - steroids are cholesterol
what is the color of Salkowski reaction______which indicates presence of ________
Red color - steroids
2 mg of dry extract was dissolved in
acetic anhydride, heated to boiling,
cooled and then 1 ml of concentrated
sulphuric acid was added along the
sides of the test tube.
- LIEBERMANN-BURCHARD’S TEST:
2 mg of dry extract was shaken with
chloroform, to the chloroform layer
sulphuric acid was added slowly by
the sides of test tube
- SALKOWSKI REACTION:
- Hydrolysis of CE (cholesteryl esters) using
potassium hydroxide (KOH) to UE (unesterified) form - Extraction of UE form with petroleum ether
- ABELL-KENDALL
- Measurement with Liebermann-Burchard reagent
- ABELL-KENDALL
acids of abell kendall
o Sulfuric acid, acetic acid, and acetic anhydride
Use smaller sample volume, no preliminary extraction
step
B. ENZYMATIC METHODS
- Rapid and precise compared to chemical methods
B. ENZYMATIC METHODS
Forms a quinoneimine dye which will create a
colored reaction and that will be detected by your
device, usually a spectrophotometer
B. ENZYMATIC METHODS
color of enzymatic method and read at _______
red color = read at 500 nm
give me the characteristics of enzymatic method
- smaller sample volume
- no preliminary extraction
step - rapid
- precise
Cholesterol oxidase (Equation 17-2) can react
with sterols other than cholesterol that are
present in plasma such as plant sterols, which are
present in appreciable concentrations in the
circulation of patients with β-sitosterolemia
- Plant sterol
Known for its properties as a reducing agent.
These properties allow it to compete with the
chromogenic substrates of equation 17-3 for
hydrogen peroxide.Therefore, plasma/serum samples with elevated
levels of ascorbic acid can result in lower
measured levels of TC.
Ascorbic acid
Can interfere with TC measurements because of
its own spectral properties; bilirubin absorbs
light at 500 nm (Rifai et al, 2000). This tends to
increase measured cholesterol values
Bilirubin
However, _________ is also oxidized by ________, and
as a result loses its absorbance at 500 nm. This
complicates the application of a serum blank to
correct for _________ absorbance
bilirubin oxidized by H2O2
as a result of hypertriglyceridemia
Sample turbidity
SCE
scandinavian committee on enzymes
Similar to Van Handel and Zilversmit method
- CDC - REFERENCE METHOD
limits interfering substances
- CDC - REFERENCE METHOD
Extraction of TAG by chloroform
* Isolation of TAG by silicic acid chromatography
* Release of glycerol by saponification ((alkaline
hydrolysis of triglycerides)
- CDC - REFERENCE METHOD
color of CDC
Pink end color
heparin in combination with manganese
to precipitate apo B-containing LPP
EARLIEST
sodium phosphotungstate with
magnesium
ALTERNATIVE
Dextran sulfate with magnesium
MORE SPECIFIC
elevated TAG levels
Interfering factor
assay for cholesterol content
Clear supernate
is being produced and measured
by the reaction with sulfuric acid solution of
chromotropic acid to produce pink
chromophore
Formaldehyde
3-Step Procedure in HDL (gold standard or reference method
Ultracentrifugation
Heparin Manganese Precipitation
Analysis of supernatant cholestero
IV. LABORATORY METHODS: LDL CHOLESTEROL
REFERENCE METHOD
Beta-Quantification
* Electrophoretic migration
* Ultracentrifugation + Chemical Precipitation
BEU
NOT appropriate in samples with TAG of greater
than___________ and patients with suspected
beta lipoprotein
400 mg/dl
Hydrogen peroxide by peroxidase will form _____________
quinoneimine dye complex
VLDL in SI unit
o LDL = TC - HDL - (TAG/2.175) (mmol/L)
conventional unit
LDL = TC - HDL - (TAG/5) (mg/dL)
