Lipids Flashcards
Blood Sampling and Storage
Biological variation
Fasting
Posture
Type of sample (plasma or serum)
Storage
Biological variation
– Age
(↑ Cholesterol )
Biological variation
Sex : women have____ level than men
lower
Biological variation
Seasonal what increases during winter
(Cholesterol↑ in winter)
Biological variation
Dietary intake (restricted diet for_____)
2 weeks
Biological variation
Medications (eg)
oral contraceptives
Postmenopausal estrogen
Antihypertensive drugs
Biological variation
– Age (↑ Chole)
– Sex : women have lower level than men
– Seasonal (Chole ↑ in winter)
– Dietary intake (restricted diet for 2 weeks)
– Medications (oral contraceptives)
– Medical disorders
– Lifestyle and other biological variation
• Recent MI, stroke, cardiac catheterization
TRUE/FALSE : Total Chole, HDL, LDL and TAG can be measured in fasting patients only
FALSE!
What can be measured in NON-FASTING individuals
– Total Cholesterol
– HDL
What requires FASTING individuals
Triglycerides (TAG)
LDL
Fasting
• Ideal time:
12 hours
CMs markedly ↑ TAG after meals (clearance time:_______)
6 to 9 hours
> 12h fasting = CM -> ???
False Increase CM
Some LPP physiologically ↓ after meals due to____ mediated compositional changes that occurs when CM is catabolized
CETP
Cholesteryl ester transfer protein
NCEP Adult treatment Panel III: at least____ hours
9hrs
Posture
• When standing patient reclines =
↓ 10% TC (total cholesterol), LDL, HDL
Position of the patient must be standardized for venipuncture
sitting position
Current NCEP guidelines for lipid concentration:____ minutes seated.
5mins
Prolonged venous occlusion = hemoconcentration (↑chole by____)
10-15%
Type of sample (plasma or serum)
• When applying Friedewald equation where____ is calculated -> Serum
LDL
Type of sample
_______: Ultracentrifugation & electrophoresis (LPP analysis)
Plasma
Type of sample
_____ should not remain in contact with the cells overnight
Plasma
Protein aggregation occurs less frequently using what sample?
Serum (less protein)
Plasma protein is about 3–5 g/liter greater than serum protein. (Taas protein sa plasma kay wala nag coagulate)
Type of sample (plasma or serum)
• Anticoagulant used:
_____: Large osmotic effects (Falsely low)
_____: Alter electrophoretic mobility
_____: preferred
Blue top (sodium citrate)
Green top (heparin)
Purple top (EDTA)
Storage
– TC, TAG, HDL, Apo
Long-term
Short-term
Long-term (>2 mos) -70C
Short-term (1-2 mos.) -20C
Estimation of Plasma Lipids
Cholesterol
A. Chemical Methods
a) Liebermann-Burchard
b) Salkowski
c) Abell-Kendall
B. Enzymatic methods
Cholesterol
A. Chemical Methods
a) Liebermann-Burchard
b) Salkowski
c) Abell-Kendall
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
Liebermann-Burchard’s test:
Formation of______ colour indicates the presence of steroids.
GREEN
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
Salkowski reaction
Formation of ____ colour indicated the presence of steroids.
RED
Formation of green colour indicates the presence of steroids.
Liebermann-Burchard’s test
Formation of red colour indicated the presence of steroids.
Salkowski reaction
Abell - Kendall
• Hydrolysis of CE using alcoholic KOH (potash) to UE form
• Extraction of UE form with petroleum ether
• Measurement with______ reagent
Liebermann-Burchard rgt.
Liebermann-Burchard rgt.
Sulfuric acid, acetic acid, and acetic anhydride
Abell - Kendall
Color indicator
BLUISH GREEN COLOR
Yung gigawa during lab kay chemical or enzymatic method?
Enzymatic
Benefits of enzymatic mtds:
• Smaller sample volume
• NO preliminary extraction step
• Rapid
• Precise
Enzymatic method
_____ COLOR = READ AT____nm
RED
500nm
Enzymatic method
Interfering Substances:
Plant sterols, Ascorbic acid, Bilirubin, Sample turbidity, Hgb
+ H2SO4 =_____ END COLOR
PINK
Quinoneimine dye
Faint Pink
HDL-CHOLESTEROL MEASUREMENT
• Separated by chemical precipitation
Precipitating Agents
•– heparin in combination with manganese to precipitate apo B-containing LPP
•– sodium phosphotungstate with magnesium
•– Dextran sulfate with magnesium
•Interfering factor: elevated____ levels
•– assay for cholesterol content
Earliest
Alternative
More specific
TAG
Clear supernate
HDL-CHOLESTEROL MEASUREMENT
• Gold standard / Reference Method
– 3-Step Procedure
– Ultracentrifugation – to remove VLDL
– Heparin Manganese Precipitation – to remove LDL
– Analysis of supernatant cholesterol by the AbellKendall Assay
HDL-CHOLESTEROL MEASUREMENT
• Gold standard / Reference Method
– 3-Step Procedure
– – to remove VLDL
– – to remove LDL
– Analysis of supernatant cholesterol by the
Ultracentrifugation
Heparin Manganese Precipitation
AbellKendall Assay
LDL-CHOLESTEROL MEASUREMENT
• Reference Method
•Electrophoretic migration
•Ultracentrifugation + Chemical Precipitation
Beta-Quantification
LDL-CHOLESTEROL MEASUREMENT
Routine
Friedewald Calculation
Estimation of Plasma Lipids FRIEDEWALD EQUATION/CALCULATION
mmol/L
LDL = TC – HDL – Plasma TAG/ 2.175 (mmol/L)
Estimation of Plasma Lipids FRIEDEWALD EQUATION/CALCULATION
mg/dL
LDL = TC – HDL – Plasma TAG/5 (mg/dL)
It is recommended that lipoprotein measurements be made no sooner than________ after any form of trauma or acute bacterial or viral infection, and_______ after childbirth.
8 weeks
3 to 4 months
________ can be used when only cholesterol, TG, and HDL are measured
Plasma or serum
In frozen samples =
TC, TAG, and HDL can be easily presereved
Frozen samples are not appropriate for
ultra-centrifugal analysis
continues to be the reference method for cholesterol used by the Centers for Disease Control and prevention or (CDC)
Abell-Kendel method
Cholesterol measurement
This method can be accurate within about 0.5% of true value.
Enzymatic methods
can be read photometrically at
500nm(Red Color)
Quinoneimine dye
CHEMICAL METHODS FOR TAG
• Van Handel
• Zilversmit Method
Measurement for TAG
Limits interfering substances
CDC - reference method
TAG measurement
CDC - reference method
- Extraction of TAG by_____
- Isolation of TAG by_____
- Release of glycerol by____
chloroform
salicylic acid chromatography
saponification
TAG Measurement
+ H2S04 =_____ END COLOR
PINK
Triglycerides
(Enzyme)
Glycerol + Fatty acids
Lipase
2H1,0, + Phenol +4-aminoantipyrine
(Enzyme)
Quinoneimine dye + 4H,O
Peroxidase
ADP + Phosphoenol pyruvate
(Enzyme)
- ATP + Pyruvate
Pyruvate kinase
Pyruvate + NADH + H+
(Enzyme)
- › Lactate + NAD*
Lactate dehydrogenase
HDL-CHOLESTEROL MEASUREMENT
- Precipitating Agents
• Earliest -_______ in combination with_____ to precipitate apo B-containing LPP
heparin
manganese
Precipitating agents
Alternative - _______with magnesium
sodium phosphotungstate
Precipitating agents
More specific -______ with magnesium
Dextran sulfate
HDL-CHOLESTEROL MEASUREMENT
Interfering factor:
elevated TAG levels
HDL-CHOLESTEROL MEASUREMENT
- assay for cholesterol content
Clear supernate
Gold standard / Reference Method
- 3-Step Procedure
- Ultracentrifugation - to remove_____
- Heparin Manganese Precipitation - to remove____
- Analysis of supernatant cholesterol by the_______
VLDL
LDL
Abell-Kendall Assay