Laboratory Methodology (TOPIC 1) Flashcards
PARAMETERS USED IN THE DIAGNOSIS OF LIPOPROTEIN DISORDERS
- Lipid Profile test
a. Triglycerides
b. Total Cholesterol
i.HDL-cholesterol determination
ii. LDL-cholesterol determination
2.Plasma Appearance Test
3.Lipoprotein electrophoresis
4.Ultracentrifugation
neutral fats because fatty acids are tied up in ester linkages
Triglycerides
depot fat or storage material of adipose tissue and main form of lipid storage in man
Triglycerides
major constituents of chylomicrons and prebeta lipoprotein
Triglycerides
higher value in serum than in plasma
Triglycerides
also known as “triacyl glycerol”
Triglycerides
Triglycerides also known as
“triacyl glycerol”
Time for fasting for triglycerides
12-14 hours
what are the Types of measurement of Triglycerides
Chemical Method
Hantzsh/Lutidine reaction
Van Handel and Zilversmit
Enzymatic Method
Weiland Method
Trinder’s Method
Eggstan and Kreutz Method
Chemical Method
Step 1: Extraction
What is the process
Removal of lipid from protein by treatment of organic solvent such as CHLOROFORM, ISOPROPANOL, ETHER
Chemical Method
Step 2: Purification by adsorption
What is the process
Removal of
PHOSPHOLIPID
MONOGLYCERIDES
DIGLYCERIDES
GLUCOSE
BILIRUBIN
by addition of
SILICIC ACID
ZEOLITE
FLOROSIL
Chemical Method
Step 3: Hydrolysis or saponification
What is the process
Breaking down of
TRIGLYCERIDES into GLYCEROL and FATTY ACIDS
by addition of
KOH or SODIUM METHOXIDE
Chemical Method
Step 4: Oxidation
What is the process
Conversion of
GLYCEROL to FORMALDEHYDE and FORMIC ACID (measurable compound)
by SODIUM PERIODATE
Chemical Method
Step 5: Colorimetric measurement
What is the process
FORMALDEHYDE is added with color reagents and absorbance is measured in spectrophotometer.
Mostt common chemical method
Hantzsh/Lutidine Reaction
Hantzsh/Lutidine Reaction principle
Formaldehye + Acetylacetone + NH4 — 3,5 diacetyl-1,4-dihydrolutidine (YELLOW at 410nm)
Van Handel and Zilversmit principle
Formaldehyde + H2SO4 + CTA (chromotropic acid) — pink chromopore
Triglyceride chemical method color reagents
Chromotrophic acid and sulfuric acid
Diphenylhydrazone
Acetylacetone and ammonia
Chromotrophic acid and sulfuric acid
Color and measured at?
pink color
500-600nm
Diphenylhydrazone
measured at?
500-600 nm
Acetylacetone and ammonia
Color and measured at?
yellow color
412nm
Triglyceride Enzymatic method
Initial Reaction
Triglyceride –(Lipase)–> glycerol + 3 fatty acids
Glycerol + ATP –(Glycerol Kinase)–> Glycerol phosphate + ADP
Weiland Method Principle
Glycerol phosphate + NAD –(Glycerophosphate dehydrogenase)–> dihydroacetone phosphate + NADH + H
NADH + tetrazolium dye –(Diaphorase)–> formazan + NAD
Weiland Method: The absorbance of NADH can be measured at ___ nm after GCPD reaction.
The absorbance of formazan can be measure at ___ nm
340
500-600
Trinders Method Principle
Triglyceride enzymatic method
Glycerophosphate + O2 –(Glycerophosphate oxidase)–> dihydroacetone + H2O2
H2O2 + phenol + 4-aminoantipyrine –(Peroxidase)–> quinoneimine dye
Trinders Method: Quinonimine dye is measured at ___ nm
500-505
EGGSTAN AND KREUTZ METHOD
Principle
ADP + phosphoenol pyruvate –(Pyruvate Kinase)–> ATP + Pyruvate
Pyruvate + NADH + H –(Lactate Dehydrogenase)–> Lactate + NAD
EGGSTAN AND KREUTZ METHOD: NAD is measured at __ nm
340
Normal Values of Triglycerides
Acceptable
Borderline
High Risk
and Coversion factor to mmol/l
<250 mg/dl
250-500 mg/dl
>500 mg/dl
0.011
Cholesterol Determination
Chemical Methods
Zlatkis, Zak, Boyle
Carr-Drekter
Abell-Kendall
Babson
Schoenheimer-Sperry
Lieberman-Burchard reaction
Salkowski reaction
Cholesterol Determination
Enzymatic Method
Trinders reaction
Oxygen consumption
General Steps in Cholesterol Chemical Method
Extraction - cholesterol is seperated from protein
Saponification - cholesterol ester is broken down to free cholesterol and fatty acids
Purification - cholesterol is precipitated
Colorimetry - addition of color reagent to be measure in spectrophotometer
Cholesterol Chemical Method
What is the Procedure for the Method
Zlatkis, Zak, Boyle
One step method
(colorimetry)
Cholesterol Chemical Method
What is the Procedure for the Method
Carr-Drekter
Two step method
Extraction-Colorimetric
Cholesterol Chemical Method
What is the Procedure for the Method
Abell-Kendall Babson
Three step method
(Saponification-Extraction-Colorimetric)
Cholesterol Chemical Method
What is the Procedure for the Method
Schoenheimer-Sperry
Four-Step Method
(Hydrolysis-Extraction-Precipitation-Colorimetric)
Cholesterol :One step method (colorimetric)
What are the 2 reactions
Lieberman-Burchard Reaction
Salkowski Reaction
Lieberman-Burchard reaction
Principle
Result
Stability
Cholesterol(from chloroform extract of serum) + acetic anhydride + sulfuric acid
Cholestapolyene sulfonic acid (GREEN)
Less Stable
Salkowski Reaction
Principle
Result
Stability
chole + HAc + FE(III)
Cholesapolyene carbonium ion (RED PURPLE)
More Stable
Cholesterol Enzymatic Method
Trinders Reaction Principle
Measure at ___ nm
Cholesterol ester + H2O –(Cholesteryl ester hydrolase)–> free cholesterol + fatty acids
Free cholesterol + O2 –(Cholesterol Oxidase)–> Cholestene-3-one + H2O2
H2O2 + Phenol + aminoantipyrine –(peroxidase)–> quinoneimine dye (Pink)
500
Cholesterol Enzymatic Method
Oxygen Consumption Principle
Cholesterol ester + H2O –(Cholesteryl ester hydrolase)–> free cholesterol + fatty acids
Free cholesterol + O2 –(cholesterol oxidase)–> Cholestene-3-one + H2O2
H2O2 + Peroxidase –(Peroxidase)–> O2 + H2O
O2 electrode the O2 releases from H2O2 decomposition
Trinders Reaction CHOLESTEROL
- Cholesterol ester + H2O (CHOLESTEROL ESTER HYDROLASE) = Free cholesterol + Fatty acids
- Free Cholesterol + O2 (CHOLESTEROL OXIDASE) = cholestene-3-one + H2O2
- H2O2 + phenol + aminoantipyrine (PEROXIDASE) = Quinoneimine Dye (PINK)
- Color produces measured at 500nm
Trinders Reaction TRIGLYCERIDES
complete
- Triglycerids (LIPASE) = Glycerol + 3 Fatty acids
- Glycerol + ATP (GLYCEROL KINASE) = glycerol phosphates + ADP
- Glycerophosphate + O2 (GLYCEROPHOSPHATE OXIDASE)= dihydroacetone + H2O2
- H2O2 + phenol + 4-aminoantipyrine (PEROXIDASE) = quinoneimine dye + 2H2O2
- Dye measured at 500-505nm
Cholesterol Values
Desirable
Borderline
High Risk
<200 mg/dl
200-239 mg/dl
>240 mg/dl
Methods for HDL cholesterol
Precipitation method
Immunoassays
Precipitation method for HDL cholesterol (DEFINITION)
Precipitating reagent is added to precipitate VLDL and LDL, supernatant is for HDL
Immunoassays for HDL cholesterol
ELISA
Immunonephelometric
HDL-cholesterol values
Male
Female
High risk of heart disease
28-62
37-77
<35 mg/dl
Friedwald Formula
LDL-C mg/dl = TC - HDL - C (Triglyceride/5)
Gold standard of LDL quantification
Ultracentrifugation
Normal Values for LDL-Cholesterol
Desirable
Borderline High-Risk
High Risk
<130 mg/dl
130-159 mg/dl
>160 mg/dl
Phospholipids Chemical Method
Extraction
Oxidation- phospholipid phosphorus is converted to inorganic phosphorus
Colorimetry - inorganic phosphorus + molybdate blue = molybdenum blue
Total phosphorus X 25= phospholipid mass
Phospholipids Enzymatic Method
Phospholipase D- phospholipid is hydrolyzed to choline
Choline oxidase- choline is oxidized to betaine and H2O2
Peroxidase –H2O2 is added with phenol and 4-aminoantipyrine = quinonimine dye
Color produced is measured at 500 nm
Fatty Acids Normal Value
Adult
Children and obese
0.3-0.9 mmol/l
>1.1 mmol/l
Fatty acids methods
Gas-layer chromatography (GLC)
Dole titration method
GUIDELINES TO REDUCE RISK OF CORONARY HEART DISEASE(From the National Cholesterol Education Program)
Cholesterol (mg/dl)
Desirable
Borderline
High Risk
<200
200-239
> or = 240
GUIDELINES TO REDUCE RISK OF CORONARY HEART DISEASE(From the National Cholesterol Education Program)
Triglycerides (mg/dl)
Desirable
Borderline
High Risk
<200
200-499
> or = 500
GUIDELINES TO REDUCE RISK OF CORONARY HEART DISEASE(From the National Cholesterol Education Program)
Chol/HDL Ratio
Desirable
Borderline
High Risk
<3.9
4.0-4.9
> or = 5.0
GUIDELINES TO REDUCE RISK OF CORONARY HEART DISEASE(From the National Cholesterol Education Program)
LDL-Chol (mg/dl)
Desirable
Borderline
High Risk
<130
130-159
> or = 160
GUIDELINES TO REDUCE RISK OF CORONARY HEART DISEASE(From the National Cholesterol Education Program)
HDL-Chol (mg/dl)
Desirable
Borderline
High Risk
> 40
35-40
<35
used to diagnose dysbetalipoproteinemia and familial combined hyperlipidemia.
Apolipoprotein B analysis
Requested in addition to lipid profile test
Apolipoprotein B analysis
Specimen for Apolipoprotein B analysis
Non-Fasting Serum
Apolipoprotein B analysis
Value for high risk of Cardiovascular Disease (CVD)
> or = 130 mg/dl
