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Laboratory Methodology (TOPIC 1) Flashcards

1
Q

PARAMETERS USED IN THE DIAGNOSIS OF LIPOPROTEIN DISORDERS

A
  1. 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
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2
Q

neutral fats because fatty acids are tied up in ester linkages

A

Triglycerides

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3
Q

depot fat or storage material of adipose tissue and main form of lipid storage in man

A

Triglycerides

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4
Q

major constituents of chylomicrons and prebeta lipoprotein

A

Triglycerides

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5
Q

higher value in serum than in plasma

A

Triglycerides

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6
Q

also known as “triacyl glycerol”

A

Triglycerides

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7
Q

Triglycerides also known as

A

“triacyl glycerol”

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8
Q

Time for fasting for triglycerides

A

12-14 hours

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9
Q

what are the Types of measurement of Triglycerides

A

Chemical Method
Hantzsh/Lutidine reaction
Van Handel and Zilversmit

Enzymatic Method
Weiland Method
Trinder’s Method
Eggstan and Kreutz Method

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10
Q

Chemical Method
Step 1: Extraction

What is the process

A

Removal of lipid from protein by treatment of organic solvent such as CHLOROFORM, ISOPROPANOL, ETHER

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11
Q

Chemical Method
Step 2: Purification by adsorption

What is the process

A

Removal of
PHOSPHOLIPID
MONOGLYCERIDES
DIGLYCERIDES
GLUCOSE
BILIRUBIN

by addition of
SILICIC ACID
ZEOLITE
FLOROSIL

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12
Q

Chemical Method
Step 3: Hydrolysis or saponification

What is the process

A

Breaking down of
TRIGLYCERIDES into GLYCEROL and FATTY ACIDS

by addition of
KOH or SODIUM METHOXIDE

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13
Q

Chemical Method
Step 4: Oxidation

What is the process

A

Conversion of
GLYCEROL to FORMALDEHYDE and FORMIC ACID (measurable compound)

by SODIUM PERIODATE

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14
Q

Chemical Method
Step 5: Colorimetric measurement

What is the process

A

FORMALDEHYDE is added with color reagents and absorbance is measured in spectrophotometer.

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15
Q

Mostt common chemical method

A

Hantzsh/Lutidine Reaction

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16
Q

Hantzsh/Lutidine Reaction principle

A

Formaldehye + Acetylacetone + NH4 — 3,5 diacetyl-1,4-dihydrolutidine (YELLOW at 410nm)

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17
Q

Van Handel and Zilversmit principle

A

Formaldehyde + H2SO4 + CTA (chromotropic acid) — pink chromopore

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18
Q

Triglyceride chemical method color reagents

A

Chromotrophic acid and sulfuric acid
Diphenylhydrazone
Acetylacetone and ammonia

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19
Q

Chromotrophic acid and sulfuric acid

Color and measured at?

A

pink color

500-600nm

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20
Q

Diphenylhydrazone

measured at?

A

500-600 nm

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21
Q

Acetylacetone and ammonia

Color and measured at?

A

yellow color

412nm

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22
Q

Triglyceride Enzymatic method
Initial Reaction

A

Triglyceride –(Lipase)–> glycerol + 3 fatty acids

Glycerol + ATP –(Glycerol Kinase)–> Glycerol phosphate + ADP

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23
Q

Weiland Method Principle

A

Glycerol phosphate + NAD –(Glycerophosphate dehydrogenase)–> dihydroacetone phosphate + NADH + H

NADH + tetrazolium dye –(Diaphorase)–> formazan + NAD

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24
Q

Weiland Method: The absorbance of NADH can be measured at ___ nm after GCPD reaction.

The absorbance of formazan can be measure at ___ nm

A

340

500-600

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25
Trinders Method Principle Triglyceride enzymatic method
Glycerophosphate + O2 --(Glycerophosphate oxidase)--> dihydroacetone + H2O2 H2O2 + phenol + 4-aminoantipyrine --(Peroxidase)--> quinoneimine dye
26
Trinders Method: Quinonimine dye is measured at ___ nm
500-505
27
EGGSTAN AND KREUTZ METHOD Principle
ADP + phosphoenol pyruvate --(Pyruvate Kinase)--> ATP + Pyruvate Pyruvate + NADH + H --(Lactate Dehydrogenase)--> Lactate + NAD
28
EGGSTAN AND KREUTZ METHOD: NAD is measured at __ nm
340
29
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
30
Cholesterol Determination Chemical Methods
Zlatkis, Zak, Boyle Carr-Drekter Abell-Kendall Babson Schoenheimer-Sperry Lieberman-Burchard reaction Salkowski reaction
31
Cholesterol Determination Enzymatic Method
Trinders reaction Oxygen consumption
32
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
33
Cholesterol Chemical Method What is the Procedure for the Method Zlatkis, Zak, Boyle
One step method (colorimetry)
34
Cholesterol Chemical Method What is the Procedure for the Method Carr-Drekter
Two step method Extraction-Colorimetric
35
Cholesterol Chemical Method What is the Procedure for the Method Abell-Kendall Babson
Three step method (Saponification-Extraction-Colorimetric)
36
Cholesterol Chemical Method What is the Procedure for the Method Schoenheimer-Sperry
Four-Step Method (Hydrolysis-Extraction-Precipitation-Colorimetric)
37
Cholesterol :One step method (colorimetric) What are the 2 reactions
Lieberman-Burchard Reaction Salkowski Reaction
38
Lieberman-Burchard reaction Principle Result Stability
Cholesterol(from chloroform extract of serum) + acetic anhydride + sulfuric acid Cholestapolyene sulfonic acid (GREEN) Less Stable
39
Salkowski Reaction Principle Result Stability
chole + HAc + FE(III) Cholesapolyene carbonium ion (RED PURPLE) More Stable
40
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
41
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
42
Trinders Reaction CHOLESTEROL
1. Cholesterol ester + H2O (CHOLESTEROL ESTER HYDROLASE) = Free cholesterol + Fatty acids 2. Free Cholesterol + O2 (CHOLESTEROL OXIDASE) = cholestene-3-one + H2O2 3. H2O2 + phenol + aminoantipyrine (PEROXIDASE) = Quinoneimine Dye (PINK) 4. Color produces measured at 500nm
43
Trinders Reaction TRIGLYCERIDES complete
1. Triglycerids (LIPASE) = Glycerol + 3 Fatty acids 2. Glycerol + ATP (GLYCEROL KINASE) = glycerol phosphates + ADP 3. Glycerophosphate + O2 (GLYCEROPHOSPHATE OXIDASE)= dihydroacetone + H2O2 4. H2O2 + phenol + 4-aminoantipyrine (PEROXIDASE) = quinoneimine dye + 2H2O2 5. Dye measured at 500-505nm
44
Cholesterol Values Desirable Borderline High Risk
<200 mg/dl 200-239 mg/dl >240 mg/dl
45
Methods for HDL cholesterol
Precipitation method Immunoassays
46
Precipitation method for HDL cholesterol (DEFINITION)
Precipitating reagent is added to precipitate VLDL and LDL, supernatant is for HDL
47
Immunoassays for HDL cholesterol
ELISA Immunonephelometric
48
HDL-cholesterol values Male Female High risk of heart disease
28-62 37-77 <35 mg/dl
49
Friedwald Formula
LDL-C mg/dl = TC - HDL - C (Triglyceride/5)
50
Gold standard of LDL quantification
Ultracentrifugation
51
Normal Values for LDL-Cholesterol Desirable Borderline High-Risk High Risk
<130 mg/dl 130-159 mg/dl >160 mg/dl
52
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
53
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
54
Fatty Acids Normal Value Adult Children and obese
0.3-0.9 mmol/l >1.1 mmol/l
55
Fatty acids methods
Gas-layer chromatography (GLC) Dole titration method
56
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
57
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
58
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
59
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
59
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
60
used to diagnose dysbetalipoproteinemia and familial combined hyperlipidemia.
Apolipoprotein B analysis
60
Requested in addition to lipid profile test
Apolipoprotein B analysis
61
Specimen for Apolipoprotein B analysis
Non-Fasting Serum
62
Apolipoprotein B analysis Value for high risk of Cardiovascular Disease (CVD)
> or = 130 mg/dl

CC 1 Midterms (14 decks)

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