LAB Exam 1 Flashcards
What are the different methods for testing glucose?
- Oxidation Reduction
- Condensation reaction
- Enzymatic reactions (3 types)
What are the three glucose enzymatic reactions?
- Hexokinase (reference method)
– Glucose oxidase-colorimetric (Influenced by heparin)
– Glucose oxidase- polargraph (Influenced by heparin)
-Type of glucose Redox reaction that is not often used.
-Copper sulfate + Glucose –> Copper II oxide + Glucose-Na
-(In alkaline conditions & heat)
Benedicts- semi quantitative
-Glucose redox reaction
-Copper sulfate + Glucose —–> Copper II oxide + Glucose-Na
-Copper II oxide + phosphomolybdate —-> molybdenum blue
Folin Wu
Folin Wu reaction:
protein precipitation is….
Tungstic acid
Glucose redox reaction that uses Zinc sulfate and barium hydroxide
Somogyi Nelson
-Glucose redox reaction
-Ferric cyanide + Glucose —-> Ferrous cyanide (red color)
Automated Hagedorn Jensen
Condensation Reaction:
___________ condenses w/ Glucose in presence of Glacial acetic (hot)
producing blue green color (630 nm)
Ortho Toluidine
The condensation reaction for glucose can react with other aldoses like….
galactose and mannose
What kind of reactions are the most commonly used for testing glucose?
Enzymatic Reactions
Enzymatic Reaction:
Glucose + ATP ! G-6-P + ADP
G-6-P + NADP+ —> NADPH + 6-phosphogluconase
Hexokinase (reference method)
Enzymatic Reaction:
-Glucose + O2 —-> gluconic acid + H2O2
-H2O2 + Chromogenic peroxidase —-> Color + H20
Glucose Oxidase-colorimetric method (influenced by heparin)
-what we use in lab
Enzymatic Reaction:
Glucose + O2 —-> gluconic acid + H2O2
Glucose Oxidase- Polarography (influenced by heparin)
-needs electric current
What are the four lipid tests?
- TRIGLYCERIDES
- CHOLESTEROL
- HDL ASSAYS
- PHOSPHOLIPIDS
Why is TG testing done?
To calculate LDL using [LDL= total cholesterol-HDL-VLDL]
“ Friedewald equation”
-Assessment for CHD risk
-Determine whether increased TG are depressing HDL
High TG increases the risk of…
pancreatitis & numerous other clinical manifestations
How is TG and HDL related?
inversely related
What are the assays done for TG?
✓Hantzsch condensation
✓Sulfuric acid reaction
✓Pyruvate kinase/lactate dehydrogenase
✓Glycerol phosphate dehydrogenase/Diaphorase
What is the most frequently used “chemical” reaction for TG?
HANTZSCH CONDENSATION
Hantzsch condensation involves quantification of ________ using multiple steps
❖Organic extraction of TG
❖Chemical hydrolysis of TG to glycerol & FFA
❖Oxidation of glycerol to formaldehyde
glycerol
formaldehyde condenses with NH4+-Ac & acetylacetone to
form product which is measured between _____ and _____nm.
400 and 485nm
product measured is 3,5-DIACETYL-1,4-DIHYDROTOLUDINE
What are the first three steps of the Sulfuric acid reaction?
extraction, acidic/alkaline hydrolysis, & oxidation of glycerol
Sulfuric acid reaction:
utilizes chromotropic acid & sulfuric acid to produce a colored product upon reaction with formaldehyde
Eegriwe’s reaction
TRIGLYCERIDES —> GLYCEROL + 3 FATTY ACIDS (LIPASE)
GLYCEROL + ATP —> GLYCEROL-3-PHOSPHATE + ADP (GLYCEROL KINASE)
ADP + PEP —-> ATP + PYRUVATE (PYRUVATE KINASE)
PYRUVATE + NADH + H+ —-> LACTATE + NAD+ (LACTATE DEHYDROGENASE)
PYRUVATE KINASE/LACTATE DEHYDROGENASE
-frequently used assay
-enzymatic method for quantifying glycerol is multistep and measures NADH consumption
PYRUVATE KINASE/LACTATE DEHYDROGENASE:
NADH is measured at _____ nm.
340
PYRUVATE KINASE/LACTATE DEHYDROGENASE:
What are the advantages?
1) Rapid and specific
2) Eliminates the need for extraction and caustic solvents
PYRUVATE KINASE/LACTATE DEHYDROGENASE:
What are the disadvantages?
1) Instability of enzymes and reagents in working solution.
2) Serum blank must be included to subtract nonspecific absorption of various serum
components.
PYRUVATE KINASE/LACTATE DEHYDROGENASE:
When is a serum blank needed?
How are results calculated?
Needed when sample: Hemolysis, Icteric, lipemic conditions
water blank results - sample blank results = true O.D.
enzymatic method which employs the use of the same first two steps as the NADH consumption method.
FORMAZAN (COLORIMETRIC)
GLYCEROL-3-PHOSPHATE + NAD+ —> DHAP + NADH (GLYCEROL PHOSPHATE DEHYDROGENASE)
NADH + OXIDIZED TETRAZOLIUM —-> REDUCED TETRAZOLIUM (DIAPHORASE)
FORMAZAN (COLORIMETRIC)
employs only lipase/protease step to produce glyceride glycerol
FLUORIMETRIC METHOD
GLYCEROL-3-PHOSPHATE + NAD+ —> DHAP + NADH (GLYCEROL PHOSPHATE DEHYDROGENASE)
NADH + RESAZURIN —-> RESORUFIN (DIAPHORASE)
FLUORIMETRIC METHOD
TG:
Plasma or serum samples must be from fasting individuals that have not ingested ALCOHOL for greater than ____ hours
36
_______ TG values are 2 to 4% lower than serum because of dilution effect of
drawing water from red blood cells by the anticoagulant.
Plasma
-remove the RBC from the plasma immediately
What is the anticoagulant of choice for TG testing?
What should not be used?
EDTA
Do not use glycerin coated vacutainers & care should be taken to avoid hand and
body creams containing glycerol
What may interfere with TG assays?
Hemolysis, icterus and lipemia
What is the sample storage for TG testing?
-samples should be analyzed ASAP
-or freeze at -60 degrees Celsius
What are the cholesterol methods?
- LIEBERMAN BURCHARD
- SALKOWSKI
- CHOLESTEROL OXIDASE/PEROXIDASE
- Described in 1885 (modified in 1889)
- Common method and still in use today
LIBERMAN BURCHARD
Cholesterol Extraction,
* React with sulfuric acid & acetic anhydride [sequential oxidation of cholesterol]
* yields a blue-green cholesta-hexene-sulfonic acid
LIBERMAN BURCHARD
LIBERMAN BURCHARD:
FREE CHOLESTEROL=
TOTAL CHOLESTEROL - ESTERIFIED CHOLESTEROL
LIBERMAN BURCHARD:
What are the disadvantages?
1) DIFFERENT ESTERIFIED CHOLESTEROLS YIELD INCREASED COLOR INTENSITIES OF THE
CHROMAGENS.
2) INTERFERENCES INCLUDE BILIRUBIN & UNREACTED DIGOXIN
-Chemical reaction first described in 1872
* Sample is extracted with chloroform
-Addition of sulfuric acid changes the solution from BLUISH RED to VIOLET RED
CHOLESTEROL: SALKOWSKI
Salkowski:
addition of sulfuric acid changes the solution from __________ to _________.
bluish red, violet red
Ferric chloride in Sulfuric acid was added to the Cholesterol in Acetic acid
Zlatkis and Boyle (1953) automated procedure
Zlatkis and Boyle (1953) automated procedure:
Ferric chloride in Sulfuric acid was added to the Cholesterol in Acetic acid to give…
a more intense and stable magenta color
‣ Modification is 7x more sensitive than the Burchard method
What is the most popular and accurate method for measuring cholesterol?
CHOLESTEROL OXIDASE / PEROXIDASE
CHOLESTEROL OXIDASE / PEROXIDASE:
ESTERIFIED CHOLESTEROL –> CHOLESTEROL + FA (__________)
CHOLESTEROL + O2 —> CHOLEST-4-EN-3-ONE + PEROXIDE (__________)
PEROXIDE + PHENOL + 4-AMINOPHENAZONE —> QUINOEIMINE + H2O
CHOLESTEROL ESTERASE
CHOLESTEROL OXIDASE
CHOLESTEROL OXIDASE / PEROXIDASE:
advantages?
INCREASED SPECIFICITY & NO HARSH REAGENTS
CHOLESTEROL OXIDASE / PEROXIDASE:
disadvantages?
PEROXIDASE REACTION IS INHIBITED BY BILIRUBIN & SURFACTANTS
What specimen type is used for cholesterol testing?
anticoagulant?
Serum or plasma
EDTA
-Others tend to cause large water shifts from the RBC to
the plasma
Cholesterol testing:
Should it be a fasting sample?
-12 hour fasting sample suggested
-Cholesterol NOT directly affected by diet, except
in patients with very high TG
What is important during the cholesterol sample draw?
-Posture is important during the draw because there can be as much as a 10 to 15% decrease after patient switches from standing to reclined position
- Prolonged tourniquet use increases lipids.
What is the sample storage for cholesterol?
- PERFORM ASSAYS AS SOON AS POSSIBLE
- IF THERE IS A DELAY, SAMPLE MUST BE FROZEN AND STORED AT -60°C (VORTEX SAMPLE WELL UPON THAWING)
HDL cholesterol is quantified by what two methods?
- APOLIPOPROTEIN B PRECIPITATION
- ELECTROPHORESIS
- Lipoproteins are precipitated using low-density apoB lipoproteins (except HDL) with ________ solutions
polyanion
HDL- APO B PRECIPITATION:
agents include…
▪ HEPARIN-MANGANESE CHLORIDE
▪ DEXTRAN SULFATE- MAGNESIUM CHLORIDE
▪ SODIUM PHOSPHOTUGSTATE
▪ POLYETHYLENE GLYCOL
▪ VARIATION-DEXTRAN-SULFATE WITH IRON ATTACHED (PPT W/MAGNET)
HDL- APO B PRECIPITATION:
What is the reference method?
HEPARIN-MANGANESE CHLORIDE
HDL- APO B PRECIPITATION:
Good Specificity for HDL
DEXTRAN SULFATE- MAGNESIUM CHLORIDE
HDL- APO B PRECIPITATION:
Problem w/ Reagent Stability
SODIUM PHOSPHOTUGSTATE