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
HDL- APO B PRECIPITATION:
Problems w/ Accuracy
POLYETHYLENE GLYCOL
HDL- APO B PRECIPITATION:
advantages?
SIMPLE, FAST, AND INEXPENSIVE
HDL- APO B PRECIPITATION:
disadvantages?
TENDENCY TO UNDERESTIMATE HDL
What are the HDL-CHOLESTEROL: ELECTROPHORESIS methods?
❖ STARCH BLOCK & GEON-PEVIKON BLOCK ELECTROPHORESIS
❖ AGAROSE GEL ELECTROPHORESIS
❖ POLYACRYLAMIDE GEL ELECTROPHORESIS
- SEPARATION OF LIPOPROTEINS BASED ON SIZE & NET CHARGE
✓HDL MIGRATES THE FASTEST
✓USED FOR ISOLATING LARGE QUANTITIES OF HDL BUT RARELY USED CLINICALLY
STARCH BLOCK & GEON-PEVIKON BLOCK ELECTROPHORESIS
✓SAME PRINCIPLE AS BLOCK ELECTROPHORESIS, but WITH A DIFFERENT RESOLUTION MATRIX
* AFTER SEPARATION, AGAROSE GEL OVERLAYED WITH ENZYME REAGENTS FOR DETECTING
CHOLESTER. AGAROSE STRIPS ARE SCANNED USING DENSITOMETER
* NOT USED FOR ROUTINE ANALYSIS BECAUSE OF LOW RESOLUTIONS
AGAROSE GEL ELECTROPHORESIS
❖DATA WITH THIS TECHNIQUE CORRELATES WELL WITH ULTRACENTRIFUGATION
POLYACRYLAMIDE GEL ELECTROPHORESIS
Specimen collection and handling for HDL cholesterol?
- Patient should fast for 12 hours, although in most cases fasting does not affect HDL cholesterol
- All other conditions are the same as for total cholesterol
What are the two types of liver function tests?
- Intravascular injection of dye (indocyanine green) that is processed solely through
liver (bile).
i. Serum clearance of dye monitored - Injection of drugs to monitor liver’s ability to metabolize drugs
- Appearance of modified drugs in serum or urinary excretion
Intravascular injection of dye liver test:
Normal is ____ % dye remaining after 45 minutes.
<5
Intravascular injection of dye liver test:
Higher retention times are indicative of…
hepatocellular disease or obstructive liver disease.
Why is Bromsulphalein (BSP) no longer used for intravascular injections?
due to “ANAPHYLAXIS”
Analytes for liver function tests?
▪ Enzymes
▪ Bilirubin
▪ Cholesterol
▪ Bile acids
▪ Serum proteins
▪ Urea and ammonia
Analyte that is helpful in differentiating the cause of jaundice*
BILIRUBIN
- 1) increased unconjugated bilirubin due to hemolysis; 2) serum bilirubin may be slightly increased, but liver will excrete large amounts of bile. 3) Increased urobilinogen in urine and feces. Urinary bilirubin will be absent.
Pre hepatic cause of jaundice
intermediate pattern, 1) unconjugated & conjugated bilirubin increased.
2) Fecal concentration of urobilinogen decreased but 3) conjugated bilirubin
increased in urine.
Hepatic cause of jaundice
-Hepatocellular damage / Bile excretion defect
1) increased conjugated bilirubin; serum conjugated bilirubin increased because of decreased biliary excretion. 2) Urobilinogen is decreased but bilirubin can now be detected in urine.
Post hepatic cause of jaundice
BILIRUBIN METHODOLOGY, what are the different types?
▪ Evelyn-Mallory
▪ Jendrassik-Groff
▪ Bilirubin oxidase
▪ HPLC
▪ Jackson Bilirubinometer
▪ Urine Bilirubin
▪ Bilirubin in amniotic fluid
Van den Bergh first discovered that bilirubin in serum reacted with ________ reagents
diazo
Reactions conducted directly on serum were hence called “direct” bilirubin measurements
(actually measures ____________ bilirubin and δ-bilirubin)
hydrophilic conjugated
1883: Ehrich: Bilirubin (Urine) + ____________ —-> color
Diazotized Sulfanilic acid
- Unconjugated bilirubin (associated with albumin) required an “accelerator” (ex. Methanol) to dissociate from protein and solubilize bilirubin. Bilirubin that requires solvent is known as…
“indirect” bilirubin
Bilirubin + diazotized sulfanilic acid —-> 2 molecules azobilirubin
BILIRUBIN METHODOLOGY: EVELYN-MALLORY
“Direct” reaction performed at pH 1.2-azobilirubin (red purple) absorbs at 560nm in absence of solvent
BILIRUBIN METHODOLOGY: EVELYN-MALLORY
EVELYN-MALLORY:
Total measurement conducted in presence of…
Methanol, urea, or DMSO
Total – “direct” = “indirect”
What are the interferences with EVELYN-MALLORY method?
hemoglobin
What is the modified test of Evelyn-Mallory?
What is different?
JENDRASSIK-GROFF
“Direct” reaction carried out at pH 6.5 (NaOH used to alkalinize reaction) yields more intense blue color at 600nm
JENDRASSIK-GROFF:
Total measurement conducted in presence of…
sodium benzoate, caffeine, methanol, urea or DMSO.
JENDRASSIK-GROFF:
Total, indirect….
Serum (plasma) + Sodium acetate with caffeine-sodium benzoate
Enzymatic method that uses bilirubin oxidase to catalyze oxidation of bilirubin to biliverdin
BILIRUBIN OXIDASE
What absorbance is Bilirubin oxidase testing measured at?
decreased absorbance monitored at 405-460 nm
BILIRUBIN OXIDASE:
Total bilirubin measured upon addition of detergent (____ or _______) added to
dissociate bilirubin
SDS or Na-cholate
-pH of reaction manipulated to monitor conjugated bilirubin.
▪ Relatively new method, with promise of improved specificity and high precision
BILIRUBIN OXIDASE
What does HPLC stand for?
What is it used for?
High Pressure Liquid Chromatography developed to separate methyl esters of
conjugated & unconjugated bilirubin.
HPLC:
Column elute is measured at ____ nm
430
Only means for accurate quantitation of bilirubin fractions.
Interfering compounds are removed.
HPLC
HPLC:
α:
β:
α: unconjugated
β: monoglucuronide
HPLC:
γ:
δ:
γ: diglucuronide
δ: irreversibly bound to albumin
Direct spectrophotometric measurement of bilirubin at 454 nm
JACKSON BILIRUBINOMETER
JACKSON BILIRUBINOMETER:
What is the drawback?
HbO2 absorbs at 454 nm, but HbO2 also absorbs at 540 nm
Bilirubin level obtained by subtracting HbO2 contribution.*
Good screening method for neonates to 3 mo.; do not have significant carotenoids
JACKSON BILIRUBINOMETER
JACKSON BILIRUBINOMETER:
Interference?
carotenoids absorb in same region and falsely elevate bilirubin levels.
In urine the only concern is __________ bilirubin
“direct”
What are the two methods to measure urine bilirubin?
- Dipstick- Ehrlich diazo reagent
- Ictotest- uses p-nitrobenzene diazonium p-toluenesulfonate
What is the most sensitive test to measure urine bilirubin?
Ictotest (2-4 times more sensitive than dipstick)
Conjugated Bilirubin + diazonium salt —-> azobilirubin (brown)
Dipstick- Ehrlich diazo reagent (2,4-dichloroaniline diazonium salt)
Bilirubin in amniotic fluid is measured by ________ spectrophotometric technique
DIRECT
High levels of bilirubin in Amniotic Fluid can indicate…
hemolytic disease
-Most often associated with Rh incompatibility
Specimen storage for bilirubin testing?
Samples are stable in dark for 2 days at RT, 4 days at 4 degree C and indefinitely at -20 °C.
Bilirubin testing:
Specimen should be protected from direct light.
▪ ___________ and ________ may effect determinations.
Hemolysis, lipidemia
Why is serum preferred for bilirubin testing?
proteins in plasma are likely to interfere with Evelyn-Malloy procedure
Infections precipitate ____% of hyperosmolar hyperglycemic states (HHS)
40-60
Major differences between HHS and other metabolic crises associated with diabetes?
-HHS is typically in older pts
-HHS is often precipitated by an infection
-acidosis does not occur in HHS, but still present with severe hyperglycemia
symptoms of HHS and DKA?
HHS- extreme thirst, dry mouth, dehydration
DKA- nausea vomiting
What is the diagnostic criteria for hyperosmolar hyperglycemic state?
-Hyperglycemia- plasma glucose of grater than 600 mg/dl
-plasma osmolality greater than 320 mOsm/kg
-absence of ketoacidosis
-dehydration
-altered mental status
-takes days to weeks to develop
What is the mechanism behind the pseudohyponatremia seen in hyperglycemic hyperosmolar state?
In HHS, blood glucose can be elevated to such a degree that osmolality increases as well. When this occurs, water moves from the intracellular space into the extracellular space, diluting the sodium concentration. In actuality, the concentration of sodium has not changed.
How do you calculate corrected sodium concentrations in the context of significant hyperglycemia?
-Methods such as the ion-selective electrode or using a corrected sodium formula that takes into account the serum glucose level.
Corr. Na+ = [(Glucose – 100 mg/dl) * 0.016] + Na+
HHS is associated with what type of diabetes?
type 2
What enzymes are generally tested to determine liver function?
-Alanine transaminase (ALT)
-Aspartate aminotransferase (AST)
-Alkaline phosphatase (ALP)
-Bilirubin
Released into bloodstream from damaged hepatocytes.
Alanine transaminase (ALT) and Aspartate aminotransferase (AST
Possible indicator of liver disease although can be present from damage to other tissues, like bone or the intestines.
Alkaline phosphatase (ALP)
Waste product produced when RBCs are broken down. Normally removed by the liver from the body through bile excretion into the GI tract. Declining liver function cannot remove bilirubin effectively so hyperbilirubinemia can occur.
Bilirubin
What are the expected values for direct bilirubin in adults?
Adults and infants (over one month): 0 – 0.5 mg/dl
What are the tests done to diagnose diabetes?
A1C, Fasting plasma glucose, Oral glucose tolerance test, and random plasma glucose test
What is the A1C threshold for diabetes mellitus?
6.5% or higher
measures the average blood glucose of the past several months.
A1C
Diabetes diagnosis:
Fasting Plasma Glucose (FPG) that is ____ mg/dl or higher. The patient must fast for a minimum of 8 hours.
126
Diabetes diagnosis:
Oral Glucose Tolerance Test (OGTT) that is ____ mg/dl or higher. This tests how well the patient can process sugar. It is done by testing blood glucose 2 hours before and after drinking a specific sweet drink.
200
Diabetes diagnosis:
Random (Casual) Glucose Test that is ___ mg/dl or higher. This is a blood glucose test at any time of the day.
200
What chronic diseases are related to diabetes?
Cardiovascular disease, stroke, chronic kidney disease, diabetic neuropathy, vision loss.
What are the normal levels for fasting blood triglycerides in adults.
44-148 mg/dl (0.50-1.67 mmol/L)^9 (Pointe Scientific manual)
Hepatitis B
Most common chronic viral infection
42nm DnA virus hepadna virus family
Made from RNA template (reverse transcription)—-> prone to mutation
Mutant prevents
HbeAg
Mutant resistance to Reverse transcription inhibitor
HbsAg is
Common tests for Hep B
AntiHbcAg is also common
For testing Hep B
Liver function tests identify
Liver disease without jaundice
Monitor serum clearance of what
Indocyanine which is solely processed through liver(bile) and normal is 5% dye remaining after 45 minutes
High retention time indicates liver disease
In the past Bromsulphalein was used but
Not today because of anaphylaxis toxicity
Bilirubin in four types
Alpha bilirubin= unconjagated bilirubin
Beta bilirubin= 1 gluconic acid monocojagated bilirubin
Gamma bilirubin= 2 glucocnic acid binding
Delta bilirubin= reversely bound irreversibly to bilirubin
Alkaline phosphatase hydrolyzes
Highest activity in
Hydrolyzes monophosphate esters
Liver bone intestine and kidneys
Placental obstruction increase activity about 10 fold
Hepatocytes damage but no release of
Alkaline phosphatase
Y- glutamyltransferase
Microsomal enzyme induced by drugs or alcohol
Catalysts transfer of glutamate from glutathione to peptide
5’ nucleotidase-
Microsomal enzyme ( not elevated by drugs or alcohol
Activity is solely in obstructive disease similar with AP
5’ nucleotidase can be used to exclude
Bond disease and damage
Lactate dehydrogenase
Cytosolic enzymes lactate-pyruvate
Released after cell damage
Viral/ toxic hepatitis, extra hepatic biliary obstructio, acute hepatic necrosis, and cirrhosis
5 types of lactate dehydrogenase
LD1- heart
LD2- renal disease
LD3- lung
LD4- Skelton muscle
LD5-liver
AST
Aspartate aminotransfersse and ALT alanine transaminase
Converts aspartate and alanine to oxaloacetate and pyruvate respectively
Most useful for detecting liver damage
AST and ALT are also in the heart
Three types of jaundice
Pre hepatic
Hepatic
Post hepatic
Pre hepatic jaundice
Increase unconjugated bilirubin due to hemolysis
Liver will secrete large amount of bile
Increased urobilinogen in urine and feces
Urinary bilirubin is absent
Hepatic jaundice
Uncojugated and conjugated bilirubin increased.
Fecal concentration of urobilinogen decreased
Conjugated bilirubin increase in urine
Post hepatic jaundice
Increased conjugated bilirubin, serum congated bilirubin increased because of decrease in bilirary excretion aka blockage
Urobilinogen is decreased but bilirubin is in urine
