[M1] Enzyme Methodologies Flashcards by Marlo Roque

Liver enzymes is also called as

Hepatic enzymes

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Enumerate the Liver Enzymes

Alkaline Phosphatase
Aspartate Aminotransferase
Alanine Aminotransferase
Gamma-Glutamyltransferase
5’-Nucleotidase

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ALP

ALKALINE PHOSPHATASE

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E.C OF ALP

E.C. 3.1.3.1

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Catalyze the hydrolysis of phosphomonoesters at an alkaline pH (9.0-10.0)

ALKALINE PHOSPHATASE

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ALP catalyzes the hydrolysis of _________________ at an (acidic/alkaline) pH (_____________)

ALP

phosphomonoesters
alkaline
9.0-10.0

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Common name of ALP

alkaline orthophosphoric monoester phosphohydrolase

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ALP

Function:
remove (organic/inorganic) phosphate from an (organic/inorganic) phosphate ester with the concomitant production of ___________

inorganic
organic
alcohol

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T/F: ALP is a non-specific enzyme

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Activators of ALP

Mg2+
Co2+
Mn2+

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inhibitors of ALP

phosphate
borate
oxalate

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ALP is useful for evaluation of ___________ and ________ disorders

hepatobiliary
bone

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RF of ALP in 20-50 MALE

53-128 U/L

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RF of ALP in 20-50 FEMALE

42-98 U/L

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RF of ALP in ≥60 MALE

56-119 U/L

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RF of ALP in ≥60 FEMALE

53-141 U/L

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RF of ALP in 4-15

54-369 U/L

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What are the major ALP isoenzymes

Liver ALP
Bone ALP
Placental ALP
Intestinal ALP

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Half life of Liver ALP

3 days

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Half life of Bone ALP

1 day

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Half life of Placental ALP

7 days

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Half life of Intestinal ALP

1 day

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Rank the ALP isoenzymes from most ANODAL to least ANODAL

Liver ALP
Bone ALP
Placental ALP
Intestinal ALP

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Rank the ALP isoenzymes from most CATHODAL to least CATHODAL

Intestinal ALP
Placental ALP
Bone ALP
Liver ALP

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Rank the ALP isoenzymes from most heat stable to least heat stable

1. Placental ALP 2. Intestinal ALP 3. Liver ALP 4. Bone ALP

Rank the ALP isoenzymes from most heat-labile to least heat-labile

1. Bone ALP 2. Liver ALP 3. Intestinal ALP 4. Placental ALP

inhibitor of Liver ALP

Levamisole

inhibitor of Bone ALP

Levamisole & 3M urea

Inhibitor of Placental ALP

Phenylalanine

Inhibitor of Intestinal ALP

Phenylalanine

BEFORE HEAT Placental ALP Liver ALP Bone ALP

100% 100% 100%

AFTER HEAT Placental ALP Liver ALP Bone ALP

100% >20% <20%

Heat Fractionation/ Stability Test is measured @ ____ for ____ minutes

56°C 10 minutes

T/F: ALP isoenzymes are NOT measured before and after heating

F; Measured before and after heating

separates liver and bone ALP

Neuraminidase Wheat Germ Lectin

Steps in Heat Fractionation/ Stability Test

1. Measure ALP 2. Heat sample @ 56°C for 10 minutes 3. Measure ALP

Heating sample @ 56°C for 10 minutes significance

To inactivate other isoenzymes

Abnormal ALP isoenzyme associated with neoplasms or cancer

Carcinoplacental ALPs

Carcinoplacental ALPs Abnormal ALP isoenzyme associated with __________ or _________

neoplasms cancer

2 types of Carcinoplacental ALPs

Regan ALP Nagao ALP

Detected in gynecological CA (ovarian, breast), lung CA, and colon CA.

Regan ALP

Regan ALP Detected in ________ (ovarian, breast), ___ CA, and ____ CA.

gynecological CA lung CA colon CA

Migrates to the same position as the Bone ALP

Regan ALP

Regal ALP migrates to the same position as ___________

Bone ALP

Most heat stable ALP isoenzyme

Regan ALP

T/F: Regan ALP is more heat stable than placental ALP

Regan ALP is stable @ ______for ____minutes

65°C 30

Inhibitor of Regan ALP

phenylalanine

Detected in metastatic carcinoma of pleural surfaces, and adenocarcinoma of pancreas and bile ducts

Nagao ALP

Nagao ALP Detected in _______________ of pleural surfaces, and ____________ of pancreas and bile ducts

metastatic carcinoma adenocarcinoma

Nagao ALP Detected in metastatic carcinoma of _________________, and adenocarcinoma of _______ and _________

pleural surfaces pancreas and bile ducts

Variant of Regan ALP

Nagao ALP

Inhibitor of Nagao ALP

phenylalanine L-Leucine

Method of Analysis for ALP

Bowers and McComb

A continuous monitoring method allowing calculation of ALP activity based on the molar absorptivity of _________________

BOWERS AND MCCOMB p-nitrophenol

The most specific method; IFCC recommended method

Bowers and McComb

Enumerate the Other Methodologies for ALP

1. Bodansky 2. Shinowara 3. Jones 4. Reinhart 5. King and Armstrong 6. Bessy, Lowry, and Brock 7. Bowers and McComb 8. Huggins and Talalay 9. Moss 10. Klein, Babson and Read

Substrate for 1. Bodansky 2. Shinowara 3. Jones 4. Reinhart

Beta-Glycerol Phosphate

End Product for 1. Bodansky 2. Shinowara 3. Jones 4. Reinhart

Inorganic phosphate + glycerol

Substrate for King and Armstrong

Phenylphosphate

End product for King and Armstrong

Phenol

Substrate for 6. Bessy, Lowry, and Brock 7. Bowers and McComb

p-nitrophenyl phosphate

End product for 6. Bessy, Lowry, and Brock 7. Bowers and McComb

p-nitrophenol/ yellow nitrophenoxide ion

Substrate for Huggins and Talalay

Phenolphthalein phosphate

End product for Huggins and Talalay

Phenolphthalein red

Substrate for Moss

Alpha-naphthol phosphate

End product for Moss

Alpha-naphthol

Substrate for Klein, Babson, and Read

Buffered phenolphthalein phosphate

End product for Klein, Babson, and Read

Free phenolphthalein

False Increase of ALP

Hemolysis Diet (Fatty meals) Stored at low temperature (4°C)

FALSE INC. IN ALP Hemolysis - ALP in RBC is ___ more concentrated than serum or plasma

FALSE INC. IN ALP Diet (fatty meals) - ALP is ____ higher

25%

Diagnostic Significance of ALP ALP - #1 marker in ______________

obstructive jaundice

Increased ALP

● Osteitis deformans ● Obstructive Jaundice ● Osteomalacia ● Rickets ● Osteoblastic bone tumors ● Sprue ● Hyperparathyroidism ● Hepatitis and Cirrhosis

Osteitis deformans is also known as

“Paget’s disease”

AST

ASPARTATE AMINOTRANSFERASE

E.C of ASPARTATE AMINOTRANSFERASE

E.C. 2.6.1.1

Old name of ASPARTATE AMINOTRANSFERASE

Serum Glutamic Oxaloacetate Transaminase

ASPARTATE AMINOTRANSFERASE Catalyze the transfer of amino groups between _________ (substrate) and ___________

aspartate a-ketoacids

ASPARTATE AMINOTRANSFERASE Catalyze the transfer of amino groups between aspartate (substrate) and a-ketoacids with the formation of ________ and ___________

oxaloacetate glutamate

ASPARTATE AMINOTRANSFERASE Coeznyme

Pyridoxal phosphate

ASPARTATE AMINOTRANSFERASE Major Tissue Source

Cardiac tissues liver and skeletal muscle

ASPARTATE AMINO TRANSFERASE RR

5-35 U/L

ASPARTATE AMINOTRANSFERASE Isozenzymes

Cytoplasmic AST Mitochondrial AST

Predominant in the circulation of a healthy individual

Cytoplasmic AST

Method of Analysis for AST

Karmen Method

Karmen Method is what type of enzymatic method

Coupled enzymatic method

Indicator enzyme for Karmen method

Malate dehydrogenase (MD)

KARMEN METHOD (AST) It monitors the (increase/decrease) in absorbance at _____ nm

decrease 340

KARMEN METHOD (AST) Storage: stable for ____ days at __________

3-4 refrigerated temp.

Variables for Karmen Method

Hemolysis

Hemolysis in Karmen Method results in false (increase/decrease) up to ___

increase 10x

Exhibits highest level AST

Acute Hepatocellular Disorder

ALT

ALANINE AMINOTRANSFERASE

E.C OF ALANINE AMINOTRANSFERASE

● E.C 2.6.1.2

old name for Alanine Aminotransferase

Serum Glutamic Pyruvic Transaminase

ALANINE AMINOTRANSFERASE Catalyze transfer of amino group between _________ (substrate) and ______________

alanine a-ketoglutarate

ALANINE AMINOTRANSFERASE is significant in the evaluation of what disorders

Hepatic disorders

ALANINE AMINOTRANSFERASE (ALT) Coenzyme

Pyridoxal phosphate

ALANINE AMINOTRANSFERASE (ALT) Major Tissue Source

Liver

ALANINE AMINOTRANSFERASE (ALT) RR

7-45 U/L

Method of Analysis for ALANINE AMINOTRANSFERASE (ALT)

1. Coupled Enzymatic Method

ALT Indicator Enzyme for Coupled Enzymatic Method

Lactate Dehydrogenase (LDH)

Coupled Enzymatic Method (ALT) The change in absorbance at ______ nm measured ________________ is directly proportional to ALT activity (pH _________).

340 continuously 7.3-7.8

Coupled Enzymatic Method (ALT) Storage

3-4 days at 4°C

T/F: Hemolysis does NOT affect ALT

True

Increase Aminotransferases (AST, ALT)

1. Toxic Hepatitis 2. Acute Myocardial Infarction (AST) 3. Wolff-Parkinson White Syndrome 4. Trichinosis (AST) 5. Chronic alcoholism 6. Dermatomyositis 7. Hepatic cancer 8. Reye’s Syndrome 9. Viral Hepatitis 10. Muscular dystrophy (AST) 11. Acute pancreatitis (AST)

Which disorders/diseases causes Increase in AST only

Acute Myocardial Infarction (AST) Trichinosis (AST) Muscular dystrophy (AST) Acute pancreatitis (AST)

Acute Myocardial Infarction (AST) Rise Peak Normal

6-8 hours 24 hours within 5 days

Trichinosis is caused by

Trichinella spiralis

unknown source in GGT is caused by pancreas

Acute pancreatitis

AST/ALT ratio

DE RITIS RATIO

Used to differentiate the cause of hepatic disorder

DE RITIS RATIO

DE RITIS RATIO >1: __________

non-viral cause

DE RITIS RATIO <1: __________

viral

GGT

GAMMA-GLUTAMYLTRANSFERASE

E.C of GAMMA-GLUTAMYLTRANSFERASE

E.C. 2.3.2.2

GAMMA-GLUTAMYLTRANSFERASE catalyzes ____________________

transpeptidation

The transfer of ɣ-glutamyl residue from ɣ-glutamyl peptides to amino acids, water and other peptides

transpeptidation

Used for diagnosis of hepatobiliary disorders and alcoholism

GAMMA-GLUTAMYLTRANSFERASE (GGT)

GAMMA-GLUTAMYLTRANSFERASE (GGT) is sed for diagnosis of _____________ and _____________

hepatobiliary disorders alcoholism

Useful for differentiating source of serum ALP elevation

GAMMA-GLUTAMYLTRANSFERASE (GGT)

LIVER DISORDER ALP GGT

Increased Increased

BONE DISEASE ALP GGT

Increased Normal

Critical for the intracellular maintenance of reduced glutathione

GAMMA-GLUTAMYLTRANSFERASE (GGT)

GAMMA-GLUTAMYLTRANSFERASE (GGT) Tissue source

Liver (epithelial cell lining of biliary ducts and bile canalicular) Kidneys Brain Pancreas, Intestine Prostate.

Method of Analysis for GAMMA-GLUTAMYLTRANSFERASE (GGT)

Szasz Assay

Method for Szasz Assay

Fixed-point or continuous monitoring

Szasz Assay Substrate

γ-glutamyl-p-nitroanilide

Szasz Assay End product

p-nitroaniline

Szasz Assay wavelength

405-420 nm

Szasz Assay Preferred specimen

Serum EDTA Plasma

Szasz Assay Storage:

4°C (1 week) -20°C (1 month)

Other Method for GGT

Rosalki and Tarrow, Orlowski

T/F; GGT is not effected by hemolysis

Why is GGT not affected by hemolysis?

GGT is not found in RBC

GGT RR: Male

6-55 U/L

GGT RR: female

5-38 U/L

Most sensitive marker of acute alcoholic hepatitis

GAMMA-GLUTAMYLTRANSFERASE (GGT)

Normal levels in patients with bone disease and during pregnancy

GAMMA-GLUTAMYLTRANSFERASE (GGT)

GGT Normalize: ___ weeks after consumption

2-3

5’N

5’-NUCLEOTIDASE

E.C. of 5'-NUCLEOTIDASE

E.C. 3.1.3.5

5'-NUCLEOTIDASE Other name

5’ ribonucleotide phosphohydrolase

A phosphoric monoester hydrolase reacting only on nucleoside-5’-phosphates (_______, __________) releasing (inorganic/organic) phosphate

5'-NUCLEOTIDASE AMP adenylic acid inorganic

5'-NUCLEOTIDASE Major Source

Liver

Marker of hepatobiliary disease and infiltrative lesions of the liver

5'-NUCLEOTIDASE

5'-NUCLEOTIDASE Marker of _____________ and ____________________ of the liver

hepatobiliary disease infiltrative lesions

Secondary Marker for Obstructive Jaundice

5'-NUCLEOTIDASE

Method for 5'-NUCLEOTIDASE

Dixon & Purdon Campbell Belfield & Goldberg

Method for 5'-NUCLEOTIDASE

Dixon & Purdon Campbell Belfield & Goldberg

5'-NUCLEOTIDASE RR

0-1.6 U/L

5'-NUCLEOTIDASE Storage

4°C (4 days) -20°C (4 months)

Enumerate the Pancreatic Enzymes

1. Amylase 2. Lipase

AMS

Amylase

E.C for Amylase

E.C. 3.2.1.1

AMYLASE Other name

alpha-1,4-glucan-4-glucohydrolase

AMYLASE Catalyzes the hydrolysis of __________________ in polysaccharides (_____, _____)

𝛂-1,4-glycosidic bonds starch, glycogen

Earlier pancreatic marker

Amylase

Smallest enzyme (freely filtered by the _________)

AMYLASE glomerulus

AMYLASE Activators

Calcium, Chloride

AMYLASE Major Tissue Source

Pancreas (acinar cells) Salivary Glands

AMYLASE (AMS) RR; Serum

28-100 U/L

AMYLASE (AMS) RR; Urine

1-15 U/h

Enumerate the isoenzymes of Amylase

1. S-type/Ptyalin/Salivary 2. P-type / Amylopsin / Pancreatic

↑ parotitis/mumps

1. S-type/Ptyalin/Salivary

The most anodal AMY isoenzyme

1. S-type/Ptyalin/Salivary

S-type/Ptyalin/Salivary is secreted by __________________

salivary glands

Inhibitor of S-type/Ptyalin/Salivary (inhibits activity of salivary glands)

Wheat Germ Lectin

↑ in acute pancreatitis

P-type / Amylopsin / Pancreatic

the most predominant subtype of P-type / Amylopsin / Pancreatic

● Secreted by the pancreas and also by the fallopian tube & lungs

P-type / Amylopsin / Pancreatic

T/F: In the laboratory, we measure the Total Amylase

TRUE

Specimen for Amylase

Serum, Heparinized Plasma

Substrate for Amylase

Starch

Enumerate the different Method of Analysis of Amylase

1. Saccharogenic 2. Amyloclastic 3. Chromogenic 4. Coupled-Enzyme

AMYLASE Classic reference method

Saccharogenic

It measures the amount of __________ produced by the hydrolysis of ______ by the usual glucose method

SACCHAROGENIC reducing sugars starch

Enumerate the glucose methods used in Saccharogenic

Hexokinase Glucose oxidase

Glucose methodology reference method

Hexoklnase

End product for Saccharogenic

Glucose

AMYLASE inverse method

Amyloclastic

It measures decrease in starch substrate

Amyloclastic

Amyloclastic Substrates are coupled with_____

iodine

Iodine + Starch = _____________

Dark-blue color

Why Dark Blue Color in Amyloclastic?

e iodine is trapped within the structure of the starch

T/F: In amyloclastic, decrease in color is proportional to AMS activity

Measures the formation of soluble starch fragments coupled with chromogenic dyes.

Chromogenic

Color intensity is proportional to AMS activity

Chromogenic

Measured amylase activity by a continuous-monitoring/kinetic technique.

Coupled-Enzyme

Substrate for Couple-Enzyme (Amylase)

Starch

Wavelength for Couple-Enzyme (Amylase)

340 nm

pH for Couple-Enzyme (Amylase)

6.9

Storage for Couple-Enzyme (Amylase)

Room temp (1 week) 4°C (2 months)

False Decrease in AMS

● Ca2+ Chelating Anticoagulant ● Triglycerides

False Increase in AMS

● Morphine, other opiates

amylase bound to immunoglobulin

Macroamylasemia

asymptomatic amylasemia

Macroamylasemia

In Macroamylasemia, (increase/decrease) serum AMS & (increase/decrease) N Urine AMS

Increase Decrease

↑ serum AMS & ↑ urine AMS

Hyperamylasemia

Amylase/Creatinine Ratio: Normal

1-4% (0.01-0.04)

Amylase/Creatinine Ratio: Acute Pancreatitis

>4%-15%

LPS

Lipase

E.C of Lipase

E.C. 3.1.1.3

LIPASE Other name

Triacylglycerolacylhydrolase

Catalyzes hydrolysis of glycerol esters of complex lipids to produce ___________ and ________.

LIPASE alcohol fatty acid.

Catalyzes partial hydrolysis of ________ to________________, with production of long-chain fatty acids.

LIPASE dietary TAG 2-monoglyceride intermediate

More pancreatic specific

LIPASE

LIPASE Cofactor

Colipase (coenzyme) Bile salts

LIPASE Major Tissue Source

Pancreas

LIPASE RR

<38 U/L

Method of Analysis for Lipase Specimen

Serum

Method of Analysis for Lipase Storage

room temp (1 week) 4°C (3 weeks)

Method of Analysis for Lipase Interferences

Hemolysis

Enumerate the Methods for Lipase

1. Cherry-Crandall 2. Tietz 3. Peroxidase Coupling

Substrate for Cherry-Crandall and Tietz

50% Olive oil (Triolein)

Titrating Agent for Cherry-Crandall and Tietz

0.4N NaOH

Indicator for Cherry-Crandall

Phenolphthalein

Indicator for Tietz

Thymolphthalein + Veronal

Endpoint for Cherry-Crandall and Tietz

Fatty Acids (Oleic acid)

End color of Cherry-Crandall

Pink

End color of Tietz

Blue

Most commonly used method (LPS)

Peroxidase Coupling

Does not use 50% olive oil (LPS)

Peroxidase Coupling

most specific pancreatic marker

Lipase

In chronic pancreatitis: → _____________ are destroyed → Loss of_____ and _____

Acinar cells AMS and LPS

AMYLASE Rise Peak Normalize

5-8 HOURS 24 HOURS 3-5 DAYS

LIPASE Rise Peak Normalize

4-8 HOURS 24 HOURS 8-14 DAYS

Enumerate the Cardiac Markers

1. Creatine Kinase 2. Lactate Dehydrogenase

Creatine Kinase

E.C for Creatine Kinase

● E.C. 2.7.3.2

CREATINE KINASE Other Name

ATP-Creatine-N-phosphotransferase

Catalyzes phosphorylation of creatine to form _____________

CREATINE KINASE creatine phosphate.

Involved in the storage of high-energy creatine phosphate in ___________

CREATINE KINASE muscle cells

contains two subunits

CREATINE KINASE Dimeric Molecule

2 subunits of CREATINE KINASE

→ M subunit (Muscle) → B subunit (Brain)

CREATINE KINASE Major Tissue Source

brain muscles (smooth, skeletal, cardiac)

CREATINE KINASE RR: Male

46-171 U/L

CREATINE KINASE RR: Female

35-145 U/L

CREATINE KINASE RR: CK-MB

<5% of total CK

Enumerate the Normal Isoenzymes of Creatine Kinase

CK-BB CK-MB CK-MM

Rank the CK isoenzymes from most ANODAL to least ANODAL

1. CK-BB 2. CK-MB 3. CK-MM

Rank the CK isoenzymes from most CATHODAL to least CATHODAL

1. CK-MM 2. CK-MB 3. CK-BB

Brain type CK Isoenzyme

CK-BB

Hybrid type CK Isoenzyme

CK-MB

Muscle type CK Isoenzyme

CK-MM

Dominant in brain, intestines, and smooth muscles

CK-BB

CK-BB is dominant in:

brain intestines smooth muscles

Present in significant concentration in the cardiac muscles

CK-MB

CK-MB is present in significant concentration in the ___________________

cardiac muscles

Abundantly present in striated muscles

CK-MM

CK-MM is abundantly present in _________

striated muscles

Rarely found in Serum

CK-BB

Why is CK-BB rarely found in serum?

it cannot pass through the blood-brain barrier

Serodiagnostic tool for AMI

CK-MB

Major Isoenzyme in normal individual (______)

CK-MM 94-100%

Useful Non-Specific Tumor Marker

CK-BB

RV of CK-MB

<5% of Total CK

Myocardial Damage/AMI:

● Elevated CK-MB ● >6 of Total CK

earliest enzymatic cardiac marker

CK-MB

increased CREATINE KINASE

1. Acute Myocardial Infarction 2. Duchenne-type Muscular Dystrophy 3. Rhabdomyolysis 4. Cerebrovascular Accident 5. Seizures 6. Nerve Degeneration 7. CNS Shock 8. Hypothyroidism 9. Malignant Hyperpyrexia 10. Reye’s Syndrome

Highest elevation of total CK

Duchenne-type Muscular Dystrophy

Method of Analysis for CK Specimen

Serum Heparinized Plasma

Inhibitor of sulfhydryl group oxidation

→ N-acetylcysteine → Mercaptoethanol → Thioglycerol → Dithiothreitol

T/F: Serum activity in CK is stable

FALSE; VERY UNSTABLE

Why is serum in CK very unstable?

because it is easily be inactivated by sulfhydryl group by oxidation

Variables for CK

Hemolysis Light Non-heparinized Anticoagulant Physical Activity IM Injection

VARIABLES FOR CK Hemolysis hgb level

>320 mg/L HGB

Total CK in Hemolysis is falsely elevated due to ______________ (enzyme) that is present in RBC

Adenylate kinase

To prevent false elevation because of adenylate kinase, add __________________

Adenosine monophosphate

Why is light a variable in CK

because CK is inactivated if exposed to light

Methods for CK

1. Tanzer-Gilvarg Assay 2. Oliver-Rosalki Method

Methods for CK Forward/Direct Method

1. Tanzer-Gilvarg Assay

Tanzer-Gilvarg Assay is coupled with _______________________________________________

pyruvate kinase-lactate dehydrogenase-NADH system

Tanzer-Gilvarg Assay Optimal pH

9.0

Tanzer-Gilvarg Assay Wavelength

340 nm

Methods for CK Reverse/indirect Method

Oliver-Rosalki Method

Most commonly performed method for CK

Oliver-Rosalki Method

Oliver-Rosalki Method is couple with _______________________________________________________

Hexokinase-Glucose-6- Phosphate-Dehydrogenase-NADP system

Oliver-Rosalki Method Optimal pH

6.8

Oliver-Rosalki Method Wavelength

340 nm

LDH

Lactate Dehydrogenase

E.C of Lactate Dehydrogenase

E.C. 1.1.1.27

Catalyze the interconversion (reversible) of ________ and __________

LACTATE DEHYDROGENASE lactic acid pyruvic acid

LDH Coenzyme

NAD+

LDH Inhibitor

Ethylenediamine tetraacetic acid (EDTA)

contain A-peptide or M-peptide unit

LACTATE DEHYDROGENASE Tetrametic Molecule

● Widely distributed

LACTATE DEHYDROGENASE (LDH)

LDH Highest Activity

Heart RBCs skeletal muscles

LDH RV

125-220 u/L

Enumerate the LDH Isoenzymes

LD 1 LD 2 LD 3 LD 4 LD 5 LD 6

Most predominant LDH isoenzyme

LD 2

increased in pulmonary involvement and various carcinomas

LD3

LD3 is increased in ______________ and _____________________

pulmonary involvement various carcinomas

LDH Isoenzyme Seen in muscular injuries

LD 4

LD 6 a.k.a

Alcohol dehydrogenase

Present in Arteriosclerotic cardiovascular failure

LD 6

LD6 is present in _________________________

Arteriosclerotic cardiovascular failure

SUBUNITS LD 1 LD 2 LD 3 LD 4 LD 5 LD 6

HHHH HHHM HHMM HMMM MMMM none

Tissue source of LD 1

Heart, RBC

Tissue source of LD 2

Heart, RBC

Tissue source of LD 3

Liver Spleen, Lymphocytes Pancreas

Tissue source of LD 4

Liver

Tissue source of LD 5

Skeletal muscle

Percentage based on Total LDH LD 1

14-26%

Percentage based on Total LDH LD 2

29-39%

Percentage based on Total LDH LD 3

20-26%

Percentage based on Total LDH LD 4

8-16%

Percentage based on Total LDH LD 5

6-16%

used as tumor markers

LD 2 LD 3 LD 4

LD2, LD3, LD4 -used as tumor markers for:

→ acute leukemia → germ cell tumor → breast cancer → lung cancer

Most to least dominant cases of: (Descending Order) Normal Pattern

LD 2>1>3>4>5

Method of Analysis LDH Specimen

Serum

Method of Analysis LDH Substrate

→ Lactate → Pyruvate → α-hydroxybutyrate (LD1)

Method of Analysis LDH Storage: Total LD

25°C (48 hours)

Method of Analysis LDH Storage: LD isoenzymes

25°C (24 hours)

Variables for LDH

→ Plasma specimen → Hemolysis → Cold storage

Plasma specimen and Hemolysis in LDH causes false (increase/decrease) because of the presence of ____________

increase plaelets

Cold Storage of LDH causes false (increase/decrease) in _____ and _____

Decrease LD 4 and LD 5

Enumerate the methods used in LDH

1. Wacker Method 2. Wrobleuski La Due

Method of Analysis LDH Forward/Direct Method

Wacker Method

Method of Analysis (LDH)\ Most commonly used method

Wacker Method

pH in Wacker Method

8.3-8.9

Wavelength in Wacker Method

340 nm

Method of Analysis LDH Reverse/Indirect Method

Wrobleuski La Due

Wrobleuski La Due is ____ the rate of forward method

thrice

● Preferred Method for Dry-Slide Technology

Wrobleuski La Due

Example of Dry-Slide Techbology

Vitros Analyzer

Uses less costly cofactor and it has a smaller specimen volume requirement

Wrobleuski La Due

Wrobleuski La Due pH

7.1-7.4

Increased LDH

1. Acute Myocardial Infarction 2. Hemolytic anemia 3. Pernicious anemia 4. Pulmonary infarction 5. Muscle dystrophy 6. Hepatic carcinoma, toxic hepatitis, cirrhosis, viral hepatitis 7. Blood transfusion 8. Pneumocystis jirovecii infection

INCREASED LDH Acute Myocardial Infarction exhibits what type of pattern

→ Flipped pattern → LD 1>2>3>4>5

INCREASED LDH Pulmonary Infarction exhibits what pattern

LD 3>4>2>1>5

INCREASED LDH Muscle dystrophy what pattern

LD 5>4>3>2>1

Which disorders/diseases in Increased LDH exhibits increased LD 4

Hepatic carcinoma, toxic hepatitis, cirrhosis, viral hepatitis

__ & __ = LD is increased but normalize after ______

6 and 7 24 hours

T/F: Increase in Total LDH is insignificant

Why is an increase in Total LDH insignificant?

because LDH is present almost virtually in cells

Acute Myocardial infarction ______ of total CK ______

>6% 1:20:0

Acute Myocardial infarction Enumerate the Enzymes involved

Aspartate Aminotransferase Creatine Kinase-MB Lactate Dehydrogenase

ACUTE MYCOCARDIAL INFARCTION Enzyme not liver specific

Aspartate aminotransferase

ACUTE MYOCARDIAL INFARCTION Enzyme cardiac specific

Creatine Kinase-MB

ASPARTATE AMINOTRANSFERASE Rise Peak Normalize

6-8 hours 24 hours Within 5 days

CREATINE KINASE-MB Rise Peak Normalize

4-8 hours ' 12-24 hours 48-72 hours

LACTATE DEHYDROGENASE Rise Peak Normalize

12-24 hours 48-72 hours After 10-14 days

Enumerate the other Clinically Significant Enzymes

1. Acid Phosphatase

ACP

Acid Phosphatase

E.C of ACID PHOSPHATASE

E.C. 3.1.3.2

ACID PHOSPHATASE Other name

Acid orthophosphoric monoester phosphohydrolase

Catalyze the hydrolysis of phosphomonoesters at an acidic pH

ACID PHOSPHATASE (ACP)

ACID PHOSPHATASE Major Tissue Source

Prostate

Useful for evaluation of metastatic-prostatic carcinoma

ACID PHOSPHATASE (ACP)

ACID PHOSPHATASE (ACP) i useful for evaluation of _____________________

metastatic-prostatic carcinoma

ACID PHOSPHATASE TOTAL ACP RR: Male

2-5-11.7 U/L

ACID PHOSPHATASE PROSTATIC ACP RR: Male

0.2-5.0 U/L

ACID PHOSPHATASE TOTAL ACP RR: Female

0.3-9.2 U/L

ACID PHOSPHATASE PROSTATIC ACP RR: Female

0.0-0.8 U/L

T/F: Total ACP is measured in the laboratory

True

Method of Analysis (ACP) Specimen

Serum

Method of Analysis ACP Substrates

Thymolphthalein monophosphate α-naphthyl phosphate

Substrate to use in ACP for end-point methods

Thymolphthalein monophosphate

Specific substrate for prostatic ACP

Thymolphthalein monophosphate

ACP substrate for continuous monitoring methods/kinetic assay

α-naphthyl phosphate

Methods of Analysis ACP INHIBITORS

L-Tartrate 2% formaldehyde, cupric sulfate solution

Inhibitor in ACP that inhibits prostatic ACP

L-Tartrate

Inhibitor in ACP that also Inhibit lysosomal ACP

L-Tartrate

Inhibitor in ACP that inhibits RBC ACP/TRAP

2% formaldehyde, cupric sulfate solution

Method of Analysis ACP Storage

Frozen Acidified

ACP is acidified in what pH

< pH of 6.5

When ACP is acidified, ACP is stable for ______ @ ________

2 days Room temp

Methods of Analysis ACP Variables

Room temp Bilirubin Hemolysis Heparin, Oxalate, Fluorides

VARIABLES (ACP) Room temp -ACP stable within ________ @ RT because CO2 from blood sample is released which (increases/decreases) pH of the sample

1-2 hrs increases

false (decrease/increase) due TRAP/RBC ACP

Bilirubin - decrease Hemolysis - increase

Heparin, Oxalate, Fluorides - false (decrease/increase)

decrease

in measuring prostatic ALP what is the inhibitor

(L. tartrate -

Enumerate the steps in measurign prostatic ACP

1. Measure total ACP 2. Add L-tartrate 3. Measure total ACP (TRAP) ○ Prostatic ACP = Total ACP - TR

Enumerate the different kind of ACP methods

1. Gutman and Gutan 2. Shinowara 3. Babson, Read & Philips 4. Roy and Hillman

GUTMAN AND GUTAN Substrate

Phenyl phosphate

GUTMAN AND GUTAN End product

Inorganic phosphate

SHINOWARA Substrate

P-nitrophenylphosphate

SHINOWARA End Product

p-nitrophenol

Babson, Read & Philips Substrate

α-naphthylphosphate

BABSON, READ & PHILIPS End product

α-naphthol

ROY AND HILLMAN Substrate

Thymolphthalein monophosphate

ROY AND HILLMAN End product

Free thymolphthalein

[M1] Enzyme Methodologies Flashcards

(385 cards)