Chemistry week 2 Flashcards
Also called Thyroxine-Binding prealbumin or Trashthyretin
Prealbumin
Proteins that are not synthesized in the liver
Immunoglobulins from activated B cell and Coag factor such as VWF
Transports Vitamin A by binding with the retinal-binding protein
Prealbumin
Prealbumin is increased in?
Chronic renal failure, Alcoholism, Steroid streatment
Prealbumin is decreased in
Malnourished
Most abundant protein in the plasma
Albumin
Serves as a mobile respiratory of amino acids for incorporation into other proteins
Albumin
Major transport protein
Albumin
Negative acute phase reactant
Albumin
Good indicator of Cystic Fibrosis
Albumin
17 days half-life
Albumin
barely 2 days half-life
Prealbumin
Important in interpreting calcium and magnesium levels because these ions are bound to these protein.
Albumin
Elevation of albumin is seen in?
Dehydration
Prolonged application of tourniquet for venipuncture
Presence of this protein is abnormal in urine
Albumin
Major component of the a1-globulins
Alpha1-antitrypsin
Alpha1-antitrypsin is increased in
Inflammation (APR), pregnancy, and contraceptives user
Alpha1-antitrypsin is decreased in
Emphysema, Hepatic cirrhosis (juvenile), and other pulmonary disorders
Largest major non-immunoglobulin protein in plasma
Alpha 2 - macroglobulin
Rises 10-fold or more in Nephrotic syndrome
Alpha 2 - macroglobulin
Alpha 2 - macroglobulin is also increased in
Diabetes, and liver disease
Laboratory methods of determination of Alpha 2 - macroglobulin
Immunodiffusion (radial type)
Immunonephelometry
ELISA
Latex Agglutination
Forms a complex by binding to prostate specific antigen
Alpha 2 - macroglobulin
Associated with Alzheimer’s disease
Alpha-1-Antichymotrypsin
Serine proteinase with Cathepsin G
Alpha-1-Antichymotrypsin
Binds and inactivate PSA
Alpha-1-Antichymotrypsin
Alpha-1-Antichymotrypsin is increased in
infection, malignancy, burns, Myocardial infarction
Alpha-1-Antichymotrypsin is decreased in
Liver disease
Marker of kidney function specifically the tubular function
β-2 Microglobulin
because it can readily pass to the glomerulus but it is reabsorbed by the tubule
Light chain component of the major histocompatibility complex (HLA)
β-2 Microglobulin
Responsible for causing Dialysis-associated amyloidosis
β-2 Microglobulin
kasi it forms a beta plated sheath kaya nag foform siya ng formation ng amyloid
β-2 Microglobulin is increased in
Renal failure, Multiple myeloma, HIV, and inflammatory disease such as RA and SLE
Method of determination for β-2 Microglobulin
Immunoassay
Carrier of hemoglobin and an APR
Haptoglobin
Major protein migrating in the alpha-2 region
Haptoglobin
Combines with hemoglobin released by lysed red cells in order to preserve body iron and protein stores
Haptoglobin
Haptoglobin is increased in
Stress, Infection, myoglobinuria, inflammation, and tissue necrosis
Haptoglobin is decreased in
Intravascular hemolysis, and hemoglobinuria
Also known as siderophilin
Transferrin
Major beta globulin transport protein for ferric ions
Transferrin
Great impact in hemoglobin production
Transferrin
Low of this protein causes anemia
Transferrin
Transferrin is increased in
pregnant women, IDA, Hemochromatosis
Transferrin is decreased in
Liver disease, Nephrotic syndrome, and malnutrition
Most abundant of the coag factor
Fibrinogen
Marker for CVD
Fibrinogen
Fibrinogen is increased in
Inflammation, elevated with other APR, pregnancy, and the use of contraceptive
Fibrinogen is decrease in
Extensive coagulation kasi niuuse up niya yung fibrinogen
Marker for wilson’s disease
Ceruloplasmin
Copper binding protein for target tissues and organs
Ceruloplasmin
Responsible in oxidizing iron from ferrous to ferric through the ferroxidase enzyme
Ceruloplasmin
Kayser-Fleishcer ring in the cornea
Copper toxicity
Menke’s disease that causes curly hair called kinky-hair syndrome and skin is ligher
Copper deficiency
Ceruloplasmin is increased in
Inflammation, Cancer, Pregnancy
Ceruloplasmin is decreased in
Wilson’s disease, Malnutrition, Malabsorption, Nephrotic conditions, and Menke’s disease
Binds heme released by degradation of hemoglobin
Hemopexin
Early marker of hemolytic condition
Hemopexin
Profoundly decreased in intravascular hemolysis
Hemopexin
Also known as orosomucoid
α1-Acid Glycoprotein
Binds to progesterone and could be important in its transport or metabolism
α1-Acid Glycoprotein
Binds to quinidine
α1-Acid Glycoprotein
Can be used in bacterial infection in neonatal population
α1-Acid Glycoprotein
α1-Acid Glycoprotein is increased in
Pregnancy, Cancer (neoplasia), other increased cell proliferation condition, Rheumatoid arthritis, Pneumonia
Contains carbohydrate molecule and sialic acid
α1-Acid Glycoprotein
Major acute phase reactant
C-Reactive Protein
Marker for CVD
C-Reactive Protein
Binds with C-polysaccharide of the pneumococcal bacteria and precipitate with the C substance
C-Reactive Protein
General scavenger molecule
C-Reactive Protein
CRP is ____ in bacterial infection and ____ in viral infection
High, Low
C-Reactive Protein is increased in
pneumococcal infections, Rheumatic fever, MI, Gout, RA
Derived from the fetal yolk sac and most abundant protein in the fetal serum
Alpha-I-Fetoprotein (AFP)
Alpha-I-Fetoprotein (AFP) is increase at what week of gestation?
13th week
Alpha-I-Fetoprotein (AFP) peaks at ___ month
7-8 months
AFP in adult means
Liver carcinoma
Alpha-I-Fetoprotein (AFP) is increased in
Amniotic fluid and serum in neural tube defect (spina bifida)
Alpha-I-Fetoprotein (AFP) is decreased in
Down’s syndrome
used as gold markers for myocardial
infarction since it increases during heart attack
Troponin I and T
Inhibitory subunit of troponin
Troponin I
Tropomyosin-binding subunit of troponin
Troponin T
Calcium-binding subunit of troponin
Troponin C
established firmly as the gold standard in the diagnosis of myocardial damage
Cardian troponin (cTN )
Most abundant troponin subunit
Troponin C
Marker for congestive heart failure
BNP and NTBNP
Released by the Myocardium of the Heart that regulates sodium
from cardiac muscles
BNP and N-Terminal-BNP
Induces natriuresis which affects the excretion of sodium through the kidney or renal excretion
BNP and N-Terminal-BNP
Marker for possible premature delivery
Fibronectin
Marker of HAVING cardiovascular diseases
Adiponectin
Lower levels of adiponectin correlate with an increased risk of?
Heart disease , type 2 DM, and metabolic syndrome, and obesity
Marker of renal function
Beta trace protein
Marker of CSF leakage
Beta trace protein
Diagnosis of perilymphatic fluid distulas
Beta trace protein
Marker for the early assessment of changes to GFR
Cystatin C
Cystatin C is increased in
Renal disease, Liver disease, Obesity, muscular diseases
Used to differentiate which type of condition is causing the increase of Creatinine.
Cystatin C
Considered as a nephrotoxin
Myoglobin
2% of total muscle protein
Myoglobin
Early marker of cardiac condition. Immediately increases in case of MI but not specificity
Myoglobin
pero not specific kasi pwede siya manggaling sa skeletal muscles
Myoglobin is increased in
MI, chest pain, skeletal disorders, Rhabdomyolysis, Strenuous exercise, IM injection, Myoglobinuria
Protein polysaccharide complex produced and deposited in tissue during some chronic infections, malignancies, and rheumatologic disorders
Amyloid
Amyloid is readily stained by
Congo red
o Digestion of protein; Measurement of Nitrogen Content
o Reference method; Assume average nitrogen content of 16%
Kjedahl
o Measurement of refractive index due to solutes in serum
o Rapid and Simple; Assume nonprotein solids are present in
same concentration as in the calibrating serum
Refractometry
o Formation of Violet-colored chelate between Cu2+ ions and
peptide bonds
o Routine Method; Requires at least two peptide bonds and an
alkaline medium
Biuret
o Protein binds to dye and causes a spectral shift in the
absorbance maximum of the dye
o For Research Use
Dye binding
o Globulins are precipitated in high salt concentrations; Albumin
in supernatant is quantitated by biuret reaction
o Labor Intensive
Sal precipitation
o Albumin binds to dye; causes shift in absorption maximum
o Organic and Nonspecific for albumin
Methyl orange
o Albumin binds to dye; causes shift in absorption maximum
o Many interferences (salicylates, bilirubin)
HABA (2,4’-Hydrixyazobenzene-Benzoic Acid)
o Albumin binds to dye; causes shift in absorption maximum
o Sensitive; Overestimates low albumin levels
o Most commonly used dye
BCG (Bromcresol Green)
o Albumin binds to dye; causes shift in absorption maximum
o Specific, Sensitive, Precise
BCP (Bromcresol Purple)
o Proteins separated based on electric charge
o Accurate; Gives overview of relative changes in different protein
fractions
Electrophoresis
A-1 globulin’s specific proteins
α-1-antitrypsin, α-acid glycoprotein
α-2-Globulin’s specific protein
Ceruloplasmin, haptoglobin, α-2-microglobulin
β-Globulin’s specific protein
LDL, VLDL, Transferrin, Hemopexin, Complement,
Fibrinogen
γ-Globulin’s specific protein
Immunoglobulin
Electrophoresis pattern
Prealbumin > Albumin > A1 globulin > a2 globulin > b globulin > gamma globulin
Monoclonal peak in gamma-globulin region which is a marker of multiple myeloma
Bence-jones protein
Tall spike pattern which shows only one variation of protein
Monoclonal peak
Tall and curve pattern which shows many variation of proteins are high
Polyclonal peak
Polyclonal peak is typically seen in
Infections
Nephrotic syndrome electrophoretic pattern
Increase alpha 2 macroglobulin
Agammaglobulinemia
Depleted or absence immunoglobulin in immunosuppressed patients
Globulins are precipitated using Sodium Sulfate, Sodium Sulfite,
Ammonium Sulfate, or Methanol leaving albumin in solution
precipitation
Order of protein elution is by molecular weight or size from largest first to smallest last
Gel filtration
Based on the charge of proteins which bind to heads of a charged support medium
Ion exchange chromatography
Samples are applied at high salt and eluted with low salt
Hydrophobic chromatography
Based on specific binding between a protein of interest and another protein that has been covalently linked to the solid medium of a column
Affinity chromatography
Separation method based on flow through a capillary tube that can be tailored to resolution of different molecules based on size, hydrophobicity, or stereospecificity
Capillary electrophoresis
Reference method of protein detection and quantitation
Kjeldahl technique
The ammonium can then be quantitated by conversion to ammonia gas and titration as a base or be nesslerization
Kjeldahl technique
Total serum protein is obtained by multiplying the value of TPN by?
6.25
Nessler reagent contains?
Double iodide of potassium and mercury to form a mercuric ammonium iodide which is YELLOW in color
Accurate for measure serum protein concentration as dissolved solute for levels above 2.5 g/dL
Refractive index
Cannot be used for urine protein measurements because of excess amounts of solutes in relation to the protein
Refractive index
Used widely as a screening test for Hemoglobin concentration in whole blood
Specific Gravity
Used to measure protein concentration in CSF or urine
Turbidimetric method
Turbidimetric method reference ranges
Negative
Clear sample. No turbidity
<20mg/dL
Turbidimetric method reference ranges
Trace
Very faint precipitate
20-200 mg/dL
Turbidimetric method reference ranges
1+
Small degree turbidity
100-1000 mg/dL
Turbidimetric method reference ranges
2+
Moderate turbidity
1000-2500 mg/dL
Turbidimetric method reference ranges
3+
Heavy turbidity
2500-4500 mg/dL
Turbidimetric method reference ranges
4+
Heavy flocculation/Clumping
>4500 mg /dL
Measure peptide linkage or peptide bond
Biuret method
Reagents are copper sulfate, Tartrate salt, Potassium Iodide
Biuret method
Presence of two or more peptide bonds in which form purple complex with copper, salts in alkaline solution
Biuret method
Involves oxidation of phenolic compounds such as tyrosine, tryptophan, and histidine to give a deep blue color
Folin-Ciocaltou reagent
Used for CSF proteins because they have less than 1% protein
Coomasie brillant dye
Produces a violet color by reacting with primary AMINES
Ninhydrine
Total protein =
Albumin + Globulins
Total protein reference range
6.5-8.5 g/dL
Total protein is ____ in these circumstances
o Dehydration
o Severe Exercise
o Infection
o Cancer
Increased
Total protein is ____ in these circumstances
o GI Cancers
o Liver Disease
o Malnutrition
o Low Thiamine
o Glomerulonephritis
Decreased
Albumin reference range
3.5-5.0 g/dL
Albumin is ______ in these circumstances
o Dehydration
o Sunstroke
o Exercise
o Multiple Sclerosis
o Hypothyroidism
Increased
Albumin is ______ in these circumstances
o Pregnancy
o Malnutrition
o Malabsorption
o Liver disease
o Kidney disease
o Burns
Decreased
- Rare inherited disorders of amino acid metabolism
- Causes deficiency, specifically enzymes
Aminoacidopathies
- Most common aminoacidopathy that is tested in newborn screening
- Autosomal recessive trait and occurs approximately in 1/15,000 births
- Absence of PAH (Phenylalanine-4-Monooxygenase) which catalyzes
the conversion of phenylalanine to tyrosine
Phenylketonuria
Reference method of phenylketonuria
High performance liquid chromatograph
Used for inhibition with B2-thienylalanine in phenylketonuria
Bacillus subtilis
- Characterized by excretion of tyrosine catabolites in urine
- Defects in fumarylacetoacetase
- Causes accumulation of Tyrosine crystals in the urine which are
needle-shaped urine crystals
Tyrosinemia
Laboratory test for Tyrosinemia
Ion-exchange column chromatography
- Deficiency of homogentisate oxidase in tyrosine catabolic pathway
- Build up of homogentisic acid
o Effect: Homogentisic acid accumulates in connective tissue causing generalized pigmentation and arthritis-like degeneration. Homogentisic acid causes to darken upon
exposure to air.
Alkaptonuria
Laboratory test for alkaptonuria
Blue color when ferric chloride is added to urine
- Deficiency of branched chain keto-acid decarboxylase
- Build up of leucine, isoleucine, and valine
o Effects: Mental retardation, convulsion, acidosis, hypoglycemia,
and death. Burnt-sugar odor of urine, breathe, and skin
Maple syrup urine disease
Screening test for MSUD
Modified Guthrie test that detects elevated plasma leucine
Uses as antagonist for the bacteria in MSUD
4-azaleucine
- Impaired activity of the enzyme Cystathionine β-Synthase
- Results in elevated plasma and urine levels of the precursors
homocysteine and methionine
o Effects: Late Childhood – thrombosis, osteoporosis, dislocated
lenses
Hemocystinuria
Neonatal screening test for hemocystinuria
Guthrie test using L-methionine sulfoximine
Confirmatory test for hemocystinuria
High performance liquid chromatography >2mg/dL
Lab test for Isovaleric acidemia
Chromatography, Mass spectrometry
Lab test for cystinuria
Test urine cyanide-nitroprusside (+) red color to purple
Marker for arginosuccinic aciduria and citrullinemia
Citrulline
Lab test for arginosuccinic aciduria and citrullinemia
Mass spectrometry
Urea in plasma
45-50%
Urea in urine
86%
Amino acids in plasma
25%
Uric acid in plasma
10%
Uric acid in urine
1.7%
Creatinine in plasma
5%
Creatinine in urine
4.5%
Creatine in plasma
1-2%
Ammonia in plasma
0.2%
Ammonia in urine
2.8%
Removal of the substance from plasma into the urine
Clearance test
Plasma concentration and clearance is
Inversely proportional
Gold standard and reference method for GFR clearance test
Inulin clearance
Excellent measure of renal function since this is freely filtered by glomeruli but not reabsorbed
Creatinine clearance test
Waste product of muscle metabolism
Creatinine
Creatinine is directly proportional to?
Muscle Mass
Denotes damage of the PCT because it is reabsorbed by the PCT
Cystatin C
Earliest GFR test
Urea clearance
Not affected by muscle mass, age, and gender
Cystatin C
First metabolite to elevate in kidney disease
BUN
Major NPN
Urea
45% of total NPN
BUN
Urea formula
BUN x 2.14
Methods of determination of BUN
Chemical method = Fearon’s reaction/Diacetyl monoxime
Enzymatic method = Uses urease
Fearon’s reaction end color
Yellow
Reference method for ALL NPN
Isotope Dilution mass spectrometry (IDMS)
End product of muscle metabolism
Creatinine
Index of overall renal function
Creatinine
Creatinine is composed of? (GAM)
Glycine, Arginine, Methionine
Method of determination of creatinine
Chemical method = Jaffe reaction
Jaffe reaction end color
Orange-red color
Marked elevation of plasma urea and other NPNs accompanied by acidemia and hyperkalemia
Uremia
PBS = (+) burr cells
Uremia
Very high plasma urea concentration accompanied by renal failure
Uremia
Elevations of concentration of nitrogenous substances such as urea and creatinine in the blood
Azotemia
Reduced renal blood flow
Increased creatinine and urea that is before kidneys
Problem in systemic circulation problem
Prerenal azotemia
Very high plasma urea concentration accompanied by renal failure
Renal azotemia
Caused by obstruction anywhere in the urinary tract
Postrenal azotemia
Major end product of purine (adenine and guanin) metabolism
Uric Acid
Method of determination for BUA
Chemical method = Caraway method
Enzymatic method = uricase (most common)
Reagent in caraway method
Phosphotungstic acid
End color of caraway method
(+) Blue
Uric acid is increased in
Gout
Lesch-nyhan syndrome (Hypoxanthine guanin phosphotibosyltransferase deficiency)
Chronic renal disease
Uric acid is decreased in (OLD)
Liver disease
Overtreatment with allopurinol
Defective tubular reabsorption - eg. Fanconi syndrome (defective PVT reabsorptiom)
Striking proteinuria
Decreased serum albumin and total protein
Relative increased in alpha 2 macroglobulin and beta-globulin fractions
Increases in serum creatinine, BUN, and Uric acid
Nephrotic syndrome
Alpha2- globulin band spike
Nephrotic syndrome
Beta gamma bridging
Hepatic cirrhosis
Alpha1-globulin flat curve
Juvenile cirrhosis
Lipiduria is present in
Nephrotic syndrome (cholesterol crystals, oval fat bodies, waxy casts may be present)
Elevated urine protein
Increased BUN and CREATININE (2)
Red cells and red cell casts in urine
Glomerulonephritis
Oliguria
increase in BUN and creatinine
Electrolyte and acid/base alteration
Renal failure
Specific alteration in excretion of amino acids, electrolytes, or other specific biochemicals
Tubular defects
Acute kidney injury biochemical changes: INC OR DEC
Calcium, Bicarbonate, Sodium
Decreased (CABISO)
Acute kidney injury biochemical changes: INC OR DEC
Urea, creatinine, potassium, phosphate, magnesium, hydrogen ion
Increased
Early biomarker for diagnosis acute kidney injury
Neutrophil gelatinase associated Lipocalin
pag walang NGAL = Cystatin C
Produced from the deamination of amino acids during protein metabolism
Ammonia
Removed from the circulation and converted urea in the liver
Ammonia
Methods of determination of ammonia
Berthelot reaction
Nesslerization reaction
Enzymatic
End color of Berthelot reaction
Blue
Enzymatic method of determination of ammonia uses?
Glutamate dehydrogenase
Enzymatic method is the most common method
Ammonia is increased in
Hepatic failure, Reye’s syndrome, inherited deficiencies of urea cycle enzymes
NPN from highest to lowest UA-UC-CA
Urea > Amino acid > Uric acid > Creatinine > creatine > ammonia
Major end product of protein metabolism
Urea
Oldest method that uses less than 1mL of serum sample to measure urea through nitrogen concentration
N → NH4 + alk. K2HgI4 → NH2HgI3
Micro-kjedahl nessler
- Urease catalyzes the breakdown of urea into separate carbon dioxide
and ammonium ion that is usually collected from Jack Beans
o Urea → CO2 + NH3
o NH3 + Nessler’s Reagent → NH2HgI3
Urease-nessler method
Normal value of urea
7-18 mg/dL
2.5-6.4 mmol/L
Conversion factor for urea
0.357
Ratio of BUN to creatinine
10-20:1
- Available on Ektachem analyzer wherein creatinine is hydrolyzed to N-methyldantolin and ammonia by creatinase. The ammonia is then
made to react with alpha-ketoglutarate and NADH in the presence of
glutamate dehydrogenase forming glutamate and NAD. The decrease
in NADH is measured fluorometrically. - Measures the oxidation of NADH into NAD at 340 nm
Creatinase method
- Creatinine is hydrolyzed to creatine by creatinine aminohydrolase
followed by a series of coupled enzyme reactions in which creatine
reacts with creatinine kinase, pyruvate kinase, and lactate
dehydrogenase, culminating in the oxidation of the NADH. - Pyruvate + NADH → Lactate Dehydrogenase → Lactate + NAD
- Measure the oxidation at 340 nm
- Alternatives:
o Trinder’s Reaction can also be used where product is hydrogen
peroxide (H2O2)
▪ Sarcosine reacts to Creatinine which also produce
hydrogen peroxide (H2O2)
Creatinine aminohydrolase
Normal value of creatinine
0.6-1.2 mg/dL
53-106 umol/L
- Jaffe Method:
o Male: 0.9–1.3 mg/dL (80–115 µmol/L)
o Female: 0.6–1.1 mg/dL (53–97 µmol/L) - Enzymatic Method:
o Male: 0.6–1.1 mg/dL (53–97 µmol/L)
o Female: 0.5–0.8 mg/dL (44–71 µmol/L
Conversion factor for creatinine
88.4
End product of nucleic acid metabolism
BUA
How many % of the filtered uric acid is reabsorbed?
89%
Modification of caraway’s method
UA + PTA → tungsten blue
Uses Na2CO3 (Sodium Carbonate) as color stabilizer
Henry’s method
Addition if this will increase BUA’s stability to bacterial destruction
Thymol
Anticoagulant than cannot be used in BUA determination
potassium oxalate
Normal values for BUA
o Men – 4.0 - 8.5 mg/dL (0.25 - 0.50 mmol/L)
o Women – 2.7 - 7.3 mg/dl (0.16 - 0.43 mmol/L)
Conversion factor for BUA
0.0595
BUA is _____ in these circumstances:
Von Gierke’s disease
Lactic acidosis
Toxemia of pregnancy
Increased
BUA is _____ in these circumstances:
Fanconi’s syndrome
Wilson’s disease
Hodgkin’s disease
Decreased
Carry blood towards glomerulus
Afferent arterioles
Carry blood away from glomerulus
Efferent arterioles
Collect the filtrate
Bowman’s capsule
o Regulates the reabsorption and excretion of Sodium
o Produced by the adrenal cortex; influence of the renin–
angiotensin mechanism (RAAS)
o Triggered by decreased blood flow or blood pressure in the
afferent renal arteriole
o Decreased plasma sodium
o Stimulates sodium reabsorption in the distal tubules and
potassium and hydrogen ion secretion
o When absorbing sodium, water is also reabsorbed in exchange
for potassium and hydrogen molecules which are excreted.
Aldosterone
o Arginine Vasopressin
o Regulates the excretion of water
o Peptide hormone secreted by the posterior pituitary
o Response to increased blood osmolality
o Released when blood volume decreases by more than 5%–10%
o Stimulates water reabsorption
o Water diffuses passively from the lumen of the tubules resulting
in more concentrated urine and decreased plasma osmolality
ADH
measures the plasma flow in the kidney
Para-amino hippurate test (PAH) or Diodrast Test
measures of the excretion of dye which is directly proportional to the renal mass to determine the renal blood flow
Phenosufophthalein (PSP) dye excretion test
measures the renal concentrating capability
of the kidney using the ff:
▪ Reagent Strip
▪ Urinometer
SG
Measure the balance between solutes and solvent
Osmolality
Osmolality formula
1.86 x Sodium + (Glucose/18) + (BUN/2.8)
Measures and determine the capability of the kidney to balance out, concentrate, and filter out the solutes coming from the renal blood flow
Fishberg concentration test
Considered as biochemical catalysts (speed up chemical reactions)
Enzymes
Site where substrates attached and undergo chemical reaction
Active site
Site opposite to the active site where regulator molecules attached (inhibitory)
Allosteric site
Nonprotein molecules that maybe necessary for enzyme activity
Cofactors
Inorganic cofactor (does not have carbon)
Activator
Organic cofactor
Coenzyme
Coenzyme that is bound tightly to the enzyme is called?
Prosthetic group
Enzymes existing in different forms within the same individual
Isoenzyme
Forms a complete and active system
Compose of apoenzyme (enzyme itself) and coenzyme
Holoenzyme
Enzymes that are originally secreted from the organ of production in a structurally inactive form
Zymogens and proenzymes
High specific: Acid phosphatase (ACP)
Rbcs, prostate gland
High specific: Alanine aminotransferase
Liver
High specific: Amylase
Pancreas, salivary glands
High specific: Lipase
Pancreas
Moderate specific: Aspartate aminotransferase (AST)
Liver, heart, skeletal muscle
Moderate specific: Creatine kinase
Heart, Skeletal muscle, Brain
Low specificity: Alkaline phosphatase (ALP)
Liver, bone, kidney, Placenta, Intestine
Low specificity: Lactate dehydrogenase
All tissues
Least specific enzyme
LDH
Catalyzes an oxidation-reduction reaction between two substrate
Uses dehydrogenase as the suffix
Oxidoreductase
Catalyze the transfer of a group other than hydrogen from one substrate to another
Uses transferase and kinase
Transferase
Catalyzes hydrolysis of various bonds
Suffix ase and in
Hydrolase
Catalyzes the removal of substrates WITHOUT hydrolysis
Contains double bonds
Lyase
Catalyze the interconversion of geometric, optical, or positional isomers
suffix isomerase
Isomerase
Catalyze the joining of two substrate molecules, coupled with breaking of the pyrophosphate bond in ATP
suffix -synthase
Ligases
First order kinetics:
Reaction rate is __________ to substrate concentration
Directly proportional
Zero order kinetics:
Reaction rate depends only on _______________
enzyme concentration
The __________ the enzyme level, the faster the reaction will proceed because ____ enzymes are present to bind with the substrate
Higher, more
pH of enzyme
7.0 - 8.0
Increasing the temperature will ____ the rate of chemical reaction
Increase
For each ___ degree increase in temperature, the rate of the reaction will approx. double
10 degree
Physically bind to the active site of an enzyme and compete with the substrate for active site
Reversible because enzymes is not damaged
Competitive inhibitors
Bind an enzyme at a place other than the active (allosteric site) and may be reversible or irreversible
Noncompetitive inhibitors
Is another kind of inhibition in which inhibitor binds to the Enzyme -substrate complex
Uncompetitive inhibitors
Has the capability to bind either on the enzyme or the enzyme-substrate complex at their active sites
It would not wait for a substrate to bind with enzyme
Mixed inhibitor
Optimal temperature for enzymes
37C
Increased temp for enzymes
40-50C
Temp of denaturation
56C
Stop reaction temperature
-20C
Phenomenon that states that a certain enzyme has the ability to adapt to their biochemical systems
Enzyme induction
Reaction that is initiated by addition of substrate
Reaction is allowed to proceed for a period of time
Measurement is done at the END of the reaction
Endpoint analysis / Fixed time analysis
Change in concentration of the indicator substance at several intervals
Continuous measurement of change in concentration as function of time
Multi-point and Kinetic assay/ Continuous monitoring
Units used to denote/express enzyme activity
IU and KATAL
Conventional unit
IU/L
Equivalent to the amount of enzyme that has catalyzes the conversion of 1 MICROMOLE of substrate per MINUTE under controlled condition
IU/L
System internationale unit
Katal unit
Equivalent to the amount of enzyme that has catalyzes the conversion of 1 MOLE of substrate per SECOND under controlled conditions
Mol/s
Enzymes that has already undergone post translational mutations
Isoforms
Enzymes whose metal ions are intrinsically part of the molecule
Metalloenzyme
Defines the class to which the enzyme belongs
First number
Indicates subclass and sub class to which the enzyme is assigned
Two middle numbers
A specific serial number to which the enzyme is assigned
Last number
- Gives the means to determine total enzyme concentration in serum
and other body fluids - Accurately describes virtually all single-substrate enzyme-catalyzed
reactions and many bisubstrate reactions in which the concentration
of one substrate is constant throughout the course of the reaction.
Michaelis-Menten equation
Another means of showing how the speed of the reaction occurs based
on the given Michaelis-Menten Constant, Maximum Velocity, and
Substrate Concentration
LINEWEAVER-BURKPLOT EQUATION
Enzymes combine with only one substrate and catalyzes only one corresponding reaction
Absolute specificity
Enzymes combining with all substrates containing a particular chemical group
Group specificity
Enzymes are specific to chemical bonds
Bond specificity
Enzymes that predominantly combine with one optical isomer of a certain compound
Stereoisomeric specificity
Based on the rigid enzyme molecule which the substrate fit
Emils Fisher’s Lock and Key theory
Based on the attachment of a substrate to the active site of an enzyme, which then causes conformational changes in the enzyme.
More acceptable theory because the protein molecule is flexible enough to allow changes
Koshland’s induced fit theory
Enzymes are stable at ____ for at least 24 hours`
6C
Involved in the reversible phosphorylation of creatine by ATP
Creatine Kinase
Predominant physiologic function occurs in muscle cells, where it is involved in the storage of high-energy creatine phosphate
Creatine Kinase
Heart specific creatine kinase
CK-MB
Reference value of creatine kinase
- Male: 15-160 U/L
- Female: 15-130 U/L
o CK-MB: 6% of total CK
Electrophoresis pattern of Creatine kinase
CK-BB - Most rapidly moving isoenzyme which is a product of chromosome 14 (anodal)
CK-MB - Hybrid which is from chromosome 19
CK-MM - slowest and most common form (most cathodal, least anodal)
is the first enzyme to increase
after onset signs and symptoms after 4-6 hours (Peaks at 24 hrs.)
Creatine Kinase
Highest elevation of total CK
Duchenne’s muscular dystrophy 50-100X than normal value
Methods of determination of CK
Tanzer-Gilvarg assay (forward direct method)
Oliver-Rosalki Method (Reverse/Indirect method)
o pH 9.0 at 340 nm
o Creatine + ATP → CPK → Creatine PO4 + ADP
o ADP + phosphoenolpyruvate → PK → Pyruvate + ATP
o Pyruvate + NADH → LD → Lactate + NAD
Tanzer-Gilvarg assay
o pH 6.8 at 340 nm;
o most commonly used; faster reaction by 6 times
o Creatine PO4 + ADP → CPK → Creatine + ATP
o ATP + Glucose→ HK → ADP + glucose-6-PO4
o Glucose-6-PO4 + NADP → G-6-PD → 6-phosphogluconate +
NADPH
Oliver-rosalki method
Anticoagulant that inhibits CK action
Oxalates and fluoride
CK-BB bounds to
IgG
CK-MM bounds to
IgA
An enzyme that catalyzes the interconversion of lactic and pyruvic acid
LDH
LD1
HHHH:H4
LD2
HHHM:H3M
LD3
HHMM:H2M2
LD4
HMMM:HM3
LD5 (PINAKA MASARAP)
MMMM:M4
LD6
Alcohol dehydrogenase
LD flip pattern
LD2 is always higher than LD1 eg. Heart attack, hemolysis in pernicious anemia, and kidney infarction
LD flipped pattern in bacterial meningitis and meningococcemia
LD4 and LD5 is flipped
LD1 and LD2 are seen in?
Heart and RBC
LD3 is seen in?
Lungs, pancreas, spleen, lymphocytes
LD4 and LD5
Skeletal muscle, liver, intestine
Reference Values of LDH
o 100-225 U/L (forward reaction)
o 80-280 U/L (reverse reaction)
Electrophoresis of LDH from fastest to slowest
LD1 > LD2 >LD3 > LD4 > LD5
Normal LDH in serum
LD 2 > LD 1 >LD 3 > LD4 > LD5
LDH if px has heart attack
LD1 > LD 2 > LD 3 > LD4 >LD 5
LDH if px has bacterial infection
LD2> LD1 > LD3 > LD5 > LD4
LD6 (alcohol dehydrogenase will only be seen if the patient is
Close to death
Highest level of LDH is seen in
Pernicious anemia and hemolytic disorder
10 fold increase especially LD5 which is markedly increased
Hepatic carcinoma and toxic hepatitis
2-3x increased
Viral hepatitis and cirrhosis
Methods of determination of LDH
Wacker method (forward/direct method)
Wrobleuski-Ladue (reverse reaction)
Involved in the transfer of an amino group between aspartate and alpha ketoacids with the formation of oxaloacetate and glutamate
AST
Highest concentration of AST is found in the
liver and cardiac tissue and skeletal muscle
Reference value of AST
5-37 U/L
Method of determination of AST
Karmen method - uses malate dehydrogenase and monitors the change in absorbance
Catalyzes the transfer of amino group from ALANINE to ALPHA KETOGLUTARATE
ALT
Reference value of ALT
6-37 U/L
Method of determination of ALT
Reitman-Frankel method
Highest concentration of ALT
Liver
Enzyme involved in the cleavage of phosphate containing compounds in alkaline pH
ALP
Placental isoenzyme is highest at ___-____the week of gestation
16-20th
Reference value of ALP
30-90
Liver ALP is inhibited by
Levamisole
Most heat labile and inhibited by 5M urea and levamisole
Bone ALP
Most heat stable (60-65C) and inhibited by phenylalanine reagent
Placental ALP
Inhibited by phenylalanine reagent
Intestinal ALP
lung, breast, ovarian and gynecological cancers; bone ALP co-migrator; most heat stable, inhibited by phenylalanine reagent
Regan ALP
Adenocarcinoma of the pancreas and bile duct, pleural cancer, variant of Regan; inhibited by L-leucine and phenylalanine
Nagao ALP
Hepatoma/Hepatocellular carcinoma
Kasahara ALP
ALP is highest in?
Paget’s disease (bone disease)
Method of determination of ALP
Bowers and Mc Comb - ph 10.15 405nm
End product and substrate of Klein, Babson, and Reid
o Substrate: Buffered phenolphthalein phosphate
o End Product: Free phenolphthalein
End product and substrate of Moss
o Substrate: Alpha-naphthol phosphate
o End Product: Alpha-naphthol
End product and substrate of Huggins and Talalay
o Substrate: Phenolphthalein diphosphate
o End Product: Phenolphthalein red
End product and substrate of Bessey, Lowry and Brock, Bowers and Mc Comb
o Substrate: p-nitrophenyl phosphate
o End Product: p-nitrophenol or yellow nitrophenoxide ion
End product and substrate of King and armstrong
o Substrate: Phenylphosphate
o End Product: Phenol
End product and substrate of Bodansky, Shinowara, Jones, Reinhart
Substrate: Beta-glycerophosphate
o End Product: Inorganic phosphate + glycerol
Heat stable to heat labile
PlaINLiBo (Promise ikaw lang baby)
Placenta, Intestine, Liver, Bone
Electrophoresis pattern of ALP
LiBoPlaIn
Most important activator of ALP
Magnesium
Reference method for measurement of total ALP
Bower’s mccomb PNPP
Diagnostic significance of ACP
Detection of prostatic carcinoma
Used in the investigation of rape
ACP
Method of determination of ACP
Shinowara
Babson, read, and phillips
Roy and HIllman
Gutman and gutman
Test for alcoholic abuse
GGT
Major source of GGT
Liver damage (Chronic cholestasis)
Obstructive jaundice increases GGT by
10 times
Hepatobillary damage
ALP and GGT
Hepatocellular damage
ALT and AST
Catalyzes the starch into dextrin
Amylase/Diastase
Smallest enzyme and earliest pancreatic marker
Amylase
Salivary amylase
Ptyalin (s-type)
Acinar cells of the pancrease for conversion of dextrine to maltose
Amylopsin (P-type)
Reference method for amylase determination
Saccharogenic method
AMYLASE DETERMINATION
- Saccharogenic – measures the amount of reducing sugars produced by
the hydrolysis of starch - Amyloclastic – amylase activity is evaluated by following the decrease
in substrate concentration - Chronometric – measures the time required for amylase to be
completely hydrolyzed - Amylometric – measures the amount of starch hydrolyzed in a fixed
period of time
Activators of amylase
Calcium and Chloride
Hydrolyzes the ester linkage of fats
Lipase
Most specific pancreas marker
Lipase
Lipase determination method
Cherry Crandall Method uses olive oil
Bilirubin transport deficit
Gilbert’s syndrome
defective ligandin = increase B1
Conjugation deficit syndrome
Crigler-Najjar syndrome
B1 cannot be converted to b2
Increase B1
Bilirubin excretion deficit
Dubin-Johnson syndrome
B2 cannot be excreted to bile
Elevated b2
Familial form of unconjugated hyperbilirubinemia
Lucey-Driscoll syndrome
UDPGT inhibitor is circulating
Elevated B1
Mixed hyperbilirubinemia
Rotor syndrome
Autosomal recessive
Elevated B1 and B2
Deficiency of enzyme glucoronyl transferase (low UDGPT)
Physiologic jaundice of the newborn
cause kernitcerus
Elevated b1
Nawawala din kasi normal
