Chapter 4 Analytical Technique and Instrumentation

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Chapter 4 Analytical Technique and Instrumentation
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• Analytic technique and instrumentation is the
  foundation for all measurements in the clinical
  chemistry department.
• 4 Basic types of analytic technique
• Spectrophotometry
• Luminescene
• Electroanalytical methods
• chromotography

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Spectrophotometry

• A photometric instrument that measures light
  intensity without the consideration of
  wavelength.
• Most instruments today use filters, prisms or
  gratings to select a wavelength
• Plank formula E hv, describes the relationship
  between wavelength and energy, the frequency of a
  wave is inversely proportional to the wave
  length.
• Measures either the absorption or emission of
  radiant energy to determine the concentration of
  atoms or molecules.

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• Absorption or emission of energy by atom results
  in a line spectrum.
• Beers law based on the relationship between
  absorption of light by solution and the
  concentration of that solution.
• Beers states that the concentration of a
  substance is directly proportional to the amount
  of light absorbed or inversely proportional to
  the logarithm of the transmitted light.

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• Percent transmitted the ratio of the radiant
  energy transmitted divided by the radiant energy
  incident on the sample.
• Absorbance
• According to Beers law absorbance is directly
  proportional to the concentration of the solution.

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• Spectrophotometer is used to measure the light
  transmitted by a solution to determine the
  concentration of the light-absorbing substance in
  the solution.
• Components
• Light source
• Monochromator
• Sample cell
• Photo detector

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• Atomic Absorption Spectrophotometer
• Used to measure concentrations by detecting
  absorption of electromagnetic radiation by atoms.
• Light source- hollow-cathode lamp
• The amount of light absorbed is proportional to
  the concentration of analyte.
• The light detector must be able to distinguish
  between the light beam emitted by the hollow
  cathode lamp and that emitted by excited atoms
  in the flame.
• Beers law is used to calculate concentration
• Method is sensitive and precise.

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Flame photometry

• Flame-emission photometer- light emitted by
  excited atoms old method used to determine
  concentration of Na, K, or Li.
• Ion selective electrodes replaced Flame-Emission

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Fluorometry

• Measures wavelength that is emitted by the
  different analytes
• Measures the concentration of solutions that
  contain fluorescing
• Uses high-pressure xenon lamp-necessary for
  determining excitation spectra.
• Measures concentrations are related to molar
  absorptivity of the compound, intensity of the
  incident radiation, quantum absorbed, and length
  of the light paths.
• Technique used to detect therapeutic and abused
  drugs.

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Chemiluminescence

• Oxidation reactions of luminol, acridinium
  esters, and dioxetanes.
• Advantages - speed
• Disadvantage- background signals interfere with
  sensitivity and specificity.

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Ion selective electrode (ISE)

• Potentiometric method of analysis involves the
  direct measurement of electrical potential due to
  the activity of free ions.
• pH- utilizes silver wire coated AgCl- immersed
  into internal solution of HCL.
• Membrane sensitive to hydrogen ions that
  selectively measures Hydrogen that consist of
  specific quantities of lithium, cesium, lanthnum,
  barium, or aluminum oxides in silicate.

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• Reference electrodes- commonly used, is a calomel
  stabilizing electrode.
• 3 major ISE types
• Inert-metal standard H electrode
• Metal
• Membrane (solid, liquid or special)
• Note Potassium selective liquid membranes use
  antibiotic valinomycin as the ion-selective
  carrier.
• Other electrodes include gas-sensing and enzyme.

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Chromatography

• Techniques used to separate complex mixtures on
  the basis of different physical interactions
  between the individual compounds and stationary
  phase of the system.
• Techniques include Mobile phase and stationary
  phase
• Thin-layer (TLC)
• High performance Liquid (HPLC)
• Gas

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Chapter 5Principles of Clinical Chemistry
Automation
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• Clinical Chemistry is very automated today.
• Three basic approaches with instruments
• Continuous flow
• Centrifugal analysis
• Discrete
• With automation there is still some very basic
  steps such as
• Specimen preparation and Identification
• Specimen measurements and delivery
• Reagents systems and delivery
• Chemical reaction
• Measurement phase
• Signal processing and data handling

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• Laboratory personnel must perform and observe
• Quality Assurance
• Quality Control
• Calculations
• Procedures
• Testing factors
• Preanalytical
• Analytical
• Post analytical

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Chapter 8 Amino acids and Proteins
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• What are Amino Acids?
• They are small bimolecules that contain at least
  1 amino group (-NH2) and 1 carboxyl group (COOH).
• Differ on the R group.
• 20 different building blocks for protein
• 9 nutrionally essential A.A. supplied by dietary
  intake in the form of protein.
• Valine, leucine, isoleucine, methionine,
  tryptophan, phenylalanine, thereronine, lysine,
  histidine.
• All are synthesized by proteolytic enzymes
• Pepsin and Trypsin and dumped in the A.A. pool to
  be utilized for the synthesis of non-protein
  nitrogen compounds (purines, pyrimidines,
  porphyrins, creatine, histamines, thyroxine,
  epinephrine and CoEnzyme NAD.

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• As proteins are broken down they provide 12-20
  of bodies total energy.
• During the breakdown of AA certain groups are
  removed by
• Deamination
• Transamination
• Degrade the AA to acetyl CoA or Actoactyl CoA
  which are precursors to ketone bodies-ketogenic
  vs glucogenic.

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• Aminoacidopathies disease involving AA breakdown
  or lack thereof.
• Phenylketonuria (PKU)
• Def. of enzyme phenylalanine hydroxylase
• Leads to a build up of phenylalanine in the body
  system
• Cause retardation
• Treat by diet, avoid the intake of phenylalanine.

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• Hyperphenylaninemia
• Def. of enzyme needed to regenerate synthesis of
  BH4
• Increases blood levels of phenyalanine and
  deficient production of neurotransmitters from
  tyrosine and tryptophan.
• No treatment available.
• Both can be detected by the PKU- Guthrie test
  measured at 360nm and 530 nm.

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• Maple Syrup Disease MSUD
• Build up of enzyme in urine, breath, skin, CSF
  that has a characteristic maple sugar or burnt
  sugar odor.
• Causes retardation, convulsions, acidosis and
  death
• Detected by the modified Guthrie test at 450 nm
  and 360nm wavelength.

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• Proteins
• Essential compound
• 50-70 of cellular dry weight
• Found in all fluid, secretions and excretions.
• Macromoles
• Composed of covalent link of polymers of A.A.
  with the removal of H2O creating a peptide bond.
• Dipeptide, tripeptide, polypeptide.

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• Protein structures (4)
• Primary
• Secondary
• Tertiary
• Quaternary
• Determines the shape and effects the functions of
  a protein.
• If the structure is disturbed protein looses its
  function and molecular characteristics.

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• Denature proteins by
• Hydrolysis
• Enzymatic activity
• Exposure to a substance
• Exposure to U.V. light
• Proteins are described by their content, charge,
  solubility, immunogenicity, and synthesis
  capability.

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• Proteins are described by
• Nitrogen Content set apart from carbohydrates
  and lipids according to their nitrogen content.
• Charge the reactive acid or basic group involved
  in the peptide linkage exist in 3 charge forms
  based on pH of the surrounding environment.
• pI isoelectric point which is the pH at which an
  A.A. or protein has no net charge.
• pH less than pI a () charge exist
• Measure for specific proteins based on its known
  pI.

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• Solubility
• Immunogenicity (antigens and antibody production)
• Synthesis helps balance the body chemistry.
  Rate of synthesis varies and is based on the
  content.
• Types of synthesis
• Disintergration
• Catabolism

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• Nitrogen Balance helps in the balance of body
  substances by the utilization and synthesis of
  proteins.
• 2 types of Nitrogen balance
• Negative Nitrogen balance- leads to decreased
  protein levels in the serum.
• Positive Nitrogen Balance- Leads to increased
  protein in the serum.

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• Classification of proteins (2 major groups)
• Simple proteins- peptide chain when it is
  hydrolyzed- yields A.A.
• Conjugated protein contains a protein and a
  nonprotein

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• Functions of Proteins
• Tissue nutrition (unique to proteins)
• H2O distribution
• Maintain pH
• Transportation of other substances
• Antibody production
• Hormone receptors
• Structure
• Act as catalyst
• Hemostasis and coagulation

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• Plasma Proteins
• Most frequently analyzed portion of the blood
• Over 500 kind
• Indicators of many diseases and disease processes

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• Prealbumin
• Migrates ahead of albumin on electrophoresis
  analysis.
• Rich in trytophan 0.5 carbohydrates
• Transports thyroid hormones
• Decrease levels associated with hepatic damage,
  acute phase inflammation and tissue damage.
• Increased levels associated with steroid use,
  alcoholism and chronic renal failure.

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• Albumin
• High concentration found in serum
• Synthesized in the liver- diagnostic test for
  liver function.
• 2 methods for detecting albumin
• Kjeldahl
• Biuret- Dye binding
• Albumin functions as
• Colloid
• Binding
• Specimen of choice- Serum

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• Decreased levels of albumin associated with
• Malnutrition
• Muscle waste
• Liver disease
• Hepatitis
• Renal disease

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• Globulins
• Proteins that consist of fractions or sections.
• Based on the number of different proteins with
  different fractions.
• Fractions include
• Alpha 1 , Alpha 2, Beta, and Gamma

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• Alpha 1-Antitrypsin
• Acute phase reactant
• Major component of serum protein (90)
• Migrates behind albumin
• Associated with pulmonary disease, juvenile
  hepatic cirrhosis.
• Most common phenotype MM
• Abnormal levels observed by a lack of band
  electrophoretically.

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• Alpha-feto Protein (AFP)
• Synthesized by fetal yolk sac and the parenchymal
  cells of the liver
• Normally peaks _at_ 13 weeks gestation, decreases _at_
  34 weeks
• Used to detect neural tube defect
• Tumor marker for gonadal tumors and heptocellular
  carcinoma.
• Increased levels seen in spina bifida, fetal
  distress, neural tube defect, twins
• Decreased levels a3-4 fold decrease indicates
  risk of Down Syndrome.

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• Alpha 1- Acid glycoprotein
• 5 carbohydrate unit attached to a polypeptide
  chain.
• Involved with formation of membranes and fibers
  in collagen
• May inactivate hormones
• See increased levels in inflammation, R.A.,
  pregnancy, pneumonia and cancer.

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• Alpha 1 Antichymotrypsin
• A serine proteinase with cathepsin G
• Found in pancreatic elastase, mast cell, chymase
  and chymotrypsin ( its target enzyme.)
• Migrates between Alpha 1 and 2.
• Inter- alpha trypsin inhibitor
• 3 polypeptid subunits

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• Ceruloplasmin
• Copper containing glycoprotein with enzymatic
  activities
• Synthesized in the liver
• 90 of total serum copper
• Increased levels seen in pregnancy, malignancies,
  contraceptive use, oral estrogen.
• Decreased levels seen in Wilsons disease,
  Malnutrition, Malabsorption, Severe liver damage,
  nephrotic Syndrome.

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• Alpha 2 Macroglobulin
• Large protein
• Synthesized by heptocytes
• Seen in Renal, liver and diabetes diseases.
• Nephrosis seen, also see an increase 10X the
  normal range with nephrosis.

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• Transferrin
• Glycoprotein synthesized by the liver
• Binds ferric iron when plasma is saturated
• Transports iron
• Increased levels in Anemia's
• Decreased levels seen in liver disease,
  malnutrition, nephrotic syndrome
• May cause Bronze skin syndrome, cirrhosis or
  diabetes mellitus.

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• Lipoproteins
• Complex of proteins and lipids that function to
  transport cholesterol, triglycerides and
  phospholipids in the blood
• Subclasses HDL, LDL, VLDL, Pre-beta.

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• Beta Microglobulin (B2M)
• Light chain component of the major HLA complex
• Found on the surface of most nucleated cells and
  in high concentration on the lymphocytes.
• Small in size filtered by the renal glomerulus
• Increased levels indicate impaired kidney
  function, inflammation, R.A. and SLE.

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• Complement collective term for a combination of
  proteins that participate in the bodies immune
  response.
• See increased levels in inflammation, DIC and
  recurrent infections.
• Fibrinogen
• One of the largest proteins
• Synthesized in the liver
• Carbohydrate content glycoprotein
• Distinct band between Beta and Gamma region
• Functions to form clots (not found in serum)
• Increased levels seen in pregnancy and the use of
  oral birth control
• Decreased levels in coagulation diseases.

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• C-reactive protein CRP
• Non-detectable in healthy individuals.
• Synthesized in the liver
• Increased in inflammatory process
• Immunoglobulin- 5 Major groups
• IgA
• IgG
• IgM
• IgD
• IgE

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• IgA
• Increased in liver disease, various infections
  and autoimmune disorders
• Decreased in disease of depressed protein
  synthesis.
• IgM
• 1St immunoglobulin to appear in response to
  antigenic stimulation.
• Increased levels found in viral and bacterial
  diseases.
• Waldenstoms macroglobulnemia

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• IgE Idiotypic immunoglobulin usually increases
  in response to allergic and anaphylactic
  reactions
• IgD increased levels observed in liver disease,
  connective tissue disease and multiple myeloma.

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• Myoglobulin- protein found in skeletal and
  myocardial muscle. It is released when damage
  occurs to muscle. Indicator used to identify
  AMI-increased 3-4 hrs onset of the attack.
• Troponin 3 protein form that binds to the thin
  filaments of striated muscle (cardiac and
  skeletal)
• Cardiac Troponin (TnT) increased up to 4 hrs of
  AMI remains increased 10-14 days post damage.

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• Total protein (2 Disease states)
• Hypoproteinuria- decreased levels of protein and
  Negative nitrogen balance.
• Caused by nephrotic syndrome, gastrointestinal
  leakage, blood loss, malnutrition, extensive
  burn.
• 2. Hypernaturemia- increased protein levels,
  related to dehydration and monoclonal disorders

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• Methods of analysis
• Total nitrogen content
• Measures all the chemical nitrogen bonds in the
  substance.
• Utilizes chemilumenscence technology.
• Total Protein
• Kjeldahl Quantitative, uses organic acid
  (Trichoracetic acid or tungstic acid to
  precipitate the protein in the sample),
• Biuret Most commonly used, utilizes Cupric ions,
  measures absorbance _at_ 540nm
• Dye Binding Based on protein ability to bind.
  Uses Bromphenol Green or Bromphenol purple dye.

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• Electrophoresis separates proteins based on
  charge.
• Example is Serum Protein Electrophoresis (SPE)
  migrate in the following pattern normally
  albumin, Alpha 1, Alpha 2, Beta and Gamma
• Certain diseases have certain electrophoretic
  patterns.

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• Samples analyzed
• Serum most often.
• Urine Reagent strip method or 24 hr urine
  collection.
• CSF usually utilizes the turbidmetric method.

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Chapter 10 Enzymes
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• Enzymes substances that end in ase
• Catalyze chemical reactions
• Do not alter or change in composition in a
  chemical reaction
• Reactions are specific and essential to its
  physiological function.
• Found in serum and plasma
• Made up of A.A. sequences that have an active
  site, substrate and allosteric site

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• Isoenzyme a different form which originate from
  genetic or nongenetic enzyme based on physical
  properties.
• International Union of Biochemistry
• Enzyme Commission
• 4 digit classification of enzymes
• 1st digit assigns enzyme to 1 of 6 major classes
• Oxireductase
• Transferase
• Hydrolase
• Lyase
• Isomerase
• Ligase

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• Enzymatic Kinectics the process of the reaction
• Reaction proceeds toward a lower energy
• Enzymes catalyze reaction by lowering activation
  energy that the reactant must reach
• Factors that influence Enzyme reactions
• 1.Substrate concentration- Michaelis and Menten
  constant (km). Theorectically vMax and km can be
  determine from a plot based on the maximum
  velocity of a reaction rate and concentration of
  enzyme.
• Lineweaver-Burk Plot- theory that says double
  reciprocal plot of km constant yields straight
  line. Enzyme concentration effects the rate of
  the reaction. Increase enzyme increase the rate
  of the reaction.
• 2. pH (7-8 optimal pH)
• 3. Temperature
• 4. Cofactor- activators like Mg, Ca, Fe, Zn, K,
  Br, Cl
• 5. Coenzymes
• 6. Inhibitors

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• Enzyme activity measured
• Increase or decrease of enzymes signal damage in
  tissue and cellular form.
• Measure photometrically
• -NADH most frequently measured coenzymes absorbs
  light _at_ 340nm.
• Quantitation of Enzymes must have a controlled
  environments.
• Methods
• Fixed-Time (End-Point)
• Continuous (Multi-point)
• Kinetic

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• Calculations reported as activity unit using-
  International Units amount of enzyme that will
  catalyze the reaction of one micromole of
  substrate per minute under specific conditions.
• Lab reference value reported as (U/L)
• katal (mol/s) amount of enzyme which converts
  1mol of substrate per second.

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• Enzymes as Reagent
• Use enzyme to measure enzymatic physiological
  constituents of unknown substace.
• Readily available
• Creatine Kinase
• Associated with ATP regeneration.
• Catalyzes phosphorlation.
• Creatine ATP Creatine Phosphate ADP, requires
  Mg for full activity to occur.

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• Creatine Kinase
• Associated with ATP regeneration
• Catalyzes phosphoralation
• Muscle cells
• Increased in cardiac and skeletal tissue damage.
• Indicator for AMI and MD
• 3 Fractions
• CKBB brain- fast
• CKMB hybrid
• CKMM Muscle- slow

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• Atypical CK-isoenzymes
• Macro-CK migrates midway between CKMM and CKMB
• CK-Mi (mitochondrial CK)
• Assay catalyzed in forward and reverse reaction
  involving phosphorlation of creatine or ADP.
  Measured _at_ 340 nm
• Forward coupled with pyruvate kinase-lactate
  dehydrogenase-NADH, occurs 2-6X faster, pH 9.0.
• Reverse Coupled with hexokinase
  glucose-6-phosphate dehydrogenase-NADP, pH 6.8.

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• Lactate Dehydrogenase LD
• Catalyzes the interconversion of lactic and
  pyruvic acids
• Utilizes coenzyme NAD
• Tissue source
• Increased levels associated with cardiac, renal,
  neoplasm an hematological disorders.
• AMI increase 12-24 hrs of attack, peaks _at_ 48-72
  hrs, returns to normal within 10 days

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• 5 major fractions or Isoenzymes
• LD1- LD5
• LD1 and LD2 fastest look for heart disease see a
  pattern change LD2 before LD1 (flipped pattern).
• LD4 and LD5 seen in liver disease
• Normal migration LD2, LD1, LD3, LD4, Ld5
• Flipped pattern LD1, LD2, LD3, LD4, LD5.

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• Aspartate Aminotransferase (AST or SGOT)
• Transfer group
• Coenzyme pyridoxal phosphate
• Tissue source- highest levels involve liver
  damage.
• Assayed Karmen Method - a coupled enzyme
  reaction.
• Hemolysis causes a ten fold increase in values.

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• Alanine Aminotransferase ALT or SGPT
• Catalyze the transfer of amino groups alpha
  ketoglutartrate to glutamate and pyruvate.
• Specific for liver
• Assay coupled enzyme reaction

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• Alkaline Phosphatase ALP
• Catalyze hydrolysis of phosphomonoesters at pH of
  9-10.
• Requires Mg as an activator
• Tissue source
• Increased in obstructive disorders
• Increased in Bone diseases
• 4 isoenzymes bone, liver, Intestine and placenta.

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• Abnormal fractions
• Ragan and Nagao
• ALP assayed by
• No specific substrate, using continuous
  monitoring at 405nm
• Source of errors hemolysis and delay in
  processing, diet and Blood group.

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• Acid Phosphatase ACP
• Similar activity as ALP, pH 5.0
• Related to prostrate, bone, liver, spleen,
  kidney, platelets and RBC regeneration.
• Highest level seen in prostrate.
• Assay colorless reaction, pH 5.0
• Errors delay in centrifugation and separation of
  serum and cells, hemolysis and storage.

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• Gamma-glutamyltransferase (GGT)
• Enzyme involved in transfer of glutamyl residue
  from gamma-glutamyl peptide to A.A.
• Liver source
• Sensitive to cholestasis
• Indicator for early liver disease related to
  alcohol consumption.
• Assay- chromogenic, 405 420nm, continuous fixed
  point.

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• Amylase
• 4 method of analysis
• Amyloclastic
• Sacchrogenic
• Chromogenic
• Continous
• Hydrolase
• Catalyze breakdown of starch and glycogen
• Requires calcium and chloride ions for
  activation.
• Tissue source pancreas and salivary glands

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• Lipase
• Hydrolyzes ester linkage of fats to produce
  alcohols and fatty acid
• Involve with hydrolysis of triglycerides in the
  intestines.
• Tissue source- pancreas
• Stays elevated 14 days
• Assayed by 1. Liberation of fatty acids
• 2. Turbidmetric, 3. Cherry-Crandall

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• Cholinesterase
• Enzyme catalyze hydrolysis of choline esters to
  form choline and fatty acids.
• 2 Types 1. Acetylcholinesterase and 2. Psuedo
  cholinesterase
• Tissue sources
• ACHE RBC, Brain and nerve cells
• PCHE serum, liver, pnacreas, heart, white matter
• Method of analysis Manometric, Electrometric and
  photometric.

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• Glucose-6-phosphate dehydrogenase
• Catalyzes the oxidation of glucose 6 phosphate to
  6-phosphogluconate or lactone.
• G-6-PD important due to its reaction is the first
  step in the pentose-phosphate shunt of glucose
  metabolism with the final product of NADPH.
• Tissue source adrenal cortex, spleen, thymus,
  lymph nodes, mammary glands and RBC. Little is
  found in normal serum

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• Focused role is in RBC
• Functions to maintain NADPH in reduced form to
  assist in maintenance of glutathione in reduced
  form, which protects Hgb from oxidation by agents
  in cells.
• Decreased levels of G-6-PD can result in RBC
  fragility leading to hemolysis- anemia.
• Increased levels seen in MI and megloblastic
  anemia
• Assay uses red cell hemolysate.