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Chapter 4 Analytical Technique and Instrumentation

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Title: Chapter 4 Analytical Technique and Instrumentation


1
Chapter 4 Analytical Technique and Instrumentation
2
  • 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

3
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.

4
  • 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.

5
  • 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.

6
  • 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

7
  • 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.

8
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

9
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.

10
Chemiluminescence
  • Oxidation reactions of luminol, acridinium
    esters, and dioxetanes.
  • Advantages - speed
  • Disadvantage- background signals interfere with
    sensitivity and specificity.

11
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.

12
  • 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.

13
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

14
Chapter 5 Principles of Clinical Chemistry
Automation
15
  • 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

16
  • Laboratory personnel must perform and observe
  • Quality Assurance
  • Quality Control
  • Calculations
  • Procedures
  • Testing factors
  • Preanalytical
  • Analytical
  • Post analytical

17
Chapter 8 Amino acids and Proteins
18
  • 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.

19
  • 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.

20
  • 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.

21
  • 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.

22
  • 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.

23
  • 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.

24
  • 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.

25
  • 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.

26
  • 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.

27
  • 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

28
  • 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.

29
  • 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

30
  • 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

31
  • Plasma Proteins
  • Most frequently analyzed portion of the blood
  • Over 500 kind
  • Indicators of many diseases and disease processes

32
  • 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.

33
  • 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

34
  • Decreased levels of albumin associated with
  • Malnutrition
  • Muscle waste
  • Liver disease
  • Hepatitis
  • Renal disease

35
  • 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

36
  • 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.

37
  • 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.

38
  • 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.

39
  • 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

40
  • 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.

41
  • 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.

42
  • 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.

43
  • Lipoproteins
  • Complex of proteins and lipids that function to
    transport cholesterol, triglycerides and
    phospholipids in the blood
  • Subclasses HDL, LDL, VLDL, Pre-beta.

44
  • 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.

45
  • 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.

46
  • 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

47
  • 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

48
  • 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.

49
  • 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.

50
  • 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

51
  • 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.

52
  • 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.

53
  • Samples analyzed
  • Serum most often.
  • Urine Reagent strip method or 24 hr urine
    collection.
  • CSF usually utilizes the turbidmetric method.

54
Chapter 10 Enzymes
55
  • 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

56
  • 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

57
  • 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

58
  • 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

59
  • 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.

60
  • 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.

61
  • 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

62
  • 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.

63
  • 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

64
  • 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.

65
  • 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.

66
  • Alanine Aminotransferase ALT or SGPT
  • Catalyze the transfer of amino groups alpha
    ketoglutartrate to glutamate and pyruvate.
  • Specific for liver
  • Assay coupled enzyme reaction

67
  • 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.

68
  • 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.

69
  • 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.

70
  • 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.

71
  • 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

72
  • 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

73
  • 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.

74
  • 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

75
  • 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.
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