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Review of Analytical Methods Part 1: Spectrophotometry

Review of Analytical Methods Part 1: Spectrophotometry. Roger L. Bertholf, Ph.D. Associate Professor of Pathology Chief of Clinical Chemistry & Toxicology University of Florida Health Science Center/Jacksonville. Analytical methods used in clinical chemistry. Spectrophotometry

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Review of Analytical Methods Part 1: Spectrophotometry

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  1. Review of Analytical MethodsPart 1: Spectrophotometry Roger L. Bertholf, Ph.D. Associate Professor of Pathology Chief of Clinical Chemistry & Toxicology University of Florida Health Science Center/Jacksonville

  2. Analytical methods used in clinical chemistry • Spectrophotometry • Electrochemistry • Immunochemistry • Other • Osmometry • Chromatography • Electrophoresis

  3. Introduction to spectrophotometry • Involves interaction of electromagnetic radiation with matter • For laboratory application, typically involves radiation in the ultraviolet and visible regions of the spectrum. • Absorbance of electromagnetic radiation is quantitative.

  4. E A H Wavelength () Electromagnetic radiation Velocity = c

  5. Wavelength, frequency, and energy • E = energy • h = Plank’s constant • = frequency c = speed of light  = wavelength

  6. Wavelength (, cm) 10-11 10-9 10-6 10-5 10-4 10-2 102   x-ray UV visible IR Rf 1021 1019 1016 1015 1014 1012 108 Frequency (, Hz) Inner shell electrons Outer shell electrons Molecular vibrations Molecular rotation Nuclear Spin Nuclear The Electromagnetic Spectrum

  7. Wavelength (nm) 390 450 520 590 620 780 Increasing Energy UV IR  Increasing Wavelength Visible spectrum “Red-Orange-Yellow-Green-Blue”

  8. * or * molecular orbital s or p atomic orbital n* n * non-bonding orbital Energy * * n n • or  molecular orbital Molecular orbital energies

  9. Singlet E4 E3 E2 Triplet VR E1 IC A F 10-6-10-9 sec P 10-4-10 sec E0 Molecular electronic energy transitions

  10. I0 (radiant intensity) I (transmitted intensity) Absorption of EM radiation

  11. Manipulation of Beer’s Law Hence, 50% transmittance results in an absorbance of 0.301, and an absorbance of 2.0 corresponds to 1% transmittance

  12. Error (dA/A) 0.0 0.434 2.0 Absorbance  Beer’s Law error in measurement

  13. Design of spectrometric methods • The analyte absorbs at a unique wavelength (not very common) • The analyte reacts with a reagent to produce an adduct that absorbs at a unique wavelength (a chromophore) • The analyte is involved in a reaction that produces a chromophore

  14. Measuring total protein • All proteins are composed of 20 (or so) amino acids. • There are several analytical methods for measuring proteins: • Kjeldahl’s method (reference) • Direct photometry • Folin-Ciocalteu (Lowery) method • Dye-binding methods (Amido black; Coomassie Brilliant Blue; Silver) • Precipitation with sulfosalicylic acid or trichloracetic acid (TCA) • Biuret method

  15. Hot H2SO4 digestion Correction for non-protein nitrogen NH4+ Titration or Nessler’s reagent (KI/HgCl2/KOH) Protein nitrogen Multiply by 6.25 (100%/16%) Total protein Kjeldahl’s method Specimen

  16. Direct photometry • Absorption at 200–225 nm can also be used (max for peptide bonds) • Free Tyr and Trp, uric acid, and bilirubin interfere at 280 nm max= 280 nm

  17. Protein (Tyr, Trp) Folin-Ciocalteu (Lowry) method • Sometimes used in combination with biuret method • 100 times more sensitive than biuret alone • Typically requires some purification, due to interferences Phosphotungstic/phosphomolybdic acid Reduced form (blue)

  18. Biuret method • Sodium potassium tartrate is added to complex and stabilize the Cu++ (cupric) ions • Iodide is added as an antioxidant

  19. Measuring albumin • Albumin is the most abundant protein in serum (40-60% of total protein) • Albumin is an anionic protein (pI=4.0-5.8) • Enriched in Asp, Glu •  Albumin reacts with anionic dyes • BCG (max= 628 nm), BCP (max= 603 nm) • Binding of BCG and BCP is not specific, since other proteins have Asp and Glu residues • Reading absorbance within 30 s improves specificity

  20. 30 s Specificity of bromocresol dyes BCG (pH 4.2) Albumin green or purple adduct BCP (pH 5.2) Absorbance  Time 

  21. Glucose oxidase Glucose + O2 Gluconic acid + H2O2 Peroxidase o-Dianiside Oxidized o-dianiside max= 400–540 (pH-dependant) Measuring glucose • Glucose is highly specific for -D-Glucose • The peroxidase step is subject to interferences from several endogeneous substances • Uric acid in urine can produce falsely low results • Potassium ferrocyanide reduces bilirubin interference • About a fourth of clinical laboratories use the glucose oxidase method

  22. Glucose isomers • The interconversion of the  and  isomers of glucose is spontaneous, but slow • Mutorotase is added to glucose oxidase reagent systems to accelerate the interconversion

  23. Measuring creatinine • The reaction of creatinine and alkaline picrate was described in 1886 by Max Eduard Jaffe • Many other compounds also react with picrate

  24. Modifications of the Jaffe method • Fuller’s Earth (aluminum silicate, Lloyd’s reagent) • adsorbs creatinine to eliminate protein interference • Acid blanking • after color development; dissociates Janovsky complex • Pre-oxidation • addition of ferricyanide oxidizes bilirubin • Kinetic methods

  25. A t 20 80 Kinetic Jaffe method Fast-reacting (pyruvate, glucose, ascorbate) Absorbance ( = 520 nm) Slow-reacting (protein) creatinine (and -keto acids) 0 Time (sec) 

  26. Enzymatic creatinine method • H2O2 is measured by conventional peroxidase/dye methods

  27. Enzymatic creatinine method • H2O2 is measured by conventional peroxidase/dye methods

  28. Measuring urea (direct method) • Direct methods measure a chromagen produced directly from urea • Indirect methods measure ammonia, produced from urea

  29. Measuring urea (indirect method) • The second step is called the Berthelot reaction • In the U.S., urea is customarily reported as “Blood Urea Nitrogen” (BUN), even though . . . • It is not measured in blood (it is measured in serum) • Urea is measured, not nitrogen

  30. Conversion of urea/BUN

  31. Measuring uric acid • Tungsten blue absorbs at  = 650-700 nm • Uricase enzyme catalyzes the same reaction, and is more specific • Absorbance of uric acid at   585 nm is monitored • Methods based on measurement of H2O2 are common

  32. Measuring total calcium • More than 90% of laboratories use one or the other of these methods. • Specimens are acidified to release Ca++ from protein, but the CPC-Ca++ complex forms at alkaline pH

  33. Molybdenum blue H+ Red. (NH4)3[PO4(MoO3)12] H3PO4 + (NH4)6Mo7O24 max= 600-700 nm Measuring phosphate • Phosphate in serum occurs in two forms: • H2PO4- and HPO4-2 • Only inorganic phosphate is measured by this method. Organic phosphate is primarily intracellular. max= 340 nm

  34. Measuring magnesium • Formazan dye and Xylidyl blue (Magnon) are also used to complex magnesium • 27Mg neutron activation is the definitive method, but atomic absorption is used as a reference method

  35. Fe++ max= 534 nm Fe++ max= 562 nm Measuring iron • The specimen is acidified to release iron from transferrin and reduce Fe3+ to Fe2+ (ferrous ion)

  36. Measuring bilirubin • Diazo reaction with bilirubin was first described by Erlich in 1883 • Azobilirubin isomers absorb at 600 nm

  37. Evolution of the diazo method • 1916: van den Bergh and Muller discover that alcohol accelerates the reaction, and coined the terms “direct” and “indirect” bilirubin • 1938: Jendrassik and Grof add caffeine and sodium benzoate as accelerators • Presumably, the caffeine and benzoate displace un-conjugated bilirubin from albumin • The Jendrassik/Grof method was later modified by Doumas, and is in common use today

  38. Bilirubin sub-forms • HPLC analysis has demonstrated at least 4 distinct forms of bilirubin in serum: • -bilirubin is the un-conjugated form (27% of total bilirubin) • -bilirubin is mono-conjugated with glucuronic acid (24%) • -bilirubin is di-conjugated with glucuronic acid (13%) • -bilirubin is irreversibly bound to protein (37%) • Only the , , and  fractions are soluble in water, and therefore correspond to the direct fraction • -bilirubin is solubilized by alcohols, and is present, along with all of the other sub-forms, in the indirect fraction

  39. Measuring cholesterol by L-B • The Liebermann-Burchard method is used by the CDC to establish reference materials • Cholesterol esters are hydrolyzed and extracted into hexane prior to the L-B reaction

  40. Cholesteryl ester hydroxylase Cholesterol Cholesterol oxidase Choles-4-en-3-one + H2O2 Phenol 4-aminoantipyrine Peroxidase Quinoneimine dye (max500 nm) Enzymatic cholesterol methods Cholesterol esters • Enzymatic methods are most commonly adapted to automated chemistry analyzers • The reaction is not entirely specific for cholesterol, but interferences in serum are minimal

  41. Dextran sulfate HDL, IDL, LDL, VLDL HDL + (IDL, LDL, VLDL) Mg++ Measuring HDL cholesterol • Ultracentrifugation is the most accurate method • HDL has density 1.063 – 1.21 g/mL • Routine methods precipitate Apo-B-100 lipoprotein with a polyanion/divalent cation • Includes VLDL, IDL, Lp(a), LDL, and chylomicrons • Newer automated methods use a modified form of cholesterol esterase, which selectively reacts with HDL cholesterol

  42. Lipase Glycerokinase ATP Glycerol + FFAs Glycerophosphate + ADP Glycerophasphate oxidase Peroxidase Dihydroxyacetone + H2O2 Quinoneimine dye (max 500 nm) Measuring triglycerides Triglycerides • LDL is often estimated based on triglyceride concentration, using the Friedwald Equation: [LDL chol] = [Total chol] – [HDL chol] – [Triglyceride]/5

  43. Spectrophotometric methods involving enzymes • Often, enzymes are used to facilitate a direct measurement (cholesterol, triglycerides) • Enzymes may be used to indirectly measure the concentration of a substrate (glucose, uric acid, creatinine) • Some analytical methods are designed to measure clinically important enzymes

  44. Enzyme kinetics The Km(Michaelis constant) for an enzyme reaction is a measure of the affinity of substrate for the enzyme. Km is a thermodynamic quantity, and has nothing to do with the rate of the enzyme-catalyzed reaction.

  45. Enzyme kinetics

  46. The Michaelis-Menton equation The Lineweaver-Burk equation is of the form y = ax + b, so a plot of 1/v versus 1/[S] gives a straight line, from which Km and Vmax can be derived.

  47. Vmax ½Vmax v [S]  Km The Michaelis-Menton curve

  48. 1/v  1/Vmax 1/[S]  -1/Km The Lineweaver-Burk plot

  49. Enzyme inhibition • Competitive inhibitors compete with the substrate for the enzyme active site (Km) • Non-competitive inhibitors alter the ability of the enzyme to convert substrate to product (Vmax) • Un-competitive inhibitors affect both the enzyme substrate complex and conversion of substrate to product (both Km and Vmax)

  50. Vmax Vmax(i) v Competitive Non-competitive [S]  Km Km(i) M-M analysis of an enzyme inhibitor

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