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Review of Analytical Methods Part 1: Spectrophotometry

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1 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

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 Electromagnetic radiation
H A Velocity = c Wavelength ()

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

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

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

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

9 Molecular electronic energy transitions
Singlet A VR Triplet IC F P sec sec E0

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

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

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

16 Direct photometry max= 280 nm 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

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

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

21 max= 400–540 (pH-dependant)
Measuring glucose Glucose + O2 Gluconic acid + H2O2 Glucose oxidase Peroxidase o-Dianiside Oxidized o-dianiside max= 400–540 (pH-dependant) 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 creatinine (and -keto acids)
Kinetic Jaffe method 80 20 t A (pyruvate, glucose, Fast-reacting ascorbate) Absorbance ( = 520 nm) Slow-reacting (protein) creatinine (and -keto acids) 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 Measuring phosphate Phosphate in serum occurs in two forms:
H3PO4 + (NH4)6Mo7O24 H+ (NH4)3[PO4(MoO3)12] Molybdenum blue max= nm Red. max= 340 nm Phosphate in serum occurs in two forms: H2PO4- and HPO4-2 Only inorganic phosphate is measured by this method. Organic phosphate is primarily intracellular.

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 Measuring iron Fe++ max= 534 nm Fe++ max= 562 nm 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 Enzymatic cholesterol methods
Cholesterol esters Cholesterol Cholesteryl ester hydroxylase Choles-4-en-3-one + H2O2 Cholesterol oxidase Quinoneimine dye (max500 nm) Phenol 4-aminoantipyrine Peroxidase 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 Measuring HDL cholesterol
Ultracentrifugation is the most accurate method HDL has density – 1.21 g/mL Routine methods precipitate Apo-B-100 lipoprotein with a polyanion/divalent cation Includes VLDL, IDL, Lp(a), LDL, and chylomicrons HDL, IDL, LDL, VLDL HDL + (IDL, LDL, VLDL) Dextran sulfate Mg++ Newer automated methods use a modified form of cholesterol esterase, which selectively reacts with HDL cholesterol

42 Measuring triglycerides
Glycerol + FFAs Lipase Glycerophosphate + ADP Glycerokinase ATP Dihydroxyacetone + H2O2 Glycerophasphate oxidase Peroxidase Quinoneimine dye (max 500 nm) 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 The Michaelis-Menton curve
Vmax v  [S]  ½Vmax Km

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

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

51 L-B analysis of an enzyme inhibitor
Non-competitive Competitive 1/[S]  1/v  1/Vmax -1/Km

52 Measuring enzyme-catalyzed reactions
The progress of an enzyme-catalyzed reaction can be followed by measuring: The disappearance of substrate The appearance of product The conversion of a cofactor

53 Measuring enzyme-catalyzed reactions
When the substrate is in excess, the rate of the reaction depends on the enzyme activity When the enzyme is in excess, the rate of the reaction depends on the substrate concentration

54 Enzyme cofactors Nicotinamide adenine dinucleotide (NAD+, oxidized form)

55 Enzyme cofactors NADH (reduced form) Phosphate attachment
(NADP+ and NADPH) NADH (reduced form)

56 NAD UV absorption spectra
Absorbance  250 300 350 400  (nm) NAD+ NADH max= 340 nm

57 Enzyme reaction profile
Product  Time  Lag phase Linear phase Substrate depletion Mix

58 Measuring glucose by hexokinase
The hexokinase method is used in about half of all clinical laboratories Some hexokinase methods use NADP, depending on the source of G-6-PD enzyme A reference method has been developed for glucose based on the hexokinase reaction

59 Measuring bicarbonate
The specimen is alkalinized to convert all forms of CO2 to HCO3-, so the method actually measures total CO2 Enzymatic methods for total CO2 are most common, followed by electrode methods

60 Measuring lactate dehydrogenase
Both PL and LP methods are available At physiological pH, PL reaction if favored LP reaction requires pH of LD (sometimes designated LDH) activity will vary, depending on which method is used

61 Measuring creatine kinase (CK)
Both creatine and phosphocreatine spontaneously hydrolyze to creatinine The reverse (PCrCr) reaction is favorable, although the reagents are more expensive All methods involve measurement of ATP or ADP

62 Measuring creatine kinase
Potential sources of interferences include: Glutathione (Glutathione reductase also uses NADPH as a cofactor) Adenosine kinase phosphorylates ADP to ATP (fluoride ion inhibits AK activity Calcium ion may inhibit CK activity, since the enzyme is Mg++-dependent.

63 Measuring creatine kinase
Since the forward (Cr PCr) reaction is slower, the method is not sensitive Luminescent methods have been developed, linking ATP to luciferin activation

64 Measuring alkaline phosphatase
The natural substrate for ALKP is not known

65 Measuring transaminase enzymes
Pyridoxyl-5-phosphate is a required cofactor Oxaloacetate and pyruvate are measured with their corresponding dehydrogenase enzymes, MD and LD

66 Measuring gamma glutamyl transferase
Method described by Szasz in 1969, and modified by Rosalki and Tarlow

67 Measuring amylase (14) Hydrolysis of both (14) and (1 6) linkages occur, but at different rates. Hence, the amylase activity measured will depend on the selected substrate There are more approaches to measuring amylase than virtually any other common clinical analyte

68 Amyloclastic amylase method
Red complex Starch + I2 Blue complex The rate of disappearance of the blue complex is proportional to amylase activity Starch also can be measured turbidimetrically Starch-based methods for amylase measurement are not very common any more

69 Saccharogenic amylase method
Starch Amylase Glucose + Maltose Reduced substrate Several methods can be used to quantify the reducing sugars liberated from starch Somogyi described a saccharogenic amylase method, and defined the units of activity in terms of “reducing equivalents of glucose” Alternatively, glucose or maltose can be measured by conventional enzymatic methods

70 Chromogenic amylase method
Small dye-labeled fragments Dye-labeled starch Photometric measurement of dye Separation step J&J Vitros application allows small dye-labeled fragments to diffuse through a filter layer Abbott FP method uses fluorescein-labeled starch

71 Defined-substrate amylase method
4-NP-(Glucose)4,3,2 4-NP-(Glucose)7 -Glucosidase 4-NP-(Glucose)4 + Glucose + NP max= 405 nm -Glucosidase does not react with oligosaccharides containing more than 4 glucose residues A modification of this approach uses -2-chloro-4-NP, which has a higher molar absorptivity than 4-NP

72 Measuring lipase (direct)
The Cherry/Crandall procedure involves lipase degradation of olive oil and measurement of liberated fatty acids by titration Alternatively, the decrease in turbidity of a triglyceride emulsion can be monitored For full activity and specificity, addition of the coenzyme colipase is required

73 Measuring lipase (indirect)
Indirect methods for lipase measurement focus on: Enzymatic phosphorylation (Glycerol kinase) and oxidation (L--Glycerophosphate oxidase) of glycerol, and measurement of liberated H2O2 Dye-labeled diglyceride that releases a chromophore when hydrolyzed by lipase Several non-triglyceride substrates have been proposed, as well

74 Post-test Identify the methods proposed by the following: Folin-Wu
Jendrassik-Grof Somogyi-Nelson Kjeldahl Lieberman-Bourchard Rosalki-Tarlow Jaffe Bertholet Fisk-Subbarrow Glucose Bilirubin Glucose/Amylase Total protein Cholesterol GGT Creatinine Urea Phosphate

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