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Review of Analytical Methods Part 1: Spectrophotometry Roger L. Bertholf, Ph.D. Associate Professor of Pathology Chief of Clinical Chemistry & Toxicology.

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Presentation on theme: "Review of Analytical Methods Part 1: Spectrophotometry Roger L. Bertholf, Ph.D. Associate Professor of Pathology Chief of Clinical Chemistry & Toxicology."— Presentation transcript:

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 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 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. 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 E H A Wavelength ( ) Velocity = c

5 Wavelength, frequency, and energy E = energy h = Planks constant = frequency c = speed of light = wavelength

6 The Electromagnetic Spectrum x-ray UVvisibleIR Rf Wavelength (, cm) Frequency (, Hz) Nuclear Inner shell electrons Outer shell electrons Molecular vibrations Molecular rotation Nuclear Spin

7 Visible spectrum Wavelength (nm) IR UV Increasing Energy Increasing Wavelength Red-Orange-Yellow-Green-Blue

8 Molecular orbital energies or molecular orbital s or p atomic orbital * or * molecular orbital non-bonding orbital n n * * Energy

9 Molecular electronic energy transitions E0E0 E4E4 E3E3 E2E2 E1E1 Singlet Triplet A VR F IC P sec sec

10 Absorption of EM radiation I 0 (radiant intensity) I (transmitted intensity)

11 Manipulation of Beers Law Hence, 50% transmittance results in an absorbance of 0.301, and an absorbance of 2.0 corresponds to 1% transmittance

12 Absorbance Error (dA/A) Beers Law error in measurement 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 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: –Kjeldahls 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 All proteins are composed of 20 (or so) amino acids. There are several analytical methods for measuring proteins: –Kjeldahls 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 Kjeldahls method Specimen Hot H 2 SO 4 digestion Correction for non-protein nitrogen NH 4 + Titration or Nesslers reagent (KI/HgCl 2 /KOH) Protein nitrogenTotal protein Multiply by 6.25 (100%/16%)

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

18 Biuret method Sodium potassium tartrate is added to complex and stabilize the Cu ++ (cupric) ions Iodide is added as an antioxidant 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 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 Albumin BCG (pH 4.2) BCP (pH 5.2) green or purple adduct Absorbance Time 30 s

21 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 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 Glucose + O 2 Gluconic acid + H 2 O 2 Glucose oxidase Peroxidase o-Dianiside Oxidized o-dianiside max = 400–540 (pH-dependant)

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 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 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 Fullers Earth (aluminum silicate, Lloyds reagent) –adsorbs creatinine to eliminate protein interference Acid blanking –after color development; dissociates Janovsky complex Pre-oxidation –addition of ferricyanide oxidizes bilirubin Kinetic methods Fullers Earth (aluminum silicate, Lloyds 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 Kinetic Jaffe method Absorbance ( = 520 nm) Time (sec) Fast-reacting (pyruvate, glucose, ascorbate) Slow-reacting (protein) t A creatinine (and -keto acids)

26 Enzymatic creatinine method H 2 O 2 is measured by conventional peroxidase/dye methods

27 Enzymatic creatinine method H 2 O 2 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 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 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 = 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 H 2 O 2 are common Tungsten blue absorbs at = 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 H 2 O 2 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 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: –H 2 PO 4 - and HPO 4 -2 Only inorganic phosphate is measured by this method. Organic phosphate is primarily intracellular. Phosphate in serum occurs in two forms: –H 2 PO 4 - and HPO 4 -2 Only inorganic phosphate is measured by this method. Organic phosphate is primarily intracellular. H 3 PO 4 + (NH 4 ) 6 Mo 7 O 24 H+H+ (NH 4 ) 3 [PO 4 (MoO 3 ) 12 ] max = 340 nm Molybdenum blue max = nm Red.

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

35 Measuring iron The specimen is acidified to release iron from transferrin and reduce Fe 3+ to Fe 2+ (ferrous ion) Fe ++ max = 534 nm Fe ++ max = 562 nm

36 Measuring bilirubin Diazo reaction with bilirubin was first described by Erlich in 1883 Azobilirubin isomers absorb at 600 nm 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 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 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 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 Enzymatic methods are most commonly adapted to automated chemistry analyzers The reaction is not entirely specific for cholesterol, but interferences in serum are minimal Enzymatic methods are most commonly adapted to automated chemistry analyzers The reaction is not entirely specific for cholesterol, but interferences in serum are minimal Cholesterol esters Cholesterol Cholesteryl ester hydroxylase Choles-4-en-3-one + H 2 O 2 Cholesterol oxidase Quinoneimine dye ( max 500 nm) Phenol 4-aminoantipyrine Peroxidase

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 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 LDL is often estimated based on triglyceride concentration, using the Friedwald Equation: [LDL chol] = [Total chol] – [HDL chol] – [Triglyceride]/5 LDL is often estimated based on triglyceride concentration, using the Friedwald Equation: [LDL chol] = [Total chol] – [HDL chol] – [Triglyceride]/5 Triglycerides Glycerol + FFAs Lipase Glycerophosphate + ADP Glycerokinase ATP Dihydroxyacetone + H 2 O 2 Glycerophasphate oxidase Peroxidase Quinoneimine dye ( max 500 nm)

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 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 K m (Michaelis constant) for an enzyme reaction is a measure of the affinity of substrate for the enzyme. K m 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 K m and V max can be derived.

47 v [S] The Michaelis-Menton curve V max ½V max KmKm

48 The Lineweaver-Burk plot 1/[S] 1/v 1/V max -1/K m

49 Enzyme inhibition Competitive inhibitors compete with the substrate for the enzyme active site (K m ) Non-competitive inhibitors alter the ability of the enzyme to convert substrate to product (V max ) Un-competitive inhibitors affect both the enzyme substrate complex and conversion of substrate to product (both K m and V max ) Competitive inhibitors compete with the substrate for the enzyme active site (K m ) Non-competitive inhibitors alter the ability of the enzyme to convert substrate to product (V max ) Un-competitive inhibitors affect both the enzyme substrate complex and conversion of substrate to product (both K m and V max )

50 M-M analysis of an enzyme inhibitor v [S] V max KmKm K m (i) Competitive V max (i) Non-competitive

51 L-B analysis of an enzyme inhibitor 1/[S] 1/v 1/V max -1/K m Competitive Non-competitive

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 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 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)

56 NAD UV absorption spectra Absorbance (nm) NAD + NADH max = 340 nm

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

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 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 CO 2 to HCO 3 -, so the method actually measures total CO 2 Enzymatic methods for total CO 2 are most common, followed by electrode methods The specimen is alkalinized to convert all forms of CO 2 to HCO 3 -, so the method actually measures total CO 2 Enzymatic methods for total CO 2 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 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 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. 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 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 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 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 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 (1 4)

68 Amyloclastic amylase method 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 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 Starch + I 2 Blue complex Amylase Red complex

69 Saccharogenic amylase method 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 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 Starch Amylase Glucose + Maltose Reduced substrate

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

71 Defined-substrate amylase method -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 -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 4-NP-(Glucose) 7 Amylase 4-NP-(Glucose) 4,3,2 -Glucosidase 4-NP-(Glucose) 4 + Glucose + NP max = 405 nm

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 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 H 2 O 2 –Dye-labeled diglyceride that releases a chromophore when hydrolyzed by lipase Several non-triglyceride substrates have been proposed, as well Indirect methods for lipase measurement focus on: –Enzymatic phosphorylation (Glycerol kinase) and oxidation (L- -Glycerophosphate oxidase) of glycerol, and measurement of liberated H 2 O 2 –Dye-labeled diglyceride that releases a chromophore when hydrolyzed by lipase Several non-triglyceride substrates have been proposed, as well

74 Post-test Folin-Wu Jendrassik-Grof Somogyi-Nelson Kjeldahl Lieberman-Bourchard Rosalki-Tarlow Jaffe Bertholet Fisk-Subbarrow 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 Identify the methods proposed by the following:


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