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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 University of Florida Health Science Center/Jacksonville Analytical methods used in clinical chemistry • • • • Spectrophotometry Electrochemistry Immunochemistry Other – Osmometry – Chromatography – Electrophoresis 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. Electromagnetic radiation E A Velocity = c H Wavelength () Wavelength, frequency, and energy E h hc E = energy h = Plank’s constant = frequency c = speed of light = wavelength The Electromagnetic Spectrum 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) Nuclear Inner shell electrons Outer shell electrons Molecular Molecular vibrations rotation Nuclear Spin Visible spectrum 390 UV 450 Wavelength (nm) 520 590 620 Increasing Energy Increasing Wavelength “Red-Orange-Yellow-Green-Blue” 780 IR Molecular orbital energies * or * molecular orbital Energy s or p atomic orbital n* n * non-bonding orbital or molecular orbital n n * * Molecular electronic energy transitions Singlet E4 E3 E2 Triplet VR E1 IC A F 10-6-10-9 sec P E0 10-4-10 sec Absorption of EM radiation I0 (radiant intensity) dI kI0 ; dn I I (transmitted intensity) N dI I I I k 0 dn ; ln I 0 kN ; log T k bc ; A abc 0 Manipulation of Beer’s Law 1 100 A abc log T log log T %T 100 log log( 100) log(% T ) 2 log(% T ) %T A 2 log(% T ) and , %T 10 ( 2 A) Hence, 50% transmittance results in an absorbance of 0.301, and an absorbance of 2.0 corresponds to 1% transmittance Error (dA/A) Beer’s Law error in measurement 0.0 0.434 Absorbance 2.0 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 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 Kjeldahl’s method Specimen 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 Direct photometry H2 C H H2 C COOH C H COOH C NH2 OH HN Tyrosine CH NH2 Tryptophan 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 Folin-Ciocalteu (Lowry) method Protein Phosphotungstic/phosphomolybdic acid (Tyr, Trp) Reduced form (blue) • Sometimes used in combination with biuret method • 100 times more sensitive than biuret alone • Typically requires some purification, due to interferences Biuret method H N H2N O NH2 O or . . . Cu++ O - H N OH H C C Blue adduct ( = 540 nm) C N H O • Sodium potassium tartrate is added to complex and stabilize the Cu++ (cupric) ions • Iodide is added as an antioxidant 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 Specificity of bromocresol dyes BCG (pH 4.2) Albumin green or purple adduct Absorbance BCP (pH 5.2) 30 s Time Measuring glucose Glucose + O2 Glucose oxidase Gluconic acid + H2O2 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 Glucose isomers CH2OH CH2OH O H OH H H OH OH OH H O H OH -D-glucose (36%) OH H H OH H OH -D-glucose (64%) • The interconversion of the and isomers of glucose is spontaneous, but slow • Mutorotase is added to glucose oxidase reagent systems to accelerate the interconversion Measuring creatinine O- NH O2 N H3C - NO2 OH + CH3 - O Creatinine NH O2N - NH O H N O NO2 NO2 Picric acid O2N Janovski complex max= 485 nm • The reaction of creatinine and alkaline picrate was described in 1886 by Max Eduard Jaffe • Many other compounds also react with picrate 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 Fast-reacting (pyruvate, glucose, ascorbate) Absorbance ( = 520 nm) 0 A t creatinine (and -keto acids) 20 Time (sec) 80 Slow-reacting (protein) Kinetic Jaffe method A rate t Enzymatic creatinine method CH3 CH3 Creatinine iminohydrolase N NH COOH N C O H N H N Sarcosine oxidase H2O CH3 NH3 + CO2 N-Carbamoylsarcosine COOH H3C N-Carbamoylsarcosine CH3 O N-Methylhydantoin N-Carbamoylsarcosine amidohydrolase H N C N H O Creatinine H N O N H O COOH H N-Methylhydantoin amidohydrolase N H2O2 Sarcosine • H2O2 is measured by conventional peroxidase/dye methods H2N COOH C H2 Glycine + CH2O Enzymatic creatinine method CH3 CH3 Creatinine amidohydrolase N Creatine NH amidohydrolase N H N NH O COOH H3C N H COOH NH2 Urea Creatinine Creatine H N COOH Sarcosine oxidase H2N O2 COOH C H2 H3C Sarcosine Sarcosine H2O2 + CH2O Glycine • H2O2 is measured by conventional peroxidase/dye methods Measuring urea (direct method) O O O O CH3 + H CH3 H3C H3C + H2N NOH + H , N NH2 O H3C Diacetyl monoxime N Diacetyl Urea CH3 Diazone max= 540 nm • Direct methods measure a chromagen produced directly from urea • Indirect methods measure ammonia, produced from urea Measuring urea (indirect method) OH H2N NH2 Urease OH 2 NH4+ + - O Urea N - O Phenol O Indophenol max = 560 nm • 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 Conversion of urea/BUN BUN mg / dL urea (mg / L) 28 mg N / mmol urea 0.1 dL / L 60 mg urea / mmol Urea mg / L BUN (mg / dL) 60 mg urea / mmol 10 L / dL 28 mg N / mmol urea Measuring uric acid O N HN O Phosphotungstic acid Tungsten blue N H Uric Acid H N H2N ON H O O2 H2O2 O O N H N H Allantoin • 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 Measuring total calcium O AsO3H2 OH OH N H2O3As CH3 CH3 HO - O OH O- O N N N - - N N O O3 S SO3 - O O O O- O Arsenazo III max= 650 nm o-Cresolphthalein complexone max= 570 - 580 nm • 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 Measuring phosphate H3PO4 + (NH4)6Mo7O24 H+ (NH4)3[PO4(MoO3)12] max= 340 nm Red. Molybdenum blue max= 600-700 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. Measuring magnesium H3C CH3 H3C CH3 O O OH HO O - - O N O N N SO3 H3C HO Calmagite max= 530 - 550 nm N - O O- CH3 CH3 SO3- - O O Methylthymol blue max= 600 nm • 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 Measuring iron Bathophenanthroline Ferrozine SO3Na SO3H N N N N Fe++ Fe++ max= 534 nm max= 562 nm • The specimen is acidified to release iron from transferrin and reduce Fe3+ to Fe2+ (ferrous ion) Measuring bilirubin OH O Azobilirubin (Isomer II) O OH HO O HO3S N + N Cl - HO3S N N N H N H O O Diazotized sulfanilic acid N H N H N H N H HO Azobilirubin (Isomer I) Bilirubin (unconjugated) O N H N H N N • Diazo reaction with bilirubin was first described by Erlich in 1883 • Azobilirubin isomers absorb at 600 nm SO3H 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 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 Measuring cholesterol by L-B L-B reagent H2SO4/HOAc HO HOO2S Cholesterol Cholestahexaene sulfonic acid max = 620 nm • 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 Enzymatic cholesterol methods Cholesterol esters Cholesteryl ester hydroxylase Cholesterol Cholesterol oxidase Choles-4-en-3-one + H2O2 Quinoneimine dye (max500 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 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 HDL, IDL, LDL, VLDL Dextran sulfate Mg++ HDL + (IDL, LDL, VLDL) • Newer automated methods use a modified form of cholesterol esterase, which selectively reacts with HDL cholesterol Measuring triglycerides Triglycerides Lipase Glycerol + FFAs Glycerokinase ATP Glycerophosphate + ADP Glycerophasphate oxidase Dihydroxyacetone + H2O2 Quinoneimine dye (max 500 nm) Peroxidase • LDL is often estimated based on triglyceride concentration, using the Friedwald Equation: [LDL chol] = [Total chol] – [HDL chol] – [Triglyceride]/5 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 Enzyme kinetics E+S k1 ES k2 E+P k-1 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. E S Km ES E Etotal ES Etot ES S k 1 Km ES k1 Enzyme kinetics E+S k1 ES k2 E+P k-1 v k 2 ES Etot S substituting for ES , v k 2 K m S when enzyme is saturated , Etot ES , and v Vmax Vmax S so, v K m S The Michaelis-Menton equation rearrangin g v Vmax v K m we get S v Vmax S K m S Michaelis Menton Vmax S or , taking the reciprocal of v K m S 1 1 1 Km we get v Vmax S Vmax ( Lineweaver Burk ) 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. The Michaelis-Menton curve v Vmax Vmax S v K m S ½Vmax Vmax when v , K m S 2 Km [S] 1 Km 1 1 v Vmax S Vmax 1/v The Lineweaver-Burk plot 1/Vmax -1/Km 1/[S] 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) M-M analysis of an enzyme inhibitor v Vmax Vmax(i) Competitive Non-competitive Km Km(i) [S] L-B analysis of an enzyme inhibitor 1/v Non-competitive Competitive 1/Vmax -1/Km 1/[S] Measuring enzyme-catalyzed reactions Enzyme Substrate Cofactor Product 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 Measuring enzyme-catalyzed reactions Enzyme Substrate Cofactor Product Cofactor* • 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 Enzyme cofactors O NH2 O - O P NH2 O - O N N O O P CH2 H H OH N+ O H H OH O N H2C N OH OH O H H Nicotinamide adenine dinucleotide (NAD+, oxidized form) Enzyme cofactors H H O NH2 O - O P NH2 O - O N N O O P CH2 H N O H OH H H OH O N H2C N OH OH O H H Phosphate attachment (NADP+ and NADPH) NADH (reduced form) NAD UV absorption spectra NAD+ Absorbance NADH max= 340 nm 250 300 (nm) 350 400 Enzyme reaction profile A ES t Mix Time Substrate depletion Lag phase Product Linear phase Measuring glucose by hexokinase Glucose ATP Hexokinase Glucose-6-phosphate ADP Glucose-6-phosphate dehydrogenase NAD+ 6-Phosphogluconate NADH • 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 Measuring bicarbonate O- O O + C HO P O PEP carboxylase O - Bicarbonate H2C C H2C O C COO- O COO- COO Oxaloacetate Malate dehydrogenase COO- HO CH H2C - NADH NAD + COO- Malate Phosphoenolpyruvate • 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 Measuring lactate dehydrogenase O OH3C O Pyruvate OH Lactate dehydrogenase OH3C NADH NAD+ O Lactate • Both PL and LP methods are available – At physiological pH, PL reaction if favored – LP reaction requires pH of 8.8-9.8 • LD (sometimes designated LDH) activity will vary, depending on which method is used Measuring creatine kinase (CK) O HN NH2 H N HN P - O Creatine kinase O N H3C N CH2 COO Creatine ATP - ADP H3C CH2 - COO Phosphocreatine • Both creatine and phosphocreatine spontaneously hydrolyze to creatinine • The reverse (PCrCr) reaction is favorable, although the reagents are more expensive • All methods involve measurement of ATP or ADP Measuring creatine kinase CK pH 6.7 Creatine phosphate Creatine ADP ATP ADP Glucose-6-phosphate Glucose G-6-PDH 6-Phosphogluconate HK + NADP NADPH • 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. Measuring creatine kinase CK pH 9.0 Creatine Creatine phosphate ATP ADP ATP Pyruvate Phosphoenolpyruvate PK NADH LD Lactate NAD+ • Since the forward (Cr PCr) reaction is slower, the method is not sensitive • Luminescent methods have been developed, linking ATP to luciferin activation Measuring alkaline phosphatase O- O N + p-Nitrophenoxide - O O + N - - O O + N Alkaline phosphatase ++ pH 10.3, Mg O O P O O H2O PI - O O Benzoid (colorless) Quinonoid (max= 404 nm) - p-Nitrophenol phosphate • The natural substrate for ALKP is not known Measuring transaminase enzymes O H2N CH C OH CH3 COO L-Aspartate + C O OH COO C - COO Aspartate transaminase - HC Oxaloacetate + Alanine transaminase - O 2-Oxyglutarate OH O CH2 CH2 CH2 C COO C CH - CH2 - O H2N COO COO - C O NH2 CH2 CH2 CH3 - COO L-Glutamate Pyruvate L-Alanine • Pyridoxyl-5-phosphate is a required cofactor • Oxaloacetate and pyruvate are measured with their corresponding dehydrogenase enzymes, MD and LD Measuring gamma glutamyl transferase COOH H N O C CH2 CH2 NH + C CH2 NH2 O -glutamyl-p-nitroanalide NH2 Glycylglycine p-Nitroanaline max= 405 nm HN CH2 CH2 C CH2 NH HC CH2 NH2 COOH C -Glutamyl transferase pH 8.2 NO2 + HC O NO2 NH2 COOH O CH2 COOH -Glutamylglycylglycine • Method described by Szasz in 1969, and modified by Rosalki and Tarlow Measuring amylase CH2OH CH2OH O O -Amylase OH OH ++ Ca O OH (14) Glucose, Maltose OH -Amylose • Hydrolysis of both (14) 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 Amyloclastic amylase method Starch + I2 Blue complex Amylase Red 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 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 Chromogenic amylase method Dye-labeled starch Amylase Small dye-labeled fragments Photometric measurement of dye Separation step • J&J Vitros application allows small dyelabeled fragments to diffuse through a filter layer • Abbott FP method uses fluorescein-labeled starch Defined-substrate amylase method 4-NP-(Glucose)7 Amylase 4-NP-(Glucose)4,3,2 -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-4NP, which has a higher molar absorptivity than 4-NP Measuring lipase (direct) H2C OFA H2C OH Lipase HC OFA H2C OFA Triglyceride FA H2C OH HC OH Lipase HC OFA H2C OFA OH HC OH H2C OH Lipase H2C OFA FA ,-Diglyceride H2C FA -Monoglyceride Glycerol • 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 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 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