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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

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review of analytical methods part 1 spectrophotometry

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

analytical methods used in clinical chemistry
Analytical methods used in clinical chemistry
  • Spectrophotometry
  • Electrochemistry
  • Immunochemistry
  • Other
    • Osmometry
    • Chromatography
    • Electrophoresis
introduction to spectrophotometry
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

H

Wavelength ()

Electromagnetic radiation

Velocity = c

wavelength frequency and energy
Wavelength, frequency, and energy
  • 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)

Inner shell

electrons

Outer shell

electrons

Molecular

vibrations

Molecular

rotation

Nuclear

Spin

Nuclear

The Electromagnetic Spectrum
visible spectrum

Wavelength (nm)

390

450

520

590

620

780

Increasing Energy

UV

IR 

Increasing Wavelength

Visible spectrum

“Red-Orange-Yellow-Green-Blue”

molecular orbital energies

* or *

molecular

orbital

s or p

atomic

orbital

n*

n *

non-bonding

orbital

Energy

*

*

n

n

  • or 

molecular

orbital

Molecular orbital energies
molecular electronic energy transitions

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
manipulation of beer s law
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

design of spectrometric methods
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
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

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

direct photometry
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

folin ciocalteu lowry method

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)

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

30 s

Specificity of bromocresol dyes

BCG (pH 4.2)

Albumin

green or purple adduct

BCP (pH 5.2)

Absorbance 

Time 

measuring glucose

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
glucose isomers
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
measuring creatinine
Measuring creatinine
  • 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
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
kinetic jaffe method

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) 

enzymatic creatinine method
Enzymatic creatinine method
  • H2O2 is measured by conventional peroxidase/dye methods
enzymatic creatinine method1
Enzymatic creatinine method
  • H2O2 is measured by conventional peroxidase/dye methods
measuring urea direct method
Measuring urea (direct method)
  • Direct methods measure a chromagen produced directly from urea
  • Indirect methods measure ammonia, produced from urea
measuring urea indirect method
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
measuring uric acid
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
measuring total calcium
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
measuring phosphate

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

measuring magnesium
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
measuring iron

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)
measuring bilirubin
Measuring bilirubin
  • Diazo reaction with bilirubin was first described by Erlich in 1883
  • Azobilirubin isomers absorb at 600 nm
evolution of the diazo method
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
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
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
enzymatic cholesterol methods

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
measuring hdl cholesterol

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
measuring triglycerides

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

spectrophotometric methods involving enzymes
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
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.

the michaelis menton equation
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.

enzyme inhibition
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

Vmax

Vmax(i)

v

Competitive

Non-competitive

[S] 

Km

Km(i)

M-M analysis of an enzyme inhibitor
l b analysis of an enzyme inhibitor

Non-competitive

Competitive

1/v 

1/Vmax

1/[S] 

-1/Km

L-B analysis of an enzyme inhibitor
measuring enzyme catalyzed reactions
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
measuring enzyme catalyzed reactions1
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
enzyme cofactors
Enzyme cofactors

Nicotinamide adenine dinucleotide (NAD+, oxidized form)

enzyme cofactors1

Phosphate attachment

(NADP+ and NADPH)

Enzyme cofactors

NADH (reduced form)

nad uv absorption spectra

NAD+

NADH

max= 340 nm

Absorbance 

250

300

350

400

 (nm)

NAD UV absorption spectra
enzyme reaction profile

Linear phase

Lag phase

Product 

Substrate depletion

Time 

Mix

Enzyme reaction profile
measuring glucose by hexokinase
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
measuring bicarbonate
Measuring bicarbonate
  • The specimen is alkalinized to convert all forms of CO2 to HCO3-, so the method actually measures totalCO2
  • Enzymatic methods for total CO2 are most common, followed by electrode methods
measuring lactate dehydrogenase
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 8.8-9.8
  • LD (sometimes designated LDH) activity will vary, depending on which method is used
measuring creatine kinase ck
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
measuring creatine kinase
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.
measuring creatine kinase1
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
measuring alkaline phosphatase
Measuring alkaline phosphatase
  • The natural substrate for ALKP is not known
measuring transaminase enzymes
Measuring transaminase enzymes
  • Pyridoxyl-5-phosphate is a required cofactor
  • Oxaloacetate and pyruvate are measured with their corresponding dehydrogenase enzymes, MD and LD
measuring gamma glutamyl transferase
Measuring gamma glutamyl transferase
  • Method described by Szasz in 1969, and modified by Rosalki and Tarlow
measuring amylase
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

(14)

amyloclastic amylase method

Amylase

Blue complex

Red complex

Amyloclastic amylase method

Starch + I2

  • 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

Amylase

Starch

Glucose + Maltose

Reduced substrate

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
chromogenic amylase method

Amylase

Small dye-labeled fragments

Separation

step

Photometric measurement of dye

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

Dye-labeled starch

defined substrate amylase method

Amylase

4-NP-(Glucose)4,3,2

-Glucosidase

4-NP-(Glucose)4 + Glucose + NP

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

4-NP-(Glucose)7

max= 405 nm

measuring lipase direct
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
measuring lipase indirect
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
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

Post-test

Identify the methods proposed by the following:

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