Review of analytical methods part 1 spectrophotometry
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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


Absorption of em radiation

I0 (radiant intensity)

I (transmitted intensity)

Absorption of EM radiation


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


Beer s law error in measurement

Error (dA/A)

0.0

0.434

2.0

Absorbance 

Beer’s Law error in measurement


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


Conversion of urea bun

Conversion of urea/BUN


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.


Enzyme kinetics1

Enzyme kinetics


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.


The michaelis menton curve

Vmax

½Vmax

v

[S] 

Km

The Michaelis-Menton curve


The lineweaver burk plot

1/v 

1/Vmax

1/[S] 

-1/Km

The Lineweaver-Burk plot


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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