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
Mar 30, 2015
Review of Analytical MethodsPart 1: SpectrophotometryReview 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
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
• Spectrophotometry
• Electrochemistry
• Immunochemistry
• Other– Osmometry– Chromatography– Electrophoresis
Introduction to spectrophotometryIntroduction 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.
Electromagnetic radiationElectromagnetic radiation
E
H
A
Wavelength ()
Velocity = c
Wavelength, frequency, and energyWavelength, frequency, and energy
hc
hE E = energyh = Plank’s constant = frequencyc = speed of light = wavelength
The Electromagnetic SpectrumThe Electromagnetic Spectrum
x-ray UV visible IR Rf
10-11 10-9 10-6 10-5 10-4 10-2 102
Wavelength (, cm)
Frequency (, Hz)
108101210141015101610191021
NuclearInner shellelectrons
Outer shellelectrons
Molecularvibrations
Molecularrotation
NuclearSpin
Visible spectrumVisible spectrum
390 780450 520 590 620
Wavelength (nm)
IR UV Increasing Energy
Increasing Wavelength
“Red-Orange-Yellow-Green-Blue”
Molecular orbital energiesMolecular orbital energies
or molecular
orbital
s or patomicorbital
* or *
molecularorbital
non-bondingorbital
nn
n*
n *
*
*
Energy
Molecular electronic energy transitions
Molecular electronic energy transitions
E0
E4E3
E2
E1
Singlet
Triplet
A
VR
F
IC
P
10-6-10-9 sec
10-4-10 sec
Absorption of EM radiationAbsorption of EM radiation
I0 (radiant intensity) I (transmitted intensity)
abcAbckTkNI
Idnk
I
dIkI
dn
dI I
I
N
;log;ln;;00
0
0
Manipulation of Beer’s LawManipulation of Beer’s Law
)2(10%,
)log(%2
)log(%2)log(%)100log(%
100log
%
100log
1loglog
ATand
TA
TTT
TTTabcA
Hence, 50% transmittance results in an absorbance of 0.301, andan absorbance of 2.0 corresponds to 1% transmittance
Absorbance
Err
or (
dA/A
)
0.0 2.0
Beer’s Law error in measurementBeer’s Law error in measurement
0.434
Design of spectrometric methodsDesign 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
Measuring total proteinMeasuring 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
• 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 methodKjeldahl’s method
SpecimenHot H2SO4 digestionCorrection for non-protein nitrogen
NH4+
Titration or Nessler’sreagent (KI/HgCl2/KOH)
Protein nitrogen
Total protein
Multiply by 6.25 (100%/16%)
Direct photometryDirect 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
C
NH2
H2C COOH
H
OH
C
NH2
H2C COOH
H
HN CH
Tyrosine Tryptophan
max= 280 nm
Folin-Ciocalteu (Lowry) methodFolin-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 acidProtein
(Tyr, Trp)
Biuret methodBiuret 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
H2NHN NH2
OO
HC
C NH
C
O
HN
O
or . . .Cu++
OH-Blue adduct ( = 540 nm)
Measuring albuminMeasuring 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
• 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 dyesSpecificity of bromocresol dyes
AlbuminBCG (pH 4.2)
BCP (pH 5.2)green or purple adduct
Abs
orba
nce
Time 30 s
Measuring glucoseMeasuring 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 + O2 Gluconic acid + H2O2
Glucoseoxidase Peroxidase
o-DianisideOxidized o-dianiside
max= 400–540 (pH-dependant)
Glucose isomersGlucose 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
OH
OH
H
OH
H
OHH
OH
CH2OH
OH
OH
OH
H
H
OHH
OH
CH2OH
-D-glucose (36%) -D-glucose (64%)
Measuring creatinineMeasuring 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
NH
NH
H3C
O
O-
NO2O2N
NO2
OH-
-O
O2N
O2N
NO2
HN
NH
CH3
-O
+
Creatinine Picric acidJanovski complex
max= 485 nm
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
• 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 methodKinetic Jaffe methodA
bsor
banc
e (
= 5
20 n
m)
Time (sec) 0 8020
Fast
-rea
ctin
g(p
yruv
ate,
glu
cose
,as
corb
ate)
Slow
-rea
ctin
g(p
rote
in)
t
A
ratet
A
creatinine (and -keto acids)
Enzymatic creatinine methodEnzymatic creatinine method
NH
N
O
CH3
NH
NH
N
O
CH3
O
H3C
HN COOH
H2O H2O2
NH
C
COOH
O
HN
CH3
H2NCH2
COOH
NH
C
COOH
O
HN
CH3
+ CH2O
Creatinine N-Methylhydantoin N-Carbamoylsarcosine
Sarcosine Glycine
Creatinineiminohydrolase
N-Methylhydantoinamidohydrolase
N-Carbamoylsarcosineamidohydrolase
Sarcosineoxidase
NH3 + CO2N-Carbamoylsarcosine
• H2O2 is measured by conventional peroxidase/dye methods
• H2O2 is measured by conventional peroxidase/dye methods
Enzymatic creatinine methodEnzymatic creatinine method
• H2O2 is measured by conventional peroxidase/dye methods
• H2O2 is measured by conventional peroxidase/dye methods
NH
N
O
CH3
NHN
CH3
NH
NH2COOH
H3C
HN COOH
O2 H2O2
H2NCH2
COOH+ CH2O
H3C
HN COOH
Creatinine
Creatinineamidohydrolase
CreatineUrea
Sarcosine
Sarcosine
Sarcosineoxidase
Glycine
Creatineamidohydrolase
Measuring urea (direct method)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
H3CCH3
O
NOH
H+
H3CCH3
O
OH2N NH2
O
N N
H3C CH3
O
+H+,
Diacetyl monoxime Diacetyl Urea Diazone
max= 540 nm
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
• 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
H2N NH2
O
Urease2 NH4
+ +
OH
OH-N
-O O
Urea Phenol Indophenol
max = 560 nm
Conversion of urea/BUNConversion of urea/BUN
dLLureammolNmg
mmolureamgdLmgBUNLmgUrea
LdLmmolureamg
ureammolNmgLmgureadLmgBUN
/10/28
/60)/(/
/1.0/60
/28)/(/
Measuring uric acidMeasuring 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
• 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
HN
NH
NH
N
O
O
O-
O2 H2O2NH
HN
NH
H2N
O
O
O
Phosphotungstic acid Tungsten blue
Uric Acid Allantoin
Measuring total calciumMeasuring 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
NN N
N
AsO3H2OH OH
H2O3As
SO3--O3S
O
CH3
HO
CH3
OH
N
O
-O
N
-O O O-O
O
O-
O
Arsenazo III
max= 650 nm
o-Cresolphthalein complexone
max= 570 - 580 nm
Measuring phosphateMeasuring 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.
• Phosphate in serum occurs in two forms:– H2PO4
- and HPO4-2
• Only inorganic phosphate is measured by this method. Organic phosphate is primarily intracellular.
H3PO4 + (NH4)6Mo7O24
H+
(NH4)3[PO4(MoO3)12]
max= 340 nm
Molybdenum blue
max= 600-700 nm
Red.
Measuring magnesiumMeasuring 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
• 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
N
N
H3C
OH
HO
SO3-
SO3-
CH3
HO
H3C CH3 H3C CH3
O
N
O
O-
O-OCH3
N
O
-O
O O-
Calmagite
max= 530 - 550 nm
Methylthymol blue
max= 600 nm
Measuring ironMeasuring iron
• The specimen is acidified to release iron from transferrin and reduce Fe3+ to Fe2+ (ferrous ion)
• The specimen is acidified to release iron from transferrin and reduce Fe3+ to Fe2+ (ferrous ion)
N N N N
SO3H
SO3NaBathophenanthroline Ferrozine
Fe++
max= 534 nm
Fe++
max= 562 nm
Measuring bilirubinMeasuring 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
NH
O NH
HO
O
NH
O
OH
NH
HO3S N N+Cl-NH
O
OH
NH
HO3S N N
NH
ONH
HO
O
SO3HNN
Diazotized sulfanilic acid
Bilirubin (unconjugated)
Azobilirubin (Isomer II)
Azobilirubin (Isomer I)
Evolution of the diazo methodEvolution 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
Bilirubin sub-formsBilirubin 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
Measuring cholesterol by L-BMeasuring 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
HO
H2SO4/HOAc
HOO2S
Cholesterol Cholestahexaene sulfonic acid
max = 620 nm
L-B reagent
Enzymatic cholesterol methodsEnzymatic 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
Cholesterylester
hydroxylase
Choles-4-en-3-one + H2O2
Cholesteroloxidase
Quinoneimine dye (max500 nm)
Phenol4-aminoantipyrinePeroxidase
Measuring HDL cholesterolMeasuring 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
• 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 HDL + (IDL, LDL, VLDL)Dextran sulfate
Mg++
• Newer automated methods use a modified form of cholesterol esterase, which selectively reacts with HDL cholesterol
• Newer automated methods use a modified form of cholesterol esterase, which selectively reacts with HDL cholesterol
Measuring triglyceridesMeasuring 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 + FFAsLipase
Glycerophosphate + ADPGlycerokinase
ATP
Dihydroxyacetone + H2O2
Glycerophasphateoxidase
PeroxidaseQuinoneimine dye (max 500 nm)
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
• 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 kineticsEnzyme kinetics
E + S ES E + Pk1
k-1
k2
1
1
k
k
ES
SESEK
ESEE
ES
SEK
totm
total
m
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 kineticsEnzyme kinetics
E + S ES E + Pk1
k-1
k2
SK
SVvso
VvandESEsaturatedisenzymewhen
SK
SEkvESforngsubstituti
ESkv
m
tot
m
tot
max
max
2
2
,
,,
,
The Michaelis-Menton equationThe Michaelis-Menton equation
)(111
,
maxmax
max
max
max
BurkLineweaverVSV
K
vgetwe
SK
SVvofreciprocalthetakingor
MentonMichaelisS
K
v
vVgetwe
SK
SVvgrearrangin
m
m
m
m
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.
v
[S]
The Michaelis-Menton curveThe Michaelis-Menton curve
Vmax
½Vmax
Km
SKV
vwhen
SK
SVv
m
m
,2max
max
The Lineweaver-Burk plotThe Lineweaver-Burk plot
1/[S]
1/v
1/Vmax
-1/Km
maxmax
111
VSV
K
vm
Enzyme inhibitionEnzyme 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)
• 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
M-M analysis of an enzyme inhibitor
v
[S]
Vmax
Km Km(i)
Competitive
Vmax(i)
Non-competitive
L-B analysis of an enzyme inhibitor
L-B analysis of an enzyme inhibitor
1/[S]
1/v
1/Vmax
-1/Km
CompetitiveNon-competitive
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
• 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
Substrate ProductEnzyme
Cofactor Cofactor*
Measuring enzyme-catalyzed reactions
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
Substrate ProductEnzyme
Cofactor Cofactor*
Enzyme cofactorsEnzyme cofactors
N+CH2
HHOH OH
H HO
OP
-O
O
O
NH2
O
P
O
-OO
H2C
N
N
N
N
NH2
H
OH OHO
H
Nicotinamide adenine dinucleotide (NAD+, oxidized form)
Enzyme cofactorsEnzyme cofactors
NCH2
HHOH OH
H HO
OP
-O
O
O
NH2
O
P
O
-OO
H2C
N
N
N
N
NH2
H
OH OHO
H
H H
NADH (reduced form)
Phosphate attachment(NADP+ and NADPH)
NAD UV absorption spectraNAD UV absorption spectraA
bsor
banc
e
250 300 350 400
(nm)
NAD+
NADHmax= 340 nm
Lag
pha
se
Enzyme reaction profileEnzyme reaction profileP
rodu
ct
Time Mix
Sub
stra
te d
eple
tion
Linear phase
ESt
A
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
• 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
ATP ADP NAD+ NADH
Glucose Glucose-6-phosphate 6-PhosphogluconateHexokinase
Glucose-6-phosphatedehydrogenase
Measuring bicarbonateMeasuring 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
• 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
C
O
O-HOC
O
COO-H2C
P
O-
-O O
H2C
CCOO-O
COO-NADH NAD+
H2C
CHCOO-HO
COO-
+
Malatedehydrogenase
Bicarbonate
Phosphoenolpyruvate
Oxaloacetate Malate
PEPcarboxylase
Measuring lactate dehydrogenaseMeasuring lactate dehydrogenase
• 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
• 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
H3CO-
O
O NADH NAD+
H3CO-
OH
O
Pyruvate Lactate
Lactatedehydrogenase
Measuring creatine kinase (CK)Measuring creatine kinase (CK)
• 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
• 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
N
HN NH2
CH2H3C
COO-
ATP ADP
N
HNHN
CH2H3C
COO-
P
O
O
O-
Creatine kinase
PhosphocreatineCreatine
Measuring creatine kinaseMeasuring 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.
ADP ATP ADP
NADP+ NADPH
Creatine phosphate Creatine
CKpH 6.7
Glucose Glucose-6-phosphateG-6-PDH
6-PhosphogluconateHK
Measuring creatine kinaseMeasuring 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
ATP ADP
PK
ATP
NADH NAD+
Creatine Creatine phosphate
CKpH 9.0
Phosphoenolpyruvate PyruvateLD
Lactate
Measuring alkaline phosphataseMeasuring alkaline phosphatase
• The natural substrate for ALKP is not known• The natural substrate for ALKP is not known
N+O O-
O
P
O-
OO H2O PI
N+O O-
O-
N+
-O O-
O
p-Nitrophenolphosphate
Alkaline phosphatase
pH 10.3, Mg++
p-Nitrophenoxide
Benzoid(colorless)
Quinonoid
(max= 404 nm)
Measuring transaminase enzymesMeasuring 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
H2N CH C
CH3
OH
O
H2N CH C
CH2
OH
O
C
OH
O
COO-
C O
CH2
CH2
COO-
COO-
C O
CH2
COO-
COO-
C O
CH3
COO-
HC NH2
CH2
CH2
COO-
+ +
L-Aspartate
L-Alanine
2-OxyglutaratePyruvate
Oxaloacetate
L-Glutamate
Aspartatetransaminase
Alaninetransaminase
Measuring gamma glutamyl transferase
Measuring gamma glutamyl transferase
• Method described by Szasz in 1969, and modified by Rosalki and Tarlow
• Method described by Szasz in 1969, and modified by Rosalki and Tarlow
C
CH2
CH2
HC
COOH
NH2
HNO
NO2
COOH
CH2
NH
C
CH2
O
NH2
NO2
NH2 COOH
CH2
NH
C
CH2
O
HNCO
CH2
CH2
HC
COOH
NH2
-glutamyl-p-nitroanalide Glycylglycine p-Nitroanaline
max= 405 nm
-Glutamylglycylglycine
+ +
-Glutamyltransferase
pH 8.2
Measuring amylaseMeasuring amylase
• 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
• 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
O
OH
OH
CH2OH
O
OH
OH
CH2OH
O
-Amylose
-Amylase
Ca++Glucose, Maltose
(14)
Amyloclastic amylase methodAmyloclastic 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 + I2 Blue complexAmylase
Red complex
Saccharogenic amylase methodSaccharogenic 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
StarchAmylase
Glucose + Maltose Reduced substrate
Chromogenic amylase methodChromogenic 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 starchAmylase
Small dye-labeled fragments
Photometric measurement of dyeSeparation
step
Defined-substrate amylase methodDefined-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
Amylase4-NP-(Glucose)4,3,2
-Glucosidase
4-NP-(Glucose)4 + Glucose + NPmax= 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
• 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
H2C OFA
HC
H2C
OFA
OFA
H2C OH
HC
H2C
OFA
OFAFA FA
H2C OH
HC
H2C
OH
OFAFA
H2C OH
HC
H2C
OH
OH
Lipase Lipase Lipase
Triglyceride ,-Diglyceride -Monoglyceride Glycerol
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
• 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-testPost-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
GlucoseBilirubinGlucose/AmylaseTotal proteinCholesterolGGTCreatinineUreaPhosphate
GlucoseBilirubinGlucose/AmylaseTotal proteinCholesterolGGTCreatinineUreaPhosphate
Identify the methods proposed by the following: