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/J acksonville
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
H
A
Wavelength ()
Velocity = c
Wavelength, frequency, and energy

v
hc
h E = =
E = energy
h = Planks constant
v = frequency
c = speed of light
= wavelength
The Electromagnetic Spectrum

x-ray
UV visible IR Rf
10
-11
10
-9
10
-6
10
-5
10
-4
10
-2
10
2

Wavelength ( , cm)
Frequency (v, Hz)
10
8
10
12
10
14
10
15
10
16
10
19
10
21

Nuclear
Inner shell
electrons
Outer shell
electrons
Molecular
vibrations
Molecular
rotation
Nuclear
Spin
Visible spectrum
390 780 450 520 590 620
Wavelength (nm)
IR UV
Increasing Energy
Increasing Wavelength
Red-Orange-Yellow-Green-Blue
Molecular orbital energies
o or t
molecular
orbital
s or p
atomic
orbital
o
*
or t
*

molecular
orbital
non-bonding
orbital
on
tn
no
*

n t
*

oo
*

tt
*

E
n
e
r
g
y

Molecular electronic energy
transitions
E
0

E
4

E
3

E
2

E
1

Singlet
Triplet
A
VR
F
IC
P
10
-6
-10
-9
sec

10
-4
-10 sec

Absorption of EM radiation
I
0
(radiant intensity) I (transmitted intensity)
abc A bc k T kN
I
I
dn k
I
dI
kI
dn
dI
I
I
N
=
'
= = = =
} }
; log ; ln ; ;
0 0
0
0
Manipulation of Beers Law
) 2 (
10 % ,
) log(% 2
) log(% 2 ) log(% ) 100 log(
%
100
log
%
100
log
1
log log
A
T and
T A
T T
T
T T
T abc A

=
=
= =
= = = =
Hence, 50% transmittance results in an absorbance of 0.301, and
an absorbance of 2.0 corresponds to 1% transmittance
Absorbance
E
r
r
o
r

(
d
A
/
A
)

0.0 2.0
Beers Law error in measurement
0.434
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:
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
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 nitrogen
Total protein
Multiply by 6.25 (100%/16%)
Direct photometry
Absorption at 200225 nm can also be used
(
max
for peptide bonds)
Free Tyr and Trp, uric acid, and bilirubin
interfere at 280 nm
C
NH
2
H
2
C COOH
H
OH
C
NH
2
H
2
C COOH
H
HN
CH
Tyrosine Tryptophan

max
= 280 nm
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
Reduced form (blue) Phosphotungstic/phosphomolybdic acid
Protein
(Tyr, Trp)
Biuret method
Sodium potassium tartrate is added to
complex and stabilize the Cu
++
(cupric) ions
Iodide is added as an antioxidant
H
2
N
H
N NH
2
O O
H
C
C N
H
C
O
H
N
O
or . . .
Cu
++
OH
-
Blue adduct ( = 540 nm)
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
Albumin
BCG (pH 4.2)
BCP (pH 5.2)
green or purple adduct
A
b
s
o
r
b
a
n
c
e

Time
30 s
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 + O
2
Gluconic acid + H
2
O
2

Glucose
oxidase
Peroxidase
o-Dianiside
Oxidized o-dianiside

max
= 400540 (pH-dependant)
Glucose isomers
The interconversion of the o and | isomers of
glucose is spontaneous, but slow
Mutorotase is added to glucose oxidase reagent
systems to accelerate the interconversion
O
H
OH
H
OH
H
OH H
OH
CH
2
OH
O
H
OH
OH
H
H
OH H
OH
CH
2
OH
o-D-glucose (36%) |-D-glucose (64%)
Measuring creatinine
The reaction of creatinine and alkaline picrate
was described in 1886 by Max Eduard Jaffe
Many other compounds also react with picrate
NH
NH
H
3
C
O
O
-
NO
2
O
2
N
NO
2
OH
-
-
O
O
2
N
O
2
N
NO
2
H
N
NH
CH
3
-
O
+
Creatinine Picric acid
Janovski complex

max
= 485 nm
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
Kinetic Jaffe method
A
b
s
o
r
b
a
n
c
e

(

=

5
2
0

n
m
)

Time (sec)
0 80 20
F
a
s
t
-
r
e
a
c
t
i
n
g

(
p
y
r
u
v
a
t
e
,

g
l
u
c
o
s
e
,

a
s
c
o
r
b
a
t
e
)

S
l
o
w
-
r
e
a
c
t
i
n
g

(
p
r
o
t
e
i
n
)

At
AA
rate
t
A
=
A
A
creatinine (and o-keto acids)
Enzymatic creatinine method
N
H
N
O
CH
3
NH
N
H
N
O
CH
3
O
H
3
C
H
N COOH
H
2
O H
2
O
2
N
H
C
COOH
O
H
N
CH
3
H
2
N
C
H
2
COOH
N
H
C
COOH
O
H
N
CH
3
+ CH
2
O
Creatinine N-Methylhydantoin N-Carbamoylsarcosine
Sarcosine Glycine
Creatinine
iminohydrolase
N-Methylhydantoin
amidohydrolase
N-Carbamoylsarcosine
amidohydrolase
Sarcosine
oxidase
NH
3
+ CO
2
N-Carbamoylsarcosine
H
2
O
2
is measured by conventional
peroxidase/dye methods
Enzymatic creatinine method
H
2
O
2
is measured by conventional
peroxidase/dye methods
N
H
N
O
CH
3
NH
N
CH
3
NH
NH
2
COOH
H
3
C
H
N COOH
O
2
H
2
O
2
H
2
N
C
H
2
COOH
+ CH
2
O
H
3
C
H
N COOH
Creatinine
Creatinine
amidohydrolase
Creatine
Urea
Sarcosine
Sarcosine
Sarcosine
oxidase
Glycine
Creatine
amidohydrolase
Measuring urea (direct method)
Direct methods measure a chromagen produced
directly from urea
Indirect methods measure ammonia, produced
from urea
H
3
C
CH
3
O
NOH
H
+
H
3
C
CH
3
O
O
H
2
N NH
2
O
N N
H
3
C CH
3
O
+
H
+
, A
Diacetyl monoxime Diacetyl Urea Diazone

max
= 540 nm
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

H
2
N NH
2
O
Urease
2 NH
4
+
+
OH
OH
-
N
-
O O
Urea Phenol Indophenol

max
= 560 nm
Conversion of urea/BUN
( )
( ) dL L
urea mmol N mg
mmol urea mg
dL mg BUN L mg Urea
L dL
mmol urea mg
urea mmol N mg
L mg urea dL mg BUN
/ 10
/ 28
/ 60
) / ( /
/ 1 . 0
/ 60
/ 28
) / ( /
=
=
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 H
2
O
2
are common
HN
N
H
N
H
N
O
O
O
-
O
2
H
2
O
2
N
H
H
N
N
H
H
2
N
O
O
O
Phosphotungstic acid Tungsten blue
Uric Acid Allantoin
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
N
N N
N
AsO
3
H
2
OH OH
H
2
O
3
As
SO
3
- -
O
3
S
O
CH
3
HO
CH
3
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 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.
H
3
PO
4
+
(NH
4
)
6
Mo
7
O
24

H
+

(NH
4
)
3
[PO
4
(MoO
3
)
12
]

max
= 340 nm
Molybdenum
blue

max
= 600-700 nm
Red.
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

N
N
H
3
C
OH
HO
SO
3
-
SO
3
-
CH
3
HO
H
3
C CH
3
H
3
C CH
3
O
N
O
O
-
O
-
O
CH
3
N
O
-
O
O O
-
Calmagite

max
= 530 - 550 nm
Methylthymol blue

max
= 600 nm
Measuring iron
The specimen is acidified to release iron from
transferrin and reduce Fe
3+
to Fe
2+
(ferrous ion)
N N N N
SO
3
H
SO
3
Na
Bathophenanthroline Ferrozine
Fe
++

max
= 534 nm
Fe
++

max
= 562 nm
Measuring bilirubin
Diazo reaction with bilirubin was first described by
Erlich in 1883
Azobilirubin isomers absorb at 600 nm
N
H
O
N
H
HO
O
N
H
O
OH
N
H
HO
3
S N N
+
Cl
-
N
H
O
OH
N
H
HO
3
S N N
N
H
O
N
H
HO
O
SO
3
H N N
Diazotized sulfanilic acid
Bilirubin (unconjugated)
Azobilirubin (Isomer II)
Azobilirubin (Isomer I)
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:
o-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%)
o-bilirubin is irreversibly bound to protein (37%)
Only the |, , and o fractions are soluble in water, and
therefore correspond to the direct fraction
o-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
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
H
2
SO
4
/HOAc
HOO
2
S
Cholesterol Cholestahexaene sulfonic acid

max
= 620 nm
L-B reagent
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
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
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 HDL + (IDL, LDL, VLDL)+
Dextran sulfate
Mg
++

Newer automated methods use a modified form of
cholesterol esterase, which selectively reacts with
HDL cholesterol
Measuring triglycerides
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)
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 ES E + P
k
1
k
-1
k
2
| || |
| |
| | | | | |
| | | | ( )| |
| |
1
1
k
k
ES
S ES E
K
ES E E
ES
S E
K
tot
m
total
m

=
=
=
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.
Enzyme kinetics
E + S ES E + P
k
1
k
-1
k
2
| |
| |
| | | |
| |
| | | |
| |
| | S K
S V
v so
V v and ES E saturated is enzyme when
S K
S E
k v ES f or ng substituti
ES k v
m
tot
m
tot
+

=
= =
+

=
=
max
max
2
2
,
, ,
,
The Michaelis-Menton equation
| |
| |
| |
( )
| |
| |
| |
) (
1 1 1
,
max max
max
max
max
Burk Lineweaver
V S V
K
v
get we
S K
S V
v of reciprocal the taking or
Menton Michaelis
S
K
v
v V
get we
S K
S V
v g rearrangin
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 K
m
and V
max
can
be derived.
v

[S]
The Michaelis-Menton curve
V
max

V
max

K
m

| |
| |
| | S K
V
v when
S K
S V
v
m
m
= =
+

=
,
2
max
max
The Lineweaver-Burk plot
1/[S]
1
/
v

1/V
max

-1/K
m

| |
max max
1 1 1
V S V
K
v
m
+
|
|
.
|

\
|
=
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
)
M-M analysis of an enzyme
inhibitor
v

[S]
V
max

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

1/V
max

-1/K
m

Competitive
Non-competitive
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
Substrate Product
Enzyme
Cofactor Cofactor*
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
Substrate Product
Enzyme
Cofactor Cofactor*
Enzyme cofactors
N+
CH
2
H
H
OH
OH
H H
O
O
P
-
O
O
O
NH
2
O
P
O
-
O
O
H
2
C
N
N
N
N
NH
2
H
OH OH
O
H
Nicotinamide adenine dinucleotide (NAD
+
, oxidized form)
Enzyme cofactors
N
CH
2
H
H
OH
OH
H H
O
O
P
-
O
O
O
NH
2
O
P
O
-
O
O
H
2
C
N
N
N
N
NH
2
H
OH OH
O
H
H H
NADH (reduced form)
Phosphate attachment
(NADP
+
and NADPH)
NAD UV absorption spectra
A
b
s
o
r
b
a
n
c
e

250 300 350 400
(nm)
NAD
+

NADH

max
= 340 nm
L
a
g

p
h
a
s
e

Enzyme reaction profile
P
r
o
d
u
c
t

Time
Mix
S
u
b
s
t
r
a
t
e

d
e
p
l
e
t
i
o
n

Linear phase
| | ES
t
A

A
A
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
ATP ADP NAD
+
NADH
Glucose Glucose-6-phosphate 6-Phosphogluconate
Hexokinase
Glucose-6-phosphate
dehydrogenase
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
C
O
O
-
HO
C
O
COO
-
H
2
C
P
O
-
-
O O
H
2
C
C
COO
-
O
COO
-
NADH NAD
+
H
2
C
CH
COO
-
HO
COO
-
+
Malate
dehydrogenase
Bicarbonate
Phosphoenolpyruvate
Oxaloacetate Malate
PEP
carboxylase
Measuring 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

H
3
C
O
-
O
O
NADH NAD
+
H
3
C
O
-
OH
O
Pyruvate Lactate
Lactate
dehydrogenase
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
N
HN NH
2
CH
2
H
3
C
COO
-
ATP ADP
N
HN
H
N
CH
2
H
3
C
COO
-
P
O
O
O
-
Creatine kinase
Phosphocreatine Creatine
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.
ADP ATP ADP
NADP
+
NADPH
Creatine phosphate Creatine
CK
pH 6.7
Glucose Glucose-6-phosphate
G-6-PDH
6-Phosphogluconate
HK
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
ATP ADP
PK
ATP
NADH NAD
+
Creatine Creatine phosphate
CK
pH 9.0
Phosphoenolpyruvate Pyruvate
LD
Lactate
Measuring alkaline phosphatase
The natural substrate for ALKP is not known
N
+
O O
-
O
P
O
-
O O
H
2
O P
I
N
+
O O
-
O
-
N
+
-
O O
-
O
p-Nitrophenol
phosphate
Alkaline phosphatase
pH 10.3, Mg
++
p-Nitrophenoxide
Benzoid
(colorless)
Quinonoid
(
max
= 404 nm)
Measuring transaminase enzymes
Pyridoxyl-5-phosphate is a required cofactor
Oxaloacetate and pyruvate are measured with their
corresponding dehydrogenase enzymes, MD and LD
H
2
N CH C
CH
3
OH
O
H
2
N CH C
CH
2
OH
O
C
OH
O
COO
-
C O
CH
2
CH
2
COO
-
COO
-
C O
CH
2
COO
-
COO
-
C O
CH
3
COO
-
HC NH
2
CH
2
CH
2
COO
-
+ +
L-Aspartate
L-Alanine
2-Oxyglutarate
Pyruvate
Oxaloacetate
L-Glutamate
Aspartate
transaminase
Alanine
transaminase
Measuring gamma glutamyl
transferase
Method described by Szasz in 1969, and
modified by Rosalki and Tarlow
C
CH
2
CH
2
HC
COOH
NH
2
H
N O
NO
2
COOH
CH
2
NH
C
CH
2
O
NH
2
NO
2
NH
2
COOH
CH
2
NH
C
CH
2
O
HN C
O
CH
2
CH
2
HC
COOH
NH
2
-glutamyl-p-nitroanalide Glycylglycine p-Nitroanaline

max
= 405 nm
-Glutamylglycylglycine
+ +
-Glutamyl
transferase
pH 8.2
Measuring amylase
Hydrolysis of both o(14) and o(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
CH
2
OH
O
OH
OH
CH
2
OH
O
o-Amylose
o-Amylase
Ca
++
Glucose, Maltose
o(14)
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
Starch + I
2
Blue complex
Amylase
Red complex
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
Starch
Amylase
Glucose + Maltose Reduced substrate
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
Amylase
Small dye-labeled fragments
Photometric measurement of dye
Separation
step
Defined-substrate amylase method
o-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

o-Glucosidase
4-NP-(Glucose)
4
+ Glucose + NP

max
= 405 nm
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
H
2
C OFA
HC
H
2
C
OFA
OFA
H
2
C OH
HC
H
2
C
OFA
OFA
FA FA
H
2
C OH
HC
H
2
C
OH
OFA
FA
H
2
C OH
HC
H
2
C
OH
OH
Lipase Lipase
Lipase
Triglyceride o,|-Diglyceride o-Monoglyceride Glycerol
Measuring lipase (indirect)
Indirect methods for lipase measurement
focus on:
Enzymatic phosphorylation (Glycerol kinase)
and oxidation (L-o-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
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
Identify the methods proposed by the following: