Download Review of Analytical Methods Part 1: Spectrophotometry

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 (max500 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 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)
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 (PCrCr) 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
(14)
Glucose, Maltose
OH
-Amylose
• 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
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