Io

Ig ------- = x B x C
I


Photometer:

I0
( . I
) ( . . )

Lambert Beer Law: A = x c x d

 I.

The amount of a substance is determined by measuring

the absorbance after the reaction is completed.
A
A1

A0
t

The resulting endpoint absorbance which is proportional to

the analyte concentration is measured at a given
wavelength.
c = (A(sample) / A(cal./std.)) * c(cal./std.)

If gone to completion, the results of endpoint reactions are

temperature independent!
 II.K

Change in absorbance is measured in a defined time

interval where the change remains constant (linear).

Substrate depletion
Linear phase

Lag phase
Abs.

A
ES
t

Time
Mix

The reaction rate is directly proportional to the amount of enzyme activity

U/L = (A/min(sample) / A/min(cal.)) * U/L(cal.)

U/L = A/min x Factor F = Vtot/ Vs * 1/ * 1/d

Absorbance can be increasing or decreasing.

The reaction rate differs with assay temperature.
 III. 2- / Fixed Time

The rate of a product formed is measured during a fixed period of time.

A
rate

Absorbance
t

A
t
Time (sec)

This reaction type is used for the determination of substrates (e.g.

Creatinine and Urea).
c = (A(sample) / A(cal./std.)) * c(cal./std.)

Difference between Kinetic and Fixed Time reaction is:

Kinetic reaction:
several measurements during the linear part of the assay (user dependent).
Fixed time:
after lag period two measurements at precisely defined time interval.
 Test Procedure With Two Reagents

Sample Start Substrate Start

Mix R1 + R2 Mix R1 + sample
(= Working Reagent) Incubate
Add sample and start reaction Add R2 and start reaction

Sample start is not always possible.

Exceptions e.g.:
Pancreatic Amylase (blocking of non-pancreatic amylase)
Bilirubin Auto Direct / Total (stabilization of high lipemic sera
sample blank necessary)
Cholesterol HDL (elimination of non HDL-Cholesterol)
Cholesterol LDL (selective protection of LDL-Cholesterol)
Iron, Ferene (Fe released from transferrin-complex)
Lipase (activation by colipase)
Uric Acid AOX (elimination of ascorbic acid)
Performance Characteristics

Linearity
Limit of Detection
Interferences
Precision
Method Comparison
Stability (On-board Stability / Calibration Stability)
 Linearity
U/L
measured
(U/L)max

U/L
(dilution steps)

The highest possible concentration of an analyte or activity of an enzyme in the

sample at the upper limit of the linear range depends on several parameters:

(U/L)max (A/min)max * 1/ * Vtot / Vs * 1/d

Vtot, Vs, d depend on the instrument and the application
Linear ranges stated in our instruction inserts usually refer to manual test
procedure with light path d = 1cm
Linearity

High linearity reduces expensive out of range reruns.

GOT on Hitachi 911

Limit of Detection

GOT on Hitachi 911

Interferences

Lipase on Hitachi 911

Precision

Alkaline Phosphatase, mod. IFCC on EPOS 5060

Method Comparison

Glucose, GOD-PAP on Hitachi 911

 Stability

Stability of reagents:
Up to expiry date if reagents are stored separately in tightly closed
bottles at proper temperature

Stability of working reagent:

R1+R2 mixed together and stored in a tightly closed bottle at proper
temperature

On board stability:
Separate reagents (or working reagent) in open vessels on the
instrument

Calibration stability on the instrument:

Time till new calibration is necessary when reagents (working reagent)
are in open vessels on the instrument
On Board / Calibration Stability

Alkaline Phosphatase, opt. DGKC on Hitachi 911

Clinical Chemistry Reagents
SUBSTRATES ENZYMES ELECTROLYTES

Albumin (BCG) Acid Phosphatase (Hillman) Calcium (Arsenazo)

Bile Acid Total (enzymatic recycling) Alkaline Phosphatase (opt. DGKC) Calcium (CPC)
Bilirubin Auto Total (DCA) Alkaline Phosphatase (mod. IFCC) Carbon Dioxide (PEP-C)
Bilirubin Auto Direct (DCA) Alpha-Amylase (mod. IFCC, ET-G7PNP) Carbon Dioxide (PEP-C, NADH-Reg.)
Bilirubin Total (Jendrassik Grof) Alpha-Amylase (CNP-G3) Chloride (Mercuric Thiocyanate)
Bilirubin Direct (Jendrassik Grof) Alpha-Amylase Pancreatic (ETG7-PNP) Iron (Ferene)
Cholesterol (CHOD-PAP) Alpha-HBDH (opt. DGKC) Magnesium (Xylidyl Blue)
Cholesterol HDL (Precipitation) Cholinesterase (Std.Meth.94 = DGKC new) Phosphorus Inorganic (Molybdate)
Cholesterol HDL, Direct (Immunoinhibition) CK-MB (opt. DGKC / IFCC)
Cholesterol LDL, Direct (Enz. Select. Protect.) CK-NAC (opt. DGKC / IFCC)
Creatinine (mod. Jaffe) Gamma-GT (SZASZ, stand. to IFCC)
Creatinine (Enzymatic, PAP) GLDH (DGKC)
Glucose (GOD-PAP) GOT (mod. IFCC)
Glucose (Hexokinase) GPT (mod. IFCC)
Homocysteine LDH-L (IFCC)
Protein Total (Biuret) LDH-P (opt. DGKC)
Protein Total in Urine/CSF (Pyrogallol Red) Lipase (Enzymatic, Colorimetric) HAEMATOLOGY
Triglycerides (GPO-PAP) G6PDH (UV, kinetic)
Urea (Urease, Colorimetric) G6PDH Deficiency Screen (qual., visual)
Urea UV Auto (Urease, GLDH) Haemoglobin Total (cyanmethaemoglobin)
Uric Acid AOX (Enzymatic, Colorimetric) HbA1c (Ion Exchange)
Uric Acid TBHBA (Enzymatic, Colorimetric) HbA1c Direct (immunoturbidimetric)
 Clinical Chemistry Clinical Significance

Heart: CK-NAC, CK-MB Pancreas: Alpha-Amylase

LDH, HBDH Pancreas Amylase
GOT Lipase

Liver: Ammonia Bone: Acid Phosphatase

Alkaline Phosphatase Alkaline Phosphatase
Bilirubin
Cholinesterase
Kidney: Creatinine
Gamma-GT
Urea
GLDH
GOT
Muscle: CK-NAC, CK-MB
GPT
LDH
LDH
GOT
 Clinical Chemistry Enzyme Assays

IFCC . International Federation of Clinical Chemistry

DGKC Deutsche Gesellschaft fr Klinische Chemie

All enzyme assays are international standard methods which

can be run at 37C and are therefore in accordance with EU
law. They correspond to the latest state of technology.
 Clinical Chemistry Applications for Automated
Systems

Abbott Spectrum Olympus AU 400, AU 600

Abbott Alcyon Synchron CX
Abbott Aeroset Targa
Advia Technicon RA
Bayer Opera Vitalab (Selectra)
Cobas Mira etc.
Dimension
Express 550
Hitachi 704, 717, 902, 911, 912,
Ilab 600, 900
Kone Optima, Delta, Progress Plus
CLINICAL CHEMISTRY

Reagents
 Protein Total

Protein Total in serum/plasma:

Biuret:
In alkaline solution, cupric ions react with protein to form a violet colored
complex.

In serum or plasma from patients who have received large intravenous amounts of
polydextrans too high values can be measured with the biuret method.

Protein Total in urine/CSF:

Pyrogallol red:
Proteins form a red complex with pyrogallol red and molybdate.
Albumin

BCG (bromcresol green):

The test procedure is based on the binding of

bromcresol green to albumin, forming a blue-green
complex:

Albumin + BCG blue-green complex

 Glucose

GOD-PAP:
In the presence of glucose oxidase, glucose is oxidized to gluconic acid and hydrogen
peroxide. The latter reacts, in the presence of peroxidase, with phenol and 4-
aminoantipyrine to form a quinoneimine dye (pink).
GOD
glucose gluconic acid + H2O2
POD
phenol + 4-aminoantipyrine + H 2O2 coloured chromogen

Hexokinase:
Glucose is phosphorylated to glucose-6-phosphate by hexokinase.
G-6-P dehydrogenase then oxidizes G-6-P and reduces NAD to NADH. Increase of
NADH is measured at 340 nm.
HK, Mg2+
glucose + ATP glucose-6-phosphate + ADP
G-6-P-DH
Glucose-6-phosphate + NAD + 6-phosphogluconate + NADPH + H +

Higher linearity
Less interferences with hemoglobin
 Cholesterol

CHOD-PAP:

Cholesterol esters are enzymaticly hydrolyzed. Free cholesterol is then

oxidized, whereby hydrogen peroxide is produced which forms coloured
quinoneimine with phenol and aminoantipyrine under catalytic action of
peroxidase.
CHE
Cholesterol ester + H2O Cholesterol + fatty acids
CHO
Cholesterol + O2 Cholesten-3-one + H2O2
POD
2 H2O2 + phenol + 4 aminoantipyrine quinonimine + 4 H2O

ATCS: minimizes turbidity caused by lipemia

 Chloride

Mercuric Thiocyanate:

Chloride ions in the sample react with mercuric thiocyanate displacing

the thiocyanate ion. The free thiocyanate ions react with ferric ions,
producing a coloured complex:

6 Cl- + 3 Hg(SCN)2 3 HgCl2 + 6 (SCN)-

6 (SCN)- + 2 Fe3+ 2 Fe(SCN)3

The reagent must not be stored in the refrigerator!

 Haemoglobin Total

Cyanmethaemoglobin-Method

Potassium ferricyanide
Haemoglobin Methaemoglobin

Methaemoglobin + Posassium Cyanide Cyanmethaemoglobin

540 nm
 Creatinine, mod. Jaff

mod. Jaff:
Creatinine reacts with alkaline picrate to form an amber-yellow solution.

H
N
O- -
O NH
NH
O2N NO2 O2N
H3C OH-
NH + CH3
-
O NO2

O
NO2

Creatinine Picric acid O2N

Janovski complex
max= 485 nm
 Iron

Ferene:

Iron bound to transferrin is released in an acidic medium as ferric iron

and is then reduced to ferrous iron in the presence of ascorbic acid.
Ferrous iron forms a blue complex with ferene.

asc.acid, buffer
transferrin-(Fe3+)2 2 Fe2+ + transferrin
Fe2+ + 3 Ferene ferrous ferene (blue complex)
 Magnesium

Xylidyl blue:

Magnesium reacts with xylidyl blue to form a coloured compound in alkaline

solution.

Magnesium + xylidyl blue pink complex

ATCS: minimizes turbidity caused by lipemia

Hemoglobin interferes because magnesium is released by erythrocytes.

Phosphorus Inorganic

Molybdate:

In acide solution phosphate reacts with ammonium

molybdate to form a yellow phosphomolybdate complex
with a maximum of absorbance at 340 nm.
 Acid Phosphatase

Hillman:

At acid pH ACP splits -Naphtyl phosphate to -Naphtol and inorganic

phosphate. -Naphtol is then coupled with an azo reagent to form a
yellow diazo dye which is measured at 405 nm.

ACP, pH 4,5-6
-naphtyl phosphate -naphtol + phosphate

-naphtol + fast red TR salt diazo dye (405 nm)

 Alkaline Phosphatase

ALP splits p-nitrophenyl phosphate to p-nitrophenol and inorganic

phosphate. Under alkaline
O
condition, colourless p-nitrophenol is
O -

converted to a yellow compound

N +
p-Nitrophenoxide
- - -
O O O O
+ +
N N

Alkaline phosphatase
pH 10.3, Mg++

O P O H2O PI
-
O O
-
O
Benzoid Quinonoid
p-Nitrophenol (colorless) (max= 404 nm)
phosphate

opt. DGKC: mod. IFCC:

Diethanolamine buffer AMP-buffer
pH = 9,8 pH = 10,4
 Alpha-Amylase
Pancreatic Amylase
-Amylase splits the substrate, a glycated aromatic compound, to free sugars
and a coloured aromate which is measured at 405 nm.
-Amylase
glycated aromatic substrate free sugars + coloured aromate

mod. IFCC: CNP-G3:

Substrate: ET-G7PNP
hemoglobin interferes even at
minimal concentrations
EDTA-plasma possible
higher linearity
Substrate: CNP-G3
less interferences with hemoglobin
no EDTA-plasma possible

-Glycosidase ensures complete digestion of substrate

strong colour response, excellent reproducibility

Pancreatic Amylase, based on mod. IFCC:

Salivary -Amylase is blocked by monoclonal antibodies present in Reagent 1
 Bile Acids

Enzymatic Recycling:
3--Hydroxysteroid dehydrogenase (3--HSD) oxidizes bile acids to 3-keto
steroids, whereby Thio-NAD is reduced to Thio-NADH. The reaction is
reversible, and the same enzyme can convert 3-keto steroids and NADH to bile
acids and NAD (recycling of bile acids).
The rate of formation of Thio-NADH is determined by measuring change of
absorbance at 405 nm.

3--HSD
bile acids + Thio-NAD Oxid. bile acids + Thio-NADH
3--HSD
oxidized bile acids + NADH bile acids + NAD

The enzymatic recycling of bile acids enables an accurate and fast measurement
even with small quantities of bile acids in the sample.
 Bilirubin Total/Direct

Direct Bilirubin:
Mono- and diglucuronides of bilirubin (= bound or direct bilirubin) are water
soluble and react with a diazotized aromate, forming a coloured diazo
compound.

DCA: Jendrassik Grof:

Substrate: diazotized dichloraniline Substrate: diazotized sulfanilic acid
ATCS: minimizes turbidity caused by
lipemia less interferences

Total Bilirubin:
Free (non bound) bilirubin is first dissolved by solvents.

DCA: Jendrassik Grof:

Solvent: specific mixture of detergents Solvent: DMSO / ethylene glycol
ATCS: minimizes turbidity caused by
lipemia less interferences
 Bilirubin Total/Direct

Sample Blank:
Yellow colour of Bilirubin has to be eliminated.

Reagent 1
+ +
Sample Reagent 2
Reagent 1 Reagent 2

Mix Mix
Incubate (3-5 min.) Incubate (5 min.)
Measure Absorbance 1 Measure Absorbance 2

A = Absorbance 2 Absorbance 1

Bilirubin is very unstable.

Protect samples, calibrators and controls from light! Use only fresh samples,
calibrators and controls!
 Calcium

Calcium forms chromophores with metal complexing dyes.

Arsenazo: CPC:
Substrate: arsenazo Substrate: cresolphtalein complexon
pH: neutral
pH: alkaline
ATCS: minimizes turbidity caused
by lipemia less interferences ATCS: minimizes turbidity caused by
lipemia O less CH
interferences
3 CH3 O
AsO3H2 H2O3As HO OH
OH OH -
O O-
N N N N
N N
O
- -
O 3S SO3 -
O O O O-
O

Arsenazo III o-Cresolphthalein complexone

max= 650 nm max= 570 - 580 nm

Do not use EDTA-plasma!

 Carbon Dioxide (CO2)

PEP-C:
Phosphoenol pyruvate carboxylase catalyzes the carboxylation (transfer of
bicarbonate) of phosphoenolpyruvate, forming oxaloacetate. Then malate
dehydrogenase catalyzes the reduction of oxaloacetate to malate and the
oxidation of NADH. Decrease in absorbance is measured at 405 nm.

O-

O -
O P O
PEP O COO- Malate HO COO-
carboxylase C dehydrogenase CH
C
+ O
HO O- H2C H2C
C COO- + COO-
NADH NAD
Bicarbonate H2C COO- Oxaloacetate Malate

Phosphoenolpyruvate
 Carbon Dioxide (CO2)

The problem:

NADH is stable in alkaline solution only. It degrades rapidly at

neutral or acid pH.

Alkaline solutions absorb CO2 from the air reduced reagent

stability
Enzymes used in this assay require neutral pH.
 Carbon Dioxide (CO2)
The Solution
Synthetic NADH
synthetic NADH analogue
shows excellent stability in
neutral solutions.
Neutral environment ensures
stability and optimal working
of enzymes
CO2 absorption from the air is
minimized
Excellent on-board and
calibration stability
NADH Recycling
slightly alkaline pH (8.05)
enables both: sufficient
activity of enzymes and
stability of NADH
enzymatic system recycles
NADH during whole shelf
life and ensures high linearity
Increased on-board and
calibration stability
 HDL-Cholesterol

Precipitation:
Non-HDL-Lipoproteins are precipitated by the addition of
Phosphotungstic Acid and Magnesium Chloride.

Direct, Immunoinhibition:
Non-HDL-Lipoproteins are inhibited by specific antibodies in R1.
highest specificity
no significant interferences
no time-consuming pretreatment
reduces variability and potential errors during
pretreatment
 HDL-Cholesterol, Direct

Ab

CHO CHE
H2O2 2 H2O2 + F-DAOS + 4 aminoantipyrine
CHE
CHO

H2O2 blue komplex + 4 H2O

CHO
CHE
H2O2
 LDL-Cholesterol, Direct

Direct, Enzymatic Selective Protection:

LDL-Lipoproteins are protected, whereas the other lipoproteins are
processed with R1.
Catalase CHE CHO
H2O2

CHE H2O2
CHO CHE
Catalase
CHO H2O2
Reagent 2 Catalase
(releasing)
2 H2O2 + H-DAOS + 4 aminoantipyrine
H2O2
CHE
CHO blue komplex + 4 H2O
Cholinesterase

Standard Method 94:

Cholinesterase catalyses the hydrolysis of butyrylthiocholine,

forming butyrate and thiocholine. Thiocholine then reduces yellow
hexacyanoferrate (III) to colourless hexacyanoferrate (II).
Decrease of absorbance is measured at 305 nm.

CHE
butyrylthiocholine butyrate + thiocholine

thiocholine
hexacyanoferrate (III) hexacyanoferrate (II)
(yellow) (colourless)
 CK-NAC / CK-MB (Creatine Kinase)

opt. DGKC / IFCC:

CK catalyzes the transfer of the phosphate group from creatine

phosphate to ADP. The resulting ATP is determined in a
combined enzymatic test using hexokinase and glucose-6-
phosphate dehydrogenase. The Increase of NADPH is measured
at 340 nm.

CK
pH 6.7
Creatine phosphate Creatine

ADP ATP ADP

G-6-PDH
Glucose Glucose-6-phosphate 6-Phosphogluconate
HK
NADP+ NADPH
 CK-MB (Creatine Kinase MB)

CK-M subunits are inhibited by specific antibodies in R1. Only CK-

B activity is measured which is half of the CK-MB activity

New antibody for improved immunoinhibition of CK-M:

higher capacity
better stability
more specific inhibition

Only use human based calibrators and controls!

 CK-MB (Creatine Kinase MB)

CK-MB Supplement
G-6PDH: Glucose-6-Phosphat Dehydrogenase
PGL: 6-Phosphogluconolactonase
6-PGDH: 6-Phosphogluconate Dehydrogenase
CK
pH 6.7
Creatine phosphate Creatine

ADP ATP ADP

G-6-PDH
Glucose Glucose-6-phosphate 6-Phosphogluconate
HK
NADP+ NADPH

6-Phospho-Gluconolactone PGL
6-Phospho-Gluconate
6-Phospho-Gluconate + NADP+ 6-PGDH
Ribulose-6-phosphate + CO2 + NADPH
 Creatinine, mod. Jaff

Creatinine and non-creatinine chromogens react at different speeds:

1st step (1 min): mostly rapid non-creatinine chromogens react
2nd step (2 min): mainly true creatinine reacts
3rd step: slow non-creatinine chromogens react
Absorbance ( = 500 nm)

A
(pyruvate, glucose,

rate
Fast-reacting

t
ascorbate)

Slow-reacting
(protein)
t
creatinine (and -keto acids)

Time (sec)

Important: very constant temperature, stable pH

Bilirubin leads to falsely reduced concentrations
Hemoglobin leads to falsely elevated concentrations
 Creatinine, mod. Jaff

Creatinine Supplement

For elimination of bilirubin interferences.

Bilirubin is oxidised by potassium ferricyanide, thus eliminated.

Creatinine Urine Diluent

For determination of Creatinine in urine by the Jaff method

together with Creatinine Supplement, Creatinine Urine Diluent
has to be used for dilution of urine.
 Creatinine, enzymatic

Enzymatic:

Creatinine + H2O <Creatininase> Creatine

Creatine + H2O <Creatinase> Sarcosine + Urea

Sarcosine + O2 + H2O <Sarcosine oxidase> Glycine + HCHO + H2O2

H2O2 + HTIB + PAP <Peroxidase> Quinone dye

high specificity
high linearity
less interferences by bilirubin
 Gamma-GT (Gamma-glutamyl Transferase)

Szasz, stand. to IFCC:

GGT catalyzes the transfer of glutymic acid from an aromatic substrate

to glycylglycine, releasing coloured amino-nitorbenzoate which is
measured at 405 nm.

GGT
L--glutamyl-3-carboxy-p-nitroanilide + glycylglycine

L--glutamyl-glycylglycine + 5-amino-2-nitrobenzoate

The Szasz and the IFCC method correlate strictly. Therefore it is possible
to use only one reagent. Szasz and IFCC results can be obtained by two
different calculation factors.
GLDH (Glutamate Dehydrogenase)

DGKC:

GLDH catalyzes the transfer of ammonium to oxoglutarate

with simultaneous oxidation of NADH to NAD. The
resulting decrease of absorbance is measured at 340 nm.

GLDH
2-oxoglutarate + NH4+ + NADH L-Glutamate + NAD+ +
H2O
 GOT (AST) / GPT (ALT)
(Glutamate Oxaloacetate Transaminase / Glutamate Pyruvate
Transaminase)

mod. IFCC:
GOT:
GOT catalyzes the transfer of the amino group from aspartate to oxoglutarate, producing glutamate
and oxaloacetate. Oxaloacetate is then reduced to malate. At the same time NADH is oxidized,
resulting in a decrease of absorbance at 340 nm.

GPT:
The determination of GPT follows the same principle, using alanine instead of aspartate as
substrate.
-
O COO

H2N CH C OH C O

CH3 CH2
- -
COO COO
L-Aspartate COO -

C O Aspartate HC NH2
O transaminase
Oxaloacetate
+ CH2 + CH2
H2N CH C OH Alanine -
COO
CH2 transaminase CH2
CH2
-
C O -
COO COO
C O
CH3 L-Glutamate
2-Oxyglutarate
OH Pyruvate
L-Alanine
 GOT (AST) / GPT (ALT)
(Glutamate Oxaloacetate Transaminase / Glutamate Pyruvate
Transaminase)

Benefits:
endogenous sample pyruvate is rapidly and completely reduced by Lactate

dehydrogenase, so it does not interfere with the assay.

top quality NADH (from micro organisms) ensures high linearity during
whole shelflife.

Pyridoxal-5-phosphate
The addition of pyridoxal-5-phosphate activates GOT- and GPT-apoenzymes.
It produces a marked increase in amino transferase activity (appr. 50% for
GOT and appr. 20% for GPT) and will yield higher results in samples and
control sera.
 Haemoglobin A1c, ion exchange

Ion Exchange:
After preparing the haemolysate, where the labile fraction is eliminated,
haemoglobins are retained by a cationic exchange resin in a column.
Haemoglobin A1c is specifically eluted after washing away haemoglobin
A1a+b fractions, and is quantified by direct reading at 415 nm.

The results obtained with this method can be converted into equivalent to the
US-NGSP certified method or IFCC standardized method, using the following
formulas:

HbA1c-NGSP (%) = 0.86 HbA1c-Dialab (%) + 0.24

HbA1c-IFCC (%) = 0.94 HbA1c-Dialab (%) 2.09
 Hemoglobin A1c Direct, Immunoturbidimetric

Normal hemoglobin and HbA1c have the same unspecific absorption rate to
latex particles. When mouse antihuman HbA1c monoclonal antibody is added
(R2), latex-HbA1c-mouse anti human HbA1c antibody complex is formed.
Agglutination is formed when goat anti-mouse IgG polyclonal antibody
interacts with the monoclonal antibody. The amount of agglutination is
proportional to the amount of HbA1c absorbed onto the surface of latex
particles. The amount of agglutination is measured as absorbance.
 Hemoglobin A1c Direct, Immunoturbidimetric

Direct Method, one assay only

no need for determination of total haemoglobin

4 level calibrator set gives results in % HbA1c
(ref. to NGSP)

Only one channel on chemistry analysers

No complicated calculation schemes, no calculation error
Time saving
Cost effective
 Homocysteine

Hcy-methyltransferase
Hcy + SAM >
> Methionine + SAH
SAH-hydrolase
SAH >
> Adenosine + Hcy
Adenosine deaminase
Adenosine >> Inosine + NH3
GLDH
NH3 + NADH + 2-Oxoglutarate >
> Glutamate + NAD+ + H2O

Oxidized Hcy is first reduced to free Hcy which then reacts with S-
adenosylmethionine (SAM) to form methionine and S-adenolylhomocysteine
(SAH). Then SAH is hydrolyzed into adenosine and Hcy by SAH-hydrolase and
Hcy is cycled into the Hcy conversion reaction by Hcy S-methyltransferase.
This forms an enzyme cycling reaction system with significant
amplification of detection signals. The formed adenosine is immediately
hydrolyzed into inosine and ammonia which reacts with glutamate
dehydrogenase with conversion of NADH to NAD+.
 LDH / -HBDH
(Lactate dehydrogenase / -Hydroxybutyrate Dehydrogenase)

LDH:
LDH catalyzes the reversible oxidation of lactate to pyruvate. The change of absorbance of NADH is
measured at 340 nm. O Lactate OH
dehydrogenase
O- O-
H3C H3C

O NADH NAD+ O
Pyruvate Lactate

LDH-L, IFCC: LDH-P, opt. DGKC:

forward reaction, substrate: lactate backward reaction, substrate: pyruvate

-HBDH, opt. DGKC:

For determining -HBDH, oxobutyrate is used as substrate:
-HBDH
2-oxobutyrate + NADH + H+ 2-hydroxybutyrate + NAD+
 Lipase

Enzymatic, colorimetric:
Lipase splits a synthetic glycerol ester, releasing a coloured compound which is
measured at 580 nm.

Benefits:
Substrate in a micellar system, colipase and bile acids simulate physiological conditions.
highly specific for pancreatic lipase, no intermediate enzyme reaction
no interferences from pancreatic cholesterase, hepatic lipase or
lipoprotein lipase

high precision / reproducibility due to high absorbance change

linear absorbance change directly proportional to the lipase activity
throughout the reaction period.
excellent sensitivity
excellent reagent blank stability
 Triglycerides

GPO-PAP:

After enzymatic splitting of triglycerides with lipoprotein lipase, released glycerol is

phosphorylated to glycerol-3-phosphate, which is then oxidized. Simultaneously
hydrogen peroxide is formed. It reacts with 4-aminoantipyrine and chlorophenol to
form a coloured quinonimine (peroxidase catalyzed reaction).

LPL
Triglycerides Glycerol + fatty acids
GK
Glycerol + ATP glycerol-3-phosphate + ADP
GPO
Glycerol-3-phosphate + O2 Dihydroxyaceton phosphate + H2O2
POD
2 H2O2 + 4-aminoantipyrine + 4-chlorophenol
quinonimine + HCl + 4 H2O

ATCS: minimizes turbidity caused by lipemia

 Urea

In a first step urease hydrolizes urea to ammonia and carbon dioxide:

urease
urea + 2 H2O 2 NH4+ + 2 HCO3-
Then ammonia is determined:
UV Auto, Urease/GLDH: Urease/Colorimetric:
GLDH catalyzes the transfer of Ammonia reacts with alkaline
Ammonia to 2-oxoglutarate, forming hypochlorite and sodium salicylate in
glutamate, and the oxidation of the presence of sodium nitroprusside
NADH. Decrease of absorbance is to form a coloured chromophore.
measured at 340 nm.
mainly for automatic systems mainly for manual test procedure
higher linearity
higher specificity

Do not use ammonium heparin as anticoagulant for collection of plasma!