Creatinine

Creatinine
Creatinine is a waste product formed in musule
by creatine metabolism.
Creatine is synthesized primarily in the liver
from:- (1)Arginine .
(2)Glycine.
(3)Methionine.
It is then transported to other tissue , such as
muscle , where it is converted to phosphocreatine
, which serves as high-energy source.
Creatine phosphate loses phosphoric acid and
creatine loses water to form creatinine , which
passes into the plasma.

Creatinine is released into the circulation at a

relatively constant rate that has been shown to
proportional to an individuals muscle mass.
It is removed from the circulation by glomerular
filtration and excreted in the urine.
Additional amounts of creatinine are secreted by
the proximal tubule.
Small amounts may also be reabsorbed by the
renal tubules.
Plasma creatinine concentration is a function of
relative muscle mass , the rate of creatine
turnover , and renal function.

The amount of creatinine in the reasonably

stable , although the protein content of the diet
dose influence the plasma concentration.
Because of the observed constancy of
endogenous production , determination of
creatinine excretion has been used as a measure
of completeness of 24-hour urine collections in a
given individual , although this method may be
unreliable.

Disease correlations

Elevated creatinine concentration is associated

with abnormal renal function , especially as it
releates to glomerular function.

Glomerular filtration rate

(GFR)

Glomerular filtration rate (GFR) is the volume of

fluid filtered from the renal(kidney) glomerular
capillaries into the Bowman's capsule per unit
time.
Glomerular filtration rate (GFR) is equal to the
Clearance Rate when any solute is freely filtered
and is neither reabsorbed nor secreted by the
kidneys.
The rate therefore measured is the quantity of
the substance in the urine that originated from a
calculable volume of blood.
Relating this principle to the below equation for the substance used, the product of urine
concentration and urine flow equals the mass of

This mass equals the mass filtered at the

glomerulus as nothing is added or removed in the
nephron.
Dividing this mass by the plasma concentration
gives the volume of plasma which the mass must
have originally come from, and thus the volume
of plasma fluid that has entered Bowman's
capsule within the aforementioned period of time.
The GFR is typically recorded in units of volume
per time, e.g., milliliters per minute ml/min.

Volume
urine flow
Time

Clearance
The definition of clearance is the volume of
plasma from which a substance (X) is completely
removed by the kidney in a given amount of time
(usually a minute).
The idealsubstancefor a clearance test should
fulfill the following criteria:-(1)It should be readily
filtered from the plasma at the glomerular filtrate.
(2)It should be neither reabsorbed nor secreted

(3)Its concentration in plasma should remain

constant throughout the period of urine
collection.
(4)The measurement if its concentration in
plasma and urine should be analytically
convenient and reliable.
Although its is not essential there are
considerable practical advantages in choosing a
substance which is normally present in plasma
when measuring the GFR.
This avoids the need to give the patient an
intravenous infusion of an exogenous substance

If a substance meets the four criteria listed the

amount of it excreted in the urine in unit time
equal the amount cleared from the plasma.

Creatinine clearance
Creatinine is an endogenous substance a
normal product of muscle metabolism. Its rate of
production is fairly constant from day to day
being determined by muscle bulk rather than
activity.
Creatinine is removed from the body mainly by
glomerular filtration and creatinine clearance is
frequently measured as an index of GFR.

Creatinine is actively secreted into the urine so

that its clearance tends to overestimate the GFR.
Inaccuracies arising from methodological
problems with creatinine measurement have
largely been overcome but the major cause of
inaccuracy in the determination of GEF by
creatinine clearance is the accurate
measurement of urine volume.
Traditionally patients have been required to
collect urine over a 24h period.
This is a convenient time but requires the
patient to void their urine completely at the

The accuracy of estimates of the GFR by

measurement of creatinine clearance may be
improved by making two or more consecutive 24h
urine collections but this is often not practical
even if it is acceptable to the patient.
There is however nothing magical about the 24h
period.
The use of shorter periods , although more
convenient for the patient may reduce accuracy
through inadequate bladder emptying but a good
compromise is to make a collection overnight.

As long as the bladder was emptied at the

beginning of the test (before retiring) and when
the final collection was made (on rising) the urine
production rate can be calculated and substituted
in the (UXV)/p formula.
A number of formulae exist for predicting
creatinine clearance ( or GFR) from
plasma[creatinine] and other readily available
information , such as age , sex and weight.
The best known
of these
is that of cockcroft and
(140 Age)
Weight(Kg)
(0.85ifema le)
Gault.CFR
72Scr (mg/dl)

1.73
U cr (mg/dl)V ur (ml/24hours)
C cr

A
P cr (mg/dl)1440minutes/24hours

WhereC cr
U cr
V cr
P cr
1.73
A

=creatinine clearance.

=urine creatinine concentration.

=urine volume excreted in24 hours.
=Serum creatinine concentration.
=normalization factor for body surface.

1.73 is the generally accepted average body

surface in square meters

It is essential that a reliable method is

employed to measure plasma and urine
creatinine concentrations.
Colorimetric assays tend to overestimate
creatinine since they detect non-creatinine
chromogens.
But even when reliable methods are used it
should be appreciated that the creatinine
clearance is based on four measurements:plasma and urine creatinine concentrations ,
urine volume and time.
Each has an inherent inaccuracy and the overall

Analytical
methods for
creatinine

Specimens
For creatinine analysis , serum or plasma and
diluted urine can be used.

Usually urine is diluted 1:100 or 1:20.

Methods
(A)Chemical methods based
on jaffe
(i)Jaffe-kinetic
reaction

(Jaffe without deproteinisation)

Principle

Creatinine forms a yellow orange compound in

alkaline solution with picric acid.

At low picric acid concentration used in this

method a precipitation of protein does not take
place.
The concentration of the dye stuff firmed over a
certain reaction time is a measure of the
creatinine and picric acid later secondary
reactions do not cause
an interference.
Reagents
This method thus distinguishes itself its high
(1)Creatinine
Picric Acid Reagent:-A solution
specificity.
containing 10 mM picric acid.
(2)Creatinine Buffer Reagent:-A solution
containing 10 mM. sodium borate, 240 mM,
sodium hydroxide and surfactant.
Important Note:-*If the Creatinine Buffer

*Warm reagent to 37 C with agitation to dissolve

all the precipitate before use.
(3)Creatinine standard (5 mg/dl):-A solution
containing creatinine in hydrochloric acid with
preservative.
(4)Acetic acid reagent:-Glacial acetic acid.

Kinetic procedure
(1)Combine equal volumes of Creatinine Picric
Acid Reagent and Creatinine Buffer Reagent , Mix
well.
(2) Set the spectrophotometer cuvette
temperature to 37 C.
(3)Pipette 1.0 ml of working reagent into test
tubes.
(4)Zero spectrophotometer with the reagent
blank at 510 nm. (Wavelength range:- 500-520
nm).
(5)Add .05 ml (50 l) of sample to reagent mix
and immediately place into cuvette.

(8)Calculate the change in absorbance

(Abs/min.) by subtracting (A2 - A1).

(ii)Jaffe with adssorbent

More accurate results are obtained when
creatinine in protein-free filtrate adsorbed onto
Fullers earth (aluminum magnesium silicate) or
Lloyds reagent (sodium aluminum silicate) then
eluted and reacted with alkaline picrate.
The most commonly used adsorbent is Lloyds
reagent , a hydrated aluminum silicate.

Protein is precipitated with tungstic acid and the

supernatant fluid is mixed with Lloyds reagent
which adsorbs creatinine in acid conditions.
After centrifugation , the pellet is resuspened in
alkaline picrate to elute the creatinine and give
the colour reaction.

Pseudocreatinine substances

(1)Protein.
(2)Glucose.
(3)Ascorbic acid.
(4)Ketone bodies.
(5)Pyruvate.
(6)Guanidine.

(7)Blood substitute products.

(8)Cephalosporins.

(iii)Jaffe without adssorbent

The creatinine in the protein free filtrate reacts
with alkaline picrate to give an orange colour
which is measured at 520nm.

Reagents

(1)Sodium tungstate solution ,100g Na2WO4 ,

2H2O/l.
(2)Sulphuric acid ,0.33mol/l.
(3)Lloyds reagent.
(4)Oxalic acid , saturated solution.

(6)Picric acid , saturated solution.

(7)Alkaline picrate solution:-Prepare freshly before
use.
*Add 5.5ml of sodium hydroxide to 27.5ml of
picric acid solution and dilute to 100 ml with
water.
(8)Standard creatrinine solution 10mmol/ll:Prepare a stock solution containine 113mg per
100ml in hydrochloric acid , 100 mmol/l , keep at
0C for not more than four weeks.
(9)Standard solution for use:-Dilute 0.5 and 1.0
ml of the stock solution to 200ml to give solutions

Step(1)Deproteinization method
0.5mlD.W+0.5ml Plasma+0.5ml
H2SO4+0.5Na2WO4 in a test tube. Mix and
centrifuge for 2min Step(2)
Supernata

Urine

1.5

0.5
0.5

1.5
0.5
0.5

1.5
0.5
0.5

1.5
0.5
0.5

nt
D.urine
Std
D.w
Na OH
Picric acid

Mix , incubate for 10min , read using green filter

(B)Enzymatic methods
(i)Creatininase
The enzyme creatininase catalyzes the
conversion of creatinine to creatine.
The creatinine is then detected with a series of
enzyme-mediated reactions involving creatine
kinase , pyruvate kinase , and lactate
dehydrogenase , with monitoring of the decrease
in absorbance at 340nm.

Creatininase
Creatinine H 2 O Creatine
Creatinine kinase
Creatine ATP creatinephosphate ADP
Pyruvatekinase
ADP Phosphoenolpyruvate Pyruvate ATP
Lactate dehydrogenase
Pyruvate NADH H lactate NAD

Initiating the reaction with creatininase allows

for the removal of edogenous creatine and
pyruvate in a preincubaion reaction.
The kinetics of the reaction are poor , and a 30
minute incubation is required to allow the
reaction to reach equilibrium.

This shortcoming can be overcome by a kinetic

approach but with a further reduction in
sensitivity.
The approach has not been popular , partly
because of poor sensitivity , poor precision , and
the relatively high cost of reagents.

(ii)Creatininase & creatinase

An alternative , more popular , approach has

been used the enzyme creatinase , which yields
sarcosine and urea , the former being measured
with further enzyme-mediated steps using
sarcosine oxidase(EC 1.5.3.1 ; yielding glycine ,
formaldehyde , and hydrogen peroxide) and

Creatininase
Creatinine H 2 O Creatine
Creatine H 2O

creatinase
Sarcosine urea

sarcosine O 2 H 2O

Sarcosineoxidase
Formaldehyde Glycine H 2O

Indicator(reduced) H 2O 2

Peroxidase

Indicator (oxidized) 2 H 2 O

The hydrogen peroxide has been detected with

a variety of methods.
Care must be taken to watch for
interference(e.g. , by bilirubin) in the final
reaction sequence.
This problem has been approached with the
addition of potassium ferricyanide(with limited

An alternative detection system involves

measuring of the reduction of nicotinamide
adenine dinuleotide (NAD) by formaldehyde in
the presence of formaldehyde dehydrogenase.

Fo rmald eh de NAD H 2 O

HCOOH NADH H

Fo rmald eh de d eh y d ro g en
ase

(iii)Creatinine Deaminase
Creatinine deaminase catalyzes the conversion
of creatinine to N-nethylhydantoin and ammonia.
Early methods concentrated on the detection of
ammonia using either glutamate dehydrogenase
or the Berthelot reaction.
An alternative approach involves the enzyme Nmethylhydantoin amidohydrolase.

creatininedeaminase
Creatinine H 2 O N methylhydantoin N H 3
l-meth y lh y dnato in ase

N meth y lh y dnay o in ATP H 2O

Carbamoy lsarcosine H 2 O

Carb amo y lsarco sin e ADP Pi

l-carbamo ylsarco sineamino hy dro

lase

Sarco sine O 2 H 2 O

In d icato r(red u ced ) H 2 O

Sarco sineo x id ase

p ero x id ase

Sarco sin e

H2 O

CO2 NH3

Gly cin e HCHO

In d icato r(Ox id ized )

2 H2 O

(iv)Dry chemistry systems

A number of multilayer dry reagent methods
have been described for the measurement of
creatinine using enzyme-mediated reactions.
An early two-slide approach employed
creatinine deaminase , with ammonia diffusing
through a emipermeable and optically opaque
layer to react with bromophenol blue to give an
increase in absorbance at 600nm.
A second multilayer film lacking the enzyme
was used to quantitate endogenous ammonia ,
enabling blank correction.
A later single-slide method used the creatinase-

The creatinine deaminase system described

above has been used and adapted for use as a
point-of-care testing device.
In all cases , the colour prodduced in the film is
quantitated by reflectance.
A dry chemistry system has been described in
which a nonenzymatic approach was used , based
on the reaction with 3,5-dinitrobenzoic acid.

(C)3,5-Dinitrobenzoic
acid(DNBA)
OH-

Creatinine 3,5 dinitrobenzoicacid

red chromogen

(D)High-performance liquid
chromatography
High-performance liquid chromatography
(HPLC) methods have been developed.
One method uses pre-treatment of the sample
with trichloroacetic acid to remove protein , ionexchange chromatography , and ultraviolet(UV)
detection of creatinine.
Another method combines separation by HPLC
with enzymatic determination of creatinine
concentration.
Both methods have been recommended as

Reference ranges