Methods of Analysis of Plasma Creatinine and Creatinine

THANKS FOR UR ATTENTION 1
METHODS OF ANALYSIS OF
PLASMA CREATININE AND
CREATININE CLEARANCE
12/08/2021 Dr Musa,I.S.
outline
2
 Aim
 Introduction
 Biological importance of Creatinine
 Methods of analysis of plasma creatinine
 Interpretation of results
 Sources of error
 Creatinine clearance
 Determination of CrC
 Clinical correlate
 conclusion
THANKS FOR UR ATTENTION 12/08/2021
aim
3
 Describe the principle of analysis of creatinine in

terms of key reagents, their role and endpoint
detection.
 Discuss the principle of analysis
 Discuss commonly encountered sources of
analytical interference of creatinine analysis.
 Re evaluate the routine methods,and
 It’s application in evaluating renal function.

introduction
4
 Creatinine is a nonprotein nitrogen compound obtained as a

waste product of creatine and phosphocreatine metabolism.
 Creatine is synthesized in the liver from methionine,
glycine, and arginine.
 In skeletal muscle, it is phosphorylated to form
phosphorylcreatine , which is an important energy store
for ATP synthesis
 The ATP formed by glycolysis and oxidative
phosphorylation reacts with creatine to form ADP and large
amounts of phosphorylcreatine. .

Intro cont’d
5
 During exercise, the reaction is reversed,

maintaining the supply of ATP, which is the
immediate source of energy for muscle contraction
 Some athletes ingest creatine as a dietary
supplement and claim that it enhances their
performance in sprints and other forms of vigorous
short-term exertion.

Interconversion of cr, crp and Cr
6
Creatine
+ATP H2O
CK creatinine
-ADP
Creatine phosphate Pi
CK-creatine kinase
Biological importance
7
 The creatinine in the urine is formed from

phosphorylcreatine.
 Creatine is not converted directly to creatinine.
 Creatine phosphate losses phosphoric acid and creatine
losses water to form creatinine.
 The rate of creatinine excretion is relatively constant
from day to day.
 Indeed, creatinine output is sometimes measured as a
check on the accuracy of the urine collections in
metabolic studies;.
Biological importance
8
 an average daily creatinine output is calculated, and

the values for the daily output of other substances
are corrected to what they would have been at this
creatinine output.
 Hence, the use in renal function assesment.
 Creatinuria occurs normally in children, in
women during and after pregnancy, and
occasionally in nonpregnant women.

Importance…
9
 There is very little, if any, creatine in the urine of

normal men, but appreciable quantities are excreted
in any condition associated with extensive muscle
breakdown.
 Thus, creatinuria occurs in starvation,
thyrotoxicosis, poorly controlled diabetes mellitus,
and
 the various primary and secondary diseases of
muscle (myopathies).

Creatinine methabolism
10
 Creatine is synthesized in the liver, pancreas, and

kidneys from the amino acids arginine, glycine, and
methionine.
 Creatine is transported through the circulatory
system to muscle, brain, and other organs, where it
is converted to phosphocreatine
 and acts as an energy reservoir much like ATP.

Cr metabolism…
11
 Creatinine is produced as a waste product of

creatine and phosphocreatine.
 Because much of the creatinine is produced in
muscle, the amount of creatinine that is measured
in blood is proportional to the patient’s lean muscle
mass.
 The waste product, creatinine, enters the blood
supply, where it is removed through the kidneys.

Methods of analysis
12
Chemical methods based on Jaffe reaction

 Jaffe-kinetic reaction;
Requires automated equipment for precision

 Jaffe with adsorbent;
Used as reference method

 Jaffe without adsorbent;
routinely applied,and used in the UPTH laboratory.

Enzymatic method
Dinitrobenzoic acid methods
HPLC
Chemical method
13
Jaffe-kinetic reaction;
 first described in 1886,adopted in 1919.the reaction is
nonspecific,and subject to positive interference by large

number of compounds
 Performed directly on sample;detection of color formation
timed to avoid interference of noncreatinine chromogens.

 Inexpensive,rapid and easy to perform.
Jaffe with adsorbent;

 More accurate results are obtained when creatinine in a
protein-free filtrate is adsorbed into aluminum magnesium

silicate/sodium aluminum silicate and then alkaline picrate.
Chemical Method…
14
Jaffe without adsorbent;

 creatinine in protein-free filtrate reacts with picric
acid in an alkaline environment to generate an

orange-red product.
 The methodology is dependent upon picric acid
concentration, alkaline pH, reaction temperature,

reaction time, and wavelength at which the product
is measured.

Chemical Methodology
15
 and the rate of change in absorbance is measured.

 Although this method eliminates some of the
nonspecific reactants but subject to interference by
ketones,glucose,gluthatione,uric acid and
cephalosporins.

Jaffe without adsorbent
16
Reagents;
 0.1M sulphuric acid(H SO )
2 4
 10% sodium tungstate

 0.75M sodium Hydroxide
 0.04M picric acid

Jaffe without adsorbent…
17
Principle;
 Sodium tungstate precipitates protein in an acid
medium.
 The creatinine in the protein free filtrate reacts with
alkaline picrate to give an orange colour which is

measured at 520nm.

18
Procedure
Stage 1(precipitation)
1.Distilled water -0.5ml
2.Serum or plasma -0.5ml
3.0.1M H2SO4 -2.5mls
4.Sodium tungstate -0.5ml
 Cap and mix thoroughly,
 stand for 10mins,
 then spin for about 10mins

19
Stage 2;
 Table 1
Standard Test blank
Distilled water - - 1.5ml
1mg standard 1.5ml - -
Supernatant - 1.5ml -
0.75M NaOH/picric 0.5ml 0.5ml 0.5ml

acid
20
 Then mix,incubate at 37oC for 5mins or at room

temperature for 10mins
 set zero with the blank
 Read absorbance at 520nm.
 Calculation;
 For serum/plasma,Cr(mg/dl)=abs of test/abs of std x conc.
of standard
 For urine sample,dilute 1:100 and commence from stage 2.
 multiply result by dilution factor.

Enzymatic Methodology
21
 Enzymatic approaches to the measurement of

creatinine have also been explored.
 Creatininase,creatinase,sarcosine
oxidase,peroxidase and creatinine deaminase have
all been used to produce a measurable product that
reflects creatinine concentration.
 Reactions are coupled with enzymatic reactions
that can be measured spectrophotometrically.

Creatinase-CK
22
 Creatinine + H2O2 creatininase creatine

 Creatine + ATP CK CreatineP + ADP
 ADP+phosphoenol pyruvate PK pyruvate+ATP
 Pyruvate + NADH +H+ LD Lactate + NAD+

Creatininase-H2O2
23
 Creatinine + H2O Crase creatine

 Creatine + H2O2 cr sarcosine + Urea
 Sarcosine + O2 + H2O2 SO glycine +CH2O +
H2O2
 H2O2 +colorless substrate peroxidases
colored product + H2O2

Enzymatic method…
24
 Serum, plasma, and urine may be measured

enzymatically.
 Fluoride and ammonium heparin should not be
used as anticoagulants in enzymatic methods.
 Reference ranges are specific to the method used.
 In general, enzymatic methods produce lower
reference ranges than nonenzymatic methods.

Enzymatic method…
25
Dinitrobenzoic acid method;

 Creatinine + DNBA -OH purple product used on
reagent strips, though coloured product is unstable.
High performance liquid chromatography;
 using the principle of reversed-phase or cation
-exchange chromatography . it is highly specific

and a proposed reference method.

Interpreting results
26
Population Jaffe Enzymatic
adult mg/dl(μmol/l) mg/dl(μmol/l)
Male 0.6-1.1(53-97) 0.5-0.8(44-71)
female 0.9-1.3(80-115) 0.6-1.1(53-97)
child 0.3-0.7(27-62) 0-0.6(0-53)

Sources of error
27
 Ascorbate,glucose,ketones and uric acid may

interfare in jaffe reaction esp at temp. above 30oC.
 Bilirubin causes a negative bias in both jaffe n
enzymatic methods.
 Px on cephalosporins may have falsely elevated
result in jaffe.
 Dopamine affects both methods.
 Lidocain interfare in enz. method

Specimen requirements
28
 Cr may be measured in serum,plasma or urine

 Hemolysed,icteric samples interare with jaffe
 Lipemic samples, n high protein diet may interfare
with assay.
 Urine should be refridgerated after collection or
frozen when longer storage is required.

Creatinine clearance
29
 Elevated creatinine concentration is associated with

abnormal renal function
 especially in relation to glomerular function.
 The clearance of a substance is the volume of
plasma from which that substance is removed per
unit time.i.e GFR
 Creatinine clearance is an imperfect measure of
glomerular filtration rate.

Creatinine clearance…
30
 use of creatinine to estimate glomerular filtration

rate is based upon three assumptions:
(1) creatinine is filtered through the glomerulus,
(2) relatively low amounts of creatinine are
reabsorbed through the nephron tubule, and
(3) creatinine production is constant over time.

Creatinine clearance
31
 Although creatinine can provide a rough estimate

of glomerular filtration rate,error may be
encountered.
 Such factors as increased tubular reabsorption of
creatinine,reduced creatinine generation from
muscle tissue, and dietary changes in nitrogenous
compounds may affect creatinine clearance.
 Up to 10% of urine creatinine is secreted by
tubules.

32
The Specimen;
 Properly collected specimens are essential for
accuracy of the creatinine clearance test.

 Two specimens are collected, a 24-hour urine and
 a blood specimen that is drawn during the 24-hour
urine collection or not more than 24 hours before

or after the urine collection.
 The laboratory may add HCl or boric acid to the
container as preservative.
33
The patient is instructed to collect the urine as follows:

1. Void and discard this urine.
2. Start timing the 24-hour period immediately after
voiding
3. Collect all the urine voided for the next 24 hours.
Keep the urine in a cool place.
4. At 24 hours, void and add this urine to the collection
container.
5. Bring the urine to the laboratory as soon as possible.
34
The Calculation;
 At the laboratory, the specimen is well mixed, the
volume is measured, and then an aliquot is taken

for testing.
 The urine specimen is diluted 1:10 with distilled
water and measured for creatinine.

 The urine creatinine is multiplied by 10 for the
dilution.

35
Creatinine clearance(ml/min) =
Ucr(mg/dL) x Uvol(mL) x average surface area (m2)
________________________________________
Pcr(mg/dL) x time (min) x patient surface area (m2)
 Plasma cr is inversely proportional to cr clearance.

 Therefore when pcr is elevated,GFR is reduced,indicating
renal damage.
 Complete collection of the 24-hour urine is necessary in
order to report the creatinine clearance accurately.
36
 Several formulas are available to estimate creatinine

clearance.
 The Schwarz formula estimates creatinine clearance from
serum creatinine.It is used for pediatric patients:

 Creatinine clearance = (k x Ht)Creat
where k 0.45 if age 1 year, k 0.55 if age 1 to 12 years, Ht

height in cm, and Creat - serum creatinine.
Reference Ranges
 Male 95–130 mL/min
 Female 80–120 mL/min

conclusion
37
 Creatine and creatinine

 Methods of determination
 Creatinine clearance
 Application to renal function

references
38
 Clinical chemistry;Michael Bishop et al

 Arneson clinical chemistry
 Review of medical physiology by Ganong…
 Tees
 www.Scribd.com

39
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THANKS FOR UR ATTENTION 1

 Describe the principle of analysis of creatinine in

THANKS FOR UR ATTENTION 12/08/2021

 Creatinine is a nonprotein nitrogen compound obtained as a

THANKS FOR UR ATTENTION 12/08/2021

 During exercise, the reaction is reversed,

THANKS FOR UR ATTENTION 12/08/2021

 The creatinine in the urine is formed from

 an average daily creatinine output is calculated, and

THANKS FOR UR ATTENTION 12/08/2021

 There is very little, if any, creatine in the urine of

THANKS FOR UR ATTENTION 12/08/2021

 Creatine is synthesized in the liver, pancreas, and

THANKS FOR UR ATTENTION 12/08/2021

 Creatinine is produced as a waste product of

THANKS FOR UR ATTENTION 12/08/2021

Chemical methods based on Jaffe reaction

Requires automated equipment for precision

Used as reference method

routinely applied,and used in the UPTH laboratory.

nonspecific,and subject to positive interference by large

timed to avoid interference of noncreatinine chromogens.

Jaffe with adsorbent;

protein-free filtrate is adsorbed into aluminum magnesium

Jaffe without adsorbent;

acid in an alkaline environment to generate an

concentration, alkaline pH, reaction temperature,

THANKS FOR UR ATTENTION 12/08/2021

 and the rate of change in absorbance is measured.

THANKS FOR UR ATTENTION 12/08/2021

 10% sodium tungstate

THANKS FOR UR ATTENTION 12/08/2021

alkaline picrate to give an orange colour which is

THANKS FOR UR ATTENTION 12/08/2021

 stand for 10mins,

 then spin for about 10mins

THANKS FOR UR ATTENTION 12/08/2021

Standard Test blank

Distilled water - - 1.5ml

1mg standard 1.5ml - -

0.75M NaOH/picric 0.5ml 0.5ml 0.5ml

 Then mix,incubate at 37oC for 5mins or at room

THANKS FOR UR ATTENTION 12/08/2021

 Enzymatic approaches to the measurement of

THANKS FOR UR ATTENTION 12/08/2021

 Creatinine + H2O2 creatininase creatine

THANKS FOR UR ATTENTION 12/08/2021

 Creatinine + H2O Crase creatine

THANKS FOR UR ATTENTION 12/08/2021

 Serum, plasma, and urine may be measured

THANKS FOR UR ATTENTION 12/08/2021

Dinitrobenzoic acid method;

-exchange chromatography . it is highly specific

THANKS FOR UR ATTENTION 12/08/2021

Population Jaffe Enzymatic

adult mg/dl(μmol/l) mg/dl(μmol/l)

Male 0.6-1.1(53-97) 0.5-0.8(44-71)

female 0.9-1.3(80-115) 0.6-1.1(53-97)

child 0.3-0.7(27-62) 0-0.6(0-53)

THANKS FOR UR ATTENTION 12/08/2021

 Ascorbate,glucose,ketones and uric acid may

THANKS FOR UR ATTENTION 12/08/2021

 Cr may be measured in serum,plasma or urine