Quantification of creatinine in biologic

Spectrochimica Acta Part A 92 (2012) 318– 324
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular
Spectroscopy
j our na l ho me p age: www.elsevier.com/locate/saa
Quantification of creatinine in biological samples based on the pseudoenzyme
activity
of copper–creatinine complex
Padmarajaiah Nagaraja∗,
Krishnegowda Avinash, Anantharaman Shivakumar, Honnur Krishna
Department of Studies in Chemistry, University of Mysore, Manasagangothri, Mysore 570006, India
a r t i c l e i n f o
Article history:
Received
20 November 2011
Received
in revised form 19 February 2012
Accepted
23 February 2012
Keywords:
Creatinine
Serum
Urine
Jaffe’s
Pseudoenzyme
a b s t r a c t
Glomerular filtration rate (GFR), the marker of chronic kidney disease can be analyzed by the concentra-
tion
of cystatin C or creatinine and its clearance in human urine and serum samples. The determination
of
cystatin C alone as an indicator of GFR does not provide high accuracy, and is more expensive, thus
measurement
of creatinine has an important role in estimating GFR. We have made an attempt to
quantify
creatinine based on its pseudoenzyme activity of creatinine in the presence of copper. Crea-
tinine
in the presence of copper oxidizes paraphenylenediamine dihydrochloride (PPDD) which couples
with
dimethylamino benzoicacid (DMAB) giving green colored chromogenic product with maximum
absorbance
at 710 nm. Kinetic parameters relating this reaction were evaluated. Analytical curves of
creatinine
by fixed time and rate methods were linear at 8.8–530 ␮mol L−1and 0.221–2.65 mmol L−1,
respectively.
Recovery of creatinine varied from 97.8 to 107.8%. Limit of detection and limit of quan-
tification
were 2.55 and 8.52 ␮mol L−1respectively whereas Sandell’s sensitivity and molar absorption
coefficient
values were 0.0407 ␮g cm−2and 0.1427 × 104L mol−1cm−1respectively. Precision studies
showed
that within day imprecision was 0.745–1.26%
and day-to-day imprecision was 1.55–3.65%. The
proposed
method was applied to human urine and serum samples and results were validated in accor-
dance
with modified Jaffe’s procedure. Wide linearity ranges with good recovery, less tolerance from
excipients
and application of the method to serum and urine samples are the claims which ascertain
much
advantage to this method.
© 2012 Published by Elsevier B.V.
1. Introduction
Estimation
of glomerular filtration rate (GFR) is the most widely
used
test for assessing renal function [1]. Functions, which mea-
sure
the GFR directly or indirectly, are major tools to assess the
extent
of impairment of renal function. The most exact technique
for
measuring the GFR requires the infusion of radioisotopes such as
51chromium-EDTA, 125I-iothalamate, 99mTc-DTPA
or radio contrast
agents
such as iohexol, or inulin [2], which do not get reabsorbed,
secreted
or metabolized by the kidney. These methods require
intravenous
and timed collection of multiple plasma and urine
samples
making the analytical methods highly cumbersome and
difficult
to apply in routine.
Abbreviations: PPDD, paraphenylenediamine dihydrochloride; DMAB, dimethy-
lamino
benzoicacid; QCM, quality control material; KP, Michaelis–Menten constants
for
PPDD; KD, Michaelis–Menten constant for DMAB; KC, Michaelis–Menten con-
stants
for copper; KCr,
Michaelis–Menten constants for creatinine; Vmax, maximum
rate
of the reaction at optimized reagent concentration.
∗Corresponding
author. Tel.: [phone]; fax: [phone].
E-mail
addresses: [email protected], [email protected] (P. Nagaraja).
Another method for the estimation of GFR is urinary measure-
ment
of cystatin C [3] an endogenous protein belonging to the type
2
cystatin gene family. Reports have shown that cystatin C levels get
altered
in patients with cancer [4], thyroid dysfunction [5]. Cystatin
C
levels are also affected by cigarette smoking and by C-reactive
proteins
[6], which might or might not reflect actual renal dysfunc-
tion
[7]. The role of cystatin C to monitor GFR during pregnancy
remains
controversial [8]. The use of cystatin C alone as a determi-
nator
of GFR does not yield high accuracy over isotope dilution mass
spectrometry-based
Modification of Diet in Renal Disease, and it is
also
more expensive [9].
Measurement
of creatinine content and its clearance in urine
and
serum samples is the legend for the GFR estimation. Although,
multi-shot
technique is more convenient, but measurement of
creatinine
and its clearance are still the methods of choice most
commonly
followed for assessing the renal, muscular, cardiac [10],
and
thyroid functions [11].
Jaffe’s
alkaline sodium picrate method is the most widely
accepted
standard method for creatinine measurement [12]. But
this
method is affected by many chemical species such as creatine,
bilirubin,
hemoglobin, cefoxitin and cephalothin [13]. Numerous
modifications
to Jaffe’s reaction have been effected to elimi-
nate
or reduce interferences, which include specific adsorption of
1386-1425/$ – see front matter © 2012 Published by Elsevier B.V.
doi:10.1016/j.saa.2012.02.104

P. Nagaraja et al. / Spectrochimica Acta Part A 92 (2012) 318– 324 319
creatinine, removal of interfering compounds, dialysis, adjusting
the
pH, and kinetic measurements. Unfortunately, none of these
modifications
were capable of completely eliminating interfer-
ences.
Spectrophotometric
method based on multi-enzyme system
has
been developed to improve the specificity of creatinine deter-
mination
[14]. The method gives accurate result but is of much
imprecise
and also expensive. Moreover, the use of multi-enzyme
system
requires caution, because of the increase in the risk of
interference
with increases in the number of the enzymes used
[15].
Other
spectrophotometric methods for the assay of cre-
atinine
concentration include 3,5-dinitrobenzoic acid [16,17],
3,5-dinitrobenzoyl
chloride [18,19], methyl-3,5-dinitrobenzoate
in
a mixture of dimethyl sulfoxide, methanol, and tetramethyl
ammonium
hydroxide [20], 1,4-naphthoquinone-2-sulfonate [21],
Sakaguchi’s
color reaction of creatinine with o-nitrobenzaldehyde
[22],
capillary electrophoresis [23–25], liquid chromatography (LC)
[26],
gas chromatography (GC) [27], GC with isotope dilution mass
spectrometry
[28], and tandem mass spectrometry [29,30]. Most
of
these methods have serious limitations with respect to their
sensitivity,
linearity, precision and applicability. In most instances,
the
conditions required for best sensitivity, stoichiometry (linear-
ity
or range), and for the elimination of interferences have not been
delineated
properly.
A
new spectrophotometric method based on enzyme like activ-
ity
of copper–creatinine complex [31] was developed to determine
creatinine
in serum and in urine samples. The performance of the
method
was evaluated and potential interferences were studied.
Results
obtained using the proposed method was compared to the
ones
achieved with a modified Jaffe’s procedure.
2.
Experimental
2.1.
Instrumentations
A
JASCO model UVIDEC-610 PC double beam spectrophotome-
ter
with 1 cm matched quartz cells was used for all measurements.
Serum
samples were centrifuged using Remi R24 (Mumbai, India)
desktop
centrifuge having 17,200 rpm and 27,440 RCF. A Remi
cyclomixer
was used for mixing of the reaction mixture. pH
of
the solution was measured using Chemlabs (Nairobi, Kenya)
pH
meter. Lab Dispo sodium heparin tubes containing 18 I.U.
of
sodium heparin obtained from Manshe Healthcare, Ahmed-
abad,
India, was used to store the blood samples drawn from
donors.
2.2.
Reagents and solutions
All
chemicals used in the assay were of analytical grade.
Solutions
were freshly prepared in double distilled water and
stored
in amber colored standard flasks and refrigerated at −4◦C
until
use. Paraphenylenediamine dihydrochloride (PPDD) (Merck,
Germany)
(1.656 mmol L−1)
was prepared by dissolving 3 mg of
this
reagent in water to prepare a 10 mL solution. Dimethylamino
benzoicacid
(DMAB) (Himedia Ltd., India) (181.18 mmol L−1) was
prepared
by dissolving 300 mg of this reagent in 0.5 mL of
0.01
mol L−1hydrochloric
acid solution and then adjusting the
volume
of solution to 10 mL with water. A 1.6 mmol L−1cop-
per
(II) sulfate pentahydrate (Himedia) solution was prepared
by
dissolving 4 mg of the salt in water to achieve a10 mL
solution.
Creatinine was purchased from S.D. Fine Laboratory,
Mumbai,
India and the required standard solutions were prepared
by
dissolving suitable amount of creatinine in double distilled
water.
2.3.
Biological samples
Blood
and urine samples were collected from volunteers follow-
ing
Institutional Human Ethical Committee guidelines (IHEC – UOM
No
22/Ph.D/2008-09). The blood samples obtained from the donors
was
centrifuged and stored in Dispo sodium heparin tubes at −4◦C
till
use.
2.4.
Quantification of creatinine by kinetic and fixed time method
The
concentration of creatinine was determined kinetically in
3
mL of the solution containing 55 ␮mol L−1PPDD,
6 mmol L−1
DMAB and 53.3 ␮mol L−1copper
in 1 mmol L−1potassium dihy-
drogen
orthophosphate/sodium hydroxide buffer of pH 7.8. The
reaction
was initiated at 25 ◦C
by adding 100 ␮L of different con-
centrations
of creatinine within the linearity range. The change in
the
absorbance was continuously recorded at 710 nm for 5 min as
a
function of reaction rate.
The
analytical curve for creatinine quantification by one time
assay
method was obtained from a final 3 mL volume of the
solution
containing 55 ␮mol L−1PPDD, 6 mmol L−1DMAB and
53.3
␮mol L−1copper in 1 mmol L−1potassium dihydrogen ortho
phosphate/sodium
hydroxide buffer at pH 7.8 and 100 ␮L of var-
ious
concentrations of creatinine within the linearity range. The
reaction
mixture was allowed to stand for 20 min at room tempera-
ture.
Absorbance of the colored solution was recorded with respect
to
blank containing all optimized reagents except creatinine.
2.5.
Evaluation of kinetic constants
Michaelis–Menten
constant values were obtained as indicated
in
the literature [32,33]. The initial velocities (Vo)
were determined
as
a function of the reagents: creatinine (Co), PPDD (Po), DMAB (Do)
and
Cu2+ (Cuo). Experiments were made in a univariate way by
varying
the concentration of one of the reagents at a time. Experi-
ments
were repeated using different concentrations of creatinine.
3.
Results and discussion
3.1.
Effect of pH
The
following buffers with concentration range of
0.1–10
mmol L−1were kinetically studied for the assay namely,
citric
acid/potassium citrate at pH 3.6–5.6, acetate/acetic
acid
at pH 3.6–5.6,
potassium dihydrogen phosphate/sodium
hydroxide
at pH 6.0–8.0,
potassium dihydrogen orthophos-
phate/dipotassium
hydrogen orthophosphate at pH 6.0–7.8 and
tris
amine/hydrochloric acid buffer at pH 7.6–10. The maximum
reaction
rate was recorded with potassium dihydrogen phos-
phate/sodium
hydroxide buffer at pH 7.8. Hence all further studies
were
carried out with potassium dihydrogen phosphate/sodium
hydroxide
buffer which offered the maximum reaction rate in its
1
mmol L−1solution.
3.2.
Temperature sensitivity
Effect
of temperature on the sensitivity of the assay was
determined
by incubating 3 ml of reaction mixture containing
55
␮mol L−1PPDD,
6 mmol L−1DMAB, 53.3 ␮mol L−1copper and
176
␮mol
L−1of creatinine in 1 mmol L−1potassium dihydrogen
ortho
phosphate/sodium hydroxide buffer at pH 7.8 at different
temperatures
(0–80 ◦C)
for 20 min. The result indicated that the
colored
product formed was stable in the temperature range of
20–35 ◦C.
Any further increase in the temperature initiated the
decomposition
process with the corresponding decrease in the
absorbance
values; decrease in temperature lowered the time

320 P. Nagaraja et al. / Spectrochimica Acta Part A 92 (2012) 318– 324
Fig. 1. Suggested reaction mechanism for the formation of green colored product.
needed for completion of reaction. Hence all analyses were carried
out
at the optimum room temperature.
3.3.
Proposed reaction mechanism
The
probable reaction mechanism involved for the
copper–creatinine
catalyzed reaction of PPDD and DMAB is
as
proposed in Fig. 1. Under optimum reaction conditions when
copper
and creatinine are present, PPDD looses two electrons
and
two protons forming electrophillic 1,4-diimine, which may
act
as the oxidative coupling species. The 1,4-diimine undergoes
electrophillic
substitution with DMAB in the free para position
to
the N,N-dimethylamino group, forming green-colored product
having
strong absorption at 710 nm
Fig. 2. Analytical curve for the quantification of creatinine by the rate method.

P. Nagaraja et al. / Spectrochimica Acta Part A 92 (2012) 318– 324 321
Table
1
Within
day and day to day imprecisions.
Within day imprecisionaDay to day imprecisiona
X (␮mol L−1) SD CV n X (␮mol L−1) SD CV n
88 0.0018 1.26 10 88 0.0049 3.623 20
265
0.0034 0.82 10 265 0.0085 2.05 20
442
0.0045 0.745 10 442 0.0096 1.553 20
X = creatinine; n = number of runs.
aDuplicate
measurement.
3.4. Analytical parameters
The
initial reaction rate obtained by kinetic method for the quan-
tification
of creatinine was plotted against the concentration of
creatinine
to get the analytical curve. The values of KCr and Vmax
for creatinine from the Lineweaver–Burk plot were found to be
3.33
␮mol L−1and 0.0668 min−1, respectively. The linear response
range
was from 0.22 to 2.65 mmol L−1of creatinine. The analytical
curve
for quantification of creatinine is depicted in Fig. 2. The lin-
ear
regression equation of the straight line as shown in Fig. 2 is rate
=0.034Ccreatinine +
0.001.
Michaelis–Menten
constants for PPDD, DMAB and copper were
determined
by double reciprocal plot in the concentrations range
of
13.75–55 ␮mol L−1, 0.6–6 mmol L−1and 5.33–53.3 ␮mol L−1,
respectively.
Creatinine concentrations of 0.44, 0.88, 1.76 and
2.65
mmol L−1in
the final volume of 3 mL were used for each
kinetic
study. The KP, KDand KCwere found to be 27.07 ␮mol L−1,
2.65
mmol L−1and 222 ␮mol L−1, respectively. Constant intercept
was
obtained in a double-reciprocal plot of Voversus Po, Doand Cuo
(panels A–C
of Fig. 3) at different concentrations of creatinine.
The
method based on measurements made after 20 min of reac-
tion
time indicated a linear response (absorbance) in function of
the
concentration of creatinine (Fig. 4) over the range from 8.8 to
530
␮mol L−1. Analytical curve equation was =0.0015Ccr + 0.0058
with
a regression coefficient of 0.999. The molar absorption coef-
ficient
was 0.1427 × 104L mol−1cm−1and the RSD was 0.00886
(n
= 10).
Sandell’s
sensitivity was studied to determine the concentra-
tion
of creatinine required to obtain the lower absorbance of 0.01;
the
study revealed that 0.04 ␮g
cm−2of creatinine in the opti-
mized
reaction mixture yields 0.01 absorbance. Limit of detection
and
limit of quantification were 2.55 ␮mol L−1and 8.52 ␮mol L−1
respectively. Recovery studies were conducted in human urine and
serum
samples by spiking standard creatinine to the calculated
diluted
samples.
The
magnitude of total imprecision of the proposed method was
studied
by analyzing reaction mixture containing known concen-
trations
of creatinine within Beer’s law range; three concentrations
Table 2
Interference
study by excipients.
Interferants Tolerance ratioa
EDTAb0.1
Iron
II, iron III, bilirubin, nitrite 0.6
Nickel,
phosphate, ascorbic acid, citrateb4
Aminophylline,
heparinbhemoglobin 8
Gentamycin,
diazepam, amoxycillin 11
Magnesium,
aluminum, nitrate, ammonium 14
Uric
acid 24
Glycine,
potassium, sodium 35
Lactose,
sucrose 50
Carbonate,
bicarbonate, calcium 75
Glucose,
acetone 90
Urea
95
Creatine,
chloride 150
aTolerance
ratio corresponds to the ratio of limit of inhibiting species concentra-
tion
to that of 152 ␮mol
L−1creatinine used.
bAnti
coagulants.
Fig. 3. Kinetic behavior of PPDD and DMAB with respect to various concentration
of
creatinine.
of creatinine ranging from lower to higher concentrations were
selected
with 10 runs in a day with a time gap of 1 h for within
day
assay and 20 runs with a time gap of 1 day for day to day assay.
Results
are shown in Table 1. Results showed that within day impre-
cision
were 0.745–1.26% (n = 10) and day to day imprecision ranged
from
1.5 to 3.6% (n = 20). These results proved that the method is
more
precise in both within day and day to day assays and is also
highly
reproducible.
Reliability
of the proposed method was carried out with QCM
containing
158 ␮mol L−1of creatinine. QCM was serially diluted for
the
analysis and the measurements were carried out in duplicate.
The
calculated creatinine concentration with respect to obtained

322 P. Nagaraja et al. / Spectrochimica Acta Part A 92 (2012) 318– 324
Fig. 4. Analytical curve for the quantification of creatinine by the fixed time method.
creatinine concentration is graphically shown in Fig. 5; the graph
yielded
y = 0.998CCr + 0.712 with linear regression coefficient of
0.999,
the results indicated that it is a much reliable method.
3.5.
Interference studies
The
extent of interference from foreign substances was
studied
in 3 mL of the solution containing 55 ␮mol L−1PPDD,
6
mmol L−1DMAB and 53 ␮mol L−1copper and 152 ␮mol L−1fixed
concentration
of creatinine in 1 mmol L−1potassium dihydrogen
ortho
phosphate/sodium hydroxide buffer at pH 7.8. A deviation
of
±3% from the original value in the absorbance reading was con-
sidered
tolerable. The resultant tolerance ratios are summarized
in
Table 2. It can be observed that compounds like creatine, glu-
cose,
ammonia, nitrate, urea and common inorganic ions showed
a
good tolerance under the given conditions, whereas hemoglobin,
ascorbic
acid, Fe (II), (III), uric acid, nitrite and some drugs showed
minimum
tolerance limit.
Table 3
Analytical
recovery of creatinine in human urine samples.
Sample
number
Proposed method Standard method
Creatinine
(␮mol
L−1)
Added
(␮mol L−1)
Found
(␮mol L−1)
Recovered
(␮mol L−1)
Recovery*
(%)
Creatinine
(␮mol L−1)
Added
(␮mol L−1)
Found
(␮mol L−1)
Recovered
(␮mol L−1)
Recovery*
(%)
1 51.2 38 90.3 39.1 102.9 54.9 38 93.6 38.7 101.2
305 366.4
315.2 103.3 305 361.2 306.3 100.4
2
73.3 76 148.2 74.9 98.5 75.0 76 152.1 77.1 101.4
190.6
265.7 192.4 100.9 190.6 267.5 192.5 100.9
3
109.6 38 146.9 37.13 97.8 114.1 38 153.9 39.8 104.7
228.8
344 234.4 102.4 228.8 346.2 232.1 101.8
4
125.53 76 200.9 75.4 99.2 123.9 76 198.1 74.2 97.6
228
355.4 229.9 100.8 228 354.4 230.5 101.1
aMean
of duplicate measurement.
*[(Found
creatinine concentration − initial creatinine concentration)/added creatinine concentration].
Table
4
Analytical
recovery of creatinine in human serum samples.
Sample
number
Proposed
method Standard method
Creatinine
(␮mol
L−1)
Added
(␮mol L−1)
Found
(␮mol L−1)
Recovered
(␮mol L−1)
Recovery*
(%)
Creatinine
(␮mol L−1)
Added
(␮mol L−1)
Found
(␮mol L−1)
Recovered
(␮mol L−1)
Recovery*
(%)
1 67.18 88.4 157.6 90.42 102.2 61.8 38 99.2 37.4 98.4
265.2
333.32 266.14 100.3 305 366.6 304.8 99.9
2
73.37 88.4 166.97 93.6 105.8 70 76 146.4 76.4 100.5
265.2
344.4 271.03
102.2 190.6 258.4 188.4 98.8
3
90.17 88.4 185.4 95.23 107.8 88.4 38 127.9 39.5 103.4
265.2
357.82 267.65 100.9 228.8 316.5 228.1 99.7
4
122.88 88.4 215.08 92.2 104.3 123.4 76 201.7 78.3 103
265.2
393.38 270.5 101.9 228 352.1 228.7 100.3
aMean
of duplicate measurement.
*[(Found
creatinine concentration −
initial creatinine concentration)/added creatinine concentration].