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Quantification of creatinine in biologic

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Spectrochimica Acta Part A 92 (2012) 318–324
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Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: 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 concentration 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. Creatinine 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−1 and 0.221–2.65 mmol L−1 ,
respectively. Recovery of creatinine varied from 97.8 to 107.8%. Limit of detection and limit of quantification were 2.55 and 8.52 ␮mol L−1 respectively whereas Sandell’s sensitivity and molar absorption
coefficient values were 0.0407 ␮g cm−2 and 0.1427 × 104 L mol−1 cm−1 respectively. 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 accordance 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 measure 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
51 chromium-EDTA, 125 I-iothalamate, 99m Tc-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, dimethylamino benzoicacid; QCM, quality control material; KP , Michaelis–Menten constants
for PPDD; KD , Michaelis–Menten constant for DMAB; KC , Michaelis–Menten constants for copper; KCr , Michaelis–Menten constants for creatinine; Vmax , maximum
rate of the reaction at optimized reagent concentration.
∗ Corresponding author. Tel.: +91 821 2412557; fax: +91 821 2421263.
E-mail addresses: [email protected], [email protected] (P. Nagaraja).
1386-1425/$ – see front matter © 2012 Published by Elsevier B.V.
doi:10.1016/j.saa.2012.02.104
Another method for the estimation of GFR is urinary measurement 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 dysfunction [7]. The role of cystatin C to monitor GFR during pregnancy
remains controversial [8]. The use of cystatin C alone as a determinator 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 eliminate or reduce interferences, which include specific adsorption of
P. Nagaraja et al. / Spectrochimica Acta Part A 92 (2012) 318–324
creatinine, removal of interfering compounds, dialysis, adjusting
the pH, and kinetic measurements. Unfortunately, none of these
modifications were capable of completely eliminating interferences.
Spectrophotometric method based on multi-enzyme system
has been developed to improve the specificity of creatinine determination [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 creatinine 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 (linearity or range), and for the elimination of interferences have not been
delineated properly.
A new spectrophotometric method based on enzyme like activity 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 spectrophotometer 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, Ahmedabad, 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−1 hydrochloric acid solution and then adjusting the
volume of solution to 10 mL with water. A 1.6 mmol L−1 copper (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.
319
2.3. Biological samples
Blood and urine samples were collected from volunteers following 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−1 PPDD, 6 mmol L−1
DMAB and 53.3 ␮mol L−1 copper in 1 mmol L−1 potassium dihydrogen orthophosphate/sodium hydroxide buffer of pH 7.8. The
reaction was initiated at 25 ◦ C by adding 100 ␮L of different concentrations 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−1 PPDD, 6 mmol L−1 DMAB and
53.3 ␮mol L−1 copper in 1 mmol L−1 potassium dihydrogen ortho
phosphate/sodium hydroxide buffer at pH 7.8 and 100 ␮L of various concentrations of creatinine within the linearity range. The
reaction mixture was allowed to stand for 20 min at room temperature. 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. Experiments 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−1 were 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 orthophosphate/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 phosphate/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−1 solution.
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−1 PPDD, 6 mmol L−1 DMAB, 53.3 ␮mol L−1 copper and
176 ␮mol L−1 of creatinine in 1 mmol L−1 potassium 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 imprecisiona
−1
X (␮mol L
88
265
442
)
Day to day imprecisiona
SD
CV
n
X (␮mol L−1 )
SD
CV
n
0.0018
0.0034
0.0045
1.26
0.82
0.745
10
10
10
88
265
442
0.0049
0.0085
0.0096
3.623
2.05
1.553
20
20
20
X = creatinine; n = number of runs.
a
Duplicate measurement.
3.4. Analytical parameters
The initial reaction rate obtained by kinetic method for the quantification 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−1 and 0.0668 min−1 , respectively. The linear response
range was from 0.22 to 2.65 mmol L−1 of creatinine. The analytical
curve for quantification of creatinine is depicted in Fig. 2. The linear 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−1 and 5.33–53.3 ␮mol L−1 ,
respectively. Creatinine concentrations of 0.44, 0.88, 1.76 and
2.65 mmol L−1 in the final volume of 3 mL were used for each
kinetic study. The KP , KD and KC were found to be 27.07 ␮mol L−1 ,
2.65 mmol L−1 and 222 ␮mol L−1 , respectively. Constant intercept
was obtained in a double-reciprocal plot of Vo versus Po , Do and Cuo
(panels A–C of Fig. 3) at different concentrations of creatinine.
The method based on measurements made after 20 min of reaction 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 coefficient was 0.1427 × 104 L mol−1 cm−1 and the RSD was 0.00886
(n = 10).
Sandell’s sensitivity was studied to determine the concentration of creatinine required to obtain the lower absorbance of 0.01;
the study revealed that 0.04 ␮g cm−2 of creatinine in the optimized reaction mixture yields 0.01 absorbance. Limit of detection
and limit of quantification were 2.55 ␮mol L−1 and 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 concentrations of creatinine within Beer’s law range; three concentrations
Table 2
Interference study by excipients.
Interferants
b
EDTA
Iron II, iron III, bilirubin, nitrite
Nickel, phosphate, ascorbic acid, citrateb
Aminophylline, heparinb hemoglobin
Gentamycin, diazepam, amoxycillin
Magnesium, aluminum, nitrate, ammonium
Uric acid
Glycine, potassium, sodium
Lactose, sucrose
Carbonate, bicarbonate, calcium
Glucose, acetone
Urea
Creatine, chloride
Tolerance ratioa
0.1
0.6
4
8
11
14
24
35
50
75
90
95
150
a
Tolerance ratio corresponds to the ratio of limit of inhibiting species concentration to that of 152 ␮mol L−1 creatinine used.
b
Anti 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 imprecision 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−1 of 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−1 PPDD,
6 mmol L−1 DMAB and 53 ␮mol L−1 copper and 152 ␮mol L−1 fixed
concentration of creatinine in 1 mmol L−1 potassium dihydrogen
ortho phosphate/sodium hydroxide buffer at pH 7.8. A deviation
of ±3% from the original value in the absorbance reading was considered tolerable. The resultant tolerance ratios are summarized
in Table 2. It can be observed that compounds like creatine, glucose, 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
Creatinine
(␮mol L−1 )
1
51.2
2
73.3
3
109.6
4
125.53
Standard method
Added
(␮mol L−1 )
Found
(␮mol L−1 )
Recovered
(␮mol L−1 )
Recovery*
(%)
38
305
76
190.6
38
228.8
76
228
90.3
366.4
148.2
265.7
146.9
344
200.9
355.4
39.1
315.2
74.9
192.4
37.13
234.4
75.4
229.9
102.9
103.3
98.5
100.9
97.8
102.4
99.2
100.8
Creatinine
(␮mol L−1 )
54.9
75.0
114.1
123.9
Added
(␮mol L−1 )
Found
(␮mol L−1 )
Recovered
(␮mol L−1 )
Recovery*
(%)
38
305
76
190.6
38
228.8
76
228
93.6
361.2
152.1
267.5
153.9
346.2
198.1
354.4
38.7
306.3
77.1
192.5
39.8
232.1
74.2
230.5
101.2
100.4
101.4
100.9
104.7
101.8
97.6
101.1
Added
(␮mol L−1 )
Found
(␮mol L−1 )
Recovered
(␮mol L−1 )
Recovery*
(%)
38
305
76
190.6
38
228.8
76
228
99.2
366.6
146.4
258.4
127.9
316.5
201.7
352.1
37.4
304.8
76.4
188.4
39.5
228.1
78.3
228.7
98.4
99.9
100.5
98.8
103.4
99.7
103
100.3
a
Mean 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
Creatinine
(␮mol L−1 )
1
67.18
2
73.37
3
90.17
4
122.88
Standard method
Added
(␮mol L−1 )
Found
(␮mol L−1 )
Recovered
(␮mol L−1 )
Recovery*
(%)
88.4
265.2
88.4
265.2
88.4
265.2
88.4
265.2
157.6
333.32
166.97
344.4
185.4
357.82
215.08
393.38
90.42
266.14
93.6
271.03
95.23
267.65
92.2
270.5
102.2
100.3
105.8
102.2
107.8
100.9
104.3
101.9
Creatinine
(␮mol L−1 )
61.8
70
88.4
123.4
a
Mean of duplicate measurement.
*[(Found creatinine concentration − initial creatinine concentration)/added creatinine concentration].
P. Nagaraja et al. / Spectrochimica Acta Part A 92 (2012) 318–324
323
Fig. 5. Reliability of the method using QCM.
3.6. Applications in human urine and serum samples
Urine and serum samples obtained from volunteer donors were
analyzed by the reference modified Jaffe’s kit method in clinical laboratory. The same samples were diluted to the linearity range and
measured in duplicate by the proposed method. Recovery studies
were carried out in both the methods by spiking standard creatinine
solution to the serum and urine samples. The recovery study exhibited minimum interference from inhibiting species studied with
good reproducibility of the assay procedure providing the recovery
of 97.8–103.3% and 97.6–104.7% in urine sample and 100.3–107.8%
and 98.4–103.4% in serum sample by proposed and by modified
Jaffe’s method respectively. The results obtained by both the methods are summarized in Tables 3 and 4.
4. Conclusion
This is the first report we are publishing on the coupling of
PPDD with DMAB in the presence of copper for the quantification of
creatinine. The coupled product gets absorbed in visible region of
710 nm. The kinetics of the system evidenced “instantaneous” color
formation even in the presence of very small quantities of colorimetric reagents. Interference from foreign substance is negligible
and is comparable to other methods. Biological samples for creatinine estimation can be applied directly without requiring any
adsorption or deproteination. The wide range of linearity simplifies the dilution factor. Enzymic action of creatinine in presence of
copper minimizes the use of enzymes making the method much
affordable. Optimization of the reaction conditions allowed the
determination of creatinine as low as 8.8 ␮mol L−1 . The enzymatic
oxidative coupling of PPDD and DMAB in the presence of copper allowed spectrophotometric determination of the Creatinine
assay achieved within the linearity range of 0.22–2.65 mmol L−1
and 8.8–530 ␮mol L−1 from the kinetic and fixed time methods,
respectively. The method requires very small amount of serum and
urine samples. This linear dependence between the concentration
of creatinine and the absorbance over a narrow range is also an
important feature for the practical application of the assay procedure. Both urine and serum samples can be analyzed for creatinine
using the same concentration of optimized reagents. The assay is
free from most interfering compounds that may be present in urine.
Fixed time assay provides the ease of manual analysis with negligible errors. Avoiding the addition of strong base or acids prevents
occurrence of multiple errors due to precipitation. Thus, the proposed method can be used for the analysis of creatinine in urine
and serum samples.
Acknowledgments
One of the authors (Krishnegowda Avinash) thanks the
University Grants Commission, India, for financial support
(DV5/373[12]/RFSMS/2008-09, dated 19-03-2009), and the
authors thank the University of Mysore for providing research
facilities.
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