Spectrochimica Acta Part A 92 (2012) 318–324 Contents lists available at SciVerse ScienceDirect 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 . 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 , 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  an endogenous protein belonging to the type 2 cystatin gene family. Reports have shown that cystatin C levels get altered in patients with cancer , thyroid dysfunction . Cystatin C levels are also affected by cigarette smoking and by C-reactive proteins , which might or might not reﬂect actual renal dysfunction . The role of cystatin C to monitor GFR during pregnancy remains controversial . 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 . 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 , and thyroid functions . Jaffe’s alkaline sodium picrate method is the most widely accepted standard method for creatinine measurement . But this method is affected by many chemical species such as creatine, bilirubin, hemoglobin, cefoxitin and cephalothin . 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 . 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 . 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 , 1,4-naphthoquinone-2-sulfonate , Sakaguchi’s color reaction of creatinine with o-nitrobenzaldehyde , capillary electrophoresis [23–25], liquid chromatography (LC) , gas chromatography (GC) , GC with isotope dilution mass spectrometry , 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  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. 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