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Homogentisic acid interference in routine urine creatinine determination
Molecular Genetics and Metabolism 100 (2010) 103–104
Contents lists available at ScienceDirect
Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme
Brief Communication
Homogentisic acid interference in routine urine creatinine determination Perry R. Loken a, Mark J. Magera a,*, Wendy Introne b, Silvia Tortorelli a, Dimitar Gavrilov a, Devin Oglesbee a, Piero Rinaldo a, Dietrich Matern a, Kimiyo Raymond a a b
Biochemical Genetics Laboratory, Mayo Clinic College of Medicine, Rochester, MN, United States National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, United States
a r t i c l e
i n f o
Article history: Received 30 December 2009 Received in revised form 13 January 2010 Accepted 13 January 2010 Available online 21 January 2010 Keywords: Homogentisic acid Alkaptonuria Homogentisate 1,2-dioxygenase Jaffe reaction Creatinine Interference
a b s t r a c t We report the artifactual elevation of homogentisic acid (HGA) in urine from alkaptonuric patients after replacing the creatinine method (Jaffe reaction) in our laboratory with an automated enzymatic method. Samples with elevated HGA by GC-MS had lower creatinine values as determined by the enzymatic method than by the Jaffe reaction. The low creatinine values were due to interference by HGA in the enzymatic method. The enzymatic method is unsuitable for creatinine determination in urine of patients with alkaptonuria. Ó 2010 Elsevier Inc. All rights reserved.
Introduction
Materials and methods
Alkaptonuria is an autosomal recessive disorder caused by deficiency of homogentisate 1,2-dioxygenase (OMIM 203500), an enzyme of the tyrosine degradation pathway. This classic inborn error of metabolism is characterized by urine that darkens on standing and alkalinization, ochronotic pigmentation of cartilage and collagenous tissues, urolithiasis, arthritis, and heart valve involvement [1]. The diagnosis is confirmed by identification of homogentisic acid (HGA), which is excreted in millimolar amounts in urine, along with its oxidation product, benzoquinone acetic acid (BQA). Currently, there is no effective treatment of alkaptonuria. Supplementation with ascorbic acid (vitamin C) and/or protein restriction have been proposed but except for a single case have not been found to be beneficial [1–3]. Clinical studies are ongoing to determine the potential benefit of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC; nitisinone; Orfadin) which blocks production of HGA [4,5].
Determination of HGA is accomplished in many laboratories by gas chromatography-mass spectrometric analysis of trimethylsilyl ethers of urinary organic acids [6]. Quantification of HGA excretion typically includes normalizing the gravimetric or molar quantity to the creatinine (CT) concentration in the same urine specimen. In high-volume laboratories manual methods for CT determination are often abandoned in favor of more automated approaches. This is the case for our laboratory, where we replaced the Jaffe reaction with an automated, enzymatic test kit method (Roche/Cobas Creatinine plus). Validation of this change included a method comparison where CT was measured by both methods in leftover random urine specimens with CT values ranging from less than 10 mg/dL to greater than 250 mg/dL. A comparison plot of these values yielded a regression of y = 0.9669x 1.67; r2 = 0.9988. A Bland-Altman analysis of the same data gave a mean difference of 5.14 mg/dL (Roche vs. Jaffe) and standard deviation of 3.4 mg/dL. Ninety-six percent (25 of 26) of paired values differed by less than ±2 SD. Results and discussion
* Corresponding author. Address: Biochemical Genetics Laboratory, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, United States. Fax: +1 507 266 2888. E-mail address: [email protected] (M.J. Magera). 1096-7192/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2010.01.006
Following implementation of the enzymatic method for CT determination, a one- to five-fold increase in urine HGA levels was discovered in samples submitted for organic acid analysis from 21 alkaptonuric patients whose treatment had not been
104
P.R. Loken et al. / Molecular Genetics and Metabolism 100 (2010) 103–104 Table 1 Titration of HGA in urine versus creatinine concentration determined by Jaffe (picrate) and enzymatic methods.
Creatinine Difference (Enzymatic - Picrate) mg/dL
-4
-32
Aliquot
Mean Difference
-60
1 2 3 4 5 6
-88
-116 0
5
10
15
20
25
30
35
40
45
HGA added
CT, picrate
CT, enzymatic
% Diff.
(mmol/L)
(mg/dL)
(mg/dL)
(mg/dL)
(mg/dL)
0 2 10 50 100 150
0 34 168 840 1680 2520
162 160 156 136 165 197
156 139 83 20 15 13
4 13 47 85 91 93
50
[Homogentisic Acid] mmol/L Fig. 1. Bland-Altman plot showing the difference between creatinine concentrations determined in urine of alkaptonuric patients (n = 21) by the Jaffe reaction (picric acid method) and an automated, enzymatic method (Roche/Cobas). The mean difference between creatinine concentrations determined by either analytical method reveals an underestimation of creatinine by the enzymatic method with increasing concentrations of homogentisic acid.
altered and who were being closely monitored in a study conducted at the National Human Genome Research Institute. The comparison data shown in Fig. 1 indicate the depression of CT values determined by the automated, enzymatic method compared to those obtained by the Jaffe reaction. When reviewing the manufacturer’s test kit insert for the automated enzymatic CT method, no mention is made of HGA or any other compound being a known interference. A literature search yielded reports mentioning HGA interfering with biochemical methods using peroxidase and hydrogen peroxide (H2O2) [7,8]. Catecholamines, such as dopamine and dobutamine, have also been reported to interfere with the generation of chromophores using peroxide and peroxidase [9,10]. The enzymatic CT method converts creatinine to creatine, creatine to sarcosine, and sarcosine to glycine. This latter reaction liberates H2O2 which is conjugated with 4-aminophenazone and 2,4,6-triiodo-3-hydroxybenzoic acid (HTIB) to form the quinone imine chromophore in a peroxidasecatalyzed reaction. The concentration of creatinine is determined by spectrophotometric response of this colored complex versus calibrators. We hypothesized that homogentisic acid competes with HTIB and 4-aminophenazone in the reaction forming the chromophore due to affinity for the peroxidase. To investigate this hypothesis, we added HGA in increasing concentration to a normal urine sample and measured the creatinine concentration by the Jaffe reaction and the automated, enzymatic method. Increasing concentrations of HGA caused no decrease in CT levels with the Jaffe reaction, but significant suppression of enzymatically determined CT values for HGA concentrations greater than 2 mmol/L (Table 1). In light of our experience with discrepancies in CT concentrations for alkaptonuric patients, we conclude that the enzymatic
determination of urinary CT is unsuitable for this patient population. Instead, CT concentrations should be determined by alternative methods, such as the Jaffe reaction. While we have not abandoned the enzymatic method in our laboratory, we have adopted an approach were CT is being re-analyzed by the Jaffe reaction in any urine sample where organic acid analysis reveals the presence of HGA. In addition, the non-specific affinity of peroxidase for HGA and other cyclic compounds, such as dopamine and dobutamine, raises concerns about the specificity of the enzymatic method for CT measurement in different clinical situations. At a minimum, these interferences should be listed in the commercially available test kits. References [1] C. Phornphutkul, W.J. Introne, M.B. Perry, I. Bernardini, M.D. Murphey, D.L. Fitzpatrick, et al., Natural history of alkaptonuria, N. Engl. J. Med. 347 (2002) 2111–2121. [2] E. Morava, G. Kosztolanyi, U.F. Engelke, R.A. Wevers, Reversal of clinical symptoms and radiographic abnormalities with protein restriction and ascorbic acid in alkaptonuria, Ann. Clin. Biochem. 40 (2003) 108–111. [3] J.A. Wolff, B. Barshop, W.L. Nyhan, J. Leslie, J.E. Seegmiller, H. Gruber, et al., Effects of ascorbic acid in alkaptonuria: alterations in benzoquinone acetic acid and an ontogenic effect in infancy, Pediatr. Res. 26 (1989) 140–144. [4] Y. Anikster, W.L. Nyhan, W.A. Gahl, NTBC and alkaptonuria, Am. J. Hum. Genet. 63 (1998) 920–921. [5] Y. Suzuki, K. Oda, Y. Yoshikawa, T. Maeda, T. Suzuki, A novel therapeutic trial of homogentisic aciduria in a murine model of alkaptonuria, J. Hum. Genet. 44 (1999) 79–84. [6] P. Rinaldo, Organic acids, in: N. Blau, M. Duran, K.M. Gibson (Eds.), Laboratory Guide to the Methods in Biochemical Genetics, vol., Springer Verlag, Heidelberg, 2008, pp. 137–170. [7] Y. Moriwaki, T. Yamamoto, Y. Nasako, H. Ohata, S. Takahashi, Z. Tsutsumi, et al., ‘Pseudohypouricosuria’ in alcaptonuria: homogentisic acid interference in the measurement of urinary uric acid with the uricase–peroxidase reaction, Ann. Clin. Biochem. 36 (Pt 4) (1999) 501–503. [8] R. Haeckel, The use of aldehyde dehydrogenase to determine H2O2-producing reactions. I. The determination of the uric acid concentration, J. Clin. Chem. Clin. Biochem. 14 (1976) 101–107. [9] B.S. Karon, T.M. Daly, M.G. Scott, Mechanisms of dopamine and dobutamine interference in biochemical tests that use peroxide and peroxidase to generate chromophore, Clin. Chem. 44 (1998) 155–160. [10] A.K. Saenger, C. Lockwood, C.L. Snozek, T.C. Milz, B.S. Karon, M.G. Scott, A.S. Jaffe, Catecholamine interference in enzymatic creatinine assays, Clin. Chem. 55 (2009) 1732–1736.
Contents lists available at ScienceDirect
Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme
Brief Communication
Homogentisic acid interference in routine urine creatinine determination Perry R. Loken a, Mark J. Magera a,*, Wendy Introne b, Silvia Tortorelli a, Dimitar Gavrilov a, Devin Oglesbee a, Piero Rinaldo a, Dietrich Matern a, Kimiyo Raymond a a b
Biochemical Genetics Laboratory, Mayo Clinic College of Medicine, Rochester, MN, United States National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, United States
a r t i c l e
i n f o
Article history: Received 30 December 2009 Received in revised form 13 January 2010 Accepted 13 January 2010 Available online 21 January 2010 Keywords: Homogentisic acid Alkaptonuria Homogentisate 1,2-dioxygenase Jaffe reaction Creatinine Interference
a b s t r a c t We report the artifactual elevation of homogentisic acid (HGA) in urine from alkaptonuric patients after replacing the creatinine method (Jaffe reaction) in our laboratory with an automated enzymatic method. Samples with elevated HGA by GC-MS had lower creatinine values as determined by the enzymatic method than by the Jaffe reaction. The low creatinine values were due to interference by HGA in the enzymatic method. The enzymatic method is unsuitable for creatinine determination in urine of patients with alkaptonuria. Ó 2010 Elsevier Inc. All rights reserved.
Introduction
Materials and methods
Alkaptonuria is an autosomal recessive disorder caused by deficiency of homogentisate 1,2-dioxygenase (OMIM 203500), an enzyme of the tyrosine degradation pathway. This classic inborn error of metabolism is characterized by urine that darkens on standing and alkalinization, ochronotic pigmentation of cartilage and collagenous tissues, urolithiasis, arthritis, and heart valve involvement [1]. The diagnosis is confirmed by identification of homogentisic acid (HGA), which is excreted in millimolar amounts in urine, along with its oxidation product, benzoquinone acetic acid (BQA). Currently, there is no effective treatment of alkaptonuria. Supplementation with ascorbic acid (vitamin C) and/or protein restriction have been proposed but except for a single case have not been found to be beneficial [1–3]. Clinical studies are ongoing to determine the potential benefit of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC; nitisinone; Orfadin) which blocks production of HGA [4,5].
Determination of HGA is accomplished in many laboratories by gas chromatography-mass spectrometric analysis of trimethylsilyl ethers of urinary organic acids [6]. Quantification of HGA excretion typically includes normalizing the gravimetric or molar quantity to the creatinine (CT) concentration in the same urine specimen. In high-volume laboratories manual methods for CT determination are often abandoned in favor of more automated approaches. This is the case for our laboratory, where we replaced the Jaffe reaction with an automated, enzymatic test kit method (Roche/Cobas Creatinine plus). Validation of this change included a method comparison where CT was measured by both methods in leftover random urine specimens with CT values ranging from less than 10 mg/dL to greater than 250 mg/dL. A comparison plot of these values yielded a regression of y = 0.9669x 1.67; r2 = 0.9988. A Bland-Altman analysis of the same data gave a mean difference of 5.14 mg/dL (Roche vs. Jaffe) and standard deviation of 3.4 mg/dL. Ninety-six percent (25 of 26) of paired values differed by less than ±2 SD. Results and discussion
* Corresponding author. Address: Biochemical Genetics Laboratory, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, United States. Fax: +1 507 266 2888. E-mail address: [email protected] (M.J. Magera). 1096-7192/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2010.01.006
Following implementation of the enzymatic method for CT determination, a one- to five-fold increase in urine HGA levels was discovered in samples submitted for organic acid analysis from 21 alkaptonuric patients whose treatment had not been
104
P.R. Loken et al. / Molecular Genetics and Metabolism 100 (2010) 103–104 Table 1 Titration of HGA in urine versus creatinine concentration determined by Jaffe (picrate) and enzymatic methods.
Creatinine Difference (Enzymatic - Picrate) mg/dL
-4
-32
Aliquot
Mean Difference
-60
1 2 3 4 5 6
-88
-116 0
5
10
15
20
25
30
35
40
45
HGA added
CT, picrate
CT, enzymatic
% Diff.
(mmol/L)
(mg/dL)
(mg/dL)
(mg/dL)
(mg/dL)
0 2 10 50 100 150
0 34 168 840 1680 2520
162 160 156 136 165 197
156 139 83 20 15 13
4 13 47 85 91 93
50
[Homogentisic Acid] mmol/L Fig. 1. Bland-Altman plot showing the difference between creatinine concentrations determined in urine of alkaptonuric patients (n = 21) by the Jaffe reaction (picric acid method) and an automated, enzymatic method (Roche/Cobas). The mean difference between creatinine concentrations determined by either analytical method reveals an underestimation of creatinine by the enzymatic method with increasing concentrations of homogentisic acid.
altered and who were being closely monitored in a study conducted at the National Human Genome Research Institute. The comparison data shown in Fig. 1 indicate the depression of CT values determined by the automated, enzymatic method compared to those obtained by the Jaffe reaction. When reviewing the manufacturer’s test kit insert for the automated enzymatic CT method, no mention is made of HGA or any other compound being a known interference. A literature search yielded reports mentioning HGA interfering with biochemical methods using peroxidase and hydrogen peroxide (H2O2) [7,8]. Catecholamines, such as dopamine and dobutamine, have also been reported to interfere with the generation of chromophores using peroxide and peroxidase [9,10]. The enzymatic CT method converts creatinine to creatine, creatine to sarcosine, and sarcosine to glycine. This latter reaction liberates H2O2 which is conjugated with 4-aminophenazone and 2,4,6-triiodo-3-hydroxybenzoic acid (HTIB) to form the quinone imine chromophore in a peroxidasecatalyzed reaction. The concentration of creatinine is determined by spectrophotometric response of this colored complex versus calibrators. We hypothesized that homogentisic acid competes with HTIB and 4-aminophenazone in the reaction forming the chromophore due to affinity for the peroxidase. To investigate this hypothesis, we added HGA in increasing concentration to a normal urine sample and measured the creatinine concentration by the Jaffe reaction and the automated, enzymatic method. Increasing concentrations of HGA caused no decrease in CT levels with the Jaffe reaction, but significant suppression of enzymatically determined CT values for HGA concentrations greater than 2 mmol/L (Table 1). In light of our experience with discrepancies in CT concentrations for alkaptonuric patients, we conclude that the enzymatic
determination of urinary CT is unsuitable for this patient population. Instead, CT concentrations should be determined by alternative methods, such as the Jaffe reaction. While we have not abandoned the enzymatic method in our laboratory, we have adopted an approach were CT is being re-analyzed by the Jaffe reaction in any urine sample where organic acid analysis reveals the presence of HGA. In addition, the non-specific affinity of peroxidase for HGA and other cyclic compounds, such as dopamine and dobutamine, raises concerns about the specificity of the enzymatic method for CT measurement in different clinical situations. At a minimum, these interferences should be listed in the commercially available test kits. References [1] C. Phornphutkul, W.J. Introne, M.B. Perry, I. Bernardini, M.D. Murphey, D.L. Fitzpatrick, et al., Natural history of alkaptonuria, N. Engl. J. Med. 347 (2002) 2111–2121. [2] E. Morava, G. Kosztolanyi, U.F. Engelke, R.A. Wevers, Reversal of clinical symptoms and radiographic abnormalities with protein restriction and ascorbic acid in alkaptonuria, Ann. Clin. Biochem. 40 (2003) 108–111. [3] J.A. Wolff, B. Barshop, W.L. Nyhan, J. Leslie, J.E. Seegmiller, H. Gruber, et al., Effects of ascorbic acid in alkaptonuria: alterations in benzoquinone acetic acid and an ontogenic effect in infancy, Pediatr. Res. 26 (1989) 140–144. [4] Y. Anikster, W.L. Nyhan, W.A. Gahl, NTBC and alkaptonuria, Am. J. Hum. Genet. 63 (1998) 920–921. [5] Y. Suzuki, K. Oda, Y. Yoshikawa, T. Maeda, T. Suzuki, A novel therapeutic trial of homogentisic aciduria in a murine model of alkaptonuria, J. Hum. Genet. 44 (1999) 79–84. [6] P. Rinaldo, Organic acids, in: N. Blau, M. Duran, K.M. Gibson (Eds.), Laboratory Guide to the Methods in Biochemical Genetics, vol., Springer Verlag, Heidelberg, 2008, pp. 137–170. [7] Y. Moriwaki, T. Yamamoto, Y. Nasako, H. Ohata, S. Takahashi, Z. Tsutsumi, et al., ‘Pseudohypouricosuria’ in alcaptonuria: homogentisic acid interference in the measurement of urinary uric acid with the uricase–peroxidase reaction, Ann. Clin. Biochem. 36 (Pt 4) (1999) 501–503. [8] R. Haeckel, The use of aldehyde dehydrogenase to determine H2O2-producing reactions. I. The determination of the uric acid concentration, J. Clin. Chem. Clin. Biochem. 14 (1976) 101–107. [9] B.S. Karon, T.M. Daly, M.G. Scott, Mechanisms of dopamine and dobutamine interference in biochemical tests that use peroxide and peroxidase to generate chromophore, Clin. Chem. 44 (1998) 155–160. [10] A.K. Saenger, C. Lockwood, C.L. Snozek, T.C. Milz, B.S. Karon, M.G. Scott, A.S. Jaffe, Catecholamine interference in enzymatic creatinine assays, Clin. Chem. 55 (2009) 1732–1736.