Clin Chem Lab Med 2015; 53(3): e81–e83
Letter to the Editor Andrew S. Davison*, Anna M. Milan, Andrew T. Hughes, John J. Dutton and Lakshminarayan R. Ranganath
Serum concentrations and urinary excretion of homogentisic acid and tyrosine in normal subjects DOI 10.1515/cclm-2014-0668 Received June 27, 2014; accepted August 25, 2014; previously published online September 25, 2014
Keywords: alkaptonuria; homogentisic acid; normal ranges; tyrosine. To the Editor, Alkaptonuria (AKU) is a rare, inherited metabolic disease (OMIM reference 203500). AKU has an autosomal recessive mode of inheritance and a reported prevalence of 1 in 250,000. However, there are countries, such as Slovakia and Dominican Republic, with increased prevalence up to approximately 1 in 19,000 [1]. AKU is a disorder of tyrosine metabolism, whereby degradation of tyrosine is blocked at the level of homogentisic acid (HGA) due to a congenital lack of the enzyme homogentisate 1,2-dioxygenase (HGD; EC 1.13.11.5). The manifestations of AKU are multiple and largely due to the consequences of increased HGA concentrations. These include severe spondyloarthropathy, renal stones, progressive kyphoscoliosis and impaired spinal and thoracic mobility [2]. Until recently, medical management of AKU has been palliative relying largely on analgesia and arthroplasty [2]. A potential HGA lowering therapy has now become available in the form of nitisinone (NTBC), an orally administered drug [3, 4]. NTBC is already licensed for the treatment of hereditary tyrosinaemia type-1 (OMIM reference 276700) and has proved to be a very efficacious mode of treatment [5]. *Corresponding author: Andrew S. Davison, Department of Clinical Biochemistry, Royal Liverpool and Broadgreen University Hospitals Trust, Liverpool, L7 8XP, UK, Phone: +44 151 706 4230, Fax: +44 151 706 4250, E-mail: [email protected] Anna M. Milan, Andrew T. Hughes, John J. Dutton and Lakshminarayan R. Ranganath: Department of Clinical Biochemistry, Royal Liverpool and Broadgreen University Hospitals Trust, Liverpool, UK
Since AKU is rare there are few laboratories that provide quantitative analysis for urine and serum HGA (u-HGA and s-HGA) and tyrosine. With the establishment of a National Alkaptonuria Centre in Liverpool, as well as the need to study NTBC in the DevelopAKUre clinical trial, there was necessity to measure HGA and tyrosine in serum and urine. Previous clinical studies on AKU have measured HGA using direct high performance liquid chromatography and indirect colorimetric assays in serum and urine, respectively [3, 4, 6]. Gas chromatography mass spectrometry has also been used for measurement of s-HGA [7]. The latter approach was employed to improve analytical specificity and sensitivity, however, this approach is time consuming. Serum and urine tyrosine (s-Tyr and u-Tyr) have traditionally been measured using cation exchange chromatography with post column ninhydrin detection [3, 4]. Recently, a liquid chromatography tandem mass spectrometry method has been validated for the simultaneous measurement of u-Tyr and HGA [8] and s-Tyr and HGA [unpublished]. This assay is sensitive and specific and allows measurement of tyrosine and HGA in a single assay. Herein we present the results from using this method to measure s-Tyr and u-Tyr and HGA in individuals that do not have AKU to generate a healthy population reference. This study was carried out following an amendment to the ethical application for the Natural History Study of Alkaptonuria (NRES No 07/H1002/111). Participants were recruited from the Royal Liverpool and Broadgreen University Hospitals Trust and the University of Liverpool after informed written consent was obtained. Volunteers comprised of 11 females and 11 males subjects. The age range was 25–61 years in females, and 32–63 years in males. None of the volunteers had AKU. Medication and health issues were documented for all recruits. A fasting blood sample and an acidified 24-h urine sample (25 mL of 5 N sulphuric acid added to 2.5 L urine collection bottle) were collected from all 22 volunteers. Blood samples were collected into plain serum tubes (Sarstedt, Germany) from all subjects in the fasting state,
Brought to you by | University of Georgia Libraries Authenticated Download Date | 5/30/15 2:55 PM
e82 Davison et al.: Serum concentrations and urinary excretion of homogentisic acid and tyrosine Table 1 Serum and urine reference ranges for tyrosine and homogentisic acid in the individuals (n = 22) recruited into the study and reference ranges quoted in previous studies. Serum [HGA], μmol/L
Letter to the Editor Andrew S. Davison*, Anna M. Milan, Andrew T. Hughes, John J. Dutton and Lakshminarayan R. Ranganath
Serum concentrations and urinary excretion of homogentisic acid and tyrosine in normal subjects DOI 10.1515/cclm-2014-0668 Received June 27, 2014; accepted August 25, 2014; previously published online September 25, 2014
Keywords: alkaptonuria; homogentisic acid; normal ranges; tyrosine. To the Editor, Alkaptonuria (AKU) is a rare, inherited metabolic disease (OMIM reference 203500). AKU has an autosomal recessive mode of inheritance and a reported prevalence of 1 in 250,000. However, there are countries, such as Slovakia and Dominican Republic, with increased prevalence up to approximately 1 in 19,000 [1]. AKU is a disorder of tyrosine metabolism, whereby degradation of tyrosine is blocked at the level of homogentisic acid (HGA) due to a congenital lack of the enzyme homogentisate 1,2-dioxygenase (HGD; EC 1.13.11.5). The manifestations of AKU are multiple and largely due to the consequences of increased HGA concentrations. These include severe spondyloarthropathy, renal stones, progressive kyphoscoliosis and impaired spinal and thoracic mobility [2]. Until recently, medical management of AKU has been palliative relying largely on analgesia and arthroplasty [2]. A potential HGA lowering therapy has now become available in the form of nitisinone (NTBC), an orally administered drug [3, 4]. NTBC is already licensed for the treatment of hereditary tyrosinaemia type-1 (OMIM reference 276700) and has proved to be a very efficacious mode of treatment [5]. *Corresponding author: Andrew S. Davison, Department of Clinical Biochemistry, Royal Liverpool and Broadgreen University Hospitals Trust, Liverpool, L7 8XP, UK, Phone: +44 151 706 4230, Fax: +44 151 706 4250, E-mail: [email protected] Anna M. Milan, Andrew T. Hughes, John J. Dutton and Lakshminarayan R. Ranganath: Department of Clinical Biochemistry, Royal Liverpool and Broadgreen University Hospitals Trust, Liverpool, UK
Since AKU is rare there are few laboratories that provide quantitative analysis for urine and serum HGA (u-HGA and s-HGA) and tyrosine. With the establishment of a National Alkaptonuria Centre in Liverpool, as well as the need to study NTBC in the DevelopAKUre clinical trial, there was necessity to measure HGA and tyrosine in serum and urine. Previous clinical studies on AKU have measured HGA using direct high performance liquid chromatography and indirect colorimetric assays in serum and urine, respectively [3, 4, 6]. Gas chromatography mass spectrometry has also been used for measurement of s-HGA [7]. The latter approach was employed to improve analytical specificity and sensitivity, however, this approach is time consuming. Serum and urine tyrosine (s-Tyr and u-Tyr) have traditionally been measured using cation exchange chromatography with post column ninhydrin detection [3, 4]. Recently, a liquid chromatography tandem mass spectrometry method has been validated for the simultaneous measurement of u-Tyr and HGA [8] and s-Tyr and HGA [unpublished]. This assay is sensitive and specific and allows measurement of tyrosine and HGA in a single assay. Herein we present the results from using this method to measure s-Tyr and u-Tyr and HGA in individuals that do not have AKU to generate a healthy population reference. This study was carried out following an amendment to the ethical application for the Natural History Study of Alkaptonuria (NRES No 07/H1002/111). Participants were recruited from the Royal Liverpool and Broadgreen University Hospitals Trust and the University of Liverpool after informed written consent was obtained. Volunteers comprised of 11 females and 11 males subjects. The age range was 25–61 years in females, and 32–63 years in males. None of the volunteers had AKU. Medication and health issues were documented for all recruits. A fasting blood sample and an acidified 24-h urine sample (25 mL of 5 N sulphuric acid added to 2.5 L urine collection bottle) were collected from all 22 volunteers. Blood samples were collected into plain serum tubes (Sarstedt, Germany) from all subjects in the fasting state,
Brought to you by | University of Georgia Libraries Authenticated Download Date | 5/30/15 2:55 PM
e82 Davison et al.: Serum concentrations and urinary excretion of homogentisic acid and tyrosine Table 1 Serum and urine reference ranges for tyrosine and homogentisic acid in the individuals (n = 22) recruited into the study and reference ranges quoted in previous studies. Serum [HGA], μmol/L
