Thomas Lynn (Presenter)
Quest Diagnostics, Inc. - Nichols Institute
Authorship: Thomas C. Lynn, Denise Salazar, Ph.D., Nigel J. Clarke, Ph.D.
Quest Diagnostics, Inc.-Nichols Institute
| Short Abstract The cerebral creatine deficiency syndromes (CCDS), inborn errors of creatine metabolism, include the two creatine biosynthesis disorders GAMT deficiency and AGAT deficiency), and the X-linked creatine transporter [SLC6A8] deficiency. Specimens were prepared by diluting urine with ultrapure water. A minimum sample volume of 100 µL was used. The diluted sample mix was injected onto an Agilent 1200 Series HPLC system using a reverse-phase column. Analysis was performed by positive electrospray ionization using an Agilent 6410 triple quadrupole mass spectrometer. Measurements of these three analytes in urine allow for the biochemical diagnosis of CCDS. The ability to measure all three analytes directly in the urine, with a very simple method of sample preparation, offers advantages over previous methods involving derivatization and indirect calculations. |
Long Abstract
Background: Energy metabolism in the muscle and brain is modulated by the creatine–creatine phosphate system. Creatine biosynthesis, in the liver, pancreas and kidneys, is dependent upon 2 enzymes: L-arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT). Three human disorders characterized by deficiencies of AGAT, GAMT, and the X-linked creatine transporter (SLC6A8) cause secondary brain creatine depletion. These cerebral creatine deficiency syndromes (CCDS) begin between ages 3 months and 3 years. They are underdiagnosed because the presenting clinical features (eg, psychomotor delay, failure to thrive, intellectual disability, seizures, autistic behaviors, spasticity, and movement abnormalities) are non-specific. Brain creatine content is corrected with oral supplementation of creatine in AGAT and GAMT. The diagnosis is dependent upon the measurement of guanidinoacetate (GAA), creatine, and creatinine in urine and plasma. Elevated or decreased concentrations of these individual analytes in urine or plasma can differentiate the 3 diseases.
We developed an LC-MS/MS electrospray ionization assay that detects and directly quantitates GAA, creatine, and creatinine in urine; the assay offers advantages over previous methods that measure creatine indirectly and require derivatization of the analytes. Here we assessed the consistency and quantitation limits of the assay.
Methods: Specimens were prepared by diluting urine 1:50 fold with ultrapure water. A minimum specimen volume of 100 µL was used. Following dilution, specimens were spiked with an internal standard mixture and mixed. The diluted specimen mix was injected onto an Agilent 1200 Series HPLC system using a reverse-phase column. Analysis was performed by positive electrospray ionization using an Agilent 6410 triple quadrupole mass spectrometer. The run time was 10 minutes.
Results: The calibration curves showed consistency in reproducibility and linearity. Inter-assay coefficients of variation were 8.1 to 4.7% at 25-500 mg/L for guanidinoacetate, 9.9 to 6.1% at 25-500 mg/L for creatine, and 1.8 to 4.1% at 120-2300 mg/L for creatinine. The method provides linear results over a range of 0.4-2500 mg/L for guanidinoacetate and creatine and 0.8-5000 mg/L for creatinine. The lower limits of quantitation were 0.3 mg/L for guanidinoacetate and creatinine and 0.4 mg/L for creatine.
Conclusions: Our LC-MS/MS electrospray ionization assay was able to directly and consistently detect and quantitate GAA, creatine, and creatinine in human urine. Testing young children presenting with non-specific neurological findings with this rapid and reliable method for diagnosing CCDS may identify a treatable cause of developmental delay.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Quest Diagnostics, salaried employee |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |