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Keywords: Diagnostics; Enzyme replacement therapy; Metabolic disorder

G.A. Diaz 1; A. Schulze 2; M.C. McNutt 3; E.L. Teles 4; J.L. Merritt II 5; G.M. Enns 6; S.P. Batzios 7; R.L. Conway 8; S. Ferrati 9; S. Rowlinson 9; S. Alters 9; J.E. Wooldridge 9; R.T. Zori 10

1) Icahn School of Medicine at Mount Sinai, New York, NY; 2) University of Toronto and The Hospital for Sick Children, Toronto, ON, Canada; 3) UT Southwestern Medical Center, Dallas, TX; 4) Centro Hospitalar de São João, Porto, Portugal; 5) University of Washington, Seattle, WA; 6) Stanford University, Stanford, CA; 7) Great Ormond Street Hospital NHS Trust, London, UK; 8) Children’s Hospital of Michigan, Detroit, MI; 9) Aeglea BioTherapeutics, Austin, TX; 10) University of Florida, Gainesville, FL

Arginine and guanidino compounds (GCs) accumulate in the plasma of patients with arginase 1 deficiency (ARG1-D) and may contribute to the neurologic phenotype of the disease. Pegzilarginase (PZA) treatment has been shown to sustainably reduce arginine levels in a phase 1/2 study of patients with ARG1-D. To date, determination of GC levels has been challenging due to inadequately sensitive methodologies. We assessed plasma GC levels in patients with ARG1-D treated with PZA using a novel liquid chromatography-tandem mass spectrometric (LC-MS/MS) method.

Blood samples for GC (α-keto-δ-guanidinovaleric acid [GVA]; Nα-acetyl-L-arginine [NAArg]; R,S-argininic acid [ArgA]; guanidinoacetic acid [GAA]) analyses were collected from patients with ARG1-D receiving up to 7 single ascending doses of PZA every 2 weeks followed by a weekly repeat dose for up to 8 weeks before entering into a long-term extension study . Samples were collected in a tube containing dipotassium ethylenediaminetetraacetic acid (K2EDTA), Nω-hydroxy-nor-arginine (nor-NOHA) and mannitol, and processed to plasma. Various concentrations of GCs were added to calibration standards prepared in a surrogate matrix, and quality control samples were prepared in pooled K2EDTA human plasma containing nor-NOHA, PZA, and mannitol. Fifty microliters of plasma were treated with 100 µL of 1N hydrochloric acid containing internal standards, followed by 400 µL of ice-cold 10% trichloroacetic acid. Supernatant derived from vortex-mix and centrifugation was analyzed by LC-MS/MS using Shimadzu Nexera UHPLC-Applied Biosystems/MDS Sciex API 5500.

Sixteen patients were recruited for the study; 69.0% were females and median age (range) was 15 (5–31) years. Baseline mean GVA, NAArg, ArgA, and GAA levels (standard deviation) were 5.1 (1.67), 1.1 (0.48), 2.8 (1.21), and 3.1 (1.11) µmol/L, respectively, which decreased to 2.7 (1.26), 0.7 (0.46), 1.8 (0.87), and 2.0 (1.21) µmol/L, respectively, 2 weeks after the last repeat dose. The concentration range for validation of this method was 0.03–10 µM for GVA, NAArg, and ArgA, and 0.300–100 µM for GAA.

Treatment with PZA was associated with marked reduction in plasma GC levels in patients with ARG1-D. Further studies are needed to confirm the relationship between GC levels and patients’ outcomes. This LC-MS/MS method was effective in measuring GCs in this patient group and could represent a useful tool in conditions with altered GC levels.