Analysis of Globotriaosylceramide (Gb<sub>3</sub>) in Liquid Urine: A Straightforward Assay Using Tandem Mass Spectrometry
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Bibliographic record
Abstract
Abstract Fabry disease (FD) is a lysosomal storage disorder caused by variants in the GLA gene encoding α‐galactosidase A, an enzyme required for catabolism of globotriaosylceramide (Gb 3 ). Accumulation of Gb 3 in patients’ cells, tissues, and biological fluids causes clinical manifestations including ventricular hypertrophy, renal insufficiency, and strokes. This protocol describes a methodology to analyze urinary Gb 3 and creatinine. Samples are diluted with an internal standard solution containing Gb 3 (C17:0) and creatinine‐D 3 , centrifuged, and directly analyzed by ultra‐high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC‐MS/MS) using an 8.7‐min method. Eight Gb 3 isoforms [C16:0, C18:0, C20:0, C22:1, C22:0, C24:1, C24:0, and (C24:0)OH] are analyzed and the total is normalized to creatinine. Confirmation ions are monitored to detect potential interferences. The Gb 3 limit of quantification is 0.023 µg/ml. Its interday coefficients of variation (3 concentrations measured) are ≤15.4%. This method minimizes matrix effects (≤6.5%) and prevents adsorption or precipitation of Gb 3 . Urine samples are stable (bias <15%) for 2 days at 21°C, 7 days at 4°C, and 4 freeze/thaw cycles, whereas prepared samples are stable for 5 days at 21°C, and 14 days at 4°C. The Gb 3 /creatinine age‐related upper reference limits (mean + 2 standard deviations) are 29 mg/mol creatinine (<7 years) and 14 mg/mol creatinine (≥7 years). This simple, robust protocol has been fully validated (ISO 15189) and provides a valuable tool for diagnosis and monitoring of FD patients. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol : Analysis of urinary globotriaosylceramide (Gb 3 ) and creatinine by UHPLC‐MS/MS Support Protocol 1 : Preparation of the urinary quality controls Support Protocol 2 : Preparation of the urine matrix used for the Gb 3 calibration curve Support Protocol 3 : Preparation of the Gb 3 calibrators Support Protocol 4 : Preparation of the working solution containing the internal standards Support Protocol 5 : Preparation of the creatinine calibrators Support Protocol 6 : Preparation of the UHPLC solutions and mobile phases
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Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.002 | 0.006 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from it