Ann Lab Med.  2024 May;44(3):235-244. 10.3343/alm.2023.0178.

Primary Hyperoxaluria Screening and Monitoring: Quantitative Measurement of Plasma Oxalate by Gas Chromatography-Mass Spectrometry With High Sensitivity

• Yazdanpanah M
• Cameron J
• Chappel C
• Yuan L

• KMID: 2555694
• DOI: http://doi.org/10.3343/alm.2023.0178

Abstract

Background
Plasma oxalate measurements can be used for the screening and therapeutic monitoring of primary hyperoxaluria. We developed a gas chromatography-mass spectrometry (GC-MS) assay for plasma oxalate measurements with high sensitivity and suitable testing volumes for pediatric populations.
Methods
Plasma oxalate was extracted, derivatized, and analyzed by GC-MS. We measured the ion at m/z 261.10 to quantify oxalate and the 13 C 2-oxalate ion (m/z: 263.15) as the internal standard. Method validation included determination of the linear range, limit of blank, limit of detection, lower limit of quantification, precision, recovery, carryover, interference, and dilution effect. The cut-off value between primary and non-primary hyperoxaluria in a pediatric population was analyzed.
Results
The detection limit was 0.78 μmol/L, and the linear range was up to 80.0 μmol/L. The between-day precision was 5.7% at 41.3 μmol/L and 13.1% at 1.6 μmol/L. The carry-over was < 0.2%. The recovery rate ranged from 90% to 110%. Interference analysis showed that Hb did not interfere with plasma oxalate quantification, whereas intralipids and bilirubin caused false elevation of oxalate concentrations. A cut-off of 13.9 μmol/L showed 63% specificity and 77% sensitivity, whereas a cut-off of 4.15 μmol/L showed 100% specificity and 20% sensitivity. The minimum required sample volume was 250 μL. The detected oxalate concentrations showed interference from instrument conditioning, sample preparation procedures, medications, and various clinical conditions.
Conclusions
GC-MS is a sensitive assay for quantifying plasma oxalate and is suitable for pediatric patients. Plasma oxalate concentrations should be interpreted in a clinical context.

Keyword

Gas chromatography-mass spectrometry; Inborn errors of metabolism; Oxalate; Pediatrics; Primary hyperoxaluria; Plasma; Sensitivity; Specificity; Validation

Figure

• 

  Fig. 1 Oxalate metabolism and primary hyperoxaluria. 1) Glyoxylate is converted to glycine by AGXT in peroxisomes. In hyperoxaluria type I, a defect in AGXT causes the accumulation of glyoxylate, which is converted to oxalate in the cytoplasm. 2) GRHPR catalyzes the conversions of glyoxylate to glycolate and hydroxypyruvate to D-glycerate in the cytoplasm. In hyperoxaluria type II, a defect in GRHPR leads to elevated oxalate and L-glycerate concentrations. 3) In hyperoxaluria type III, a defect in HOGA1 blocks the conversion of 4-hydroxy-2-ketoglutarate to glyoxylate in mitochondria. Excessive 4-hydroxy-2-ketoglutarate in the cytoplasm leads to elevated oxalate concentrations. Abbreviations: AGXT, alanine-glyoxylate aminotransferase; GRHPR, glyoxylate reductase/hydroxypyruvate reductase; HOGA1, 4-hydroxy-2-oxoglutarate aldolase 1.

• 

  Fig. 2 GC-MS chromatograph of plasma oxalate. The peaks of oxalate (m/z 261.1) and 13C2-oxalate (m/z 263.15) elute at 13.26 mins. The oxalate peak is baseline-resolved from those of other, closely eluting molecules. No interference material co-elutes with oxalate. Abbreviations: GC-MS, gas chromatography-mass spectrometry; TIC, total ion chromatograph.

• 

  Fig. 3 Plasma oxalate linearity analysis. Linearity was assessed by analyzing eight calibrators with oxalate concentrations of 0.0, 1.0, 8.9, 16.8, 32.6, 48.4, 64.2, and 80.0 µmol/L. Each sample was run in triplicate. The ratio of the oxalate to 13C2-oxalate peak areas was calculated for each concentration to plot a linear regression line. The line was visually linear in the range of 0–80 µmol/L. The data points were close to the linear regression lines, with an R2 value of 0.9986.

• 

  Fig. 4 Comparison between GC-MS and enzymatic methods. Method comparison was performed using 12 patient samples with oxalate concentrations between 0 and 80 μmol/L. (A) Linear regression analysis showed that the two sets of measurements were in a linear relationship, with an R2 value of 0.9936. (B) Absolute difference plot and (C) relative difference plot indicating that, for the three lowest concentrations, the enzymatic assay results were significantly lower than those from GC-MS. At concentrations >40 μmol/L, the enzymatic assay results were consistently higher than those from GC-MS, with an average difference of 20%. Abbreviation: GC-MS, gas chromatography-mass spectrometry.

• 

  Fig. 5 Plasma oxalate cut-offs for HP. (A) Boxplot of plasma oxalate concentrations in primary HP (red box) and non-primary HP conditions (green box). The cut-off of 13.9 μmol/L is indicated by the red line. (B) ROC curve, AUC value, specificity, and sensitivity for the cut-off of 13.9 μmol/L. (C) Boxplot of plasma oxalate concentrations with the cut-off of 4.15 μmol/L (indicated by the red line). (D) ROC curve, AUC value, specificity, and sensitivity for the cut-off of 4.15 μmol/L. Abbreviations: GC-MS, gas chromatography-mass spectrometry; HP, primary hyperoxaluria; AUC, area under the ROC curve.

Reference

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