1-13C-propionate breath testing as a surrogate endpoint to assess efficacy of liver-directed therapies in methylmalonic acidemia (MMA)

Abstract

Purpose: To develop a safe and noninvasive in vivo assay of hepatic propionate oxidative capacity.

Methods: A modified 1-¹³C-propionate breath test was administered to 57 methylmalonic acidemia (MMA) subjects, including 19 transplant recipients, and 16 healthy volunteers. Isotopomer enrichment (¹³CO₂/¹²CO₂) was measured in exhaled breath after an enteral bolus of sodium-1-¹³C-propionate, and normalized for CO₂ production. 1-¹³C-propionate oxidation was then correlated with clinical, laboratory, and imaging parameters collected via a dedicated natural history protocol.

Results: Lower propionate oxidation was observed in patients with the severe mut⁰ and cblB subtypes of MMA, but was near normal in those with the cblA and mut^- forms of the disorder. Liver transplant recipients demonstrated complete restoration of 1-¹³C-propionate oxidation to control levels. 1-¹³C-propionate oxidation correlated with cognitive test result, growth indices, bone mineral density, renal function, and serum biomarkers. Test repeatability was robust in controls and in MMA subjects (mean coefficient of variation 6.9% and 12.8%, respectively), despite widely variable serum methylmalonic acid concentrations in the patients.

Conclusion: Propionate oxidative capacity, as measured with 1-¹³C-propionate breath testing, predicts disease severity and clinical outcomes, and could be used to assess the therapeutic effects of liver-targeted genomic therapies for MMA and related disorders of propionate metabolism.

Trial registration: This clinical study is registered in www.clinicaltrials.gov with the ID: NCT00078078. Study URL: http://clinicaltrials.gov/ct2/show/NCT00078078.

Fig. 1. Method development, reproducibility, and test performance.

(a) Studies in a single healthy adult volunteer showed similar test performance using 2- and 20-fold reduced isotopomer dose. The amount of the administered dose of 1-¹³C-sodium propionate oxidized after 2 hours was 48.8% with the 9.7 mg/kg dose (blue line), 56.03% with a 5 mg/kg, and 55.91% with a 0.5 mg/kg dose (red line). When predicted CO₂ rather than the VCO₂ measured by the metabolic cart was used for calculations with the 0.5 mg/kg dose, result were identical up to the 60-minute timepoint. (b) Healthy adult controls (n = 8, red) and heterozygote parents (n = 8, blue) volunteered for the study. At 120 minutes, controls metabolized 47.9 ± 4.26% and heterozygotes 41.6 ± 5.05% of the administered 1-¹³C-propionate dose (P = 0.01). (c) Enrichment of ¹³CO₂/¹²CO₂ in the exhaled air collected in frequent intervals in the first 15 minutes of the study showed a similarly rapid propionate oxidation in both, controls and heterozygote subjects. (d) Healthy adult volunteers (n = 7, labeled 1–7) repeated the study three times each (scatter dot plot, mean ± SD for each participant). (e) Cumulative dose oxidized at 120 minutes is depicted for repeat 1-¹³C-propionate breath tests in 15 subjects with mut subtype methylmalonic acidemia (MMA) (scatter dot plot, mean ± SD). Patient numbers (#) correlate to the IDs in Table 1 and are ranked from high to low 1-¹³C-propionate oxidation capacity. Gray-shaded band in (d, e) corresponds to the mean ± SD of healthy controls. (f) 1-¹³C-propionate breath test showed near identical propionate oxidation in selected subjects with significantly different serum methylmalonic acid concentrations at the time of testing. (g) Serum methylmalonic acid concentrations (μmol/L, mean ± SD is shown for each subject) corresponding to the subjects depicted in (e). Patients with higher oxidative capacity had lower serum methylmalonic acid concentrations. (h) Serum methylmalonic acid means (y-axis) and standard deviations (x-axis) are depicted for 57 subjects with MMA, who had more than one methylmalonic acid measurement during their week-long stay at the National Institutes of Health (NIH) Clinical Research Center (CRC). (i) A Bland–Altman plot shows near perfect agreement between the two methods employed for measuring the delta over baseline ¹³CO₂/¹²CO₂ enrichment: isotope ratio mass spectrometry (IRMS) and the Exalenz BreathID® device. The plot depicts the differences between the two techniques on the y-axis against the average of the two methods on the x-axis (****P < 0.0001. ***P < 0.001, **P < 0.01, *P < 0.05).

Fig. 2. 1-¹³C-propionate oxidation in methylmalonic acidemia (MMA) patients: correlations with subtype severity and response after organ transplantation.

(a) The cumulative percent dose of administered sodium 1-¹³C-propionate metabolized by 120 minutes is depicted for controls (n = 8) and MMA subjects, per disease subtype (mut⁰ n = 26, mut^– n = 6, cblB n = 2, cblA n = 9). The severe mut⁰ patients (marked in red) had a range of oxidative capacity with a mean ± SD of 21.77 ± 9.50% at 120 minutes (P < 0.0001 compared with the controls 47.96 ± 4.26% and cblA 47.75 ± 5.91%; P = 0.0002 compared with mut^– individuals, 42.88 ± 9.02%, and P = NS to cblB 31.78 ± 21.82%). (b) The delta over baseline ¹³C enrichment in exhaled breath samples is depicted in controls and MMA subjects per subtype. A slower time to maximum enrichment was observed in mut⁰ subjects, 46.59 ± 18.25 minutes, as opposed to 22.5 ± 11.81 in controls, 23.18 ± 6.8 in cblA patients, and 32.50 ± 11.65 in mut^–. Asterisks correspond to P values compared with controls for each time point. (c) A receiver operating curve (ROC) is depicted for the cumulative dose oxidized in 120 minutes between controls and mut⁰ (area under the curve [AUC]= 1, P < 0.0001) as well as between mut⁰ and mut^– (AUC: 0.9345 ± 0.0655, P = 0.001). Method sensitivity between controls and mut^– patients was low (AUC: 0.687, P = NS). (d) Three patients were tested before and after combined liver/kidney transplant (LT/LKT), and three before and after kidney transplant (KT). One subject was tested before and after a second KT procedure after failure of the first kidney graft. On average pre- and post-transplant values were significant even for the kidney transplant recipients. (e, f) Cumulative percent dose metabolized and enrichment curves are depicted for mut⁰ patients without and with a LT/LKT transplant (marked in red and blue, respectively) or isolated KT procedure. LT/LKT but not KT resulted in complete restoration of propionate oxidation to control values. (g) 1-¹³C-propionate oxidation breath test result in controls and MMA patients per subtype and transplant status are depicted as areas under the curve, for patients with more than one test, the average value is represented (scatter plot of individual values, boxes and error bars represent mean ± SD). (h) An adult with cblA defect unable to comply with his hydroxocobalamin intramuscular injections was tested before and 2 months after B₁₂ therapy, showing improved oxidation. (****P < 0.0001. ***P < 0.001, **P < 0.01, *P < 0.05).

Fig. 3. 1-¹³C-propionate oxidation in MMUT methylmalonic acidemia (MMA) patients: correlations with clinical and biochemical parameters.

The lowest 1-¹³C-propionate oxidation rates were mostly observed in mut MMA patients, who harbored two loss-of-function (LOF) variants in MMUT. Comparisons reached significance for the area under the curve at 120 minutes (adjusted P = 0.0154), but with significant overlap with the compound heterozygous carrying LOF/missense or two missense variants. (b) 1-¹³C-propionate oxidation rates showed a positive correlation with renal function, with higher propionate oxidation subjects having a near normal estimated glomerular filtration rate (eGFR) (mL/min/1.73 m²) calculated with the combined equation using creatinine and cystatin C values (correlation coefficient r = 0.436, R² = 0.1909, P = 0.0098). (c) Age of onset for stage 3 chronic kidney disease (CKD) (eGFR based on the bedside Schwartz equation ≤60 mL/min/1.73m²) was censored in a Kaplan–Meier curve for mut⁰ MMA patients stratified by their 1-¹³C-propionate oxidation rate. Fifty percent of patients with the lowest (<10%) propionate oxidation reached stage 3 CKD at age 8.3 years, as opposed to 15 years in patients with oxidation >20% (marked in red and blue, respectively, P = 0.0574). (d, e) Patients with the lowest 1-¹³C-propionate oxidation (<10%, red) had more severe intellectual impairment, based on standardized age-appropriate neurocognitive evaluations (full-scale IQ [FSIQ]) compared with patients with >20% oxidation rates (adjusted P = 0.0325, blue). Bivariate correlation coefficient r = 0.455, R² = 0.2078, P = 0.0129. (f–i) Significant correlations are shown between 1-¹³C-propionate oxidation rates and clinical disease-specific serum biomarkers, including serum methylmalonic acid (r = −0.739, R² = 0.547, P < 0.0001, log transformed values were used for skewed variables); acyl/free carnitine ratio (r = −0.584, R² = 0.341, P < 0.0004), as well as biomarkers of hepatic or multisystem mitochondrial dysfunction (FGF21, fibroblast growth factor 21: r = −0.486, R² = 0.237, P < 0.0064 and GDF15, growth differentiation factor 15: r = −0.664, R² = 0.4418, P < 0.0001).