1. IN VIVO MONITORING OF UREA CYCLE
WITH ORALLY ADMINISTERED 13C – SODIUM ACETATE
Zangerl K.1, Opladen T.1, Lindner M.1, Marquardt T.2, Böhm T.5, Rosenkranz B.6, Das A.M.3, Mayorandan S.3, Yudkoff M.4, Hoffmann G.F.1
1 Division of Metabolic Disorders, University Children‘s Hospital Heidelberg, Germany
2 Division of Metabolic Disorders, University Children‘s Hospital Münster, Germany
3 Division of Metabolic Disorders, Medical School Hannover, Germany
4 Division of Child Development and Metabolic Disease, Children's Hospital of Philadelphia, USA
5Cytonet GmbH & Co. KG, Albert-Ludwig-Grimm-Str. 20, Weinheim, Germany
6Division of Pharmacology, Faculty of Health Sciences, Department of Medicine, Stellenbosch University, South Africa
Inclusion criteria:
1. Healthy subjects age yrs., with no known metabolic disease
2. Genetically confirmed UCD symptomatic patients (OTC-D, CPS1-D, ASS-D, ASL-D) 0-65 yrs., with at least 1 metabolic decompensation and with clinical signs of hyperammonaemia in their medical history or genetically confirmed and prospectively treated siblings of symptomatic patients, even without clinical symptoms.
3. Asymptomatic mutation carriers: 0-65 yrs., asymptomatic carriers of UCD mutations, no dietary protein restriction, no intake of ammonia scavenging drugs, no known metabolic decompensation with clinical signs of hyperammonaemia.
Background:
Urea cycle disorders can present neonatally with severe hyperammonaemia and a high case fatality rate or as a late-onset, milder phenotype after the newborn period with variable outcome. Long-term prognosis depends on the residual activity of the defective enzyme. An easy and reliable in vivo method to estimate urea cycle activity does not exist yet. Clear correlations between genotype and enzymatic function on the one side and clinical phenotype on the other do not seem to exist. The aim of the study is the evaluation of oral (1,2) 13C-sodium acetate loading for assessment of urea cycle function (Figure 1).
(1,2)13C-Sodium Acetate
p.o.
Methods:
After a standard breakfast and 2-4 hours fasting time (age-dependent) (1,2) 13C double – labelled sodium acetate was applied orally in a single dose (55 mg/kg BW). 13C plasma urea, 13C plasma bicarbonate and 13CO2 in exhalation air were measured at points (depending on weight) for over a period of minutes (Figure 2) Samples were analysed with Isotope Ratio Mass Spectroscopy (=IRMS) by Dr. Aygen and his team at INFAI GmbH, Cologne, Germany.
TCA Cycle
NaH13CO3
13C-Urea
Application of (1,2) 13C sodium acetate
Before 13C testing
Physical examination
Safety blood sampling and establishment of peripheral i.v. line
Standard breakfast
Fasting period 2 – 4h+
Assessment of past medical history
Time (min) *
* Additional blood sampling time points for children >10kg
+ Fasting period for children <5yrs = 2hrs, for children ≥5yrs = 4hrs
Points of taking samples of 13C plasma urea, 13C plasma bicarbonate and 13CO2 in exhalation air
Figure 1 Integration and elimination of 1,2 13C sodium acetate in urea cycle.
Figure 2 Protocol of 13C test in patients, carriers and healthy subjects.
Results:
So far, the method and the production of 13C urea in plasma was examined in 10 healthy controls (mean age 39.9 yrs, range yrs), 13 patients with urea cycle disorders (mean age 11.8 yrs, range 2-33 yrs) and 9 asymptomatic mutation carriers (mean age 33.6 yrs, range 4-45 yrs). In all examined subjects an increase of 13C urea over the time was observed. The increase was highest in healthy controls and carriers, while 13C plasma urea concentration of patients remained at lower concentrations. As shown in Figure 3, 13C plasma urea values overlap substantially. Figure 4 shows individual graphs for the 13 UCD patients. Patients with neonatal onset citrullinemia show very low 13C urea in plasma. Individuals with OTC-D constitute a heterogeneous group that is distinguishable from patients with neonatal onset ASS-D. As expected, they show considerable variability. For instance, the lowest concentration in this group was found in a boy with neonatal onset type OTC-D (see Pat 1), who had a neonatal decompensation requiring dialysis. He has a hemizygous point mutation with very low residual enzyme activity. The second boy (see Pat 2), with considerable higher 13C urea concentration was found at neonatal age due to feeding problems. Hyperammonemia (350 µmol/l) was found, no dialysis was required. During clinical follow-up this patient remained stable under conservative therapy pointing to benign disease course. A single patient with ASL-D (detected by newborn screening with no history of metabolic decompensations but with developmental delay) revealed 13C plasma urea concentrations within the range of healthy controls.
(Pat 2)
(Pat 1)
(Pat 2)
(Pat 1)
Figure 4 Single graphs of 6 patients with OTC-D, 5 with ASS-D and 1 with ASL-D.
(f=female, m=male, lo=late onset, no=neonatal onset)
Figure 3 Means and min/max of patients, carriers and healthy controls.
Conclusion:
Preliminary results suggest that in vivo monitoring of urea cycle function with (1,2) 13C-labelled sodium acetate is possible and safe. Data may differentiate between healthy controls, carriers and patients, but overlap substantially. Single graph analysis reveals a clustering of different types of UCD patients. It remains to be determined whether the 13C urea assay is sensitive enough to distinguish precisely between patient groups or to predict the clinical course. Correlation with clinical data will follow.
Contact data
Kathrin Zangerl, M.D.
University Children´s Hospital Heidelberg
Literature
Tuchman, M. et al., N-carbamylglutamate markedly enhances ureagenesis in N-acetylglutamate deficiency and propionic acidemia as measured by isotopic incorporation and blood biomarkers
Pediatr Res, vol.64, no.2, p , 2008
Yudkoff, M. et al., Measuring in vivo ureagenesis with stable isotopes, MGM, S37-S41, 2010
Conflict of interest: The study was performed as a sponsor initiated trial, financially supported by Cytonet GmbH & Co KG. K. Zangerl and T. Opladen received travel support.