Establishment and evaluation of a method for measuring ornithine transcarbamylase activity in micro blood of neonates

Abstract

Background: Ornithine transcarbamylase deficiency exhibits a high degree of clinical heterogeneity, making its screening and classification challenging in some instances. In this study, we first established a simple and stable method for testing ornithine transcarbamylase activity using micro blood from newborns, rather than relying on venous blood.

Methods: The activity of ornithine transcarbamylase was assessed by measuring the concentration of citrulline produced in the reaction with carbamoyl phosphate and ornithine, using serum, plasma or micro blood. Correlation analysis was evaluated using Sangerbox Tools. The Receiver Operating Characteristic curve was used in SPSS Statistics 17.0 to evaluate the diagnostic efficiency of Ornithine transcarbamylase deficiency.

Results: A strong linear relationship was observed between ornithine transcarbamylase activity and both micro blood volume and reaction time (R² = 0.9793, 0.9922 respectively). The intra-coefficient variation and inter-coefficient variation were 11% and 12.5% with a 1-h reaction time, and 6.77% and 9.58% with a 3-h reaction time, respectively. And the inter-coefficient variation was lower than the most widely used colorimetry method (5.1-21.1%). The Limit of Blank was 0.57 nmol/mL/h. The reference interval for normal newborn population is greater than or equal to 39.6 nmol/mL/h. Notably, the method exhibited a 100% sensitivity, surpassing the sensitivity of colorimetry method (94.3%), along with and a specificity of 96.9% for diagnosing ornithine transcarbamylase deficiency.

Conclusions: We pioneered a method for testing OTC activity that normally carried on venous blood can be effectively performed on microblood heel samples. Meanwhile, our method presents a simpler, more stable and reproducible approach compared to colorimetry.

Keywords: Citrulline; Micro blood; Newborns; Ornithine transcarbamylase activity; Ornithine transcarbamylase deficiency; Tandem mass spectrometry.

Fig. 1

The activity of OTC in serum and plasma. a The concentration of Cit was measured after a 1-h enzyme activity reaction using 20 μL of serum or plasma with or without substrates, *** P < 0.001, two-tailed. b, d The concentration of Cit was measured after a 1-h enzyme activity reaction using varying volumes (10, 20, 30, 40, 50 μL) of serum or plasm. c, e The concentration of Cit was measured at intervals of 1, 6, 18 h following an enzyme activity reaction using 10 μL of serum or plasma

Fig. 2

The activity of OTC in micro blood from neonatal heel. a The concentration of Cit was measured after a 1-h enzyme activity reaction using varying volumes (10, 20, 30, 40, 50 μL) of micro blood from same neonatal heel. b The concentration of Cit was measured at intervals of 1, 6, 18 h following an enzyme activity reaction using 10 μL of micro blood

Fig. 3

Accuracy evaluation of the OTC activity testing system. a The correlation between serum OTC activity and micro blood OTC activity,r = 0.94, P = 1*10^−4, two-tailed, n = 10. b The correlation between plasma OTC activity and micro blood OTC activity,r = 0.75, P = 0.03, two-tailed, n = 10. c The concentration of Cit was measured in in micro blood samples diluted with 0.9% NaCl and ddH₂O, respectively

Fig. 4

Imprecision evaluation of the OTC activity testing system. a The OCT activity detected after 1-h reaction time, n = 20. b The OCT activity detected after 3-h reaction time, n = 20. c The OCT activity detected after 1-h reaction time, repeated 4 times with 20 points each time. d The OCT activity detected after 3-h reaction time, repeated 4 times with 20 points each time

Fig. 5

The frequency histogram of OTC activity in micro blood of normal neonates

Fig. 6

The clinical application of OTC activity testing system. The ROC curve of OTCD in normal newborns; NC = 64, OTCD = 2