Simultaneous ManNAc and Neu5Ac Quantification in Human Sera by LC-MS/MS

Abstract

N-Acetyl-D-mannosamine (ManNAc) and N-acetylneuraminic acid (Neu5Ac) are important components of glycosylation, affecting numerous physiologic processes. The effects of age, body mass index (BMI), race, or sex on serum levels of ManNAc and Neu5Ac are poorly understood. However, these associations are of substantial interest. Simultaneous quantification of ManNAc and Neu5Ac, using liquid chromatography tandem mass spectrometry (LC-MS/MS), was developed and validated for human serum samples. This method has high sensitivity, specificity, and reproducibility, with limits of detection as low as 1.02 ng/mL for ManNAc or 1.14 ng/mL for Neu5Ac. A set of 155 serum samples from the Adventist Health Study 2 (AHS-2) cohort was analyzed. Concentrations of conjugated Neu5Ac were 35.1 ± 9.4 µg/mL and 33.0 ± 9.5 µg/mL in black and white participants, respectively. Conjugated and total Neu5Ac levels were significantly higher in women, with p-values of 0.029 and 0.026, respectively. The free forms of Neu5Ac were 594 ± 421 ng/mL and 439 ± 168 ng/mL in black and white participants, respectively. Similarly, conjugated and total ManNAc levels were higher in black participants, at 1.81 ± 0.81 µg/mL and 1.90 ± 0.83 µg/mL, compared to 1.32 ± 0.52 µg/mL and 1.41 ± 0.53 µg/mL in white participants (both cases, p < 0.001). Free ManNAc was 93.1 ± 36.2 ng/mL in black and 89 ± 20.2 ng/mL in white participants. Subjects with higher BMI tend to have higher free ManNAc (p = 0.041). Furthermore, older subjects tend to have higher free (p ≤ 0.001) and total (p = 0.045) ManNAc. The improved LC-MS/MS quantification method should facilitate further investigations.