Quantitative metabolic profiles of 2nd and 3rd trimester human amniotic fluid using (1)H HR-MAS spectroscopy

Abstract

Object: To establish and compare normative metabolite concentrations in 2nd and 3rd trimester human amniotic fluid samples in an effort to reveal metabolic biomarkers of fetal health and development.

Materials and methods: Twenty-one metabolite concentrations were compared between 2nd (15-27 weeks gestation, N = 23) and 3rd (29-39 weeks gestation, N = 27) trimester amniotic fluid samples using (1)H high resolution magic angle spinning (HR-MAS) spectroscopy. Data were acquired using the electronic reference to access in vivo concentrations method and quantified using a modified semi-parametric quantum estimation algorithm modified for high-resolution ex vivo data.

Results: Sixteen of 21 metabolite concentrations differed significantly between 2nd and 3rd trimester groups. Betaine (0.00846+/-0.00206 mmol/kg vs. 0.0133+/-0.0058 mmol/kg, P < 0.002) and creatinine (0.0124+/-0.0058 mmol/kg vs. 0.247+/-0.011 mmol/kg, P < 0.001) concentrations increased significantly, while glucose (5.96+/-1.66 mmol/kg vs. 2.41+/-1.69 mmol/kg, P < 0.001), citrate (0.740+/-0.217 mmol/kg vs. 0.399+/-0.137 mmol/kg, P < 0.001), pyruvate (0.0659+/-0.0103 mmol/kg vs. 0.0299+/-0.286 mmol/kg, P < 0.001), and numerous amino acid (e.g. alanine, glutamate, isoleucine, leucine, lysine, and valine) concentrations decreased significantly with advancing gestation. A stepwise multiple linear regression model applied to 50 samples showed that gestational age can be accurately predicted using combinations of alanine, glucose and creatinine concentrations.

Conclusion: These results provide key normative data for 2nd and 3rd trimester amniotic fluid metabolite concentrations and provide the foundation for future development of magnetic resonance spectroscopy (MRS) biomarkers to evaluate fetal health and development.

Fig. 1

500 MHz ¹H MR spectra of the same amniotic fluid sample obtained using a a 4 mm HR-MAS probe and b a conventional 5 mm inverse probe. Both samples were 90% aqueous with 10% D₂O added to provide a lock signal. For HR-MAS, the sample was vortexed and an aliquot was analyzed; for the inverse probe, the sample was centrifuged and the supernatant was analyzed

Fig. 2

Representative 500 MHz ¹H HR-MAS spectra of the 0.75–4.25 ppm region of a 2nd and b 3rd trimester human amniotic fluid samples. The 2.95–3.35 ppm region of each spectrum is shown in inset. Note the significant decrease in glucose and alanine and concurrent increase in creatinine and betaine with advancing gestational age

Fig. 3

Example of HR-QUEST fitting of a 3rd trimester amniotic fluid sample. The figure demonstrates the ~3.0–4.2 ppm region of a the measured spectrum, b the modeled spectrum and c the residual spectrum. The modeled spectrum is composed only of metabolites contained in reference spectral database and a baseline correction (dotted line)