Anal. Chem. 2007, 79, 8367-8375
Potential of NMR Spectroscopy for the Study of Human Amniotic Fluid Gonc¸ alo Grac¸ a,† Iola F. Duarte,† Brian J. Goodfellow,† Anto´nio S. Barros,‡ Isabel M. Carreira,§ Ana Bela Couceiro,| Manfred Spraul,⊥ and Ana M. Gil*,†
CICECO and QOPNAA, Department of Chemistry, Campus Universita´ rio de Santiago, University of Aveiro, 3810-193 Aveiro, Portugal, Cytogenetics Laboratory and Center of Neurosciences and Cellular Biology, Faculty of Medicine, University of Coimbra, 3001-401 Coimbra, Portugal, Maternidade Bissaya Barreto, Centro Hospitalar de Coimbra, Coimbra, Portugal, and Bruker Biospin GmbH, Silberstreifen, D76287 Rheinstetten, Germany
1D and 2D 800 MHz high-resolution nuclear magnetic resonance spectroscopy of human amniotic fluid (HAF) enabled the identification of ∼50 metabolites. In addition, liquid chromatography-NMR and diffusion ordered spectroscopy (DOSY) allowed signal overlap to be reduced and the characterization of higher molecular weight (Mw) components, respectively. Indeed, the DOSY spectrum of a Mw >10 kDa HAF fraction enabled three protein families, differing in average Mw, to be detected and may therefore be of potential value in the study of disorderrelated variations in HAF protein profiles. The effects of freeze-drying, storage at -20 or -70 °C, and freezethawing cycles on HAF compositional stability were investigated, as well as stability at room temperature (to account for overnight data acquisition runs). These data are the basis for establishing statistically validated correlations between HAF NMR data and any physiological disorders of the fetus/mother. Freeze-drying caused signal loss for urea, ethanol, and compounds resonating at 2.22 and 1.17 ppm. Storage at -70 °C or lower is recommended since only minor compositional changes were observed, affecting mainly acetate and pyruvate. Freezethaw cycles did not cause significant compositional changes, and room-temperature stability studies indicated a 4-5 h maximum period of handling/acquisition time to ensure HAF stability. The study of human body fluids by nuclear magnetic resonance (NMR) spectroscopy has been recognized, for many years now, as a very powerful tool for the monitoring of drug toxicity effects and disease,1-4 with a number of recent reviews appearing in the * Corresponding author. Tel: +351 234 370707. Fax: +351 234 370084. E-mail:
[email protected]. † CICECO, Department of Chemistry, University of Aveiro. ‡ QOPNAA, Department of Chemistry, University of Aveiro. § University of Coimbra. | Centro Hospitalar de Coimbra. ⊥ Bruker Biospin GmbH. (1) Waters, N. J.; Waterfield, C. J.; Farrant, R. D.; Holmes, E.; Nicholson, J. K. Chem. Res. Toxicol. 2005, 18, 639-654. (2) Azmi, J.; Griffin, J. L.; Shore, R. F.; Holmes, E.; Nicholson, J. K. Xenobiotica 2005, 35, 839-852. (3) Solanky, K. S.; Bailey, N. J.; Beckwith-Hall, B. M.; Bingham, S.; Davis, A.; Holmes, E.; Nicholson, J. K.; Cassidy, A. J. Nutr. Biochem. 2005, 16, 236244. 10.1021/ac071278d CCC: $37.00 Published on Web 10/06/2007
© 2007 American Chemical Society
literature.5,6 Generally, NMR spectroscopy, in tandem with increasingly sophisticated data analysis methods, may successfully detect and study any type of disorder occurring in a living organism that may arise from a variety of effects, e.g., medication, disease, exposure to toxicants, or intake of certain foods. In this respect, urine and blood plasma or serum have been the most extensively studied biofluids, followed by bile and other biofluids. Human amniotic fluid (HAF) has also been previously studied due to its rich low Mw metabolite composition, which allows a number of biomarkers, either of the normal development of the fetus or of the onset and evolution of disorders affecting the fetus’s or mother’s health to be detected.7 Some of the earliest NMR studies of amniotic fluid involved the use of 31P NMR for the analysis of phospholipids.8-10 However, the larger wealth of information obtained from 1H NMR spectra has justified the increasing number of 1H NMR studies reported over the last 15 years. Initial 1H NMR studies, conducted for healthy subjects, involved the identification of a number of low Mw compounds and their quantification by comparison with the signal of an added standard.11,12 A parallel study emphasized the importance of NMR spectroscopy of HAF as an aid for the eventual routine clinical use of magnetic resonance (MR) methods for monitoring pregnancies.13 However, such MR methods have not evolved as fast as other MR applications due to some concern regarding possible effects of the radio frequency field and other characteristics of the examination on the fetus and mother. The same study describes the assignment of the main organic acids (lactate, (4) Duarte, I. F.; Goodfellow, B. J.; Barros, A.; Jones, J. G.; Barosa, C.; Diogo, L.; Garcia, P.; Gil, A. M. NMR Biomed. 2007, 20, 401-412. (5) Lindon, J. C.; Nicholson, J. K.; Holmes, E.; Everett, J. R. Concepts Magn. Reson. 2000, 12, 289-320. (6) Bollard, M. E.; Stanley, E. G.; Lindon, J. C.; Nicholson, J. K.; Holmes, E. NMR Biomed. 2005, 18, 143-162. (7) Smith, I. C.; Blandford, D. E. Anal. Chem. 1995, 67, 509-518. (8) Pearce, J. M.; Shifman, M. A.; Pappas, A. A.; Komoroski, R. A. Magn. Reson. Med. 1991, 21, 107-116. (9) Pearce, J. M.; Komoroski, R. A. Magn. Reson. Med. 1993, 29, 724-731. (10) Pearce, J. M.; Krone, J. T.; Pappas, A. A.; Komoroski, R. A. Magn. Reson. Med. 1993, 30, 476-484. (11) Nelson, T. R.; Gillies, R. J.; Powell, D. A.; Schrader, M. C.; Manchester, D. K.; Pretorius, D. H. Prenatal Diagn. 1987, 7, 363-372. (12) Sims, C. J.; Fujito, D. T.; Burholt, D. R.; Dadok, J.; Giles, H. R.; Wilkinson, D. A. Prenatal Diagn. 1993, 13, 473-480. (13) McGowan, P. E.; Reglinski, J.; Wilson, R.; Walker, J. J.; Wisdoms, S.; McKillop, J. H. J. Pharm. Biomed. Anal. 1993, 11, 629-632.
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acetate, citrate, formate), amino acids (valine, alanine, histidine), glucose, and indoxyl sulfate found in HAF and their quantitation by standard addition of acetate and alanine. The selection of possible indicators for renal function (indoxyl sulfate, histidine, formate) and diabetes (glucose) is discussed. A first NMR metabonomic study of HAF from women suffering from selected disorders addressed the effects of preeclampsia, spina bifida, gestational diabetes, and fetal trisomy 21.14 Although significant correlations were only found for preeclampsia and spina bifida, due to the limited number of samples for the remaining disorders, this work also addressed important points such as integration methods for quantification (use of added reference vs of internal reference), problems of overlap with the protein envelope, and the effects of fetal maturity and gestational age on HAF composition, the latter having been further investigated in a later report.15 In addition, a study attempting to correlate HAF 1H NMR spectra with the incidence of cystic fibrosis, decreased enzyme activities, and trisomy 2116 has noted the apparent low dependence of NMR spectra with such disorders. Other work has included a study of pregnant women suffering from insulin-dependent diabetes mellitus in their third trimester, indicating glucose and lactate as possible indicators,17 a study of HAF from spina bifida fetuses, which showed higher contents of succinic acid and glutamine,18 and, more recently, NMR detection of an increase in choline in HAF taken as a possible indicator of fetal lung maturity.19 In the latter study, the tandem use of NMR spectroscopy and MR spectroscopy in vivo measurements suggested MR as a potential noninvasive method for detection of lung maturity. The present work aims at showing how presently available state-of-the-art NMR methods, ranging from one- and twodimensional experiments to methods such as diffusion ordered spectroscopy (DOSY) and hyphenated NMR allow qualitative metabolite detection in HAF. A second matter addressed is that of HAF compositional stability over freeze-drying, storage, freezethaw cycles, and data acquisition (at room temperature). The detection of the compositional changes occurring during these processes and during sample handling procedures is the basis for enabling statistically validated variations to be obtained and correlated to specific health disorders of the fetus, the mother, or both. Once the conditions for sample handling/storage and method robustness are established, NMR can be used to achieve a rapid compositional profile evaluation of HAF and monitor the health of fetus or mother during a variety of processes (e.g., disease, medication). Similar studies relating to sample handling have been, very recently, carried out for human urine20 and blood serum and plasma.21 (14) Bock, J. L. Clin. Chem. 1994, 40, 56-61. (15) Sims, C. J.; Fujito, D. T.; Burholt, D. R.; Dadok, J.; Wilkinson, D. A. J. Matern>-Fetal Invest. 1996, 6, 62-66. (16) Le-Moyec, L.; Muller, F.; Eugene, M.; Spraul, M. Clin. Biochem. 1994, 27, 475-483. (17) McGowan, P. E.; Lawrie, W. C.; Reglinski, J.; Spickett, C. M.; Wilson, R.; Walker, J. J.; Wisdom, S.; Maclean, M. A. J. Perinatal Med. 1999, 27, 404408. (18) Groenen, P. W.; Engelkec, U. F.; Weversc, R. A.; Hendriksa, J. M.; Eskesb, T. B.; Merkusb, H. M.; Steegers-Theunissena, R. M. Eur. J. Obstetr. Gynecol. Rep. Biol. 2004, 112, 16-23. (19) Clifton, M. S.; Joe, B. N.; Zektzer, A. S.; Kurhanewicz, J, Vigneron, D. B.; Coakley, F. V.; Nobuhara, K. K.; Swanson, M. G. J. Pediatr. Surg. 2006, 41, 768-773.
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EXPERIMENTAL SECTION Samples. Amniotic fluid samples were obtained from 16 healthy pregnant women (16-22 weeks of gestation) with maternal age over 35. The use of the samples for research was carried out under ethical committee approval. After collection of 15-20 mL of HAF, the samples were centrifuged (177.4g, 22 °C, 10 min) and the supernatants collected and frozen until NMR analysis. Storage temperatures of -20 or -70 °C were used. For NMR analysis at 500 MHz, samples were thawed at room temperature and 100 µL of 0.5 M sodium phosphate buffer pH 7.2 (in D2O) were added to 500 µL of HAF sample (final pH was 7.27 ( 0.07 for all samples). After centrifugation (13400g, 25 °C, 5 min), 550 µL of this solution was transferred into a 5 mm NMR tube. For NMR analysis at 800 MHz, 1 mL of HAF was freeze-dried overnight and reconstituted into 720 µL of D2O and 80 µL of 0.24% 3-(trimethylsilyl)propionate sodium salt D2O solution. No buffering was employed, and the resulting pH was 9.0. For comparison, a second aliquot of the same HAF sample stored at -20 °C (for some days) without freeze-drying was also prepared. The 550 µL aliquots of these solutions were transferred into 5 mm NMR tubes after centrifugation. HAF samples differing in the average Mw of their components were prepared by size-selective ultrafiltration using Amicon 3 kDa and 10 kDa cutoff membrane centrifuge filters. After several washes with 0.5 M sodium phosphate buffer pH 7.2, Mw
