A different approach to the quantification of human seminal plasma metabolites using high-resolution NMR spectroscopy.

In this study, a reliable method was established for the absolute quantification of metabolite concentrations in human seminal plasma using ERETIC2, a quantification tool developed by Bruker based on the PULCON principle. The performance of the ERETIC2 was examined using an AVANCE III HD NMR spectrometer (600 MHz) equipped with a triple inverse 1.7 mm TXI probe in terms of some experimental parameters that may affect the accuracy and precision of the quantitative results. Then, the accuracy, precision, and repeatibility of ERETIC2 were determined using L-asparagine solutions at different concentrations. And it was evaluated by comparing it with the classical internal standard (IS) quantification method. The relative standard deviation (RSD) values for ERETIC2 were calculated in the range of 0.55-1.90% and the minimum recovery value was 99.9%, while the RSD values for the IS method were calculated in the range of 0.88-5.83% and recovery value was minimum 91.0%. Besides, the RSD values of the inter-day precisions for the ERETIC2 and IS methods were obtained to be in the range of 1.25 - 3.03% and 0.97 - 3.46%, respectively. Finally, the concentration values of seminal plasma metabolites were determined using different pulse programs with both methods for samples obtained from normozoospermic control and azoospermic patient groups. The results proved that this quantification method developed using NMR spectroscopy is easy to use in complex sample systems such as biological fluids and is a good alternative to the classical internal standard method in terms of accuracy and sensitivity. In addition, the improvement of the spectral resolution and sensitivity with the microcoil probe technology and the possibility of analyzing with minimum sample quantities has contributed positively to the results of this method.

Metabolomic analysis of endometrial cancer by high-resolution magic angle spinning NMR spectroscopy.

PURPOSE: To analyze endometrial metabolite profiles between patients with endometrial cancer and controls. METHODS: Seventeen (17) women with endometrium cancer and 18 controls were enrolled in this study. 1H HR-MAS (High Resolution-Magic Angle Spinning) NMR (Nuclear Magnetic Resonance) spectroscopy data obtained from endometrial tissue samples of patients with endometrial cancer and control group were analyzed with bioinformatics methods. RESULTS: Principal component analysis (PCA) and the partial least squares discriminant analysis (PLS-DA) score plots obtained with the multivariate statistical analysis of pre-processed spectral data shows a separation between the samples from patients with endometrial cancer and controls. Analysis results suggest that the levels of lactate, glucose, o-phosphoethanolamine, choline, glycerophosphocholine, phosphocholine, leucine, isoleucine, valine, glutamate, glutamine, n-acetyltyrosine, methionine, taurine, alanine, aspartate and phenylalanine are increased in patients with endometrial cancer compared to the controls. CONCLUSION: The metabolomics signature of patients with endometrial cancer is different from that of benign endometrial tissue.

Metabolomics analysis of placental tissue obtained from patients with fetal growth restriction.

AIM: The aim of this study was to determine whether there was a difference in placental metabolite profiles between patients with fetal growth restriction (FGR) and healthy controls. METHODS: The study included 10 patients with FGR diagnosis with 14 healthy controls with both matched maternal age and body mass index. 1 H HR-MAS NMR spectroscopy data obtained from placental tissue samples of patients with FGR and healthy control group were analyzed with bioinformatics methods. The obtained results of metabolite levels were further validated with the internal standard (IS) quantification method. RESULTS: Principal component analysis (PCA) and the partial least squares discriminant analysis (PLS-DA) score plots obtained with the multivariate statistical analysis of preprocessed spectral data shows a separation between the samples from patients with FGR and healthy controls. Bioinformatics analysis results suggest that the placental levels of lactate, glutamine, glycerophosphocholine, phosphocholine, taurine, and myoinositol are increased in patients with FGR compared to the healthy controls. CONCLUSIONS: Placental metabolic dysfunctions are a common occurrence in FGR.

Metabolomics analysis of follicular fluid in women with ovarian endometriosis undergoing in vitro fertilization.

The purpose of this study was to investigate whether a change in the follicular fluid metabolomics profile due to endometrioma is identifiable. Twelve women with ovarian endometriosis (aged<40 years, with a body mass index [BMI] of <30 kg/m2) and 12 age- and BMI-matched controls (women with infertility purely due to a male factor) underwent ovarian stimulation for intracytoplasmic sperm injection (ICSI). Follicular fluid samples were collected from both of groups at the time of oocyte retrieval for ICSI. Next, nuclear magnetic resonance (NMR) spectroscopy was performed for the collected follicular fluids. The metabolic compositions of the follicular fluids were then compared using univariate and multivariate statistical analyses of NMR data. Univariate and multivariate statistical analyses of NMR data showed that the metabolomic profiles of the follicular fluids obtained from the women with ovarian endometriosis were distinctly different from those obtained from the control group. In comparison with the controls, the follicular fluids of the women with ovarian endometriosis had statistically significant elevated levels of lactate, ß-glucose, pyruvate, and valine. We conclude that the levels of lactate, ß-glucose, pyruvate, and valine in the follicular fluid of the women with endometrioma were higher than those of the controls. Abbreviations: ASRM: American Society for Reproductive Medicine; BMI: body mass index; CPMG: Carr-Purcell-Meiboom-Gill; E2: estradiol; ESHRE: European Society of Human Reproduction and Embryology; ERETIC: electronic to access in vivo concentration; FF: follicular fluid; FSH: follicle-stimulating hormone; hCG: human chorionic gonadotropin; HEPES: 2-hydroxyethyl-1-piperazineethanesulfonic acid; ICSI: intracytoplasmic sperm injection; IVF: in vitro fertilization; NMR: nuclear magnetic resonance spectroscopy; PCA: principal component analysis; PCOS: polycystic ovary syndrome; PLS-DA: partial least squares discriminant analysis; ppm: parts per million; PULCON: pulse length-based concentration determination; TSP: 3-(trimethylsilyl)-1-propanesulfonic acid sodium salt; VIP: variable importance in projection.

Determination of pKa values of some novel benzimidazole salts by using a new approach with (1)H NMR spectroscopy.

Benzimidazoles and their derivatives including imidazole are studied widely because they exist in the structure of natural products and different drugs. pKa values are extremely important for drug discovery and improvement in order to determine pharmacokinetic and pharmacodynamic features such as permeation through biological barriers, interactions with the target area or side effects. Acid-base features (pKa ) have great importance not only for physiological characteristics but also for being used as a ligand or changing physico-chemical features by turning benzimidazoles into salts. Within the scope of this study, a variety of new benzimidazole salts were synthesized, and their characterizations were made by NMR spectroscopy, FTIR spectroscopy and element analysis techniques. The pKa values of synthesized benzimidazole salts were determined by inflection point approach using integration values obtained with (1) H NMR spectroscopy and Henderson-Hasselbalch analysis. pKa values of some benzimidazole salts were also determined by potentiometric methods in order to compare those of NMR spectroscopy results.