John R. Sheedy

A sample preparation protocol for 1H nuclear magnetic resonance studies of water-soluble metabolites in blood and urine.

We describe a general protocol for preparing protein-containing biofluids for (1)H nuclear magnetic resonance (NMR) metabolomic studies. In this protocol, untreated samples are diluted in deuterated solvents to precipitate proteins and recover metabolites quantitated relative to standard reference compounds such as 3-trimethylsilylpropionic acid (TSP) and 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS). The efficacy of this protocol was tested using a bovine serum albumin/metabolite mix and human serum samples. This sample preparation method can be readily applied to any protein-containing biofluid for (1)H NMR studies.

Male obesity is associated with changed spermatozoa Cox4i1 mRNA level and altered seminal vesicle fluid composition in a mouse model

The rate of obesity among men of reproductive age has tripled in the last three decades. Previously, we demonstrated that paternal obesity resulted in impaired preimplantation developmental kinetics, compromised post-compaction metabolism and decreased blastocyst cell number when embryos were generated in vivo. Subsequently, using in vitro fertilization we found embryos of obese males to have altered metabolism before compaction, reduced inner cell mass cell number and retarded fetal development--the difference between these two studies being the method of embryo generation and the presence or absence of seminal plasma, respectively. Here, we hypothesize that both sperm and seminal plasma are affected by obesity, compromising embryogenesis and pregnancy health in a cumulative manner. Epididymal sperm and seminal vesicle fluid were collected from normal and obese C57BL/6 mice. RNA and DNA were extracted from spermatozoa for qPCR and global methylation analysis, respectively. Proteomic (Luminex) and metabolomic (GC-MS) techniques were employed to analyse the composition of seminal vesicle fluid. Nuclear encoded cytochrome c oxidase subunit IV isoform 1 (Cox4i1) of the terminal enzyme in the mitochondrial respiratory chain demonstrated significantly increased RNA levels in the sperm of obese males (P< 0.05). Quantitative seminal plasma analysis identified significant changes in levels of the hormones insulin, leptin and estradiol between normal and obese males (P < 0.05). Further, the metabolite composition of seminal vesicle fluid was significantly affected by obesity. Consequently, this study has determined that obesity affects both sperm and seminal plasma composition. The interaction between sperm and seminal plasma warrants further analysis.

NMR metabolic profiling of serum identifies amino acid disturbances in chronic fatigue syndrome

Chronic fatigue syndrome (CFS) is a debilitating multisystem disorder characterised by long-term fatigue with a variety of other symptoms including cognitive dysfunction, unrefreshing sleep, muscle pain, and post-exertional malaise. It is a poorly understood condition that occurs in ~5 in every 1000 individuals. We present here a preliminary study on the analysis of blood samples from 11 CFS and 10 control subjects through NMR metabolic profiling. Identified metabolites that were found to be significantly altered between the groups were subjected to correlation analysis to potentially elucidate disturbed metabolic pathways. Our results showed a significant reduction of glutamine (P=0.002) and ornithine (P<0.05) in the blood of the CFS samples. Correlation analysis of glutamine and ornithine with other metabolites in the CFS sera showed relationships with glucogenic amino acids and metabolites that participate in the urea cycle. This indicates a possible disturbance to amino acid and nitrogen metabolism. It would be beneficial to identify any potential biomarkers of CFS for accurate diagnosis of the disorder.

1 H-NMR analysis of the human urinary metabolome in response to an 18-month multi-component exercise program and calcium-vitamin-D 3 supplementation in older men

The musculoskeletal benefits of calcium and vitamin-D3 supplementation and exercise have been extensively studied, but the effect on metabolism remains contentious. Urine samples were analyzed by (1)H-NMR spectroscopy from participants recruited for an 18-month, randomized controlled trial of a multi-component exercise program and calcium and vitamin-D3 fortified milk consumption. It was shown previously that no increase in musculoskeletal composition was observed for participants assigned to the calcium and vitamin-D3 intervention, but exercise resulted in increased bone mineral density, total lean body mass, and muscle strength. Retrospective metabolomics analysis of urine samples from patients involved in this study revealed no distinct changes in the urinary metabolome in response to the calcium and vitamin-D3 intervention, but significant changes followed the exercise intervention, notably a reduction in creatinine and an increase in choline, guanidinoacetate, and hypoxanthine (p < 0.001, fold change > 1.5). These metabolites are intrinsically involved in anaerobic ATP synthesis, intracellular buffering, and methyl-balance regulation. The exercise intervention had a marked effect on the urine metabolome and markers of muscle turnover but none of these metabolites were obvious markers of bone turnover. Measurement of specific urinary exercise biomarkers may provide a basis for monitoring performance and metabolic response to exercise regimes.

Impact of oxygen concentration on adult murine pre-antral follicle development in vitro and the corresponding metabolic profile

Oxygen concentration during in vitro culture has a significant effect on the physiology of embryos, altering metabolic profile and developmental outcome. Although atmospheric oxygen has been used routinely for the culture of ovarian follicles, oxygen concentration may also be critical for follicle growth but the optimal concentration has not been determined. In this study, mechanically isolated primary and secondary follicles (80-140 µm diameter) from adult mouse ovaries were cultured in serum-free conditions for 8 days in either 5 or 20% oxygen to determine growth (follicular diameter), morphology and viability. For each oxygen concentration, half of the medium was replaced on Days 2, 4 and 6 or on Day 4 only. In the latter group, metabolic analysis of spent follicular culture media was performed by (1)H-NMR. The proportion of viable, growing follicles was significantly (P < 0.0001) higher in 5% than in 20% oxygen (59% versus 8%). Reducing the frequency of medium replacement during culture in 5% oxygen resulted in significantly (P < 0.001) more viable follicles (79 versus 46%). In 20% oxygen, poor follicular viability was observed irrespective of the frequency of medium replacement (8 and 10% respectively). Metabolic profiles showed marked differences in amino acid and carbohydrate utilization with respect to both oxygen concentration and between Days 4 and 8 of development. Metabolites which significantly discriminated between oxygen concentration at both time points were glucose consumption, lactate utilization, alanine, alanyl-glutamine, leucine and proline. In conclusion, the poor in vitro follicular development previously observed in minimal culture conditions may reflect the use of 20% oxygen. Frequent medium replenishment is not necessary and does not overcome the detrimental effect of high oxygen on follicle viability. Further optimization of culture conditions would benefit from metabolic analyses and the use of 5% oxygen should be tested further for impact on functional aspects of follicle culture such as steroid production which is currently unknown.

Cross-Platform Urine Metabolomics of Experimental Hyperglycemia in Type 2 Diabetes

Hyperglycemia causes diabetic nephropathy, a condition for which there are no specific diagnostic markers that predict progression to renal failure. Here we describe a multiplatform metabolomic analysis of urine from individuals with type 2 diabetes, collected before and immediately following experimental hyperglycemia. We used targeted nuclear magnetic resonance spectroscopy (NMR), liquid chromatography - mass spectrometry (LC-MS) and gas chromatography - MS (GC-MS) to identify markers of hyperglycemia. Following optimization of data normalisation and statistical analysis, we identified a reproducible NMR and LC-MS based urine signature of hyperglycemia. Significant increases of alanine, alloisoleucine, isoleucine, leucine, N-isovaleroylglycine, valine, choline, lactate and taurine and decreases of arginine, gamma-aminobutyric acid, hippurate, suberate and N-acetylglutamate were observed. GC-MS analysis identified a number of metabolites differentially present in post-glucose versus baseline urine, but these could not be identified using current metabolite libraries. This analysis is an important first step towards identifying biomarkers of early-stage diabetic nephropathy.

Fathers That Are Born Small Program Alterations in the Next-Generation Preimplantation Rat Embryos

BACKGROUND Low birth weight is associated with increased risk of adult cardiovascular and metabolic disease development, with recent studies highlighting transmission to subsequent generations via both maternal and paternal lines. However, the timing of parent-specific programming of disease risk to the next generation remains to be characterized. OBJECTIVE The aim of this study was to examine how paternal low birth weight affects the cellular and molecular physiology of the next-generation [second-generation (F2)] blastocysts, before uterine implantation. METHODS Uteroplacental insufficiency was surgically induced in Wistar Kyoto pregnant rats in late gestation, giving rise to first-generation restricted (born small) and sham-operated control (normal birth weight) male offspring, respectively. First-generation restricted and control male rats were naturally mated with normal females. RESULTS Resultant F2 blastocysts derived from restricted males displayed reduced expression of growth regulatory genes of the mammalian target of rapamycin pathway compared with F2 control blastocysts (9-74%; P < 0.05). No differences were found in F2 restricted blastocyst structural characteristics, cell number, or carbohydrate utilization at the time of blastocyst retrieval or after 24 h of in vitro culture. However, histidine, methionine, pyruvate, serine, and tryosine consumption and aspartate and leucine production were greater in F2 restricted outgrowth than in controls (P < 0.05). CONCLUSIONS The findings from this study clearly indicate that male rat offspring born small, arising from uteroplacental insufficiency, have physiologic alterations that manifest as modifications in gene expression levels and nutrient metabolism of F2 blastocysts, even in the absence of overt cellular growth differences. These data demonstrate that growth restriction and associated disease risk have the capacity to be transmitted to the next generation of offspring via the male germ line and is manifest as early as the blastocyst stage of development.

Metabolite analysis of biological fluids and tissues by proton nuclear magnetic resonance spectroscopy.

NMR-based biochemical profiling of natural products has become popular due to the development of high-resolution instruments (>400 MHz) and cryogenically cooled probes/preamplifiers, by increasing the sensitivity of NMR instruments several fold and reducing instrument noise levels. NMR provides a rapid, nondestructive, high-throughput method that requires minimal sample preparation, therefore maintaining the biological integrity of the sample. One-dimensional (1D) solution-state (1)H NMR is used in untargeted sample screening (metabolomics/metabonomics) to gain insight into spectral pattern changes associated with samples of different origins. Metabolomics and metabonomics contextually explains the systematic and quantitative measurement of metabolites that are produced from the biochemical reactions of living systems. This chapter describes some commonly used (1)H NMR experiments for identification and quantification of small molecular weight, water soluble metabolites in biological samples, some considerations for choosing the correct NMR experiment, and sample preparation protocols for isolating metabolites from a number of biological sample types.

Low female birth weight and advanced maternal age programme alterations in next-generation blastocyst development

Low birth weight is associated with an increased risk for adult disease development with recent studies highlighting transmission to subsequent generations. However, the mechanisms and timing of programming of disease transmission to the next generation remain unknown. The aim of this study was to examine the effects of low birth weight and advanced maternal age on second-generation preimplantation blastocysts. Uteroplacental insufficiency or sham surgery was performed in late-gestation WKY pregnant rats, giving rise to first-generation (F1) restricted (born small) and control offspring respectively. F1 control and restricted females, at 4 or 12 months of age, were naturally mated with normal males. Second-generation (F2) blastocysts from restricted females displayed reduced expression of genes related to growth compared with F2 control (P<0.05). Following 24 h culture, F2 restricted blastocysts had accelerated development, with increased total cell number, a result of increased trophectoderm cells compared with control (P<0.05). There were alterations in carbohydrate and serine utilisation in F2 restricted blastocysts and F2 restricted outgrowths from 4-month-old females respectively (P<0.05). F2 blastocysts from aged restricted females were developmentally delayed at retrieval, with reduced total cell number attributable to reduced trophectoderm number with changes in carbohydrate utilisation (P<0.05). Advanced maternal age resulted in alterations in a number of amino acids in media obtained from F2 blastocyst outgrowths (P<0.05). These findings demonstrate that growth restriction and advanced maternal age can alter F2 preimplantation embryo physiology and the subsequent offspring growth.

Physiological oxygen culture reveals retention of metabolic memory in human induced pluripotent stem cells

Reprogramming somatic cells to a pluripotent cell state (induced Pluripotent Stem (iPS) cells) requires reprogramming of metabolism to support cell proliferation and pluripotency, most notably changes in carbohydrate turnover that reflect a shift from oxidative to glycolytic metabolism. Some aspects of iPS cell metabolism differ from embryonic stem (ES) cells, which may reflect a parental cell memory, or be a consequence of the reprogramming process. In this study, we compared the metabolism of 3 human iPS cell lines to assess the fidelity of metabolic reprogramming. When challenged with reduced oxygen concentration, ES cells have been shown to modulate carbohydrate use in a predictably way. In the same model, 2 of 3 iPS cell lines failed to regulate carbohydrate metabolism. Oxygen is a well-characterized regulator of cell function and embryo viability, and an inability of iPS cells to modulate metabolism in response to oxygen may indicate poor metabolic fidelity. As metabolism is linked to the regulation of the epigenome, assessment of metabolic responses of iPS cells to physiological stimuli during characterization is warranted to ensure complete cell reprogramming and as a measure of cell quality.