Synthesis of [3-C-13]-2,3-dihydroxy-4-methoxybenzaldehyde

An efficient synthesis of [3-13C]-2,3-dihydroxy-4-methoxybenzaldehyde, an isotopically labelled probe of a common intermediate used in the synthesis of a number of biologically relevant molecules, has been achieved in 9 steps from an acyclic, non-aromatic precursor. A 13C label for molecular imaging was introduced in a linear synthesis from the reaction of [13C]-labelled methyl iodide with glutaric monomethyl ester chloride. Cyclisation then aromatisation gave 1,3-dimethoxybenzene and an additional methoxy group was introduced by a formylation/Baeyer–Villiger/hydrolysis/methylation sequence. Subsequent ortho-formylation and selective demethylation yielded the desired [3-13C]-2,3-dihydroxy-4-methoxybenzaldehyde

1H Magnetic Resonance Spectroscopy of live human sperm.

STUDY QUESTION: Can 1H Magnetic Resonance Spectroscopy (MRS) be used to obtain information about the molecules and metabolites in live human spermatozoa? SUMMARY ANSWER: Percoll-based density gradient centrifugation (DGC) followed by a further two washing steps, yielded enough sperm with minimal contamination (<0.01%) from seminal fluid to permit effective MRS which detected significant differences (p < 0.05) in the choline/glycerophosphocholine, lipid and lactate regions of the 1H MRS spectrum between sperm in the pellet and those from the 40/80% interface. WHAT IS KNOWN ALREADY: Current methods to examine sperm are either limited in their value (e.g. semen analysis) or are destructive (e.g. immunohistochemistry, sperm DNA testing). A few studies have previously used MRS to examine sperm, but these have either looked at seminal plasma from men with different ejaculate qualities or at the molecules present in pooled samples of lyophilized sperm. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Sperm suspended in Phosphate Buffered Saline (PBS) at 37°C were examined by 1H MRS scanning using a 1H excitation-sculpting solvent suppression sequence after recovery from fresh ejaculates by one of three different methods: (i) simple centrifugation; (ii) DGC with one wash; or (iii) DGC with two washes. In the case of DGC, sperm were collected both from the pellet ('80%' sperm) and the 40/80 interface ('40%' sperm). Spectrum processing was carried out using custom Matlab scripts to determine; the degree of seminal plasma/Percoll contamination, the minimum sperm concentration for 1H MRS detection and differences between the 1H MRS spectra of '40%' and '80% sperm. MAIN RESULTS AND THE ROLE OF CHANCE: DGC with 2 washes minimized the 1H MRS peak intensity for both seminal plasma and Percoll/PBS solution contamination whilst retaining sperm specific peaks. For the MRS scanner used in this study, the minimum sperm concentration required to produce a choline/glycerophosphocholine 1H MRS peak greater than 3:1 signal to noise ratio was estimated at ~3 × 106/ml. The choline/glycerophosphocholine (GPC) and lactate/lipid regions of the 1H spectrum were significantly different by two-way ANOVA analysis (p < 0.0001; n = 20). ROC curve analysis of these region showed significant ability to distinguish between the two sperm populations: choline/GPC ROC AUC = 0.65-0.67, lactate/lipid ROC AUC = 0.86-0.87. LIMITATIONS, REASONS FOR CAUTION: Only 3-4 semen samples were used to assess the efficacy of each sperm washing protocol that were examined. The estimated minimum sperm concentration required for MRS is specific to the hardware used in our study and may be different in other spectrometers. Spectrum binning is a low resolution analysis method that sums MRS peaks within a chemical shift range. This can obscure the identity of which metabolite(s) are responsible for differences between sperm populations. Further work is required to determine the relative contribution of somatic cells to the MRS spectrum from the '40%' and '80%' sperm. WIDER IMPLICATIONS OF THE FINDINGS: 1H MRS can provide information about the molecules present in live human sperm and may therefore permit the study of the underlying functional biology or metabolomics of live sperm. Given the relatively low concentration of sperm required to obtain a suitable MRS signal (~3 × 106/ml), this could be carried out on sperm from men with oligo-, astheno- or teratozoospermia. This may lead to the development of new diagnostic tests or ultimately novel treatments for male factor infertility. LARGE SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by the Medical Research Council Grant MR/M010473/1. The authors declare no conflicts of interest

NMR spectroscopy of live human asthenozoospermic and normozoospermic sperm metabolism

Sperm motility varies between ejaculates from different men and from individual men. We studied normozoospermic and asthenozoospermic ejaculates after density gradient centrifugation washing (DCG, 80/40%) and compared high (80%) and low (40%) motility sperm populations within the same sample. Our objective was to identify differences in endogenous metabolomes and energy metabolism in relation to sperm motility. 1H-Nuclear Magnetic Resonance Spectroscopy (NMR) measured the endogenous metabolome of live human sperm. Incubating sperm with 13C-labelled substrates detected energy metabolism by 13C-NMR. The studied examined 850 ejaculates and diagnosed asthenozoospermia in 6.1%. DGC was used to wash 160 normozoospermic (N) and 52 asthenozoospermic (A) ejaculates to recover high motility sperm from the pellet (80N/80A) and low motility from the interface (40N/40A). 1H-NMR spectra, 45(N), 15(A), were binned and the integrals normalised by sperm concentration. Sperm from 126(N) and 36(A) ejaculates were incubated with either 13C-glucose, 13C-fructose or 13C-pyruvate. 13C-NMR lactate and bicarbonate integrals were normalised by motile or vital sperm concentrations. 1H-NMR spectra choline integrals from the 80A population were significantly lower than the 80N, p<0.0001. 13C-substrate conversion to lactate was significantly higher for 40A sperm than 80A sperm when normalised by motile sperm concentration. Bicarbonate integrals were sporadically observed. Sperm from asthenozoospermic ejaculates had similar glycolytic requirements to normozoospermic ones, with larger differences observed between 40% and 80% sperm populations. Higher lactate levels produced by 40% sperm may indicate that impaired sperm motility is due to dysregulated energy metabolism. The alteration in choline metabolism provides opportunities to understand the aetiology of asthenozoospermia

Kinetic modelling of dissolution dynamic nuclear polarisation 13C magnetic resonance spectroscopy data for analysis of pyruvate delivery and fate in tumours

Dissolution dynamic nuclear polarisation (dDNP) of 13C-labelled pyruvate in magnetic resonance spectroscopy/imaging (MRS/MRSI) has the potential for monitoring tumour progression and treatment response. Pyruvate delivery, its metabolism to lactate and efflux were investigated in rat P22 sarcomas following simultaneous intravenous administration of hyperpolarised 13C-labelled pyruvate (13C1-pyruvate) and urea (13C-urea), a nonmetabolised marker. A general mathematical model of pyruvate-lactate exchange, incorporating an arterial input function (AIF), enabled the losses of pyruvate and lactate from tumour to be estimated, in addition to the clearance rate of pyruvate signal from blood into tumour, Kip, and the forward and reverse fractional rate constants for pyruvate-lactate signal exchange, kpl and klp. An analogous model was developed for urea, enabling estimation of urea tumour losses and the blood clearance parameter, Kiu. A spectral fitting procedure to blood time-course data proved superior to assuming a gamma-variate form for the AIFs. Mean arterial blood pressure marginally correlated with clearance rates. Kiu equalled Kip, indicating equivalent permeability of the tumour vasculature to urea and pyruvate. Fractional loss rate constants due to effluxes of pyruvate, lactate and urea from tumour tissue into blood (kpo, klo and kuo, respectively) indicated that T1s and the average flip angle, θ, obtained from arterial blood were poor surrogates for these parameters in tumour tissue. A precursor-product model, using the tumour pyruvate signal time-course as the input for the corresponding lactate signal time-course, was modified to account for the observed delay between them. The corresponding fractional rate constant, kavail, most likely reflected heterogeneous tumour microcirculation. Loss parameters, estimated from this model with different TRs, provided a lower limit on the estimates of tumour T1 for lactate and urea. The results do not support use of hyperpolarised urea for providing information on the tumour microcirculation over and above what can be obtained from pyruvate alone. The results also highlight the need for rigorous processes controlling signal quantitation, if absolute estimations of biological parameters are required

Measurement of the acute metabolic response to hypoxia in rat tumours in vivo using magnetic resonance spectroscopy and hyperpolarised pyruvate

Purpose: To estimate the rate constant for pyruvate to lactate conversion in tumours in response to a hypoxic challenge, using hyperpolarised 13C1-pyruvate and magnetic resonance spectroscopy. Methods and materials: Hypoxic inspired gas was used to manipulate rat P22 fibrosarcoma oxygen tension (pO2), confirmed by luminescence decay of oxygen-sensitive probes. Hyperpolarised 13C1-pyruvate was injected into the femoral vein of anaesthetised rats and slice-localised 13C magnetic resonance (MR) spectra acquired. Spectral integral versus time curves for pyruvate and lactate were fitted to a precursor-product model to estimate the rate constant for tumour conversion of pyruvate to lactate (kpl). Mean arterial blood pressure (MABP) and oxygen tension (ArtpO2) were monitored. Pyruvate and lactate concentrations were measured in freeze-clamped tumours. Results: MABP, ArtpO2 and tumour pO2 decreased significantly during hypoxia. kpl increased significantly (p < 0.01) from 0.029 ± 0.002 s−1 to 0.049 ± 0.006 s−1 (mean ± SEM) when animals breathing air were switched to hypoxic conditions, whereas pyruvate and lactate concentrations were minimally affected by hypoxia. Both ArtpO2 and MABP influenced the estimate of kpl, with a strong negative correlation between kpl and the product of ArtpO2 and MABP under hypoxia. Conclusion: The rate constant for pyruvate to lactate conversion, kpl, responds significantly to a rapid reduction in tumour oxygenation