Rep 249 Sharma
Total Page：16
File Type：pdf, Size：1020Kb
Reproduction (2001) 122, 431–436 Research A proton NMR study of the effect of a new intravasal injectable male contraceptive RISUG on seminal plasma metabolites U. Sharma1, K. Chaudhury2, N. R. Jagannathan1 and S. K. Guha3* 1Department of NMR, All India Institute of Medical Sciences, New Delhi 110 029, India; 2Centre for Biomedical Engineering, Indian Institute of Technology, New Delhi 110 016, India; and 3Centre for Biomedical Engineering, Indian Institute of Technology and All India Institute of Medical Sciences, New Delhi 110 029, India Nuclear magnetic resonance (NMR) spectroscopy was subjects injected with RISUG compared with controls. used to quantify citrate, glucose, lactate, glycero- In addition, metabolite ratios such as choline:citrate, phosphorylcholine and choline in seminal plasma from citrate:lactate, choline:lactate and glycerophosphoryl- subjects injected with a new male contraceptive RISUG, a choline:choline were calculated. Citrate:lactate and copolymer of styrene maleic anhydride dissolved in glycerophosphorylcholine:choline ratios were significantly dimethyl sulphoxide, and in seminal plasma from normal lower in RISUG-injected subjects than in controls ejaculates. No significant difference in the concentration (P < 0.01), thereby indicating the occurrence of partial of citrate was observed between the groups, indicating obstructive azoospermia. The most important finding of that the prostate is not affected by the contraceptive. The the present study was that the intervention of RISUG in the concentrations of glucose, lactate, glycerophosphoryl- vas deferens even for a period as long as 8 years is choline and choline were significantly lower (P < 0.01) in absolutely safe and does not lead to prostatic diseases. al., 1990), including teratogenic safety on reversal (Sethi et Introduction al., 1992), have also been conducted. RISUG is non-toxic There is a need to develop a suitable male contraceptive for and safe, and has long-term effectiveness. The compound is population control programmes. The current unavailability retained in the vas deferens even though it is non-sclerosing of an ideal male contraceptive is due to the lack of one or and not tissue adherent. In addition, reversibility can be more essential features such as a minimally invasive drug achieved easily in a minimally invasive manner (Guha, delivery system with a one-time intervention, long-term 1999). effectiveness of the contraceptive with negligible side- Human seminal plasma contains a mixture of fluid from effects and the option of reversal whenever desired. the seminiferous tubules, epididymis and accessory glands Rigorous research has been carried out over the past 25 (bulbo-urethral, prostate and seminal vesicles). The composi- years to meet these objectives, which has led to the tion of each secretion has characteristic biochemical successful development of a reversible male contraceptive features: fructose and prostaglandins are markers for that can be injected bilaterally into the lumen of the vas seminal vesicles; prostatic fluid contains uniquely high deferens. The contraceptive, named RISUG, consists of a concentrations of citrate (Costello and Franklin, 1991), zinc, specific preparation of the copolymer styrene maleic polyamines and myo-inositol; and glycerophosphorylcholine, anhydride dissolved in dimethyl sulphoxide (DMSO). choline, neutral α-glucosidase and carnitine are markers of When spermatozoa come into contact with RISUG in the epididymal function (Cooper et al., 1990). High-resolution vas deferens, their membranes rupture and enzymes such as 1H NMR spectroscopy has emerged as a powerful tool in acrosin and hyaluronidase leach out (Guha, 1996). Hence, the analysis of biofluids (Foxall et al., 1993; Spraul et al., these damaged spermatozoa are rendered incapable of 1994; Liu et al., 1996). Recent studies have shown that fertilization. Phase I and phase II clinical trials have chemometric methods along with automated NMR-based provided guidelines for drug dose selection (Guha et al., analysis can be used for toxicological assessment of 1993) and established the effectiveness of a specific dose of biofluids (Holmes et al., 2000). Lynch et al. (1994) showed the contraceptive (Guha et al., 1997). A phase III clinical that NMR could be used successfully as a diagnostic probe trial is currently underway. Toxicological studies (Sethi et for altered male reproductive gland function. 1H NMR spectroscopy has also been used to examine seminal plasma of patients in an IVF programme (Segalen et al., *Correspondence 1995). Recently, Hamamah et al. (1998) showed that Email: [email protected] seminal plasma metabolite ratios could be used to © 2001 Journals of Reproduction and Fertility 1470-1626/2001 Downloaded from Bioscientifica.com at 09/25/2021 09:25:37PM via free access 432 U. Sharma et al. differentiate between cases of spermatogenic failure and for quantitative measurements. A constant receiver gain was obstructive azoospermia. The main advantage that NMR used for all one-dimensional experiments to minimize spectroscopy offers over other classical biochemical errors in quantitative estimation. The free induction decays analyses is that it is not biased towards a particular (FIDs) were collected with 32 K data points and line compound; it can simultaneously detect metabolites that broadening of 0.3 Hz was applied before Fourier are expected or unexpected or that are difficult to assay transformation. using standard biochemical methods (Patel et al., 1999). Connectivity between protons was established with There is also no need for specific preparation such as phase-sensitive double quantum-filtered correlation spectra derivatization of compounds. using a standard pulse sequence. Typically, the following The aim of the present study was to evaluate prostatic experimental conditions were used: spectral width 5 kHz, function in RISUG-injected subjects by determining the 2 K data points in F2 dimension with 512 t1 increments concentration of citrate in the seminal plasma ejaculates. In in F1 dimension. The number of scans was 32–64, the addition, glucose, lactate, glycerophosphorylcholine and relaxation delay was 2.5 s and the receiver gain was choline metabolites were quantified. Peak area ratios of optimized in each instance to obtain good signal:noise these metabolites were also estimated to assess the ratio. Data were zero filled to 1 K in F1 dimension. Squared occurrence of obstructive azoospermia in RISUG-injected sine bell window function was applied before Fourier subjects. transformation. Quantification of metabolites Materials and Methods Peaks or multiplets of identified metabolites, including Seminal plasma preparation that of internal standard TSP, were integrated to obtain Human semen samples were collected by masturbation signal intensity after careful baseline correction. The from (i) 20 normal human volunteers (age 32–40 years; following formula was used to estimate the concentrations controls) who had fathered children within the previous 4 of various metabolites: years and (ii) 17 subjects (aged 36–40 years) who had fathered children before the injection of RISUG given to NTSP . IX [C]X = [C]TSP them 8 years earlier. Each sample was liquefied at 37ЊC for NX . ITSP 20 min to reduce viscosity. A 0.5 ml aliquot was removed to determine semen parameters using standard methods [C]X is the concentration of biochemical X, and IX and (WHO Recommended Procedure, 1999). The remaining ITSP are the NMR signal intensities of X and TSP, semen sample was centrifuged at 1000 g for 15 min to respectively. NX is the number of protons per molecule remove cells and spermatozoa. The resulting supernatant giving rise to the integrated signal and NTSP = 9. Metabolite was separated, diluted with D2O and NMR experiments ratios were calculated from the integrated intensity of were performed immediately, within 1 min. Even after individual peaks. separation from spermatozoa by centrifugation, the composition of seminal plasma undergoes progressive Statistical analysis changes in vitro, for example, proteolysis, increase in free Concentrations of citrate, glucose, lactate, glycero- choline content due to dephosphorylation of phosphoryl- phosphorylcholine and choline are expressed as mean Ϯ SD. choline, and fructolysis. Therefore, the plasma was Concentration and peak area ratios of specific biochemical subjected to NMR analysis immediately after separation markers from both the groups were compared using of spermatozoa and cells. Identical conditions were Student’s t test. The level of significance was set at P < 0.01. maintained strictly for each sample evaluation. NMR analysis Results Seminal plasma samples were transferred into 5 mm Various amino acids, carbohydrates and lipids present in NMR sample tubes with D2O (Aldrich Company Inc., human seminal plasma were assigned in RISUG-injected Milwaukee, IL) for field/frequency lock. Sodium 3- subjects using 400 MHz 1H NMR spectroscopy. Expanded (trimethylsilyl-2,2,3,3-H)-1-propionate (TSP) (E-Merck, regions of a typical one-dimensional proton NMR spectrum Darmstadt) was added at a concentration of 2 mmol l–1 to of seminal plasma of controls and of subjects injected with the seminal plasma samples to serve as a chemical shift and RISUG are shown (Fig. 1a,b). Several intense resonances concentration reference. were observed in the aliphatic range (0.5–4.5 p.p.m.) and The spectra were recorded on a Bruker DRX-400 aromatic region (6.5–8.5 p.p.m.). A two-dimensional double spectrometer (9.4 T) operating