Applications Of NMR Spectroscopy In Biology Biology Essay
Cancer of Prostate (CaP) is most common malignancy in men above age of 40 years. Incidence rate of CaP varies widely across the world, with highest rates in Europe and United States compared to rates reported in Asia (1-3). Early detection of CaP is important for treatment success. Current methods for screening of CaP involve measurement of serum prostate specific antigen (PSA) and digital rectal examination (DRE) however; both of these methods suffer from low specificity and sensitivity (4). Therefore, other methods and molecular markers like free PSA, urine PCA3 etc. are constantly being evaluated to improve the accuracy and early detection of CaP.
Advancements in nuclear magnetic resonance (NMR) spectroscopic methods have increased the capabilities it offers for cancer detection. Magnetic resonance (MR) methods are of particular interest in prostate cancer evaluation. MR methods can provide physiological, metabolical and anatomical information. These methods include detection and quantification of metabolites obtained from NMR spectroscopy of body fluids, tissue extracts, excised intact tissues and in vivo. The present chapter is focussed on study of metabolites detected in prostatic fluid, prostate tissue extracts and samples of intact prostate tissue.
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One of the first applications of NMR spectroscopy in biology is to study the various body fluids ranging from saliva to urine. NMR could detect the metabolites present at enough concentration in biological fluids for example amino acids. It proved to be a great technique to detect and quantitate the metabolites and also monitor changes appearing due to different pathological conditions. Both in vitro and ex vivo NMR studies have been utilized in the study and characterization of tissues of various diseases (CaP, breast carcinoma, brain tumors and many more). These studies are helpful in improving our understanding the metabolic profiling (5-11). Recently, NMR became an important technique in Metabolomics, defined as the quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification (12). These studies provide tumor biomarkers, which could assist in diagnosis and therapy for a specific disease. NMR spectroscopy also has the chance of detecting unexpected or previously unknown metabolites (13). Additionally, the development of high resolution magic angle spinning (HRMAS) 1H NMR spectroscopy has provided a novel method for metabolomic/metabonomic studies of intact tissue biopsy samples (5,14) and has been showing the ability to unravel the prostatic disorders (15-17). In this article, we review the in vitro and ex vivo NMR techniques in prostate cancer. For the prostate tissues, normal prostate gland contains high level of citrate (Cit) and these levels decrease in prostate cancer (18,19). In addition to citrate, other metabolites like choline and polyamines have also been shown to alter due to malignancy (20,21).
Prostate consists of different anatomical zones. Among these, peripheral zone (PZ) is the largest zone and makes up the bulk of prostatic glandular tissue (70%). It is also the site of origin of about 70% of prostatic carcinomas. The secretory epithelial cells of PZ are the main site of production of prostatic fluid which contains extraordinarily high levels of citrate Costello and franklin 1991a. This occurs due to uniquely limiting m-aconitase activity which converts citrate to isocitrate franklin and Costello 1997. The m-aconitase activity is inhibited by the presence of high levels of mitochondrial zinc, which is another unique feature of secretory eipithelial cells of PZ Costello and franklin 2000 costello 2005. Normal prostate and BPH tissue contain citrate levels of about 8000 to 15000 nmol/g of tissue while all other tissues contain 150 to 450 nmol/g Costello and franklin 2000. In other cells of the body, citrate is oxidized via Krebs cycle to generate cellular energy. Interestingly, in prostate cancer the citrate levels are decreased to 1000 2000 nmol/g and were found equivalent to citrate levels of other non-prostate tissues when estimated on basis of malignant tissue component Marberger 1962 costello and franklin 2000. Thus, malignant prostate cells oxidize citrate and do no accumulate it. Malignant cells also contain low levels of zinc allowing m-aconitase to act and result into oxidation of citrate. In addition to decreased levels of citrate in malignant prostate cells compared to normal prostatic cells, increased choline levels are also noticed in malignant cells due to increased membrane synthesis and cell proliferation .
The metabolites of normal prostate and metabolic changes resulting due to malignant cells can be quantified by NMR techniques (other techniques like liquid-chromatrograpy-mass spectrometry may also be used, however, these are out of scope of the discussion of the present chapter). Two most widely techniques used are (a) high resolution NMR spectroscopy of prostatic fluid and extracts of prostate tissues, and (b) high resolution magic angle spinning (HR-MAS) NMR spectroscopy, which allows quantification of metabolites in solid state tissue samples and histopathological analysis from same sample.
Prostate tissue obtained by biopsy, transurethral resections or from radical prostatectomy may be used NMR studies. Biopsy does not provide enough tissue therefore these should be studied using HRMAS technique. Typically, tissues are snap frozen immediately after excision, in liquid nitrogen (N2) to arrest the metabolic activity and then stored at -70 to -80C for appropriate extraction and NMR spectroscopy.
In NMR spectroscopy, protons present within in molecule give rise to distinct resonance signals (peak) at their resonance frequency, hence, each metabolite provide a specific signature pattern of peaks by which the metabolite can be identified. The signal intensity obtained is directly proportional to the number of protons resonating at that particular frequency. Therefore, the area of the peak may be used for determining the concentration of the metabolite.
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In vitro NMR studies
In vitro NMR methods are non-invasive, non-destructive, and allow simultaneous determination of a number of metabolites and have several advantages compared to in vivo. With the advent of higher magnetic fields, in vitro NMR spectra can be acquired with increased amount of sensitivity, spectral resolution and absolute quantification of metabolites.
In vitro NMR spectral analysis of tissues relies on tissue extraction methods. However, the rapid post-mortem change in tissue metabolites is the major drawback of in vitro NMR studies of tissues. One should be cautious during tissue extraction because metabolites may be lost or degenerated during the extraction procedure.
The purpose of various extraction procedures used is to produce a high total tissue metabolite yield with low variability. In extraction procedure, the macromolecules are removed to improve upon the spectral resolution of signals from the low molecular weight metabolites (Peeling et al 1992). However, this procedure needs careful handling to avoid decomposition of metabolites. An extensive review has been presented by Beckonert et al on NMR spectroscopy of urine, plasma, serum and tissue extracts [Beckonert et al]. Amongst many tissue extraction procedures, the most ubiquitous is the perchloric acid (PCA) method, which extracts water-soluble metabolites (Le belle et al, Yacoe et al, Glonek et al, Tyagi et al). The method has been described in details elsewhere, in brief, frozen prostate tissue, ground to powder mixed with PCA and centrifued. Supernantant is collected and adjusted to pH 7 and lyophilized to remove water traces and stored. For NMR spectroscopy, sample is dissolved in D2O and 3-(trimethyl silyl)-tetradeutero sodium proprionate (TSP) is added to serve as an internal reference standard. There are other extraction methods which are based on the nature of the investigation to be carried out. For aqueous extraction, weighed frozen tissue is homogenized in 50% acetonitrile/50% H2O (vol/vol) (5 ml/g of tissue). Further, this homogenates is centrifuged at 10,000-12,000g for 10 minutes at 4 oC. Then after collecting the supernatant, it is lyophilized and stored at -80 oC for later NMR processing [Waters NJ et al].
Lipid extraction is performed by chloroform and methanol; however for simultaneous extraction of both aqueous and lipids soluble metabolites, methanol-chloroform-water extraction (M/C) is employed [Folch J, Tyagi, Lin CY et al]. In M/C extraction, weighed frozen tissue is homogenized with methanol (4 ml/g) and water (0.9 ml/g) and thoroughly mixed. Afterwards, we add chloroform (2ml/g) and water (2ml/g) and sample is vortexed. In order to prevent methanol evaporation sample is kept in fridge for 10 min. Then it is centrifuged (1000g, 10 min, 4 oC) and the mixture is separated in two phases. Lower chloroform phase is lipid extracts and upper methanol/water phase is aqueous extracts. The sample is recentrifuged if required, to separate the lower and upper phases more clearly. Solvent is removed by using stream of nitrogen and then stored at -80 oC for later NMR processing.
After lyophilisation, tissue powder is dissolved in a suitable solvent. The solvent are chosen depending upon their experimental molecules. For hydrophilic molecules- ideal solvent is deuterium oxide (D2O), for lipophilic molecule- deuterochloroform (CDCl3), for organic acids- deuterated dimethyl sulfoxide (d6-DMSO) is preferred. The optimal concentration depends on the nucleus. The lower limits of concentration depend on field strength and the type of probe.
A standard is usually added to provide a sharp peak of known chemical shift in the NMR spectrum, in a region of chemical shifts that does not interfere with the sample peaks. Sodium salt of 3-trimethylsilylpropionic acid (TSP) is used as a reference compound in aqueous media and for organic solvents organic solvents tetramethylsilane (TMS) is ideal reference. For prostatic tissue extract commonly used solvent is D2O and reference compound is TSP.
Extraction of polar metabolites from tissue can be performed using acetonitrile, whereas methanol-chloroform-water extraction (M/C) is used for lipids extraction [Folch et al, Edzes et al, Tyagi et al, Henke et al]. An in vitro 1H and 31P NMR study of PCA extracts of tissue samples of human prostate have been reported (Cornel et al). Statistically significant differences between the cancer and benign groups were seen for the various metabolites eg. Cit, creatine (Cr), and alanine (Ala) (Fowler et al, Yacoe et al).
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For the first time, 13C NMR spectroscopy was used to show the detectablibiy of Citrate in prostate using NMR (22). This prompted investigators to explore possibilities of using NMR spectroscopy for detection of citrate in prostate and subsequently use it to differentiate malignant and bening tissues. Cell strains derived from prostate cancers were studied using high resolution 1H NMR spectroscopy. The study confirmed the decrease in citrate levels in malignant cells compared to normal epithelial cells (23). Fowler et al perfomed in vitro 1H NMR spectroscopy on perchloric acid extracts of tissue samples of human prostate includeing prostate cacner, benign prostatic hypertrophy and samples containing mixture of both. Study reported statatistically significant differences between cacner and bening groups for metabolic peak area ratios of citrate, creatine and phosphorylcholine to alanine and citrate to glutamate (24). Further studies were conducted to confirm the findings low citrate concentrations in malignant samples and also explore the changes in other metabolites (25). In addition to 1H NMR spectroscopy, 31 P NMR spectroscopy was also utilised for perchloric acid extracts for investigation of human prostate samples. The citrate/lactate, citrate/total choline, phoshocholine/total creatine, choline/total creatine, alanine/total creatine, phosphoethanolamine/total phosphate, phosphocholine/total phosphate and glycerophosphoethanolamine/total phosphate ratios were statistically different for teh prostate cancer samples as copared to BPH specimens (26). Studies also focussed on NMR spectroscopy of human seminal fluid, seminal vesicle adn prostatic secretions (27). Proton MRS of human prostatatic fluid showed strong correlation between citrate and spermine, teh molar ratio was 5:1 citrate: spermine. The study also suggested significant difference between citrate to spermine rationi n prostatic fluid from men with prostate cancer (28). More rigorous statitstical analysis for classification of beningn and malignant hman prostrate tissue by NMR spectra using multivariate analysis ie linear discriminant analysis has been perfmored (29). A overall classification accuracy of 96.6% was achieved wtih a sensitivity of 100% and a specificity of 95.5% in classifying bening prostatic hyperplasia from prostatic cancer. Resonances due to citrate, glutamate, tauraine were among having diagnostic potential (29). The study confirmed teh presence of high citrate and spermine and myo-inositol in prostatic fluid. NMR has also been shown to discriminate the androgen dependency in cell lines base on relative creatine and citrate levels (30). All these studies showed detectability of citrate in normal prosate adn its decrease in malignancy. These findings led the way for invivo prostate spectroscopy.
High resolution magic angle spinning (HR-MAS) NMR Spectroscopy
The field of high resolution NMR spectroscopy of tissue samples was revolutioned by using HRMAS technique. By this method, high resolution NMR spectrum may be obtained from intact tissue can be performed. This also allowed quantitative histopathology from same tissue from which |NMR spectrum was obtained.
Conventional prostate in vitro NMR spectroscopy has two major limitations. First is the limited amount of prostatic tissue obtained from surgical procedures, second is the PCA tissue extractions which itself is time consuming, and requires good lab practice. Looking into the increasing oncolgical demand, several group sought the alternative which could provide NMR spectra of intact prostatic sample [Tomlins et al, Cheng et al FEBS]. Recently, most of the prostatic tissue NMR studies are based on HRMAS- NMR technique which had led to the development of CaP metabolomics/ metabonomics [Burns 04, Swanson 2006, Ratiney, Defeo et al] [Table 4].
Brief HRMAS theory is described here. In solids, extensive spectral-broadening effects are caused by interactions such as dipolar couplings and chemical shift anisotropy, and they show an angular dependence between the static magnetic field and the internuclear vector responsible for the interaction. Andrew et al. [Andrew] and Lowe [Lowe et al] first showed that by spinning the solid sample at high speed and at an angle of 54.7 (the magic angle) relative to the applied magnetic field, reduces the line-broadening effects and hence resulted in highly resolved NMR spectra. An extensive review on HRMAS sample preparation, experiment design and NMR parameters are described by Beckonart et al [Beckonart et al].
Tomlins AM et al (1998) were the first to analyze intact prostatic tissue from BPH and CaP patients using HRMAS. This group concluded that HRMAS of intact tissue biopsy sample provides a wider range of metabolites than conventional tissue extraction NMR spectroscopy. Afterwards various HRMAS studies were performed on prostatic sample. [Levin YS, Tomlins AM, Cheng LL 05, Swanson MG03, 06, 08Taylor JL, Zektzer et al, Wu CL, Burns MA, Burns MA, , Jordan KW ERP 2007, Tessem MB, van Asten JJA, Albers MJ, Santos CF, Stenman K, Maxeiner A, Ratiney H, DeFeo EM, Spencer NG]. It was conjectured that spectral resolution of HRMAS may be dependent on freeze thawing process of tissue [Middleton et al], which led to view the thawing effect on prostatic specimens. It was reported that storage conditions had minimal effect on spectral resolution, relative metabolite concentrations, also it was reported that the prostatic tissue metabolites could be easily quantified using HRMAS by storing the sample even for three years [Wu et al, Jordon et al BI 07]. The slow spinning methods were also evaluated in order to protect the prostate tissue pathology [Burns 05, Taylor].
For the first time, inhibitory function of spermine in human prostate was evaluated by Cheng et al in 2001 by HRMAS [Cheng LL 2001]. The impact of HR-MAS spectroscopy on the histopathologic and mRNA integrity of postsurgical, TRUS and MRSI targeted biopsy tissues were studied and quantified [Swanson 03, Santos]. New metabolic biomarkers have been observed in order to better identify and distinguish the malignant and benign prostatic disorders. Apart from several established CaP biomarkers (eg. Cho and its derivatives), lactate, alanine, polyunsaturated omega-6 fatty acids (PUFAs) have been shown to promote CaP and could be detected and quantified by HRMAS [Tessem et al, Stenman et al, Burns 04, Defeo, Swanson 06, Swanson 08, Albers-ERECtic, Ratiney et al]. Recently, Levin et al studied metabolic pathways on human CaP cells using13C-labeled substrates as metabolic probes by 13C and 1H HRMAS. It was observed that the fractional enrichments of alanine, glutamate, and aspartate were moderately higher and may characterise metabolic and bioenergetics properties of CaP cells.
HRMAS is quintessential in order to find the clinical relevance to improve upon the diagnostic information of CaP patients. Diagnostic utility and CaP aggressiveness in terms of cancer recurrence of prostate tissue metabolite had been successfully studied [Cheng 05, Van asten etl al, Maxiener et al].
The study reported linear correlation between NMR measured spermine and volume percentage of normal prostatic epithelial cells by histopathology (15). Studies focussed on evalutiono of elevation of choline upon malignant transformation also used NMR spectroscopy. Malignannt normal human prostate cells adn tumors cells derived from metastases exhibit significantly higher phoshophocholine as well as glycerophosphocholine levels compared with normal prostate epithelial adn stromal cells (31).
Effects of various methodological steps and technicaltities of HR MAS spectroscopy of prostate tissues has been addressed. Optimum Spinning rates, rotor synchronized adiabatic pulses, reduction of spinning sidebands adn effect of fresh and previously frozen tissues on HRMAS resutsl have been described (32-35). Averna et al studied human seminal plasma of 3 control subjects wtih PSA less than 1 ng/ml. When they compared the seminal plasma of pateitns iwht prostate cancer , it revealed citrate NMR signals corresponding of only 28, 24 mM. The study highlights teh importance of citrate conten in semen to diagnose prostate cancer (36). Metabolic profiles obtained can differentiate malignant from benign samples obtained from same patieint. Metabolic profiles can also delineate a subset of less aggressive tumors and predict tumor perinural invations within the subset. Hence, metabolic profiles can be used to assess prostate cancer pathologic stage adn aggressiveness which at present can be determined histopathologically only after prostectomy (37).
Quantification of metabolites
Metabolites proposed as biomarkers in prostate cancer
The most important metabolite shown to distinguish prostate cancer from BPH and normal tissue is citrate. Citrate is has been shown to decrease in malignant prostate by in vitro NMR, HR MAS and in vivo MRS. Other metabolites shown to be of interest to distinguish prostate cancer are decreased polyamines (spermine), high lactate and choline levels. Sarcosine levels also been correlated with prostate cancer progression. Sarcosine detected by LC-MS-/GC-MS in urine has been proposed a method for detecting prostate cancer .
Though all these above discussed metabolites have been shown to distinguish between malignant and bening samples, the efficacy of metabolic profiling can be improved by analysing entire metabolic profiles. The use of entire metabolomic profiles using principla component analysis, differentiation of malignant and BPH samples has been shown to hoghly significant with an accuracy more than 98%. Such an analsysis also have been shown to distinguish between confined and invasive prostate cacner. It aislo been shown to predict biochemical recuurence.
Metabolomic analysis of entire metabolic profile obtained from prostate tissue samples, urine, blood, prostatic fluid and semen.
1D and 2D NMR parameters techniques
Mainstream of NMR experiments employed for analysis of samples are one-dimensional (1D) and two-dimensional (2D) experiment [Jeener et al, Ernst et al, Aue et al]. In a simple 1D NMR spectrum, the structural information of the sample is obtained from the chemical shift and peak area. 2D NMR experiments are carried out to get more accurate assignment of overlapped resonances using two frequency scales in x- and y- axis. Commonly used 2D NMR experiments are 2D correlation spectroscopy (COSY)/ double quantum filtered correlation spectroscopy (DQF-COSY) and total correlation spectroscopy (TOCSY).
Various type NMR spectroscopic studies of prostatic disorders
The in vitro NMR spectroscopy studies on prostate till date reported in literature are provided in Tables (1, 2, 3, 4). Nuclei like 1H, 13C, and 31P had used to study CaP tissues, cell line, animal tissues, and fluids. These studies are carried out to understand the metabolic pathway of CaP and to seek potential biomarkers from NMR as an alternative of PSA screening.
Carbon (13C) NMR of prostate
The initial application of in vitro NMR spectroscopy of prostate tissues was studied using 13C nucleus. In 1988, Halliday et al and Sillerud et al independently studied rat and human prostate using 13C NMR spectroscopy (Table 1). Both authors successfully detected Cit in prostate and also observed that the prostatic tumors contained - larger amounts of triacylglycerols, smaller amounts of Cit, and acidic mucins.
Phosphorous (31P) NMR of prostate
In vitro 31P NMR spectroscopy is an excellent method for probing the phospholipid metabolites prominent in CaP. Table 2 presents the studies carried out using 31P NMR. 31P NMR spectroscopy was performed on CaP in a nude mouse model and on cell line. [Kurhanewicz et al, Cornel 93, Macdonald et al]. These studies suggested that the level of phosphocreatine (PCr) was reduced in both DU 145 xenografts and perfused DU 145 cells relative to spectra of in situ human CaP. Phosphomonesters (PMEs) were elevated in 31P spectra of both DU 145 xenografts and in situ human CaP [Kurhranewicz et al]. Recently, [Komoroski et al] reported that 31P MRS, with its ability to distinguish the major choline-containing phospholipid metabolites, including metabolites phosphoethanolamine (PE) and glycerophosphoethanolamine (GPE) may be a useful approach for diagnosing CaP.
Proton (1H) NMR of prostate diseases
1D and 2D 1H-NMR
The year 1990 is (90s was) the era in which most researchers performed studies on prostate using 1H NMR spectroscopy some of them were based on 31P NMR spectroscopy as well [Table 2, 3]. First in vitro 1H NMR study was performed by Yacoe et al and they reported that Cit was observed in less quantity in cultured CaP cells than in cultured prostate epithelial tissue however, differences were not statistically significant for both groups [Yacoe et al]. In the search of novel CaP biomarkers human prostate cell lines were studied by other groups as well [Cornell et al, Teahan et al, Ackerstaff et al]. Several preclinical studies were performed in order to assess the metabolic pathway of CaP [Kurhanewicz MRM 93, Teichert et al]. TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mouse had been useful in metabolic profiling and in the development of novel chemopreventive or chemotherapeutic interventions for CaP [Teichert et al, Raina et al].
Body fluids are easily accessible for in vitro NMR studies and had more impact on several clinical studies [Bell et al, Nicholson et al 1989, Azaroual et al]. The prostate contains high concentrations of Cit, polyamine (predominantly spermine), myo-inositol (mI), and Zn [Huggins et al, Costello 1991 et al, Tabor et al, Lewin et al, Cohen et al]. Using this knowledge several groups have studied the seminal fluid and prostatic secretions [Lynch MJ 94, 97, Averna et al, Kline et al, Serkova et al]. It was observed that prostatic fluid NMR spectra are less complex as compared to seminal fluid spectra in other male genital diseases [Lynch MJ]. Ratio of Cit to spermine in CaP patients was different compared to the control group [Lynch MJ]. Serkova et al observed that the absolute concentrations of Cit, mI, and spermine were highly predictive of CaP and inversely related to the risk of CaP [Serkova et al]. Due to limitation and disadvantages of PSA as a screening test, it was suggested that 1H NMRS could be used a rapid and non-invasive screening technique for CaP detection by measuring Cit in seminal fluid [Averna et al, Kline et al].
In order to delineate the human BPH from CaP and PIN the prostate tissue extracts had been studied using 1H NMR [Fowler et al, Schiebler et al, Hahn et al, Swindle et al 2008 van der graff et al, Koutcher et al]. Besides preclinical and fluid based studies, metabolite peak area ratios, Cit and Cho resonances were reported for prostate tissue extracts by several groups to differentiate the various prostatic disorders [Swindle 2003 et al, Fowler et al]. However, these metabolites are not enough to distinguish the various prostatic disorders [Swindle et al 2003]. Further, it was reported that using the intensity of resonances of Cho, creatine, lipid, lysine, glutamate, spermine and taurine, it was possible to diagnose CaP with a high degree of sensitivity and specificity [Hahn et al, Swindle et 2003, van der graff et al]. Proton 1D and 2D NMR spectra from glandular BPH, stromal BPH, PIN, and CaP have been analyzed and assignments are well reported [Swindle et al 2008, Mountford et al].
HRMAS of prostate
Looking into the excellent method of choice, in vitro NMR studies has great capability in understanding the prostatic disorders. Future studies would be more based on 1H- and 13C-HRMAS. Recently, lacatate and alanine are found to be metabolic biomarker for CaP and which could assist in CaP detection using hyperpolarised 13C MRSI. Moreover, it is expected that by looking the ability of NMR methods, it may be helpful in better understanding in CaP in vivo, however more studies and general consensus is required to bring the NMR techniques as a screening method for CaP detection.
In vitro NMR has huge potential as it provides the pathological classification, all low molecular weight metabolites in complex mixtures, metabolomic and metabonomics application under one roof for the prostatic disorders also distinguish between benign and malignant prostate could be possible under purview of NMR.
The authors searched PubMed database using the following terms: prostate or prostate cancer AND (NMR OR MR OR MRS OR MRI OR magnetic resonance OR magic angle spinning OR HRMAS OR metabolomics OR metabolomic). The last search was performed in July 2011.
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