Magic angle spinning NMR spectroscopic metabolic profiling of gall bladder tissues for differentiating malignant from benign disease

Gall bladder tissue specimens obtained from 112 patients were examined by high resolution magic angle spinning (HR-MAS) NMR spectroscopy. Fifty one metabolites were identified by combination of one and two-dimensional NMR spectra. To our knowledge, this is the first report on metabolic profiling of gall bladder tissues using HR-MAS NMR spectroscopy. Metabolic profiles were evaluated for differentiation between benign Chronic Cholecystitis (CC, n = 66) and xantho-granulomatous cholecystitis (XGC, n = 21) and malignant gall bladder cancer (GBC, n = 25). Increase in choline containing compounds, amino acids, taurine, nucleotides and lactate as common metabolites were observed in malignant tissues whereas lipid content was found low as compared to benign tissues. Principal component analysis obtained from the NMR data showed clear distinction between CC and GBC tissue specimens; however, 27 % of XGC tissues were classified with GBC. The partial least square discriminant analysis (PLS-DA) multivariate analysis between benign (CC, XGC) and malignant (GBC) on the training data set (CC; n = 51, XGC; n = 15, GBC; n = 19 tissues specimens) provided 100 % sensitivity and 94.12 % specificity. This PLS-DA model when executed on the spectra of unknown tissue specimens (CC; n = 15, XGC; n = 6, GBC; n = 6) classified them into the three histological categories with more than 95 % of diagnostic accuracy. Non-invasive in vivo MRS technique may be used in future to differentiate between benign (CC and XGC) and malignant (GBC) gall bladder diseases.     

Spermine and Citrate as Metabolic Biomarkers for Assessing Prostate Cancer Aggressiveness

Separating indolent from aggressive prostate cancer is an important clinical challenge for identifying patients eligible for active surveillance, thereby reducing the risk of overtreatment. The purpose of this study was to assess prostate cancer aggressiveness by metabolic profiling of prostatectomy tissue and to identify specific metabolites as biomarkers for aggressiveness. Prostate tissue samples (n = 158, 48 patients) with a high cancer content (mean: 61.8%) were obtained using a new harvesting method, and metabolic profiles of samples representing different Gleason scores (GS) were acquired by high resolution magic angle spinning magnetic resonance spectroscopy (HR-MAS). Multivariate analysis (PLS, PLS-DA) and absolute quantification (LCModel) were used to examine the ability to predict cancer aggressiveness by comparing low grade (GS = 6, n = 30) and high grade (GS≥7, n = 81) cancer with normal adjacent tissue (n = 47). High grade cancer tissue was distinguished from low grade cancer tissue by decreased concentrations of spermine (p = 0.0044) and citrate (p = 7.73·10⁻⁴), and an increase in the clinically applied (total choline+creatine+polyamines)/citrate (CCP/C) ratio (p = 2.17·10⁻⁴). The metabolic profiles were significantly correlated to the GS obtained from each tissue sample (r = 0.71), and cancer tissue could be distinguished from normal tissue with sensitivity 86.9% and specificity 85.2%. Overall, our findings show that metabolic profiling can separate aggressive from indolent prostate cancer. This holds promise for the benefit of applying in vivo magnetic resonance spectroscopy (MRS) within clinical MR imaging investigations, and HR-MAS analysis of transrectal ultrasound-guided biopsies has a potential as an additional diagnostic tool.     

Discrimination of Basal Cell Carcinoma from Normal Skin Tissue Using High-Resolution Magic Angle Spinning 1H NMR Spectroscopy

High-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) spectroscopy is a useful tool for investigating the metabolism of various cancers. Basal cell carcinoma (BCC) is the most common skin cancer. However, to our knowledge, data on metabolic profiling of BCC have not been reported in the literature. The objective of the present study was to investigate the metabolic profiling of cutaneous BCC using HR-MAS ¹H NMR spectroscopy. HR-MAS ¹H NMR spectroscopy was used to analyze the metabolite profile and metabolite intensity of histopathologically confirmed BCC tissues and normal skin tissue (NST) samples. The metabolic intensity normalized to the total spectral intensities in BCC and NST was compared, and multivariate analysis was performed with orthogonal partial least-squares discriminant analysis (OPLS-DA). P values < 0.05 were considered statistically significant. Univariate analysis revealed 9 metabolites that showed statistically significant difference between BCC and NST. In multivariate analysis, the OPLS-DA models built with the HR-MAS NMR metabolic profiles revealed a clear separation of BCC from NST. The receiver operating characteristic curve generated from the results revealed an excellent discrimination of BCC from NST with an area under the curve (AUC) value of 0.961. The present study demonstrated that the metabolite profile and metabolite intensity differ between BCC and NST, and that HR-MAS ¹H NMR spectroscopy can be a valuable tool in the diagnosis of BCC.     

A 7-day oral supplementation with branched-chain amino acids was ineffective to prevent muscle damage during a marathon

The aim of this study was to determine the effectiveness of a 7-day oral supplementation with branched-chain amino acids (BCAA) to prevent muscle damage during a marathon. Forty-six experienced runners were randomly divided into two groups, one with BCAA supplementation (n = 25, supplemented with 5 g day^?1 of powdered 1:0.5:0.5 leucine:isoleucine:valine, during the 7 days prior to the competition) and the other as a control group (n = 21, supplemented with an isocaloric placebo). Before the marathon race and within 3 min of finishing, leg muscle power was measured with a maximal countermovement jump and a urine sample was obtained. During the race, running pace was measured by means of a time-chip. Myoglobin concentration was determined in the urine samples as an indirect marker of muscle damage. A visual analog scale (0–10 points) was used to assess leg muscle pain during the race. In the BCAA group, the mean running pace during the marathon was similar to the control group (3.3 ± 0.4 vs. 3.3 ± 0.5 m s^?1, respectively, 0.98). The pre- to post-race reduction in muscle power was similar in both BCAA and control groups (?23.0 ± 16.1 vs. ?17.3 ± 13.8 %, P = 0.13). Post-race urine myoglobin concentration was similar in both BCAA and control groups (5.4 ± 7.5 vs. 4.5 ± 8.6 μg mL^?1, P = 0.70). Finally, there were no differences between groups in the perceived muscle pain during the race (6 ± 1 vs. 5 ± 1 points, P = 0.80). A 7-day supplementation of BCAA (5 g day^?1) did not increase the running performance during a marathon. Furthermore, BCAA supplementation was ineffective to prevent muscle power loss, muscle damage or perceived muscle pain during a marathon race.     

Proton HR-MAS MR spectroscopy of oral squamous cell carcinoma tissues: an ex vivo study to identify malignancy induced metabolic fingerprints

Oral squamous cell carcinoma (SCC) represents more than 90% of all head and neck cancers as reported by Hermans (Cancer Imaging, 5(Spec No A), S52–S57, 2005), which draws attention of investigative research for novel predictive metabolic biomarkers to understand the malignancy induced biochemical perturbations occurring at molecular level. In the present work, proton HR-MAS NMR spectroscopic studies have been performed on resected human oral SCC tumor tissues, its neighboring margins and bed tissues (n = 159), obtained from 36 patients (n = 27 training set; n = 9 unknown test set), for the identification of metabolic fingerprints. The proton NMR spectra were then subjected to chemometric unsupervised PCA and supervised OSC-filtered PCA and PLS-DA multivariate analysis. Application of PLS-DA on orthogonally signal corrected training data-set (n = 120 tissue specimens; 27 patients) allowed >95% correct classification of malignant tissues from benign samples with >98% specificity and sensitivity. The OSC-PLS-DA model thus constructed was used to predict the class membership of unknown tissue specimens (n = 39) obtained from nine patients. These tissue samples were correctly predicted in its respective histological categories with 97.4% diagnostic accuracy. The regression coefficients obtained from OSC-filtered PLS-DA model indicated that malignant tissues had higher levels of glutamate, choline, phosphocholine, lactate, acetate, taurine, glycine, leucine, lysine, isoleucine and alanine, and lower levels of creatine and PUFA, representing altered metabolic processes (lipidogenesis, protein synthesis, and volume regulation) during tumor progression. Thus proton HR-MAS MR spectroscopy could efficiently identify the metabolic perturbations of malignant tumor from non-malignant bed and margins tissue specimens, which may be helpful in understanding the extent of tumor penetration in neighboring tissues.     

Response of Degarelix treatment in human prostate cancer monitored by HR-MAS <Superscript>1</Superscript>H NMR spectroscopy

Introduction

The androgen receptor (AR) is the master regulator of prostate cancer cell metabolism. Degarelix is a novel gonadotrophin-releasing hormone blocker, used to decrease serum androgen levels in order to treat advanced human prostate cancer. Little is known of the rapid metabolic response of the human prostate cancer tissue samples to the decreased androgen levels.

Objectives

To investigate the metabolic responses in benign and cancerous tissue samples from patients after treatment with Degarelix by using HRMAS ¹H NMR spectroscopy.

Methods

Using non-destructive HR-MAS ¹H NMR spectroscopy we analysed the metabolic changes induced by decreased AR signalling in human prostate cancer tissue samples. Absolute concentrations of the metabolites alanine, lactate, glutamine, glutamate, citrate, choline compounds [t-choline = choline + phosphocholine (PC) + glycerophosphocholine (GPC)], creatine compounds [t-creatine = creatine (Cr) + phosphocreatine (PCr)], taurine, myo-inositol and polyamines were measured in benign prostate tissue samples (n = 10), in prostate cancer specimens from untreated patients (n = 7) and prostate cancer specimens from patients treated with Degarelix (n = 6).

Results

Lactate, alanine and t-choline concentrations were significantly elevated in high-grade prostate cancer samples when compared to benign samples in untreated patients. Decreased androgen levels resulted in significant decreases of lactate and t-choline concentrations in human prostate cancer biopsies.

Conclusions

The reduced concentrations of lactate and t-choline metabolites due to Degarelix could in principle be monitored by in vivo ¹H MRS, which suggests that it would be possible to monitor the effects of physical or chemical castration in patients by that non-invasive method.

     

Profiling whole microalgal cells by high-resolution magic angle spinning (HR-MAS) magnetic resonance spectroscopy

Microalgae are a rich source of high value compounds such as carbohydrates, lipids, proteins and bioactive compounds. In particular, microalgae have been identified as a potentially important resource for carbon-capture and as a feedstock for green biofuels. Successful cultivation of microalgae can occur under a variety of nutrient and environmental conditions with each condition producing a unique distribution of compounds. In order to steer the cultivation towards a particular distribution of compounds, rapid and accurate methods for compound identification are required. Current methods for determining the absolute quantity of each component are time consuming and arduous making cultivation optimization impractical. High-resolution magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy offers a robust and rapid screening method capable of ascertaining the absolute quantity of each component with minimal sample manipulation. Sample preparation consists of harvested, centrifuged and freeze-dried whole-cell Nannochloropsis granulata from large-scale photobioreactors being accurately weighed and rehydrated with deuterium oxide and placed in an HR-MAS rotor. One-dimensional HR-MAS NMR spectra were recorded under quantitative conditions to determine the lipid and carbohydrate profile of the microalgae. The total time per sample for preparation, data acquisition and analysis was approximately 1 h. Changes in resonance profiles corresponding to varying proportions of saturated and polyunsaturated fatty acids were correlated to the time of harvest. In addition, standard two dimensional experiments were used to identify the major carbohydrate components. HR-MAS NMR spectroscopy has been used to profile the lipid and carbohydrate content of N. granulata and we have begun to establish methodologies for quality analysis/quality control for cultivation of various microalgal strains.     

Assessment of a 1H high-resolution magic angle spinning NMR spectroscopy procedure for free sugars quantification in intact plant tissue

In most plants, sucrose is the primary product of photosynthesis, the transport form of assimilated carbon, and also one of the main factors determining sweetness in fresh fruits. Traditional methods for sugar quantification (mainly sucrose, glucose and fructose) require obtaining crude plant extracts, which sometimes involve substantial sample manipulation, making the process time-consuming and increasing the risk of sample degradation. Here, we describe and validate a fast method to determine sugar content in intact plant tissue by using high-resolution magic angle spinning nuclear magnetic resonance spectroscopy (HR-MAS NMR). The HR-MAS NMR method was used for quantifying sucrose, glucose and fructose in mesocarp tissues from melon fruits (Cucumis melo var. reticulatus and Cucumis melo var. cantalupensis). The resulting sugar content varied among individual melons, ranging from 1.4 to 7.3 g of sucrose, 0.4–2.5 g of glucose; and 0.73–2.83 g of fructose (values per 100 g fw). These values were in agreement with those described in the literature for melon fruit tissue, and no significant differences were found when comparing them with those obtained using the traditional, enzymatic procedure, on melon tissue extracts. The HR-MAS NMR method offers a fast (usually <30 min) and sensitive method for sugar quantification in intact plant tissues, it requires a small amount of tissue (typically 50 mg fw) and avoids the interferences and risks associated with obtaining plant extracts. Furthermore, this method might also allow the quantification of additional metabolites detectable in the plant tissue NMR spectrum.     

1H NMR based metabolite profiling for optimizing the ethanol extraction of Wolfiporia cocos

Metabolite profiling of Wolfiporia cocos (family: Polyporaceae) had been much advancement in recent days, and its analysis by nuclear magnetic resonance (NMR) spectroscopy has become well established. However, the highly important trait of W. cocos still needs advanced protocols despite some standardization. Partial least squares discriminant analysis (PLS-DA) was used as the multivariate statistical analysis of the ¹H NMR data set. The PLS-DA model was validated, and the key metabolites contributing to the separation in the score plots of different ethanol W. cocos extract. ¹H NMR spectroscopy of W. cocos identified 33 chemically diverse metabolites in D₂O, consisting of 13 amino acids, 11 organic acids 2 sugars, 3 sugar alcohols, 1 nucleoside, and 3 others. Among these metabolites, the levels of tyrosine, proline, methionine, sarcosine, choline, acetoacetate, citrate, 4-aminobutyrate, aspartate, maltose, malate, lysine, xylitol, lactate threonine, leucine, valine, isoleucine, uridine, guanidoacetate, arabitol, mannitol, glucose, and betaine were increased in the 95% ethanol extraction sample compared with the levels in other samples, whereas level of acetate, phenylalanine, alanine, succinate, and fumarate were significantly increased in the 0% ethanol extraction sample. A biological triterpenoid, namely pachymic acid, was detected from different ethanol P. cocos extract using ¹H-NMR spectra were found in CDCl₃. This is the first report to perform the metabolomics profiling of different ethanol W. cocos extract. These researches suggest that W. cocos can be used to obtain substantial amounts of bioactive ingredients for use as potential pharmacological and nutraceuticals agents.     

1H-NMR-based metabolomics for cancer targeting and metabolic engineering –A review

Nuclear magnetic resonance (NMR) spectroscopy acts as the best tool that can be used in tissue engineering scaffolds to investigate unknown metabolites. Moreover, metabolomics is a systems approach for examining in vivo and in vitro metabolic profiles, which promises to provide data on cancer metabolic alterations. However, metabolomic profiling allows for the activity of small molecules and metabolic alterations to be measured. Furthermore, metabolic profiling also provides high-spectral resolution, which can then be linked to potential metabolic relationships. An altered metabolism is a hallmark of cancer that can control many malignant properties to drive tumorigenesis. Metabolite targeting and metabolic engineering contribute to carcinogenesis by proliferation, and metabolic differentiation. The resulting the metabolic differences are examined with traditional chemometric methods such as principal component analysis (PCA), and partial least squares-discriminate analysis (PLS-DA). In this review, we examine NMR-based activity metabolomic platforms that can be used to analyze various fluxomics and for multivariant statistical analysis in cancer. We also aim to provide the reader with a basic understanding of NMR spectroscopy, cancer metabolomics, target profiling, chemometrics, and multifunctional tools for metabolomics discrimination, with a focus on metabolic phenotypic diversity for cancer therapeutics.     

Cholesterol and its esters as serum biomarkers in malignant obstructive jaundice: a single step 1H NMR metabolomic approach

The paucity of biomarkers for malignant obstructive jaundice results in formidable morbidity and mortality rates. Therefore, alternative diagnostic measures are required for improved clinical interpretation and better peri-operative management of patients. In the present study, ¹H NMR-based metabolomic approach has been applied to investigate serum and bile based metabolic biomarkers in benign and malignant causes of obstructive jaundice (OBJ). Serum and bile specimens from benign OBJ patients (n = 28), malignant OBJ patients (n = 36) and serum of healthy controls (n = 57) were analysed by ¹H NMR spectroscopy. Quantitation of eight serum metabolites (isobutyrate, lactate, alanine, acetone, glutamine, creatine, threonine and 1-methylhistidine) was carried out. A newer and rapid single step NMR based semi-quantitative ratio analysis of serum total cholesterol (tCho), cholesterol (Chol) and cholesterol ester (CE) were performed in deuterated dimethyl sulfoxide-d₆. In bile, total bile acids, cholesterol, phosphatidylcholine, glycerophosphatidylcholine and urea were quantified. The effect of benign and malignant OBJ on small metabolites and lipids were statistically analysed by Mann–Whitney U test and multivariate discriminant function analysis. It was found that malignancy could be differentiated from benign cases of OBJ with a correct classification of 85.7 % when eight serum metabolites in combination with ratios of serum cholesterol were analysed. Significant alterations in serum tCho, Chol, CE and serum metabolites may have potential for early and differential non-invasive diagnosis of malignant and benign OBJ cases. It will further augment the novel insights of local and systemic effects in OBJ patients.     

Effects of rumen-protected methionine on plasma amino acid concentrations during a period of weight loss for late gestating beef heifers

This study determined changes in plasma amino acid concentration in late-gestating (beginning 58 ± 1.02 days prior to calving), primiparous, winter-grazing range heifers receiving wheat middling-based supplement without (CON) or with rumen-protected methionine (MET) to provide 15 g dl-MET each day. Plasma was collected on days ?2 and 0 (start of MET supplementation just prior to individually receiving supplement at 0700 hours). Plasma was sampled again on days 40, 42 and 44 prior to supplementation at 0700 and 1100 hours (4 h after receiving daily supplement). Data were analyzed with cow as the experimental unit. Continuous variables were analyzed by the main effects of treatment, date, or time and their interaction when appropriate. Comparable BW (P = 0.32) and BCS (P = 0.83) over the 44-day metabolism trial were found between both CON- and MET-fed heifers. MET-supplemented heifers had greater (P < 0.01) plasma concentrations of methionine indicating that the rumen-protection technology successfully delivered methionine to the small intestine. Supplementation with rumen-protected dl-MET caused a significant supplement × date interaction for glutamine (P = 0.03), glycine (P = 0.02), methionine (P < 0.01), and serine (P = 0.05). In addition, trends for supplement × date interactions were detected for leucine (P = 0.07), threonine (P = 0.09), valine (P = 0.08), total amino acids (TAA; P = 0.08), non essential amino acids (NEAA; P = 0.08), branched chain amino acids (BCAA; P = 0.08), and glucogenic amino acids (GLUCO; P = 0.08). These results suggest that the BCAA (leucine and valine) were utilized more efficiently with MET supplemented heifers compared to CON supplemented heifers. Plasma AA concentrations for glutamic acid (P < 0.01), histidine (P = 0.01), tyrosine (P < 0.01), and EAA (P < 0.01), all decreased throughout the study. These results further confirm methionine is a limiting amino acid in forage fed late-gestating heifers and further suggests the limitation when grazing dormant range forages as shown by improved utilization of other plasma amino acids when supplemental methionine was provided.     

A proton nuclear magnetic resonance-based metabolomic approach in IgA nephropathy urinary profiles

Immunoglobulin A nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide. Both one-dimensional NOESY and transverse-relaxation filter CPMG NMR spectra were recorded to investigate the urine metabolome of 24 IgAN patients and to detect altered metabolic profiles in comparison with 68 healthy matched controls. The spectral data were analyzed using multivariate statistical techniques. The analysis revealed that the NMR spectra of IgAN patients were statistically different from those of the controls (P = 4 × 10^?7 for 1D-NOESY and P = 2 × 10^?7 for CPMG). The robustness of the determined statistical model was confirmed by its predictive performance (for the 1D-NOESY dataset: sensitivity = 67 %, specificity = 95 %; for the CPMG dataset sensitivity = 60 %, specificity = 94 %). For the first time we found metabolites, including betaine and citrate, that are differentially modulated in IgAN patients compared to controls and that may be directly involved in the pathogenesis of IgAN. These metabolites may influence, directly or indirectly, the TNF-α, a regulating factor of the Th1/Th2 cell balance that is relevant in the pathology. The involvement of metabolites such as betaine and citrate in TNF-α regulation supports the power of the identified metabolic profiles to discern IgAN from controls.     

Peripheral metabolic abnormalities of lipids and amino acids implicated in increased risk of suicidal behavior in major depressive disorder

Suicide is the most serious consequence of major depressive disorder (MDD), yet a vast majority of MDD patients never attempt nor commit suicide. This discrepancy suggests a predisposition to suicidal behavior independent of MDD. However, the molecular basis of this predisposition remains largely unknown, hampering development of specific and targeted treatment of MDD patients at risk for suicide. A proton nuclear magnetic resonance (¹H NMR)-based metabonomic approach was used to capture metabolic perturbations related to suicide predisposition in the context of MDD. ¹H NMR spectra of plasma sampled from drug-naïve depressed suicide attempters (n = 21), non-attempters (n = 35), and healthy controls (n = 35) were recorded and analyzed through a multivariate statistical approach. Multivariate statistical analysis demonstrated that the depressed suicide attempter group was significantly distinguishable from the depressed non-attempter group and controls group. Several key metabolites, including lipids (low-density lipoprotein, very low-density lipoprotein, cholesterol and unsaturated lipid), lipid metabolism-related molecules (glucose, pyruvate and lactate) and amino acids (alanine, glycine and taurine) responsible for discriminating depressed suicide attempters from the nonattempters and controls were identified. This study is the first to indicate that peripheral perturbations in lipid and amino acid metabolism may be implicated in the predisposition to suicide in MDD patients.     

Synthesis and hyperpolarisation of eNOS substrates for quantification of NO production by 1H NMR spectroscopy

Hyperpolarization enhances the intensity of the NMR signals of a molecule, whose in vivo metabolic fate can be monitored by MRI with higher sensitivity. SABRE is a hyperpolarization technique that could potentially be used to image nitric oxide (NO) production in vivo. This would be very important, because NO dysregulation is involved in several pathologies, including cardiovascular ones. The nitric oxide synthase (NOS) pathway leads to NO production via conversion of l-arginine into l-citrulline. NO is a free radical gas with a short half-life in vivo (≈5 s), therefore direct NO quantification is challenging. An indirect method – based on quantifying conversion of an l-Arg- to l-Cit-derivative by ¹H NMR spectroscopy – is herein proposed. A small library of pyridyl containing l-Arg derivatives was designed and synthesised. In vitro tests showed that compounds 4a–j and 11a–c were better or equivalent substrates for the eNOS enzyme (NO₂^? production = 19–46 μM) than native l-Arg (NO₂^? production = 25 μM). Enzymatic conversion of l-Arg to l-Cit derivatives could be monitored by ¹H NMR. The maximum hyperpolarization achieved by SABRE reached 870-fold NMR signal enhancement, which opens up exciting future perspectives of using these molecules as hyperpolarized MRI tracers in vivo.     

Plasma-free amino acid profiling of cervical cancer and cervical intraepithelial neoplasia patients and its application for early detection

In this study, plasma-free amino acid profiles were used to investigate pre-cancerous cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (CSCC) metabolic signatures in plasma. Additionally, the diagnostic potential of these profiles was assessed, as well as their ability to provide novel insight into CSCC metabolism and systemic effects. Plasma samples from CIN patients (n = 26), CSCC patients (n = 22), and a control healthy group (n = 35) were analyzed by high-performance liquid chromatography, and their spectral profiles were subjected to the t test for statistical significance. Potential metabolic biomarkers were identified using database comparisons that examine the significance of metabolites. Compared with healthy controls, patients with CIN and CSCC demonstrated lower levels of plasma amino acids; plasma levels of arginine and threonine were increased in CIN patients but were decreased in cervical cancer patients. Additionally, the levels of a larger group of amino acids (aspartate, glutamate, asparagine, serine, glycine, histidine, taurine, tyrosine, valine, methionine, lysine, isoleucine, leucine, and phenylalanine) were gradually reduced from CIN to invasive cancer. These findings suggest that plasma-free amino acid profiling has great potential for improving cancer screening and diagnosis and for understanding disease pathogenesis. Plasma-free amino acid profiles may have the potential be used to determine cancer diagnoses in the early stage from a single blood sample and may enhance our understanding of its mechanisms.     

NMR analysis of the CSF and plasma metabolome of rigorously matched amyotrophic lateral sclerosis,Parkinson’s disease and control subjects

Introduction

Amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD) are two severe neurodegenerative disorders for which the disease mechanisms are poorly understood and reliable biomarkers are absent.

Objectives

To identify metabolite biomarkers for ALS and PD, and to gain insights into which metabolic pathways are involved in disease.

Methods

Nuclear magnetic resonance (NMR) metabolomics was utilized to characterize the metabolite profiles of cerebrospinal fluid (CSF) and plasma from individuals in three age, gender, and sampling-date matched groups, comprising 22 ALS, 22 PD and 28 control subjects.

Results

Multivariate analysis of NMR data generated robust discriminatory models for separation of ALS from control subjects. ALS patients showed increased concentrations of several metabolites in both CSF and plasma, these are alanine (CSF fold change = 1.22, p = 0.005), creatine (CSF-fc = 1.17, p = 0.001), glucose (CSF-fc = 1.11, p = 0.036), isoleucine (CSF-fc = 1.24, p = 0.002), and valine (CSF-fc = 1.17, p = 0.014). Additional metabolites in CSF (creatinine, dimethylamine and lactic acid) and plasma (acetic acid, glutamic acid, histidine, leucine, pyruvate and tyrosine) were also important for this discrimination. Similarly, panels of CSF-metabolites that discriminate PD from ALS and control subjects were identified.

Conclusions

The results for the ALS patients suggest an affected creatine/creatinine pathway and an altered branched chain amino acid (BCAA) metabolism, and suggest links to glucose and energy metabolism. Putative metabolic markers specific for ALS (e.g. creatinine and lactic acid) and PD (e.g. 3-hydroxyisovaleric acid and mannose) were identified, while several (e.g. creatine and BCAAs) were shared between ALS and PD, suggesting some overlap in metabolic alterations in these disorders.

     

1H HRMAS-NMR based metabolic fingerprints for discrimination of cheeses based on sensory qualities

In this study, the ¹H HRMAS-NMR (High-resolution Magic Angle Spinning-Nuclear Magnetic Resonance) spectra of 52 cheese samples obtained from the South Korean dairy farms were evaluated for their metabolic profiling and intensities associating with the sensory qualities. The NMR profiles displayed a broad range of compounds comprising amino acids, carbohydrates, organic acids, and phospholipids. Afterwards, the cheese samples were categorized into three groups (more likeness - G1, moderate likeness - G2, less likeness - G3), in relating to their sensory scores. The NMR data of the samples were later investigated through multivariate statistical tools to define the variations in metabolic fingerprints of every cheese sample and their intensities hailing in individual sensory groups. The unsupervised PCA employing all cheese samples unveiled the uniqueness in metabolite profiles of the brown and cheddar type cheeses (outliers). Moreover, Gouda and other types of cheeses displayed samples positioning in respective of their metabolite profiles. The pairwise comparison of sensory groups in the supervised models perceived better separation in OPLS-DA than PLS-DA. The corresponding VIP (PLS-DA) and loading (OPLS-DA) plots revealed amino acids and organic acids (lactate, citrate) as significant variables. The discrimination of G 1 Gouda type of cheeses against G 2 and G 3 was highly associated with their citrate levels. Further investigation using heatmaps displayed clear differentiation between each sensory group in terms of the levels of amino acids, lactate, citrate, phospholipids, and glycerol, conveying these variations are likely due to proteolytic and metabolic processes in cheese ripening. This study concluded that ¹H HRMAS-NMR metabolite profile of the Korean cheeses is consistence with their sensory qualities. Further, the candidate metabolites identified in this study confers their potential application as biomarkers in cheese industries for faster and effective validation of sensory characteristics.     

Influence of triacontanol and jasmonic acid on metabolomics during early stages of root induction in cultured tissue of tomato (<Emphasis Type="Italic">Lycopersicon esculentum)</Emphasis>

Influence of n-triacontanol (TRIA) and jasmonic acid (JA) on metabolic profiling during root morphogenesis was studied in Lycopersicon esculentum (cv. PKM-1). Proton nuclear magnetic resonance (¹H NMR) based metabolomics was employed to investigate the variations in metabolic profile. Chenomx NMR suite v.8.1 was used to identify and quantify metabolites based on their respective signature spectra. The levels of 47 metabolites were monitored for 72 h at specific time intervals (0, 3, 6, 9, 12, 24, 36, 48 and 72 h). Principal component analysis was performed to determine the variations in the metabolic profile between control and treatments during in vitro rhizogenesis. TRIA was observed to promote early root emergence (24 h) and also influence the metabolic variation during rhizogenesis between 9 and 24 h post exposure. Compounds such as IAA, ATP, NADPH, UDP-N-acetylglucosamine and gallate predominated at 9 h. Unlike TRIA, JA was unable to promote an early root induction. However, it influenced the synthesis of a relatively higher concentration of IAA at 6 h when compared to ATP, NADPH and trigonelline at 9 h. In the presence of both TRIA and JA (TRIA?+?JA), significant changes in the metabolic profiles were observed 24 h post exposure and the rooting was observed only after 72 h. The study suggests that TRIA may accelerate in vitro rhizogenesis of cultured tomato tissues by mainly increasing the synthesis of other growth promoting metabolites. But in the presence of JA, TRIA’s effect appears to be reduced.     

Ruthenium(II) carbonyl complexes containing S-methylisothiosemicarbazone based tetradentate ligand: synthesis, characterization and biological applications

A series of hexa-coordinated ruthenium(II) complexes of the type [Ru(CO)(B)L ⁿ ] (n = 1–4; B = PPh₃, AsPh₃ or Py) have been synthesized by reacting dibasic quadridentate Schiff base ligands H₂L ⁿ (n = 1–4) with starting complexes [RuHCl(CO)(EPh₃)₂(B)] (E = P or As; B = PPh₃, AsPh₃ or Py). The synthesized complexes were characterized using elemental and various spectral studies including UV–Vis, FT-IR, NMR (¹H, ¹³C and ³¹P) and mass spectroscopy. An octahedral geometry was tentatively proposed for all the complexes based on the spectral data obtained. The experiments on antioxidant activity showed that the ruthenium(II) S-methylisothiosemicarbazone Schiff base complexes exhibited good scavenging activity against various free radicals (DPPH, OH and NO). The in vitro cytotoxicity of these complexes has been evaluated by MTT assay. The results demonstrate that the complexes have good anticancer activities against selected cancer cell line, human breast cancer cell line (MCF-7) and human skin carcinoma cell line (A431). The DNA cleavage studies showed that the complexes have better cleavage of pBR 322 DNA.