Altered expression of the L-arginine/nitric oxide pathway in ovarian cancer: metabolic biomarkers and biological implications.

MOTIVATION: Ovarian cancer (OC) is a highly lethal gynecological malignancy. Extensive research has shown that OC cells undergo significant metabolic alterations during tumorigenesis. In this study, we aim to leverage these metabolic changes as potential biomarkers for assessing ovarian cancer. METHODS: A functional module-based approach was utilized to identify key gene expression pathways that distinguish different stages of ovarian cancer (OC) within a tissue biopsy cohort. This cohort consisted of control samples (n = 79), stage I/II samples (n = 280), and stage III/IV samples (n = 1016). To further explore these altered molecular pathways, minimal spanning tree (MST) analysis was applied, leading to the formulation of metabolic biomarker hypotheses for OC liquid biopsy. To validate, a multiple reaction monitoring (MRM) based quantitative LCMS/MS method was developed. This method allowed for the precise quantification of targeted metabolite biomarkers using an OC blood cohort comprising control samples (n = 464), benign samples (n = 3), and OC samples (n = 13). RESULTS: Eleven functional modules were identified as significant differentiators (false discovery rate, FDR < 0.05) between normal and early-stage, or early-stage and late-stage ovarian cancer (OC) tumor tissues. MST analysis revealed that the metabolic L-arginine/nitric oxide (L-ARG/NO) pathway was reprogrammed, and the modules related to "DNA replication" and "DNA repair and recombination" served as anchor modules connecting the other nine modules. Based on this analysis, symmetric dimethylarginine (SDMA) and arginine were proposed as potential liquid biopsy biomarkers for OC assessment. Our quantitative LCMS/MS analysis on our OC blood cohort provided direct evidence supporting the use of the SDMA-to-arginine ratio as a liquid biopsy panel to distinguish between normal and OC samples, with an area under the ROC curve (AUC) of 98.3%. CONCLUSION: Our comprehensive analysis of tissue genomics and blood quantitative LC/MSMS metabolic data shed light on the metabolic reprogramming underlying OC pathophysiology. These findings offer new insights into the potential diagnostic utility of the SDMA-to-arginine ratio for OC assessment. Further validation studies using adequately powered OC cohorts are warranted to fully establish the clinical effectiveness of this diagnostic test.

High-throughput quantitation of serological dimethylarginines by LC/MS/MS: Potential cardiovascular biomarkers for rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by systemic inflammation of the joints and extra-articular tissues. The incidence of cardiovascular disease (CVD) remains the main cause of morbidity and mortality in patients with RA. Despite the development of new therapeutics targeting the articular manifestations, the relief of the cardiovascular burden is still an unmet medical need during the management of RA. So, the early prognosis of RA-associated CVD plays a crucial role in improving the clinical outcomes of RA patients. Recently, circulating dimethylarginines have gained attention as potential biomarkers for CVDs. Here, we present the development and validation of a high-throughput liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for simultaneous quantification of creatinine, arginine, and dimethylarginines in human serum within 2 mins by isotope dilution mass spectrometry. This method employed a protein precipitation method for rapid sample preparation, trichloroacetic acid (TCA)-based ion pairing chromatography for fast analyte separation, and multiple reaction monitoring (MRM) with stable isotope-labeled internal standards (ISs) for simultaneous quantitation. To assure the quality, our method was validated against the FDA guidelines for lower limit of quantitation (0.2 µM), linearity (square of coefficient correlation>0.99), precision (intra-&inter-assay imprecision < 10 %), accuracy (intra-&inter-assay inaccuracy < 10 %), sample preparation recovery (recovery ≥ 90 %), stability (instability < 10 %), matrix effect (signal suppression < 55 %), and carryover ( < 0.01 %). Afterward, we applied the validated method to a retrospective cross-sectional study. We aimed to evaluate the utility of serological dimethylarginines as potential cardiovascular biomarkers in the development of RA-associated CVD. Our results revealed that the serological ratio of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), an indicator of physiological arginine methylation status, was significantly elevated in patients with RA. This finding might provide value in detecting CVD to improve clinical outcomes in RA management.

Serological Phenotyping Analysis Uncovers a Unique Metabolomic Pattern Associated With Early Onset of Type 2 Diabetes Mellitus.

Background: Type 2 diabetes mellitus (T2DM) is a multifaceted disorder affecting epidemic proportion at global scope. Defective insulin secretion by pancreatic ß-cells and the inability of insulin-sensitive tissues to respond effectively to insulin are the underlying biology of T2DM. However, circulating biomarkers indicative of early diabetic onset at the asymptomatic stage have not been well described. We hypothesized that global and targeted mass spectrometry (MS) based metabolomic discovery can identify novel serological metabolic biomarkers specifically associated with T2DM. We further hypothesized that these markers can have a unique pattern associated with latent or early asymptomatic stage, promising an effective liquid biopsy approach for population T2DM risk stratification and screening. Methods: Four independent cohorts were assembled for the study. The T2DM cohort included sera from 25 patients with T2DM and 25 healthy individuals for the biomarker discovery and sera from 15 patients with T2DM and 15 healthy controls for the testing. The Pre-T2DM cohort included sera from 76 with prediabetes and 62 healthy controls for the model training and sera from 35 patients with prediabetes and 27 healthy controls for the model testing. Both global and targeted (amino acid, acylcarnitine, and fatty acid) approaches were used to deep phenotype the serological metabolome by high performance liquid chromatography-high resolution mass spectrometry. Different machine learning approaches (Random Forest, XGBoost, and ElasticNet) were applied to model the unique T2DM/Pre-T2DM metabolic patterns and contrasted with their effectiness to differentiate T2DM/Pre-T2DM from controls. Results: The univariate analysis identified unique panel of metabolites (n = 22) significantly associated with T2DM. Global metabolomics and subsequent structure determination led to the identification of 8 T2DM biomarkers while targeted LCMS profiling discovered 14 T2DM biomarkers. Our panel can effectively differentiate T2DM (ROC AUC = 1.00) or Pre-T2DM (ROC AUC = 0.84) from the controls in the respective testing cohort. Conclusion: Our serological metabolite panel can be utilized to identifiy asymptomatic population at risk of T2DM, which may provide utility in identifying population at risk at an early stage of diabetic development to allow for clinical intervention. This early detection would guide ehanced levels of care and accelerate development of clinical strategies to prevent T2DM.

Early-pregnancy prediction of risk for pre-eclampsia using maternal blood leptin/ceramide ratio: discovery and confirmation.

OBJECTIVE: This study aimed to develop a blood test for the prediction of pre-eclampsia (PE) early in gestation. We hypothesised that the longitudinal measurements of circulating adipokines and sphingolipids in maternal serum over the course of pregnancy could identify novel prognostic biomarkers that are predictive of impending event of PE early in gestation. STUDY DESIGN: Retrospective discovery and longitudinal confirmation. SETTING: Maternity units from two US hospitals. PARTICIPANTS: Six previously published studies of placental tissue (78 PE and 95 non-PE) were compiled for genomic discovery, maternal sera from 15 women (7 non-PE and 8 PE) enrolled at ProMedDx were used for sphingolipidomic discovery, and maternal sera from 40 women (20 non-PE and 20 PE) enrolled at Stanford University were used for longitudinal observation. OUTCOME MEASURES: Biomarker candidates from discovery were longitudinally confirmed and compared in parallel to the ratio of placental growth factor (PlGF) and soluble fms-like tyrosine kinase (sFlt-1) using the same cohort. The datasets were generated by enzyme-linked immunosorbent and liquid chromatography-tandem mass spectrometric assays. RESULTS: Our discovery integrating genomic and sphingolipidomic analysis identified leptin (Lep) and ceramide (Cer) (d18:1/25:0) as novel biomarkers for early gestational assessment of PE. Our longitudinal observation revealed a marked elevation of Lep/Cer (d18:1/25:0) ratio in maternal serum at a median of 23 weeks' gestation among women with impending PE as compared with women with uncomplicated pregnancy. The Lep/Cer (d18:1/25:0) ratio significantly outperformed the established sFlt-1/PlGF ratio in predicting impending event of PE with superior sensitivity (85% vs 20%) and area under curve (0.92 vs 0.52) from 5 to 25 weeks of gestation. CONCLUSIONS: Our study demonstrated the longitudinal measurement of maternal Lep/Cer (d18:1/25:0) ratio allows the non-invasive assessment of PE to identify pregnancy at high risk in early gestation, outperforming the established sFlt-1/PlGF ratio test.

High-throughput quantitation of serological ceramides/dihydroceramides by LC/MS/MS: Pregnancy baseline biomarkers and potential metabolic messengers.

Ceramides and dihydroceramides are sphingolipids that present in abundance at the cellular membrane of eukaryotes. Although their metabolic dysregulation has been implicated in many diseases, our knowledge about circulating ceramide changes during the pregnancy remains limited. In this study, we present the development and validation of a high-throughput liquid chromatography-tandem mass spectrometric method for simultaneous quantification of 16 ceramides and 10 dihydroceramides in human serum within 5 min. by using stable isotope-labeled ceramides as internal standards. This method employs a protein precipitation method for high throughput sample preparation, reverse phase isocratic elusion for chromatographic separation, and Multiple Reaction Monitoring for mass spectrometric detection. To qualify for clinical applications, our assay has been validated against the FDA guidelines for Lower Limit of Quantitation (1 nM), linearity (R2>0.99), precision (imprecision<15 %), accuracy (inaccuracy<15 %), extraction recovery (>90 %), stability (>85 %), and carryover (<0.01 %). With enhanced sensitivity and specificity from this method, we have, for the first time, determined the serological levels of ceramides and dihydroceramides to reveal unique temporal gestational patterns. Our approach could have value in providing insights into disorders of pregnancy.

Oral Ganglioside Supplement Improves Growth and Development in Patients with Ganglioside GM3 Synthase Deficiency.

Ganglioside GM3 synthase is a key enzyme involved in the biosynthesis of gangliosides. GM3 synthase deficiency (GM3D) causes an absence of GM3 and all downstream biosynthetic derivatives. The affected individuals manifest with severe irritability, intractable seizures, and profound intellectual disability. The current study is to assess the effects of an oral ganglioside supplement to patients with GM3D, particularly on their growth and development during early childhood. A total of 13 young children, 11 of them under 40 months old, received oral ganglioside supplement through a dairy product enriched in gangliosides, for an average of 34 months. Clinical improvements were observed in most children soon after the supplement was initiated. Significantly improved growth and development were documented in these subjects as average percentiles for weight, height, and occipitofrontal circumference increased in 1-2 months. Three children with initial microcephaly demonstrated significant catch-up head growth and became normocephalic. We also illustrated brief improvements in developmental and cognitive scores, particularly in communication and socialization domains through Vineland-II. However, all improvements seemed transient and gradually phased out after 12 months of supplementation. Gangliosides GM1 and GM3, although measureable in plasma during the study, were not significantly changed with ganglioside supplementation for up to 30 months. We speculate that the downstream metabolism of ganglioside biosynthesis is fairly active and the potential need for gangliosides in the human body is likely substantial. As we search for new effective therapies for GM3D, approaches to reestablish endogenous ganglioside supplies in the affected individuals should be considered.

Quantification of monosialogangliosides in human plasma through chemical derivatization for signal enhancement in LC-ESI-MS.

Gangliosides are found in abundance in the central nervous system of vertebrates. Their metabolic disruption and dysfunction are associated with various neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. In order to improve our understanding of the etiology of these diseases, analytical ganglioside assays with sufficient specificity and sensitivity in relevant biological matrices are required. In the present work we have developed and validated a reverse-phase ultra-performance liquid chromatography (UPLC)/tandem mass spectrometry (MS) method for determining monosialogangliosides GM1, GM2, and GM3 present in human plasma. Compared with our previous method, this method enhanced, by 15 fold, MS responses of the analytes by employing 2-(2-Pyridilamino)-ethylamine (PAEA) & 4-(4, 6-Dimethoxy-1, 3, 5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM)-based derivatization. The analytes and internal standards were derivatized with PAEA&DMTMM after extraction from plasma using a protein precipitation procedure. They were then purified using liquid-liquid partitioning. When the samples were then analyzed by UPLC-MS/MS with a multiple reaction monitoring (MRM) mode, we achieved superior sensitivity and specificity. This method was evaluated for extraction recovery, calibration linearity, precision, accuracy, and lower limit of quantification (LLOQ). The validated method was successfully applied to monitor monosialoganglioside levels in the plasma from patients with GM3 synthase deficiency. With significantly increased sensitivity, we have, for the first time, detected a significant amount of GM3 in the affected patients.

A new liquid chromatography/tandem mass spectrometry method for quantification of gangliosides in human plasma.

Gangliosides are a family of glycosphingolipids characterized by mono- or polysialic acid-containing oligosaccharides linked through 1,3- and 1,4-ß glycosidic bonds with subtle differences in structure that are abundantly present in the central nervous systems of many living organisms. Their cellular surface expression and physiological malfunction are believed to be pathologically implicated in considerable neurological disorders, including Alzheimer and Parkinson diseases. Recently, studies have tentatively elucidated that mental retardation or physical stagnation deteriorates as the physiological profile of gangliosides becomes progressively and distinctively abnormal during the development of these typical neurodegenerative syndromes. In this work, a reverse-phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay using standard addition calibration for determination of GM2, GM3, GD2, and GD3 in human plasma has been developed and validated. The analytes and internal standard were extracted from human plasma using a simple protein precipitation procedure. Then the samples were analyzed by reverse-phase ultra-performance liquid chromatography (UPLC)/MS/MS interfaced to mass spectrometry with electrospray ionization using a multiple reaction monitoring mode to obtain superior sensitivity and specificity. This assay was validated for extraction recovery, calibration linearity, precision, and accuracy. Our quick and sensitive method can be applied to monitor ganglioside levels in plasma from normal people and neurodegenerative patients.