Cell culture studies: a promising approach to metabolomic study of human aging

: With the increasing aging of the world’s population, a detailed study of the characteristics of aging, as well as the pathologies this process can lead to, is crucial. Therefore, there are several study models for the study of aging, from computational models to animals or even to cell cultures. The latter have shown high potential for aging studies as they are easier to handle, cheaper, do not require the same level of ethical consideration required for animal and human studies and present little biological heterogeneity when grown under the same conditions and in the same context population. For aging studies, these characteristics are a great advantage since cells have a huge variety of morphologic characteristics and markers that can be studies. Thus, the aim of this review is to offer an overview of the models used in studies of aging, with focus on cell culture models, presenting the advantages and disadvantages of cell culture in the study of aging, of what information can we extract of these studies and how cell studies can be compared with the other models.

Anaerobic Glycolysis and Glycogenolysis do not Release Protons and do not Cause Acidosis

Background: A metabolic pathway is composed of a series of enzymatic steps, where the product of each reaction becomes the substrate of the subsequent one. We can summarize the single reactions to obtain the overall equation of the metabolic pathway, suggesting its role in the metabolic network. Objective: In this short review, we aim at presenting our present knowledge on the biochemical features underlying the interrelation between acidosis occurring during anaerobic muscle contraction and the glycolytic and glycogenolytic pathways. We emphasize that both pathways per se are not acidifying processes. Conclusions: The review emphasizes the following points: i) The importance that single reactions, as well as the overall equation of a metabolic pathway, are balanced; ii) Unbalanced reactions lead to unbalanced overall equations, whose functions cannot be correctly understood; iii) Glycogen acts as the major fuel for muscle anaerobic contraction. Anaerobic glycogenolysis not only does not release protons, but it also consumes one proton; iv) When dealing with metabolic acidosis, it should be always recalled that protons are released by muscle ATPase activity, not by glycolysis or glycogenolysis.

Monitoring of Energy Metabolism by Organic Acid Profiling Analysis in Plasma of Type 2 Diabetic Mice

Objective:: This study was conducted to investigate energy metabolism based on changes in organic acids in diabetes and to establish a correlation between metabolites or bone microarchitecture and the glucose index in type 2 diabetic mice. Method:: Seven-week-old male C57BL/6 mice were randomly divided into a non-diabetic group and a diabetic group. The diabetic group was fed a high-fat diet (HFD) that induced insulin resistance for 5 weeks. Afterwards, diabetes was induced by a single streptozotocin injection. Both the groups were fed a normal diet and HFD diet for 9 weeks. Results:: The fasting blood glucose level glycosylated hemoglobin (HbA1c) significantly increased in diabetic mice. Bone-alkaline phosphatase activity decreased in the diabetic group. Diabetes increased the levels of ketone bodies, including 3-hydroxybutyric, acetoacetic and butyric acid, whereas it decreased Krebs cycle components, including succinic acid and malic acid, as well as levels of glycolytic products, including lactic acid. Diabetes also induced a shortage of trabecular bone mineral density (BMD) by the regulation of trabecular morphometric parameters in the femur and tibia. Correlation analysis indicated that BMD, Krebs cycle components and lactic acid levels were negatively correlated with HbA1c, whereas ketone bodies were positively correlated with HbA1c. Conclusion: : This research suggested that uncontrolled HbA1c can affect bone loss, production of ketone bodies and utilization of glucose metabolites for energy production in type 2 diabetes.

Advances in Strain Engineering for Improved Bio-fuel Production- a Perspective

Bio-fuels are ecologically sustainable alternates of fossil fuel and have attracted interest of research community in the last few decades. Microorganisms such as bacteria, fungi and microalgae have important roles to play at various steps of bio-fuel production. And therefore several efforts such as genetic engineering have been made to improve the performance of these microbes to achieve the desired results. Metabolic engineering of organisms has benefitted immensely from the novel tools and technologies that have recently been developed. Microorganisms have the advantage of smaller and less complex genome and hence are best suitable for genetic manipulations. In this perspective, we briefly review a few interesting studies which represent some recent advances in the field of metabolic engineering of microbes.

Oxaloacetate Mediates Mitochondrial Metabolism and Function

Oxaloacetate, an intermediate in the tricarboxylic acid cycle, plays important roles in regulating mitochondrial function, gluconeogenesis, the urea cycle, and amino acid syntheses. Because this compound is not stable, more information is needed about its stability profile before its medicinal potential can be realized. In this short review, we present current knowledge and understanding of oxaloacetate with a focus on its stability, degradation, quantification methods, regulation of mitochondrial function, and potential therapeutic benefits. Further, we report previously unpublished spectral data related to the stability profile of oxaloacetate. We found that oxaloacetate has a half-life of about 14 hours in biological aqueous solution at 25°C before degrading into pyruvate. This mandates careful attention to handling this compound including storage at -20 to -80°C when not in use to prolong its shelf-life. Also, the oxaloacetate stability profile needs to be taken into account when conducting experiments involving the compound either in clinical trials or evaluating it as a health supplement or for other experiments. Measuring oxaloacetate by mass-spectrometry requires cumbersome derivatization to assure stability. However, we found that NMR can be used to detect oxaloacetate quantitatively without the need for making derivatives, and the NMR method is sensitive enough to detect oxaloacetate in the micromolar range. Using this method, we showed that oxaloacetate regulates mitochondrial complex II-driven respiration by potent inhibition of succinate dehydrogenase. Moreover, a growing literature in the past few years suggests that oxaloacetate may have therapeutic benefits in treating a variety of diseases.

Qualitative and Quantitative Analysis of Resveratrol and Oxyresveratrol by Liquid Chromatography

Introduction: Resveratrol and its monooxygenated metabolite oxyresveratrol were the subject matter of intense research due to their medicinal value. Absorption, distribution, metabolism and excretion are important to understand the bioavailability and pharmacokinetic profile of resveratrol and oxyresveratrol. Quantification of resveratrol and oxyresveratrol is essential for both in vitro and in vivo studies. Methods: During in vitro drug metabolism studies, both qualitative and quantitative information are essential to understand the metabolic profile of resveratrol and oxyresveratrol. In the present study, a simple and stable method is outlined using high performance liquid chromatography to quantify both resveratrol and oxyresveratrol. This method is suitable to understand the metabolic stability, plasma stability, pharmacokinetics and toxicokinetics of resveratrol and oxyresveratrol. Results: Generally, in vitro incubation studies are performed at high concentrations and in vivo studies are carried out at both high and low concentrations, therefore high performance liquid chromatography method is demonstrated as a suitable technique to quantify resveratrol and oxyresveratrol. Conclusion: Retention time of resveratrol and oxyresveratrol from liquid chromatography qualitatively confirm its identity.

Metabolomics in Lipoid Proteinosis

Background: Lipoid proteinosis (LP) is an autosomal recessive transfer lysosomal storage disease that is characterized by the accumulation of hyalin substance in the mucous membranes, skin, internal organs, and brain. Thus far, no biochemical diagnostic method has been identified. Objective: The aim of this study was to determine the carnitine and acylcarnitine metabolic profiles of LP patients and to examine the potential of LC-MS/MS as a new biochemical method for the identification of biochemical markers. Methods: In this study, 27 carnitine and acylcarnitine esters were measured with LCMS/ MS in serum samples taken from 14 healthy control subjects and 14 patients. The patients, who presented at the Skin and Venereal Diseases Polyclinic, were diagnosed with LP on the basis of clinical, radiological, and histopathological examinations. Results: The results of the study showed that the C0 (free carnitine) C3, C4, C4:DC, C5DC, C6, C8, C14:1, C14:2, C16, and C18 acylcarnitines were statistically significantly reduced in the LP patients (p < 0.05, p < 0.01). Conclusion: It was concluded that the application of carnitine profile screening, an inexpensive, rapid, and reliable method, as a supporting laboratory test could make a contribution to the differential diagnosis for individuals with suspected LP.

A Robust Protocol for Extracting Aqueous Metabolites of High Lipid Sera

Background: With the increasing focus of metabolomic methods on obesityrelated diseases, it is important to consider how sample handling may need to be adapted for the high compositions of lipids that can occur in such subjects. Introduction: High-lipid (cloudy, milky appearances; a.k.a. lipemic) biofluids are common in very high BMI subjects. Organic extractions of biofluids are useful for removing protein backgrounds, inactivating capsid viruses, and yielding relatively stable samples with excellent spectroscopic characteristics. This work considered how acetonitrile extractions, which are widely used, perform on lipemic sera. Results: In this technical note, we report the observation and remediation of a liquid-liquid phase separation in acetonitrile extractions of many lipemic sera. This unexpected behavior can be challenging to identify, especially if working with small volumes. The liquid-liquid separation shows a high miscibility of proteins in both liquid phases that impairs NMR data quality. We also report a simple temperature-based adaption of the acetonitrile extraction procedure that consistently results in a single aqueous phase and eliminates unwanted constituents. Conclusion: A robust approach to achieving reproducible, high quality samples of aqueous metabolites from lipemic sera from very high BMI subjects should be of utility in expanding metabolomics applications to lipemic biofluids.

Non-Targeted Metabolomics Signature in the Plasma and Bone Marrow of Patients with Long Bone Injuries

Background: The contribution of long bone injury and reaming to the inflammatory response of trauma is unknown. Introduction: This study evaluated whether metabolomics can be used to (1) reveal differences in the plasma from long bone injury trauma patients before and after reaming and (2) distinguish healthy adult plasma from that of trauma patients. Methods: Prospective cohort study with enrollment from February 17, 2017 to December 5, 2017 included 15 patients with long bone injuries and 20 healthy adults. Patients with femoral or tibial fractures scheduled to undergo intramedullary nailing were identified at the Medical Center of the Rockies, (Loveland, Co), and Memorial Hospital, (Colorado Springs, CO). Pre-and post-reaming plasma and bone marrow from fifteen patients with femoral and tibial fractures and 20 heathy adult plasma were analyzed by ultra-high-performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS). Results: Trauma patients had 1259 plasma metabolites and healthy adults had 1272 plasma metabolites detected. Fifty percent (657 metabolites) were common between the bone marrow and plasma profiles, and 304 metabolites showed statistical significance for differential abundance between pre- and post-reaming (P<0.05). Post-ream lipids, fatty acids and ceramides were 1.09-1.46-fold increased and diacylglycerols were 0.73-0.82-fold decreased compared to the pre-ream patient control. Post-ream tryptophan metabolites were decreased 0.84-fold, whereas cysteine metabolites were elevated 1.42-fold. Metabolite signals associated with bone matrix remodeling, stress and inflammation were modulated in all patients. Conclusion: Plasma metabolite signatures changed in long bone fracture patients pre- and post-reaming showing distinct profiles from healthy adults without trauma injury. Metabolite signatures of long bone trauma patients have an inflammatory response reflective of healing cascades and merits additional testing for markers of individualized responses to injury.

Metabolite Profiling of Different Solvent Extracts of the Microalgae Chlorella Vulgaris Via 1H NMR-Based Metabolomics

Introduction: In the present study, profiling of the cultured Chlorella vulgaris metabolome was carried out via 1H NMR metabolite profiling of 6 different solvent extracts. The results indicated that the six solvent extracts have metabolite profiles which are clearly different from each other. Methods: Multivariate data analysis (MVA) reveal that ethyl acetate and ethanol extracts were well separated from the aqueous extract by PC1 while being well separated from each other by PC2. The same observations were seen with chloroform and 50% ethanol extracts. In contrast, the chemical shift signals for hexane extract clusters in-between that of chloroform and 50% ethanol, indicating that they have similar chemical profiles. Using partial least square discriminative analysis (PLS-DA), compounds responsible for the group separation were identified from the loading plot. Detailed examination of the loading plot shows that ethanol and ethyl acetate extracts contain significantly higher amounts of carotenoids, amino acids, vitamins and fatty acids. A total of 35 compounds were detected from the 6 different solvents upon which the ethanolic and ethyl acetate extracts was identified to contain more metabolites and in wider range than the other organic solvent extracts. Results: Hence, these two extracts would be more appropriate in metabolite extraction for analysis and for medicinal purposes. Therefore, NMR spectroscopy in compliment with right choice of solvent for extraction could be utilized by relevant industries to evaluate and obtain maximum important metabolites in a shorter time. Conclusion: In addition to possession of high diverse metabolites, the microalgae C. vulgaris could serve as an important functional food ingredient in aquaculture industry and may possibly be considered as a source of biofuel.