Liquid Chromatography Mass Spectrometry-Based Metabolite Pathway Analyses of Myeloma and Non-Hodgkin’s Lymphoma Patients

Medriano, Carl Angelo D.,Na, Jinhyuk,Lim, Kyung-min,Chung, Jin-ho,Park, Youngja H. Royan Institute 2017 Cell journal (Yakhteh) Vol.19 No.1

<P><B>Objective</B></P><P> This study attempted to identify altered metabolism and pathways related to non-Hodgkin’s lymphoma (NHL) and myeloma patients. </P><P><B>Materials and Methods</B></P><P> In this retrospective study, we collected plasma samples from 11 patients-6 healthy controls with no evidence of any blood cancers and 5 patients with either multiple myeloma (n=3) or NHL (n=2) during the preliminary study period. Samples were analyzed using quadrupole time-of-flight liquid chromatography mass spectrometry (LC-MS). Significant features generated after statistical analyses were used for metabolomics and pathway analysis.</P><P><B>Results</B></P><P> Data after false discovery rate (FDR) adjustment at q=0.05 of features showed 136 for positive and 350 significant features for negative ionization mode in NHL patients as well as 262 for positive and 98 features for negative ionization mode in myeloma patients. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis determined that pathways such as steroid hormone biosynthesis, ABC transporters, and arginine and proline metabolism were affected in NHL patients. In myeloma patients, pyrimidine metabolism, carbon metabolism, and bile secretion pathways were potentially affected by the disease.</P><P><B>Conclusion</B></P><P>The results have shown tremendous differences in the metabolites of healthy individuals compared to myeloma and lymphoma patients. Validation through quantitative metabolomics is encouraged, especially for the metabolites with significantly expression in blood cancer patients.</P>

The effects of oxytetracycline on Nitrogen Cycle in a model aquaculture system

Carl Angelo Medriano,Kartik Chandran,Samir Khanal,Jae Woo Lee(이재우),Sungpyo Kim(김성표) 한국산학기술학회 2014 한국산학기술학회 학술대회 Vol.- No.-

This study aims to investigate the effects of OTC in the response of a Nitrosomonas europaea, a model nitrifying bacterium. For this aim, the variations of nitrogen species in aquaculture water samples and Nitrosomonas europaea culture, as a function of OTC concentration, have been monitored. As a result, aquaculture samples show the nitrification level is lower by 23% from 5ppm, 43% from 50ppm, and 46% from 100ppm OTC as compared to a control (no added OTC). Also, the addition of 50ppm OTC to Nitrosomonas europaea culture showed 29% lower degradation level compared to a control. As compared to control, the 10ppm OTC showed increased nitrite level by 30% and nitrous oxide level by up to 29% after 80 hrs. RNA expression analysis was able correlate amoA expression to the reduced level of ammonia removal. The hao, nirK, and norB gene expressions did not show correlation to the oxytetracycline concentration, thus suggesting that the dose influenced in other ways perhaps somewhere during post-transcription to enzyme activity

Effects of low concentration of clarithromycin to Danio rerio after acute exposure through metabolic analysis

Ryan De Sotto,Carl Medriano,Youngja Park,Sungpyo Kim 한국방재학회 2015 한국방재학회 학술발표대회논문집 Vol.14 No.-

The presence of pharmaceuticals in the environment has led to apparent toxicity with different aquatic species. Clarithromycin, for example, is used in treating respiratory tract infections, has been recently found in the surface waters and rivers which might threaten non-targeted organisms in these matrices. In this study, a model vertebrate Danio rerio (zebrafish) was exposed to 100ppb clarithromycin for 72 hours to evaluate acute toxicity through significantly affected metabolic compounds in the fish’s pathway. Metabolites obtained from q-TOF LC/MS were identified and mapped with the zebrafish’s metabolic pathway using Metlin, and KEGG respectively. 335 compounds are believed to have been significantly altered by the acute exposure of the antibiotic with the fish. The most affected pathways are ABC transporters, steroid hormone biosynthesis, arachidonic acid metabolism, purine metabolism, and biosynthesis of amino acids. With the said findings, it can be concluded that, although concentration of some pharmaceuticals may be as low as the one used in this study, its effects on the aquatic species exposed to it might be significant and should be given immediate attention

Screening and identification of neuroprotective compounds from <i>Scrophularia buergeriana</i> using cell extraction coupled with LC–MS

Shin, Hyeji,Medriano, Carl Angelo,Park, Byoungduck,Park, Youngja H,Lee, Ki Yong Pergamon Press 2018 Journal of pharmaceutical and biomedical analysis Vol.148 No.-

<P><B>Abstract</B></P> <P>In the cell extraction_LC–MS method, when cells are incubated with natural product extracts, bioactive compounds selectively bind to extracellular or intracellular targets. The extracts and major compounds (phenylpropanoids and iridoid glycosides) of <I>Scrophularia buergeriana</I> Miquel have been reported to show neuroprotective effects both <I>in vitro</I> and <I>in vivo</I>. In this study, the cell extraction_LC–MS strategy was applied to screen and identify potential neuroprotective compounds from <I>S. buergeriana</I> by using immortalized mouse hippocampal HT22 cells. The results showed that two known compounds from <I>S. buergeriana</I> selectively bound HT22 cells. Additionally, metabolomics analyses were performed using the Mass Profiler Professional and Limma differential expression package of R to identify significant differences between HT22 cells treated with <I>S. buergeriana</I> and untreated cells. The cell extraction approach more accurately reflects <I>in vivo</I> conditions compared with other methods and can be readily used for screening bioactive components from natural products.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Cell extraction_LC–MS strategy was applied to screen and identify potential neuroprotective compounds from <I>S. buergeriana</I> by using HT22 cells. </LI> <LI> Two neuroprotective metabolites, buergeriside C<SUB>1</SUB> and buergeriside A<SUB>1</SUB>, were selectively bound HT22 cells. </LI> <LI> The significant metabolites, which are compounds that affect the statistical difference between the two groups, were identified. </LI> <LI> The cell extraction approach is a better representation of the <I>in vivo</I> situation than other methods. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Effects of solids retention time on the fate of tetracycline resistance in SBRs for the treatment of carcass leachate

De Sotto, R.B.,Medriano, C.A.D.,Salcedo, D.E.,Lee, H.,Cho, Y.,Kim, S. Academic Press 2016 Journal of Environmental Management Vol. No.

<P>In the event of a foot and mouth disease outbreak, further spread of the virus is generally prevented by culling of infected animals in burial pits. This practice may eventually lead to groundwater contamination through leaching of wastewater from the animal carcasses. Wastewater from carcass leachate often contains antibiotic resistant bacteria and genes as well as traces of pharmaceuticals, and a high nutrient content. The role of operational parameters used in activated sludge treatment of this wastewater in the spread of antibiotic resistance has not been fully understood. This study investigated the fate of tetracycline-resistant bacteria and genes in sequencing batch reactors with synthetic carcass leachate at different solid retention times. Escherichia coli DH5 alpha was used as the representative tetracycline-resistant bacteria with multiple antibiotic-resistant genes encoded in plasmid pB10. Solids retention time contributed to an increase in antibiotic resistance in SBRC (SRT = 25 days) with TRB values up to 1.25 x 107 CFU/mL which is one log higher than the influent. Microbial community analysis of the DNA samples from effluent of SBRC showed four major phyla: Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria under which are ecologically-important microbial species. It was shown that antibiotic resistance genes cannot be eliminated during treatment of synthetic carcass leachate in a lab-scale sequencing batch reactor. (C) 2016 Elsevier Ltd. All rights reserved.</P>

A pilot study identifying a potential plasma biomarker for determining EGFR mutations in exons 19 or 21 in lung cancer patients

Pamungkas, Aryo D.,Medriano, Carl A.,Sim, Eunjung,Lee, Sungyong,Park, Youngja H. D.A. Spandidos 2017 MOLECULAR MEDICINE REPORTS Vol.15 No.6

<P>The most common type of lung cancer is non-small cell lung cancer (NSCLC), which is frequently characterized by a mutation in the epidermal growth factor receptor (EGFR). Determining the presence of an EGFR mutation in lung cancer is important, as it determines the type of treatment that a patients will receive. Therefore, the aim of the present study was to apply high-resolution metabolomics (HRM) using liquid chromatography-mass spectrometry to identify significant compounds in human plasma samples obtained from South Korean NSCLC patients, as potential biomarkers for providing early detection and diagnosis of minimally-invasive NSCLC. The metabolic differences between lung cancer patients without EGFR mutations were compared with patients harboring EGFR mutations. Univariate analysis was performed, with a false discovery rate of q=0.05, in order to identify significant metabolites between the two groups. In addition, hierarchical clustering analysis was performed to discriminate between the metabolic profiles of the two groups. Furthermore, the significant metabolites were identified and mapped using Mummichog software, in order to generate a potential metabolic network model. Using metabolome-wide association studies, metabolic alterations were identified. Linoleic acid [303.23 m/z, (M+Na)<SUP>+</SUP>], 5-methyl tetrahydrofolate [231.10 m/z, (M+2H)<SUP>+</SUP>] and N-succinyl-L-glutamate-5 semialdehyde [254.06 m/z, (M+Na)<SUP>+</SUP>], were observed to be elevated in patients harboring EGFR mutations, whereas tetradecanoyl carnitine [394.29 m/z, (M+Na)<SUP>+</SUP>] was observed to be reduced. This suggests that these compounds may be affected by the EGFR mutation. In conclusion, the present study identified four potential biomarkers in patients with EGFR mutations, using HRM combined with pathway analysis. These results may facilitate the development of novel diagnostic tools for EGFR mutation detection in patients with lung cancer.</P>

Significance of metabolite extraction method for evaluating sulfamethazine toxicity in adult zebrafish using metabolomics

De Sotto, R.,Medriano, C.,Cho, Y.,Seok, K.S.,Park, Y.,Kim, S. Academic Press 2016 Ecotoxicology and environmental safety Vol.127 No.-

<P>Recently, environmental metabolomics has been introduced as a next generation environmental toxicity method which helps in evaluating toxicity of bioactive compounds to non-target organisms. In general, efficient metabolite extraction from target cells is one of the keys to success to better understand the effects of toxic substances to organisms. In this regard, the aim of this study is (1) to compare two sample extraction methods in terms of abundance and quality of metabolites and (2) investigate how this could lead to difference in data interpretation using pathway analysis. For this purpose, the antibiotic sulfamethazine and zebrafish (Danio rerio) were selected as model toxic substance and target organism, respectively. The zebrafish was exposed to four different sulfamethazine concentrations (0, 10, 30, and 50 mg/L) for 72 h. Metabolites were extracted using two different methods (Bligh and Dyer and solid phase extraction). A total of 13,538 and 12,469 features were detected using quadrupole time-of-flight liquid chromatography mass spectrometry (QTOF LC-MS). Of these metabolites, 4278 (Bligh and Dyer) and 332 (solid phase extraction) were found to be significant after false discovery rate adjustment at a significance threshold of 0.01. Metlin and KEGG pathway analysis showed comprehensive information from fish samples extracted using Bligh and Dyer compared to solid phase extraction. This study shows that proper selection of sample extraction method is critically important for interpreting and analyzing the toxicity data of organisms when metabolomics is applied. (C) 2016 Elsevier Inc. All rights reserved.</P>

Sub-lethal pharmaceutical hazard tracking in adult zebrafish using untargeted LC–MS environmental metabolomics

Sotto, Ryan B. De,Medriano, Carl D.,Cho, Yunchul,Kim, Hyuk,Chung, In-Young,Seok, Kwang-Seol,Song, Kyung Guen,Hong, Seok Won,Park, Youngja,Kim, Sungpyo Elsevier 2017 Journal of hazardous materials Vol.339 No.-

<P><B>Abstract</B></P> <P>Antibiotics in the aquatic environment are dispersed through anthropogenic activities at low concentrations. Despite their sub lethal concentration, these biologically active compounds may still have adverse effects to non-target species. This study examined the response of adult zebrafish to 0.1mg/L concentration of clarithromycin, florfenicol, sulfamethazine, and their mixture using environmental metabolomics. Embryo and larvae of the fish were also used to assess fish embryo acute toxicity and behavior tests respectively. The fish embryo toxicity test did not show any inhibition of growth and development of the embryos after 96h of exposure to the antibiotics. Changes in swimming activity were seen in 5-dpf larvae which is believed to be correlated with the length of exposure to the compounds. Meanwhile, environmental metabolomics revealed diverse metabolites and pathways that were affected after 72h of exposure of the adult fish to sub-lethal concentration of the compounds. We found that even at low concentration of the antibiotics, behavioral and metabolic effects were still observed despite the lack of visible morphological changes. Further studies involving other aquatic organisms and bioactive compounds are encouraged to strengthen the findings presented in this novel research.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Individual and mixture of antibiotics at 0.1mg/L did not impair growth of embryos. </LI> <LI> Zebrafish larvae preferred blue over yellow regardless of antibiotic’s presence. </LI> <LI> Swimming behavior of 5-dpf larvae significantly changed relative to exposure time. </LI> <LI> Metabolites choline, guanosine, and ADP were regulated in the exposed zebrafish. </LI> <LI> Antibiotics’ mechanism of action seems to play a role in zebrafish’s metabolism. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

High-resolution metabolomics to identify urine biomarkers in corticosteroid-resistant asthmatic children

Park, Youngja H.,Fitzpatrick, Anne M.,Medriano, Carl Angelo,Jones, Dean P. Elsevier 2017 The Journal of allergy and clinical immunology Vol.139 No.5

<P><B>Background</B></P> <P>Corticosteroid (CS) treatment has been established as the first anti-inflammatory treatment for adults and children with asthma. However, a subset of patients fails to respond to combined systemic and inhaled CS treatment.</P> <P><B>Objective</B></P> <P>This study was aimed at further understanding CS resistance among children with severe asthma.</P> <P><B>Methods</B></P> <P>High-resolution metabolomics was performed on urine samples from CS-respondent (n = 15) and CS-nonrespondent (n = 15) children to determine possible urine biomarkers related to CS resistance. The metabolic phenotypes of CS responders and CS nonresponders were analyzed using bioinformatics including Manhattan plot with false- discovery rate, hierarchical cluster analysis, Kyoto Encyclopedia Genes and Genomes, and Mummichog pathway analysis.</P> <P><B>Results</B></P> <P>The 2-way hierarchical cluster analysis study determined 30 metabolites showing significantly different levels between CS responders and CS nonresponders. The important metabolites annotated were 3,6-dihydronicotinic acid (126.05 <I>m</I>/<I>z</I>, RT: 106, [M+H]<SUP>+</SUP>), 3-methoxy-4-hydroxyphenyl(ethylene)glycol (185.05 <I>m</I>/<I>z</I>, RT: 155, [M+H]<SUP>+</SUP>), 3,4-dihydroxy-phenylalanine (198.07 <I>m</I>/<I>z</I>, RT: 446, [M+H]<SUP>+</SUP>), γ-glutamylcysteine (236.06 <I>m</I>/<I>z</I>, RT: 528, [M+S(34)+H]<SUP>+</SUP>), Cys-Gly, (253.06 <I>m</I>/<I>z</I>, RT: 528, [M-NH<SUB>3</SUB>+H]<SUP>+</SUP>), and reduced Flavin mononucleotide (517.0794 <I>m/z</I>, RT: 533, [M+NaCl]<SUP>+</SUP>). Tyrosine metabolism, degradation of aromatic compounds, and glutathione metabolism are suggested to be significant pathways relating to CS resistance.</P> <P><B>Conclusions</B></P> <P>High-resolution metabolomics is a promising approach in asthma research. Five candidate markers were identified to be related to CS-resistant children with severe asthma. These compounds, upon validation, may contribute further in the understanding of CS resistance among children with severe asthma through the use of urine.</P>