Uridine Glucuronosyltransferase 2B7 Polymorphism-Based Pharmacogenetic Dosing of Epirubicin in FEC Chemotherapy for Early-Stage Breast Cancer.

BACKGROUND: Epirubicin is metabolized by uridine glucuronosyltransferase 2B7 (UGT2B7). Patients homozygous for the minor allele (CC) in the UGT2B7 -161 promoter polymorphism have lower clearance and significantly higher rates of leukopenia compared to wild-type homozygote (TT) or heterozygote (CT) patients. This study was designed to determine if TT and CT genotype patients could tolerate a higher epirubicin dose compared to CC genotype patients. PATIENTS AND METHODS: We studied women with histologically confirmed non-metastatic, invasive breast cancer who were scheduled to receive at least three cycles of FE100C in the (neo)adjuvant setting. Patients received standard-dose FE100C during the first 21-day cycle. Based on genotype, the epirubicin dose was escalated in the second and third cycles to 115 and 130 mg/m2 or to 120 and 140 mg/m2 for CT and TT genotype patients, respectively. The main outcome measurements were myelosuppression and dose-limiting toxicity. These were analyzed for relationships with the three genotypes. RESULTS: Forty-five patients were enrolled (10 CC, 21 CT, and 14 TT genotypes) and received 100 mg/m2 of epirubicin in the first cycle. Twelve and 10 TT patients were dose escalated at the second and third cycles, respectively; 16 CT patients were dose escalated at the second and third cycles. Leukopenia, but not febrile neutropenia, was genotype and dose dependent and increased in patients with CT and TT genotypes as their dose was increased. However, the third-cycle leukopenia rates were comparable to patients with the CC genotype receiving standard-dose epirubicin. CONCLUSION: Pharmacogenetically guided epirubicin dosing is well tolerated and allowed dose escalation without increased toxicity.

Plasma levels of platinum-induced fatty acid [16:4n-3] do not affect response to platinum-based chemotherapy: A pilot study in non-small cell lung cancer patients.

BACKGROUND & AIMS: Pre-clinical studies suggest that 16:4(n-3) in purified form or as a component of fish oil might induce platinum-based chemotherapy resistance. Our aim was to determine plasma total and free 16:4(n-3) before and during platinum-based chemotherapy in non-small cell lung cancer (NSCLC) patients supplemented with fish oil or provided standard care, and to explore relationships between plasma 16:4(n-3) levels and tumor response to treatment. METHODS: In a retrospective, secondary data analysis of a prior clinical trial, plasma from patients with NSCLC (n = 21) who underwent platinum-based chemotherapy and were assigned to 2.2 g/day of eicosapentaenoic (EPA) plus 1.1 g DHA/day as fish oil (FO; n = 12) or received no intervention (standard care; SC; n = 9). Plasma 16:4(n-3) was quantified as free and esterified (total) fatty acid using HPLC-MS/MS. Plasma 16:4(n-3) levels were evaluated over time in relation to fish oil supplementation and response to platinum-based therapy, and compared with a group of healthy subjects (REF; n = 11). RESULTS: Plasma 16:4(n-3) was detected in all samples. The percentage change/day in plasma esterified (total) 16:4(n-3) was higher for FO versus SC group (2.7 versus -1.8%/d, U = 20, p = 0.02), but change in plasma free 16:4(n-3) was not different between FO and SC. Median plasma free and esterified 16:4(n-3) were similar between responders and non-responders to platinum-based chemotherapy. Total and free plasma 16:4(n-3) fatty acids were similar between NSCLC patients and REF (NSCLC vs REF: total 16:4(n-3): 122.9 vs. 95.2 nM and free 16:4(n-3) 23.9 vs. 27.6 nM). CONCLUSIONS: This first of its kind study that evaluated plasma 16:4(n-3) in NSCLC patients showed that 16:4 (n-3) was elevated during FO supplementation, independent of fish oil supplementation or platinum-based chemotherapy.

PRESS timings for resolving 13 C4 -glutamate 1 H signal at 9.4 T: Demonstration in rat with uniformly labelled 13 C-glucose.

MRS of 13 C4 -labelled glutamate (13 C4 -Glu) during an infusion of a carbon-13 (13 C)-labelled substrate, such as uniformly labelled glucose ([U-13 C6 ]-Glc), provides a measure of Glc metabolism. The presented work provides a single-shot indirect 13 C detection technique to quantify the approximately 2.51 ppm 13 C4 -Glu satellite proton (1 H) peak at 9.4 T. The methodology is an optimized point-resolved spectroscopy (PRESS) sequence that minimizes signal contamination from the strongly coupled protons of N-acetylaspartate (NAA), which resonate at approximately 2.49 ppm. J-coupling evolution of protons was characterized numerically and verified experimentally. A (TE1 , TE2 ) combination of (20 ms, 106 ms) was found to be suitable for minimizing NAA signal in the 2.51 ppm 1 H 13 C4 -Glu spectral region, while retaining the 13 C4 -Glu 1 H satellite peak. The efficacy of the technique was verified on phantom solutions and on two rat brains in vivo during an infusion of [U-13 C6 ]-Glc. LCModel was employed for analysis of the in vivo spectra to quantify the 2.51 ppm 1 H 13 C4 -Glu signal to obtain Glu C4 fractional enrichment time courses during the infusions. Cramér-Rao lower bounds of about 8% were obtained for the 2.51 ppm 13 C4 -Glu 1 H satellite peak with the optimal TE combination.

Identification of ABC transporters acting in vitamin B12 metabolism in Caenorhabditis elegans.

Vitamin B12 (cobalamin, Cbl) is a micronutrient essential to human health. Cbl is not utilized as is but must go through complex subcellular and metabolic processing to generate two cofactor forms: methyl-Cbl for methionine synthase, a cytosolic enzyme; and adenosyl-Cbl for methylmalonyl-CoA mutase, a mitochondrial enzyme. Some 10-12 human genes have been identified responsible for the intracellular conversion of Cbl to cofactor forms, including genes that code for ATP-binding cassette (ABC) transporters acting at the lysosomal and plasma membranes. However, the gene for mitochondrial uptake is not known. We hypothesized that ABC transporters should be candidates for other uptake and efflux functions, including mitochondrial transport, and set out to screen ABC transporter mutants for blocks in Cbl utilization using the nematode roundworm Caenorhabditis elegans. Thirty-seven mutant ABC transporters were screened for the excretion of methylmalonic acid (MMA), which should result from loss of Cbl transport into the mitochondria. One mutant, wht-6, showed elevated MMA excretion and reduced [14C]-propionate incorporation, pointing to a functional block in methylmalonyl-CoA mutase. In contrast, the wht-6 mutant appeared to have a normal cytosolic pathway based on analysis of cystathionine excretion, suggesting that cytosolic methionine synthase was functioning properly. Further, the MMA excretion in wht-6 could be partially reversed by including vitamin B12 in the assay medium. The human ortholog of wht-6 is a member of the G family of ABC transporters. We propose wht-6 as a candidate for the transport of Cbl into mitochondria and suggest that a member of the corresponding ABCG family of ABC transporters has this role in humans. Our ABC transporter screen also revealed that mrp-1 and mrp-2 mutants excreted lower MMA than wild type, suggesting they were concentrating intracellular Cbl, consistent with the cellular efflux defect proposed for the mammalian MRP1 ABC transporter.

Cancer Metabolomics and the Human Metabolome Database.

The application of metabolomics towards cancer research has led to a renewed appreciation of metabolism in cancer development and progression. It has also led to the discovery of metabolite cancer biomarkers and the identification of a number of novel cancer causing metabolites. The rapid growth of metabolomics in cancer research is also leading to challenges. In particular, with so many cancer-associate metabolites being identified, it is often difficult to keep track of which compounds are associated with which cancers. It is also challenging to track down information on the specific pathways that particular metabolites, drugs or drug metabolites may be affecting. Even more frustrating are the difficulties associated with identifying metabolites from NMR or MS spectra. Fortunately, a number of metabolomics databases are emerging that are designed to address these challenges. One such database is the Human Metabolome Database (HMDB). The HMDB is currently the world's largest and most comprehensive, organism-specific metabolomics database. It contains more than 40,000 metabolite entries, thousands of metabolite concentrations, >700 metabolic and disease-associated pathways, as well as information on dozens of cancer biomarkers. This review is intended to provide a brief summary of the HMDB and to offer some guidance on how it can be used in metabolomic studies of cancer.

MyCompoundID: using an evidence-based metabolome library for metabolite identification.

Identification of unknown metabolites is a major challenge in metabolomics. Without the identities of the metabolites, the metabolome data generated from a biological sample cannot be readily linked with the proteomic and genomic information for studies in systems biology and medicine. We have developed a web-based metabolite identification tool ( http://www.mycompoundid.org ) that allows searching and interpreting mass spectrometry (MS) data against a newly constructed metabolome library composed of 8,021 known human endogenous metabolites and their predicted metabolic products (375,809 compounds from one metabolic reaction and 10,583,901 from two reactions). As an example, in the analysis of a simple extract of human urine or plasma and the whole human urine by liquid chromatography-mass spectrometry and MS/MS, we are able to identify at least two times more metabolites in these samples than by using a standard human metabolome library. In addition, it is shown that the evidence-based metabolome library (EML) provides a much superior performance in identifying putative metabolites from a human urine sample, compared to the use of the ChemPub and KEGG libraries.

Development of an isotope labeling ultra-high performance liquid chromatography mass spectrometric method for quantification of acylglycines in human urine.

Acylglycines play a crucial regulatory and detoxification role in the accumulation of the corresponding acyl CoA esters and are an important class of metabolites in the diagnoses of inborn errors of metabolism. Sensitive quantification of a large number of acylglycines not only improves diagnosis but also enables the discovery of potential new biomarkers of diseases. We report an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS) method for quantifying acylglycines in human urine with high sensitivity. This method is based on the use of a newly developed isotope labeling reagent, p-dimethylaminophenacyl (DmPA) bromide, to label acylglycines to improve detection sensitivity. Eighteen acylglycines, namely acetylglycine, propionylglycine, isobutyrylglycine, butyrylglycine, 4-hydroxyphenylacetylglycine, 2-furoylglycine, tiglylglycine, 2-methybutyrylglycine, 3-methylcrotonylglycine, isovalerylglycine, valerylglycine, hexanoylglycine, phenylacetylglycine, phenylpropionylglycine, glutarylglycine, heptanoylglycine, octanoylglycine and suberylglycine, were measured. This method uses calibration standards prepared in surrogate matrix (un-derivatized urine) and stable-isotope labeled analytes as the internal standards. The analysis was carried out in the positive ion detection mode using multiple reaction monitoring (MRM) survey scans. The calibration curves were validated over the range of 1.0-500 nM. The method achieved a lower limit of quantitation (LLOQ) of 1-5 nM for all analytes, as measured by the standard derivations associated with calibration curves and confirmed in surrogate matrix; the signal-to-noise ratio at LLOQ ranged from 12.50 to 156.70. Both accuracy (% RE or relative error) and precision (% CV) were <15%. Matrix effects were minimized using the surrogate matrix. All eighteen analytes were stable in urine for at least 5h at room temperature, autosampler (4 °C) for 24 h, 7 weeks at -20 °C and after three freeze/thaw cycles. This surrogate matrix approach was validated using a standard addition experiment. As an example of applications, the endogenous concentrations of all eighteen analytes in urine samples of 20 healthy individuals collected in three consecutive days (i.e., 60 samples) were determined; there was no significant correlation found between the acylglycine profile and gender or body mass indices.

A method for comprehensive analysis of urinary acylglycines by using ultra-performance liquid chromatography quadrupole linear ion trap mass spectrometry.

Acylglycines are an important class of metabolites that have been used in the diagnosis of several inborn errors of metabolism (IEM). However, current analytical methods detect only a few acylglycines. There is a need to profile these metabolites in a comprehensive manner for studying their functions and improving their diagnostic values for different IEM and potentially other diseases. We describe a sensitive method that combines the chromatographic resolving power of ultra-performance liquid chromatography (UPLC) to separate closely related metabolites including isomers with tandem mass spectrometry (MS/MS). Acylglycines were extracted from urine using an anion exchange solid-phase extraction (SPE) cartridge. After UPLC separation, the acylglycines were detected on a hybrid triple quadrupole linear ion trap mass spectrometer. A set of standards were used for the development of an optimal MS acquisition method. Several acquisition modes using information derived from collision-induced dissociation breakdown curves were used to detect acylglycines. Using this method, 18 acylglycines were detected in the urine of healthy individuals and confirmed using standards, while 47 additional acylglycines were detected and tentatively identified, based on their retention and fragmentation pattern. Among the 65 acylglycines detected, only 18 of them have been previously reported in biofluids of healthy individuals. These results will be deposited in a public human metabolome database. This example illustrates that by developing a method tailored to the analysis of a class of metabolites sharing similar structural moieties, we can potentially identify many more new metabolites, thereby expanding the overall metabolome coverage.