Coexistencia de regímenes distintos Criterio para determinar el régimen

2.2.1. Serum samples preparation

Fasting blood samples were collected from lung cancer patients and normal controls in anticoagulant-free sterile tubes and left to coagulate for 30 min at room temperature. The serum was collected after centrifuged at 1800 g for 20 min (Kocijancic et al., 2014) and transferred into clean sterile Eppendorf tubes and frozen at -80ºC. 250 µl of serum samples were shipped on dry ice (Chariot et al., 1994) to Metabolon Inc., NC, USA.

At Metabolon Inc., the serum samples were centrifuged at 20,800 g, the obtained supernatants were divided into four equal aliquots and dried overnight in nitrogen vacuum drier at medium speed without heat. Dried samples were subjected to three independent analyses, two of which were for acidic and basic amino acids using UHPLC/MS optimized and the third for GC/MS.

62 2.2.2. UHPLC/MS protocol

One dried specimen was reconstituted in 50 µl of 0.1% formic acid (acidic injection) and another in 50 µl of 6.5 mM NH4HCO3 pH 8.0 (basic injection). Samples were run on Waters Acquity UHPLC coupled to an LTQ mass spectrometer equipped with an electrospray ionization source. Two independent UHPLC/MS injections were performed: acidic and basic for monitoring positive and negative ions, respectively. 2.2.3. GC/MS protocol

The third dried specimen was reconstituted in a mixture (1:1 v/v) of N,O-bis-tri- methyl-silyl-trifluoroacetamide (TFA) and a solvent containing [1(acetonitrile): 1(dichloromethane): 1(cyclohexane)] and [5% trimethylamine] at a ratio of 4:1 and heated at 60ºC for 1 h. All solvents used in sample reconstitution were also used as an internal control to monitor instrument performance. The GC/MS data were analysed using Thermo-Finnigan Trace DSQ fast-scanning single-quadrupole MS. 2.2.4. Data processing

The MS/MS2 data were analysed against the Metabolon’s reference standard library. The library contains information about the retention time/index, mass to charge ratio (m/z), and the MS/MS spectra of 1500 standards used in making the library. The library also contains the associated adducts, multi-meres and in-source fragments of the standards. These multi-parameters were used to identify the detected metabolites. All quantifications and identifications were subjected to quality control (QC) to verify the quality of peak integration and identification.

2.2.5. Metabolomics’ biomarkers validation 2.2.5.1. Serum sample handling

Fasting blood samples were collected in anticoagulant-free vacutainer tubes and allowed to clot for 30 min and centrifuged at 1800 g for 15 min. The serum was

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collected (Kocijancic et al., 2014), aliquoted, and frozen in cryovials at -80ºC until use (Chariot et al., 1994).

2.2.5.2. Pyruvate

The colorimetric method described in the Enzychrom™ pyruvate assay kit (Cat #EPYR-100) was used (Hansen JL and EF, 1978). 10 µl of sample or standard per well was mixed with 90 µl of enzyme mix working cocktail [94 µl enzyme mix (pyruvate oxidase and hydrogen peroxide) and 1µl of dye reagent] in a 96-well plate and incubated at room temperature for 30 min. OD was read at 570 nm (550-585 nm). A standard serial dilution ranging from zero (water as a blank) to 500 µM was used to construct the standard curve. Sample concentration was calculated using the following equation:

𝐏𝐲𝐫𝐮𝐯𝐚𝐭𝐞 𝐜𝐨𝐧𝐜𝐞𝐧𝐭𝐫𝐚𝐭𝐢𝐨𝐧 (µ𝐌) =𝐒𝐚𝐦𝐩𝐥𝐞 𝐎𝐃 − 𝐛𝐥𝐚𝐧𝐤 𝐎𝐃 𝐒𝐥𝐨𝐩

Samples with OD higher than the OD of 500 µM were diluted in assay buffer, measured again and the calculated values were multiplied by the dilution factor. 2.2.5.3. Free fatty acids (FFAs)

The colorimetric method described in the Enzychrom™ free fatty acid assay kit (Cat #EFFA-100) was used (Seo et al., 2011, Lu et al., 2013). 10 µl of sample or standard was mixed with 90 µl working reagent containing (1 µl Enzyme A, 1 µl Enzyme B, 1 µl co-substrate, 1 µl dye reagent and 90 µl assay buffer) in a 96-well plate, incubated at room temperature for 30 min, and OD was read at 570 nm (550-585 nm). Free fatty acid concentrations were calculated from the following equation:

𝐒𝐚𝐦𝐩𝐥𝐞 𝐅𝐅𝐀𝐬 𝐜𝐨𝐧𝐜𝐞𝐧𝐭𝐫𝐚𝐭𝐢𝐨𝐧 (µ𝐌) = 𝐒𝐚𝐦𝐩𝐥𝐞 𝐎𝐃 − 𝐛𝐥𝐚𝐧𝐤 𝐎𝐃 𝐒𝐥𝐨𝐩

Samples with OD higher than the OD of 1000 µM were diluted in assay buffer, measured again and the calculated values were multiplied by the dilution factor.

64 2.2.5.4. Ketone bodies

The colorimetric method described by Siegel, et al. (1977) for the EnzyChrom™ ketone assay kit (EKBD-100) was used for quantitative assay of ketone bodies. 2.2.5.4.1. Acetoacetic acid (AcAc)

195 µl of assay reagent containing (195 µl AcAc buffer, 8 µl reconstituted AcAc reagent and 0.5 µl 3-hydroxybutyrate dehydrogenase enzyme) was added per well and combined with 5 µl of serum sample, water or standard. Sample blank assay reagent contained no enzyme. Samples were run in duplicates, one using the assay reagent and the other with blank reagent. The reaction mixture was incubated at room temperature for 5 min and the OD of NADH was measured at 340 nm (Hansen and Freier, 1978). The AcAc concentration was calculated from the following formula:

𝐀𝐜𝐀𝐜 𝐜𝐨𝐧𝐜𝐞𝐧𝐭𝐫𝐚𝐭𝐢𝐨𝐧 (𝐦𝐌) = 𝐎𝐃 𝐬𝐚𝐦𝐩𝐥𝐞 − 𝐎𝐃 𝐛𝐥𝐚𝐧𝐤

𝐎𝐃 𝐬𝐭𝐚𝐧𝐝𝐚𝐫𝐝 − 𝐎𝐃 𝐰𝐚𝐭𝐞𝐫 𝐱 𝟖

8: samples dilution factor.

2.2.5.4.2. 3 β-hydroxybutyric acid (BOHB)

In each well, 195 µl of assay reagent containing (195 µl of AcAc buffer, 8 µl of reconstituted BOHB reagent and 0.5 µl of 3-hydroxybutyrate dehydrogenase enzyme) was added to 5 µl serum sample, water or standard. Sample blank assay reagent contained no enzyme. Samples were run in duplicates, one using the assay reagent and the other with the blank reagent. The reaction mixture was incubated at room temperature for 15 min and the OD of NADH was measured at 340 nm (Hansen and Freier, 1978). The BOHB concentration was calculated from the following formula:

𝐁𝐎𝐇 𝐜𝐨𝐧𝐜𝐞𝐧𝐭𝐫𝐚𝐭𝐢𝐨𝐧 (𝐦𝐌) = 𝐎𝐃 𝐬𝐚𝐦𝐩𝐥𝐞 − 𝐎𝐃 𝐛𝐥𝐚𝐧𝐤

𝐎𝐃 𝐬𝐭𝐚𝐧𝐝𝐚𝐫𝐝 − 𝐎𝐃 𝐰𝐚𝐭𝐞𝐫 𝐱 𝟖

65 2.2.5.5. Glutamate

Glutamate Colorimetric Assay Kit (Bio vision® Catalogue #K629-100) was used according to the manufacturer’s recommendations (Koçdor et al., 2003). 100 µl of reaction mixture containing [90 µl of glutamate assay buffer (part No. K629-100-1), 2 µl glutamate enzyme mix (part No. K629-100-2) and 8 µl glutamate developer (part No. K629-100-3)] was added to each well containing 50 µl of standard or serum sample (diluted 5 times in assay buffer). Sample background was performed by adding 100 µl of reaction mixture containing 2 µl of water instead of glutamate enzyme mix. The plate was mixed well, incubated at 37ºC for 30 min, and the OD was measured at 450 nm in a microplate reader. Samples OD values were corrected by subtracting the blank values, and the linear equation obtained from the standard curve was used to calculate glutamate concentrations in the samples. The obtained values were multiplied by the dilution factor as following:

𝐆𝐥𝐮𝐭𝐚𝐦𝐚𝐭𝐞 (𝐦𝐌) =

Sa: sample amount calculated fro the standard curve Y= a x (-/+)b (Y = sample OD, x = sample concentration, a: slop (given), b: intercept (given) and Sr: sample volume added to the well. The obtained values were multiplied by sample dilution factor.

2.2.5.6. Acetyl co-A

Human acetyl co-A sandwich ELISA kit (Wuhan Fine Biological Technology Co. Ltd.; cat. #EH0606) was used to estimate the levels of acetyl Co-A in serum control and lung cancer patients’ samples. The kit uses 96-well plates pre-coated with anti acetyl co-A capture antibodies, biotinylated anti acetyl co-A detection antibody, HRP-streptavidin and TMB as substrate for the peroxidase, which turns blue corresponding to the amount of glutamate in the sample. The reaction was terminated

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by adding 1M H2SO4 and the resultant yellow colour was measured at OD 340 nm. The acetyl co-A levels were then calculated from the standard curve equation. 2.2.5.7. Statistical analysis

Wilcoxon-Mann-Whitney test (Marx et al., 2016) was used to calculate the significance of change among the compared samples. In biomedical sciences, it is preferred over the parametric and non-parametric methods to calculate more accurate p values for comparison of two populations using permutation (Ludbrook J., 1998).

2.3. Formaldehyde-fixed paraffin-embedded tissue (FFPE) samples