An LC-MS Approach to Quantitative Measurement of Ammonia Isotopologues

Ammonia is a fundamental aspect of metabolism spanning all of phylogeny. Metabolomics, including metabolic tracing studies, are an integral part of elucidating the role of ammonia in these systems. However, current methods for measurement of ammonia are spectrophotometric, and cannot distinguish isotopologues of ammonia, significantly limiting metabolic tracing studies. Here, we describe a novel LC-MS-based method that quantitatively assesses both 14N-and 15N-isotopologues of ammonia in polar metabolite extracts. This assay (1) quantitatively measures the concentration of ammonia in polar metabolite isolates used for metabolomic studies, and (2) accurately determines the percent isotope abundance of 15N-ammonia in a cell lysate for 15N-isotope tracing studies. We apply this assay to quantitatively measure glutamine-derived ammonia in lung cancer cell lines with differential expression of glutaminase.

generates the compound indophenol from ammonia (Fig. 1A). Indophenol is highly conjugated, and absorbs strongly between 630 and 720 nm, enabling spectrophotometric detection 16 . This reaction has been applied to measure ammonia in complex matrices, such as blood, soil, and seawater [17][18][19] . This assay is also the standard analysis used in hospital Blood Urea Nitrogen (BUN) exams, in which urea is converted to ammonia via Urease and subsequently converted to indophenol for detection 20 . However, similar to the limitations of other colorimetric assays, the Berthelot reaction for ammonia detection is complicated by competing molecules that interfere with the reaction or absorb on the same spectra as indophenol, such as chlorophyll 11,21 .
We hypothesized that indophenol generated in the Berthelot reaction could be amenable to LC-MS, allowing for the direct and specific quantification of ammonia in a manner that filters out background reactions. We developed an LC-MS coupled Berthelot assay, which can be performed on polar metabolite extracts and other complex biological samples. Furthermore, we showed that the MS Berthelot assay allowed detection of isotopologues of ammonia ( 14 NH 3 and 15 NH 3 ). In this study we applied this assay to quantify the contribution of 15 N-(amide)-glutamine to total cellular ammonia.

The Berthelot Reaction Generates Indophenol and is Sensitively Detected by LC-MS.
We developed a mass spectrometry-based (MS) Berthelot assay to quantitatively measure ammonia. First, we assayed a pure indophenol standard using LC-MS (Fig. 1B,C). Next, we assayed products from the Berthelot reaction, which we adapted to be performed in 80:20 methanol:water, allowing for compatibility with polar metabolite extracts. We observed the same peak and MRM transition (198.1-167.1) as the pure indophenol standard (Fig. 1D,E). Next, we optimized the molar ratio of the reactant phenol to ammonia being measured ( Fig. 2A). In this assay, we tested 50 mM NH 4 Cl, which is far greater than any physiological concentration observed in biology 7 . To ensure reaction completion, a minimum volumetric ratio of 4:1 for each reactant solution to ammonia was required. Therefore, to ensure all measurements are quantitative, we used a volumetric ratio of 5:1 in our assays. Next, we assessed the sensitivity of this assay to low ammonia concentrations (Fig. 2B) and determined the lower limit of ammonia detection to be 500 nM, with a limit of linearity of 0.4 mM. Therefore, this assay is optimal for quantitatively measuring ammonia concentrations in physiological ranges for human plasma (~35 μM) and in fresh water, in which ammonia concentration is very low.
The MS Berthelot Assay Accurately Detects Ammonia Isotopologues ( 14 NH 3 and 15 NH 3 ). We tested whether ammonia isotopologues ( 14 NH 3 and 15 NH 3 ) can be distinguished by LC-MS (Fig. 2C). We performed the Berthelot reaction on both isotopologues of ammonia and found distinct mass spectra: 14 NH 3 generates 14 N-indophenol with an MRM transition of 198.2-167.9, and 15 NH 3 generates 15 N-indophenol with an MRM transition of 199.2-168.9 (Fig. 2D). Next, we tested whether the assay can accurately calculate the percent abundance of 14 N versus 15 N-labeled ammonia (Fig. 2E). The Berthelot reaction was performed with ratios of 500 μM 14 N-and 15 N-ammonia mixtures. The resulting standard curve was linear, R 2 = 0.9989, and therefore accurately measures ratios of 14 N: 15 N-ammonia.

The MS Berthelot Assay Accurately Measures Ammonia in Cell Lysates.
We tested whether the MS Berthelot assay could quantify ammonia from cell lysates, a more complex matrix comprised of many metabolites that may potentially interfere with the Berthelot reaction. Importantly, we performed the MS Berthelot assay using lysates extracted in 80% methanol, amenable for metabolite profiling (Fig. 3A). To ensure completion of the reaction in a cell lysate, we used a volumetric ratio of 10:1 of each solvent to lysate. First, we assayed the specificity of indophenol for the MS Berthelot assay. Indophenol was undetectable in mammalian cell lysates, and was only detected after the Berthelot reaction (Fig. 3D). Thus, as indophenol is not a mammalian metabolite, it can be distinctly measured after reaction with ammonia in the complex matrix of a polar metabolite extraction.
We also evaluated the sensitivity of ammonia detection from mammalian cells. We spiked a concentration curve of ammonia into 293T lysates, generating a linear standard curve with an R 2 value of 0.998 (Fig. 3C). Furthermore, ammonia increased linearly with a dynamic cell range of 500,000 to 2.5 million cells (R 2 = 0.9991) (Fig. 3D). To directly assay ammonia concentrations within mammalian cells, we performed the MS Berthelot assay from a cell lysate not spiked with ammonia. We used the standard addition technique to generate a standard curve by spiking ammonia into 293T cell lysates (0.0-0.8 mM ammonia) (Fig. 3E). In 293T cells, we measured 100 femtomoles of ammonia per cell (Fig. 3F), consistent with levels detected using a commercial ammonia assay.  The MS Berthelot Assay Distinctly Measures Ammonia Isotopologues in Cell Lysates. Next, we tested whether the MS Berthelot assay accurately measured ratios of 14 N/ 15 N-ammonia spiked into a cell lysate. Indeed, with R 2 = 0.995, this assay accurately measures the ratio of ammonia isotopologues (Fig. 4A). We next applied isotope-tracing studies to test whether ammonia as a by-product of metabolic reactions can be detected using a biologically relevant cellular system. Glutamine is commonly catabolized by the ubiquitous enzyme glutaminase (GLS) to generate glutamate and ammonia (Fig. 4B). Lung cancer cell lines vary in GLS expression, and therefore represent an excellent model to study glutamine-derived ammonia using this assay (Fig. 4C). We treated GLS high (Calu6, H2030) and GLS low (H23, H1299) lung cancer cell lines with the stable isotope 15 N-(amide)-glutamine, which liberates 15 N-ammonia in the process of glutaminolysis. We performed the MS Berthelot assay on media from cultured cells, as well as cell lysates. As anticipated, cell lines with high GLS expression contributed more glutamine-derived ammonia to the total ammonia pool in media (~55%) and in cells (~45%), compared to GLS low cell lines, which contributed 45% and 30% to media and cellular ammonia, respectively (Fig. 4D).

Discussion
Here we describe the development of a novel mass spectrometry assay, MS Berthelot assay, which sensitively measures ammonia in both pure solutions and cell lysates. This method utilizes the Berthelot reaction to convert ammonia into indophenol, which is directly detected by LC-MS. In samples that have complex matrices, such as cell lysates (as shown in Figs 3 and 4), the MS Berthelot assay generates a distinct molecule, indophenol, which can only be derived from ammonia. Thus, indophenol provides a direct readout of ammonia levels. Therefore, the MS Berthelot assay significantly advances current methods for ammonia detection by filtering out the interfering molecules that often complicate analysis of ammonia in complex matrices such as soil, blood, seawater, and cell lysates. We speculate that the MS-Berthelot assay can be applied to these systems for a more sensitive and direct method to measure ammonia compared to techniques that are currently available.
The MS Berthelot assay has several advantages over current ammonia detection strategies, including specificity, sensitivity, and the ability to detect isotopologues. The colorimetric Berthelot reaction is complicated by interfering background reactions that absorb in the same spectrum as indophenol. In the MS Berthelot assay, the triple quadruple mass spectrometer filters out background reactions by selectively measuring ion counts of the mass:charge ratio of indophenol. Therefore, the MS-Berthelot assay resolves one of the major hurdles of ammonia measurement by eliminating the interference of background reactants in the readout. Furthermore, the colorimetric Berthelot assay for ammonia measurement has a lower limit of detection of ~7 μM, whereas the MS-Berthelot assay measures ammonia as low as 500 nM, increasing the sensitivity for ammonia detection 14-fold 11 . This sensitivity is not only an improvement on the colorimetric Berthelot assay, but also an improvement on commercial assays, which often detect a lower limit of ~10 μM ammonia.
In this study, the MS-Berthelot assay was performed on cell lysates that were isolated with a polar metabolite extraction, showing the potential for this assay to be multiplexed with metabolite profiling studies. For this type of analysis, the Berthelot Reaction must be performed on an aliquot of the initial metabolite isolation, and metabolite profiling be performed on the remaining unreacted portion, since the highly oxidizing conditions of the Berthelot reaction will alter the contents of the metabolite isolate (Fig. 3A). The MS-Berthelot assay requires only 2% of the initial metabolite isolation, and is therefore feasible to do in parallel with steady-state metabolomics experiments without negatively impacting metabolite detection.
Finally, the MS Berthelot assay was used to distinctly measure ammonia isotopologues ( 14 NH 3 and 15 NH 3 ). The MS Berthelot assay accurately measures ratios of ammonia isotopologues by analyzing 14 N -indophenol (m/z:198) to represent 14 NH 3 and 15 N-indophenol (m/z: 199) to represent 15 NH 3 . Importantly, this analysis was performed on cellular lysates isolated by methanol extraction, and therefore, measurement of ammonia isotopologues can be integrated with nitrogen metabolic tracing studies. In this study, we provide one example of this in which the stable isotopologue of glutamine ( 15 N-(amide)-glutamine) was used. We showed that using the MS Berthelot assay, cells with high glutaminase expression generate more ammonia from glutaminolysis than cells with low glutaminase expression. We detected this in both cell lysates and the media. In addition to glutamine, this technique can be applied to wide-range of metabolic tracing experiments involving other nitrogen-containing metabolites such as branched chain amino acids and nucleotides. Beyond mammals, the MS Berthelot assay is applicable to all organisms, as ammonia is a ubiquitious by-product of metabolism, and will significantly advance mechanistic studies of ammonia metabolism.

Materials and Methods
Mass Spectrometry. Samples were analyzed on a reverse phase ion-pairing chromatography coupled to tandem mass spectrometry (Agilent LC-MS). Analytes were eluted in buffer A (97% H 2 O, 3% MeOH, 10 mM Tributylamine, 15 mM Glacial Acetic Acid, pH 5.5) and buffer B (10 mM Tributylamine, 15 mM Glacial Acetic Acid in 100% MeOH). Samples were run on a ZORBAX Extend-C18, 2.1 × 150 mm, 1.8 μm (Agilent) starting with a flow rate of 0.25 mL/min for 2.5 minutes of buffer A, followed by a linear gradient (100% buffer A to 80% buffer A) for 5 minutes, followed by a linear gradient (80% buffer A to 55% buffer A) for 5.5 minutes, followed by a linear gradient (55% buffer A to 1% buffer A) for 7 minutes, followed by 4 minutes with (1% buffer A). Following each run, an acetonitrile backwash was utilized to clean the column followed by a 8 minute re-equilibration period of 100% buffer A. Samples were ionized (with negative polarity) using Agilent Jet Spray ionization; nebulizer 45 psi, capillary -2000 V, nozzle voltage: 500 V, sheath gas temperature 325 °C, and sheath gas flow 12 L/ min. An Agilent 6470 Triple Quadrupole mass spectrometer was used for mass detection with a targeted method for indophenol. Using a 1 ug/mL indophenol standard (Sigma), the method was developed with a fragmentor and wells were quickly washed with 1 mL ice-cold PBS. Then, 1 mL of 80% HPLC-grade Methanol (BT Baker) for a 10 cm dish or 500 uL of ice-cold 80% HPLC-grade Methanol (BT Baker) for a 6-well dish were used to extract polar metabolites. Tissue culture dishes were incubated at −80 for 10 minutes, and scraped on dry ice. Lysates were centrifuged at 10,000 x g for 10 minutes and supernatants were either used for assays or stored at −80.
Metabolic tracing. Lung cancer cell lines were incubated with 2.0 mM 15 N-(amide)-glutamine for 24 hours.
Metabolites were isolated from the media in a 10:1 80% MeOH: lysate ratio and from cells as previously described. The Berthelot reaction was performed on isolates and analyzed by LC-MS.
The MS Berthelot assay. The Berthelot reaction was performed on pure ammonia standards made in 80:20 methanol:water or cellular lysates generated using the previously described method for polar metabolite extraction. Reactions in 80% MeOH were performed with a volumetric ratio of 10:1 (reactant mixture: sample). For example, 20uL of sample was reacted with 100uL Solution #1 and 100 uL Solution #2. In a cell lysate, the volumetric ratio used was 20:1 (reactant mixture: sample), in which 20 uL of sample would be reacted with 200 uL Solution #1 and 200 uL Solution #2. Solution #1 is composed of 100 mM Phenol (Sigma) and 50 mg/L sodium nitroprusside (Sigma). Solution #2 is composed of 0.38 M Dibasic Sodium Phosphate (Sigma), 125 mM NaOH, 1% sodium hypochlorite, available chlorine 10-15% (Sigma). Upon addition of the two reactant solutions, the samples are mixed and incubated at 37 °C for 40 minutes. All solutions and reactions should be stored at 4 °C until use for LC-MS analysis.
Ammonia Measurement. Ammonia was measured using the Berthelot method as described above or using a commercial colorimetric assay (Abcam #ab83360).