Highly sensitive quantification of serum malonate, a possible marker for de novo lipogenesis, by LC-ESI-MS/MS.

We describe a new sensitive and specific method for the quantification of serum malonate (malonic acid, MA), which could be a new biomarker for de novo lipogenesis (fatty acid synthesis). This method is based upon a stable isotope-dilution technique using LC-MS/MS. MA from 50 microl of serum was derivatized into di-(1-methyl-3-piperidinyl)malonate (DMP-MA) and quantified by LC-MS/MS using the positive electrospray ionization mode. The detection limit of the DMP-MA was approximately 4.8 fmol (500 fg) (signal-to-noise ratio = 10), which was more than 100 times more sensitive compared with that of MA by LC-MS/MS using the negative electrospray ionization mode. The relative standard deviations between sample preparations and measurements made using the present method were 4.4% and 3.2%, respectively, by one-way ANOVA. Recovery experiments were performed using 50 microl aliquots of normal human serum spiked with 9.6 pmol (1 ng) to 28.8 pmol (3 ng) of MA and were validated by orthogonal regression analysis. The results showed that the estimated amount within a 95% confidence limit was 14.1 +/- 1.1 pmol, which was in complete agreement with the observed X(0) = 15.0 +/- 0.6 pmol, with a mean recovery of 96.0%. This method provides reliable and reproducible results for the quantification of MA in human serum.

were conducted in accordance with the ethical standards of the Helsinki Declaration. Rat serum was prepared in our previous study ( 9 ) and had been stored at Ϫ 20°C until it was used in the present experiments.

Sample preparation
Fifty l of serum was placed in a microcentrifuge tube (1.5 ml, Eppendorf, Hamburg, Germany), and 19.2 pmol (2 ng) of [ 13 C 3 ] MA in 100 l of acetonitrile as an internal standard. The sample tube was vortexed for 1 min and centrifuged at 2,000 g for 1 min. The solution of internal standard in acetonitrile led to deproteinization of the sample and the liquid phase was collected and evaporated to dryness at 80°C under a nitrogen stream. Derivatization of MA into DMP-MA was performed according to the Shiina method for the synthesis of carboxylic esters ( 10 ) with some modifi cations. The reagent mixture for derivatization consisted of 2-methyl-6-nitrobenzoic anhydride (67 mg), 4-dimethylaminopyridine (20 mg), pyridine (900 l), and 3-hydroxy-1-methylpiperidine (100 l). The freshly prepared reagent mixture (100 l) was added to the serum extract and the reaction mixture was allowed to stand at room temperature for 30 min. After the addition of 2 ml of n-hexane, the mixture was vortexed for 30 s and centrifuged at 700 g for 2 min. The clear supernatant was collected and evaporated at 80°C under nitrogen. The residue was redissolved in 50 l of 1% formic acid in water and an aliquot (1 l) was injected into the following LC-MS/MS system.

Determination of DMP-MA by LC-P-ESI-MS/MS
The LC-MS/MS system consisted of a TSQ Quantum Ultra quadrupole mass spectrometer (Thermo Fisher Scientifi c, San Jose, CA) equipped with an H-ESI probe and a Nanospace SI-2 HPLC system (Shiseido, Tokyo, Japan). Chromatographic separation was performed using a Hypersil GOLD aQ column (150 × 2.1 mm, 3 m, Thermo Fisher Scientifi c) at 40°C. Initially, the mobile phase was comprised of 0.2% formic acid in water and was used at a fl ow rate of 200 l/min for 5 min, and it was then switched to 0.2% formic acid in acetonitrile at a fl ow rate of 300 l/min for an additional 3.5 min. The general LC-MS/MS conditions were as follows: spray voltage, 1000 V; vaporizer temperature, 350°C; sheath gas (nitrogen) pressure, 50 psi; auxiliary gas (nitrogen) fl ow, 40 arbitrary units; ion transfer capillary temperature, 350°C; collision gas (argon) pressure, 1.5 mTorr; collision energy, 15 V; and ion polarity, positive.
Although methodological reports for the quantifi cation of serum MA are not available, there have been some reports that describe the methods for the determination of urinary MA levels in patients with MCD defi ciency by gas chromatography ( 5,6 ) or gas chromatography-mass spectrometry ( 7 ). In these methods, urinary organic acids were extracted with ethyl acetate and converted into trimethylsilyl derivatives before analysis. Alternatively, blood malonylcarnitine has been measured for the diagnosis of MCD defi ciency using liquid chromatography-tandem mass spectrometry coupled with electrospray ionization mode (LC-ESI-MS/MS) ( 8 ). However, because all of these methods were developed to diagnose markedly elevated MA levels in patients with MCD defi ciency, the authors did not pay signifi cant attention to the sensitivities of the methods.
The aim of this study was to measure serum MA concentrations in normal human subjects with suffi cient sensitivity and specifi city. For this purpose, serum MA was derivatized into di-(1-methyl-3-piperidinyl)malonate (DMP-MA) and quantifi ed using positive LC-ESI-MS/MS (LC-P-ESI-MS/MS).

Sample collection
Blood samples were collected from healthy human volunteers. After coagulation and centrifugation at 1,500 g for 10 min, serum samples were stored at Ϫ 20°C until analysis. Informed consent was obtained from all subjects, and the experimental procedures late the values, orthogonal regression analysis was performed in the recovery study by using JMP software. For all analyses, significance was accepted at the level of P < 0.05.

Selected reaction monitoring
A typical ESI positive mass spectrum of the DMP-MA is shown in Fig. 1 . This DMP ester derivative exhibited [M+H] + ion at m/z 299 as the base peak. In the MS/MS spectrum using m/z 299 as a precursor ion, the [C 6 H 11 N+H] + ion was observed at m/z 98 as the most prominent peak. The selected reaction monitoring (SRM) was conducted using m/z 299 → m/z 98 for the DMP-MA and m/z 302 → m/z 98 for the [ 13 C 3 ] variant. We also monitored m/z 299 → m/z 202, a product ion containing the MA molecule

Determination of MA by LC-N-ESI-MS/MS
LC-negative (N)-ESI-MS/MS analysis of MA was carried out using the same LC-MS/MS instrument described above. Hypersil GOLD column (150 × 2.1 mm, 3 m, Thermo Fisher Scientifi c) was used at 40°C. The mobile phase consisted of methanol-water (5:95, v/v) containing 0.2% formic acid and was used at a fl ow rate of 200 l/min. The general LC-MS/MS conditions were as follows: spray voltage, 4000 V; vaporizer temperature, 350°C; sheath gas (nitrogen) pressure, 50 psi; auxiliary gas (nitrogen) fl ow, 30 arbitrary units; ion transfer capillary temperature, 300°C; collision gas (argon) pressure, 1.5 mTorr; collision energy, 15 V; and ion polarity, negative.

Statistics
Data are reported as the mean ± SD. Linearity of the calibration curve was analyzed by simple linear regression. Reproducibility was analyzed by one-way ANOVA (JMP software, SAS Institute, Inc., Cary, NC). The estimated amount ± 95% confidence limit was obtained as an index of precision ( 11 ). To calcu- to-noise ratio of 10, whereas the conventional LC-N-ESI-MS/ MS was barely able to detect 480 fmol of MA ( Fig. 2B ).

Calibration curve
A calibration curve was established for MA ( Fig. 3 ). Each of different amounts (2.4, 4.8, 9.6, 19.2, 38.5, and 76.9 pmol) of authentic MA was mixed with 19.2 pmol of [ 13 C 3 ]MA, derivatized to the DMP ester and quantifi ed as described in the Materials and Methods. The weight ratio of MA, relative to the corresponding 13 C-labeled internal standard, was plotted on the abscissa and the peak-area ratio of the DMP-MA to the [ 13 C 3 ] variant measured by LC-P-ESI-MS/MS was plotted on the ordinate. The linearity of the standard curve, as determined by simple linear regression, was excellent for weight ratios between 0.125 and 4.0 (n = 6; r = 1.000; P < 0.0001). Figure 4 shows typical SRM chromatograms for DMP-MA and the [ 13 C 3 ] variant obtained with 50 l sera from a normal human (A) and a control rat (B). The peak-area ratio of the DMP-MA to the [ 13 C 3 ] variant was calculated from the chromatograms, and MA amount was determined by applying the ratio to the calibration curve. The peaks of DMP-MA in chromatograms A and B correspond to ‫ف‬ 0.66 pmol (0.66 M) and ‫ف‬ 4.43 pmol (4.43 M), respectively.

Precision and accuracy of the LC-P-ESI-MS/MS method
The following studies were performed to determine the precision and accuracy of the present method using the same serum obtained from a normal human subject. Reproducibility was investigated by analyzing four samples in triplicate by LC-P-ESI-MS/MS ( Table 1 ). The results were analyzed by a one-way ANOVA in which the analytical errors were divided into two sources, sample preparation but the former showed much better signal-to-noise ratio than the latter.
By N-ESI mode, authentic MA exhibited [M-H]ion at m/z 103 as the base peak. In the MS/MS spectrum, the CH 3 COO ion was observed at m/z 59 as the most prominent peak. The SRM was conducted using m/z 103 → m/z 59 for the MA.

Comparison of the sensitivities between P-ESI and N-ESI methods
To  as shown in Fig. 2, the sensitivity of N-ESI was not suffi cient to quantify MA concentrations in a small volume of normal human serum. Recently, we derivatized another organic acid, mevalonate, into mevalonyl-(2-pyrrolidin-1-yl-ethyl)-amide and measured it using LC-P-ESI-MS/MS ( 12 ). In this method, mevalonate was lactonized into mevalonolactone and then a tertiary amine moiety was introduced by a characteristic amidation reaction with a primary alkylamine. As a result, the tertiary amine moiety markedly promoted protonation and attomole levels of mevalonate were detected. In the present study, tertiary amine moieties were successfully introduced to MA by esterifi cation with 3-hydroxy-1-methylpiperidine. Thus, the reaction for the synthesis of carboxylic esters by Shiina et al. ( 10 ) appears to be useful not only for the derivatization of alcohols ( 13 ) but also for that of carboxylic acids. This derivative, DMP-MA, exhibited [M+H] + as the base peak by P-ESI-MS and the detection limit by SRM was more than 100 times lower than that of underivatized MA by SRM with N-ESI mode.
The derivatization and purifi cation steps in this method are very simple but it should be mentioned that there are two pitfalls to obtaining reliable and reproducible results. First, use of the anion exchange column cartridge gave unexpectedly high values of MA concentrations. Serum MA was extracted by this cartridge and interfering peaks on SRM chromatograms were markedly reduced by the addition of this purifi cation step. However, the recoveries of known amounts of MA from this cartridge were always more than 100%, and additional experiments suggested that a signifi cant amount of MA was produced from unknown substance(s) in organic solvents by this anion exchange column (data not shown). Plasma methylmalonic acid (MMA) and its isomer succinic acid (SA) are also known to be extracted by this column ( 14 ). We have derivatized MMA and SA into DMP-MMA and DMP-SA, respectively, and analyzed them by the same HPLC condition as that for DMP-MA. The SRM was conducted using m/z 313 → m/z 98 for both DMP-MMA and DMP-SA. The results showed that DMP-MMA and DMP-SA were much more hydrophobic than DMP-MA and both compounds were eluted during washout phase with 0.2% formic acid in acetonitrile (after 6 min).
Second, pH of the fi nal sample solution should not be more than 7 because an alkaline condition easily hydrolyzes DMP-MA. After the derivatization step, most of the excess reagents and hydrophilic impurities were and SRM measurement. The variances were not considered to be attributable to the sample preparation because the errors during sample preparation were not signifi cantly larger than those between the measurements ( Table 2 ). The inter-assay coeffi cients of variation for the betweenand within-sample variations were 4.4% and 3.2%, respectively.
For the recovery experiments, known amounts of MA (a, 2a, 3a; a = 9.6 pmol) were spiked into 50 l aliquots of the serum samples (n = 2). After the clean-up and derivatization procedures, SRM was carried out in triplicate for each sample. The recoveries of the known spiked amounts of MA ranged from 94.5% to 99.0%, with a mean of 96.0% ( Table 3 ). In addition, the amount of endogenous MA found in unspiked 50 l serum aliquots was within the 95% confi dence limit for the estimated amount of MA calculated by orthogonal regression analysis, which also constituted an index for the precision and accuracy of the present method. Figure 5 depicts the circadian rhythm of the serum concentrations of MA in a healthy male. Postprandial increases of MA concentrations (maximum 235% after dinner) were observed and the levels peaked between 2.5 and 6.5 h postmeal. The increase of MA concentration disappeared after skipping breakfast on the second day, which supports the idea that the diurnal pattern of serum MA concentrations is controlled mainly by food intake.

DISCUSSION
We describe a sensitive new LC-P-ESI-MS/MS method for the quantifi cation of MA in serum. LC-N-ESI-MS/MS may be more suitable for the determination of negatively charged compounds, such as organic acids because the method does not require a derivatization step. However, precipitated by the addition of n-hexane but signifi cant amounts of 3-hydroxy-1-methylpiperidine and 4-dimethylaminopyridine were recovered with DMP-MA in the fi nal residue of the extract. Therefore, it was necessary to dissolve the fi nal residue in 1% formic acid in water to keep the pH of the solution less than 7. The mobile phase of the HPLC (0.2% formic acid in water) was not suffi cient to neutralize the fi nal extract.
The highly sensitive quantifi cation of serum MA can be useful for monitoring of de novo FAS, also called de novo lipogenesis, in normal humans. The diurnal variation of serum MA levels in a healthy human ( Fig. 5 ) was similar to the variation of de novo FAS determined in humans by continuous intravenous infusion of sodium [1-13 C]acetate and mass isotopomer distribution analysis ( 15,16 ). According to Timlin et al. ( 16 ), de novo FAS peaked 4.2 h after ingestion of a meal whereas lipoprotein-triacylglycerol concentrations peaked at 2.0 h postmeal. Another study, by Hudgins et al. ( 15 ), showed that the maximum values of de novo FAS occurred in the evening, 3.0-9.0 h after the last meal, although the peak after every meal was not detected because a limited number of postprandial data points were obtained. In our data, postprandial increases of MA concentrations peaked between 2.5 h and 6.5 h after meals and the maximum value was observed in the night 6.0 h after dinner. In addition, the increase of MA concentration disappeared after skipping the meal. Thus, serum MA concentrations are regulated by food intake and appear to be a good marker that refl ects de novo FAS in normal humans.
Because serum MA concentrations correlate well with de novo FAS, the most important enzyme that determines serum MA concentration is thought to be ACC, the ratelimiting enzyme in the fatty acid biosynthesis. In mammals, two ACC isoforms exist. Cytosolic ACC1 synthesizes malonyl-CoA, which participates in both de novo FAS and negative regulation of ␤ -oxidation. In contrast, malonyl-CoA synthesized by mitochondrial ACC2 acts mainly as an inhibitor of ␤ -oxidation ( 17 ). We cannot clarify at present which ACC contributes to serum MA concentration but both ACCs regulate de novo lipogenesis in a coordinated and complementary manner ( 18 ). Known amounts of MA were spiked into 50 l of normal human serum before sample preparation. a The value was obtained from Table 1 . b Recovery (%) = (amount found -X 0 ) / amount added × 100. c The estimated amount was calculated by orthogonal regression. Under special conditions, however, other enzymes, MCD, and FAS can also be determinants of tissue malonyl-CoA levels and serum MA concentrations. For example, when MCD activity is reduced, such as with MCD deficiency, serum MA concentrations are elevated. Alternatively, when FAS is blocked by any drugs, such as C75 and cerulenin ( 2 ), MA concentrations increase in spite of reduced de novo FAS. Therefore, it is important to rule out the presence of such special conditions when we use serum MA as a biomarker for de novo FAS.
In summary, we developed a new method for the quantifi cation of MA in human serum, which can be a good marker for de novo FAS. Derivatization of MA into DMP-MA allowed it to be quantifi ed by LC-P-ESI-MS/MS with excellent sensitivity. Recovery and reproducibility experiments verifi ed that this method provided highly reliable and reproducible analytical results.