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Clinical Chemistry 51: 1487-1492, 2005. First published May 26, 2005; 10.1373/clinchem.2004.046995
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Right arrow Automation and Analytical Techniques
(Clinical Chemistry. 2005;51:1487-1492.)
© 2005 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

Stable-Isotope Dilution Liquid Chromatography–Electrospray Injection Tandem Mass Spectrometry Method for Fast, Selective Measurement of S-Adenosylmethionine and S-Adenosylhomocysteine in Plasma

Henkjan Gellekink1, Dinny van Oppenraaij-Emmerzaal1, Arno van Rooij1, Eduard A. Struys3, Martin den Heijer2 and Henk J. Blom1,a

1 Laboratory of Pediatrics and Neurology (424) and 2 Departments of Endocrinology (531) and Epidemiology and Biostatistics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
3 Metabolic Unit, Department of Clinical Chemistry, VU University Medical Centre Amsterdam, Amsterdam, The Netherlands.

aAddress correspondence to this author at: Laboratory of Pediatrics and Neurology (424), Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Fax 31-24-3688754; e-mail h.blom{at}cukz.umcn.nl.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: It has been postulated that changes in S-adenosylhomocysteine (AdoHcy), a potent inhibitor of transmethylation, provide a mechanism by which increased homocysteine causes its detrimental effects. We aimed to develop a rapid and sensitive method to measure AdoHcy and its precursor S-adenosylmethionine (AdoMet).

Methods: We used stable-isotope dilution liquid chromatography–electrospray injection tandem mass spectrometry (LC-ESI-MS/MS) to measure AdoMet and AdoHcy in plasma. Acetic acid was added to prevent AdoMet degradation. Solid-phase extraction (SPE) columns containing phenylboronic acid were used to bind AdoMet, AdoHcy, and their internal standards and for sample cleanup. An HPLC C18 column directly coupled to the LC-MS/MS was used for separation and detection.

Results: In plasma samples, the interassay CVs for AdoMet and AdoHcy were 3.9% and 8.3%, and the intraassay CVs were 4.2% and 6.7%, respectively. Mean recoveries were 94.5% for AdoMet and 96.8% for AdoHcy. The quantification limits were 2.0 and 1.0 nmol/L for AdoMet and AdoHcy, respectively. Immediate acidification of the plasma samples with acetic acid prevented the observed AdoMet degradation. In a group of controls (mean plasma total Hcy, 11.2 µmol/L), plasma AdoMet and AdoHcy were 94.5 and 12.3 nmol/L, respectively.

Conclusions: Stable-isotope dilution LC-ESI-MS/MS allows sensitive and rapid measurement of AdoMet and AdoHcy. The SPE columns enable simple cleanup, and no metabolite derivatization is needed. The instability of AdoMet is a serious problem and can be prevented easily by immediate acidification of samples.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Increased plasma total homocysteine (tHcy) 1 is a risk factor for many pathologic conditions, including cardiovascular disease, congenital abnormalities, certain malignancies, and neurologic disorders (1)(2). However, whether increased homocysteine itself is causally related to these disease states or is a marker of impaired 1-carbon metabolism remains a subject of debate.

Homocysteine is a sulfur-containing amino acid derived from demethylation of the essential amino acid methionine. After condensation of methionine and ATP, S-adenosylmethionine (AdoMet), the principal methyl donor in the human body, is formed. The methyl group can be donated to a variety of macromolecules, such as DNA, RNA, proteins, and lipids, as well as to (small) precursor molecules such as guanidinoacetate and catechol(amine)s. The demethylated product of AdoMet is S-adenosylhomocysteine (AdoHcy), which is hydrolyzed to homocysteine and adenosine in a reversible reaction catalyzed by AdoHcy hydrolase.

Efficient removal of adenosine and homocysteine is essential for cellular function because the equilibrium of the reaction catalyzed by AdoHcy hydrolase strongly favors the formation of AdoHcy, a strong inhibitor of most cellular methylation reactions. In vivo studies have demonstrated that increased tHcy is associated with increased plasma AdoHcy and a lower AdoMet/AdoHcy ratio (3), also called the methylation index, which correlates well with global DNA hypomethylation in patients with cardiovascular disease (4) as well as in different tissues, including lymphocytes, brain, and liver (5)(6). It has been suggested that AdoHcy-mediated hypomethylation provides an alternative mechanism for the pathogenesis of diseases related to hyperhomocysteinemia. Moreover, several studies have shown that AdoHcy is a stronger risk factor for cardiovascular disease than homocysteine (4)(7), a finding that makes the determination of AdoMet and AdoHcy an important tool to evaluate the clinical conditions associated with hyperhomocysteinemia.

Because the concentrations of AdoMet and AdoHcy in body fluids are low (~10–100 nmol/L), their measurement is time-consuming and difficult. In addition, AdoMet is unstable and partially degrades into AdoHcy in untreated samples (this study). We therefore aimed to develop a sensitive and rapid high-throughput method for simultaneous measurement of AdoMet and AdoHcy in biological samples by liquid chromatography–electrospray injection tandem mass spectrometry (LC-ESI-MS/MS).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
sample collection and storage
Blood samples were drawn from the antecubital vein into 4.5-mL evacuated glass tubes containing EDTA (BD Vacutainer Systems), placed on ice immediately, and centrifuged at 3500g for 5 min with minimal delay. The plasma was separated and stored at –20 °C until analysis. For AdoMet and AdoHcy measurements, 500 µL of plasma was directly acidified with 50 µL of 1 mol/L acetic acid to a final acetic acid concentration of 0.091 mol/L, mixed thoroughly, and then stored at –20 °C. All study participants gave informed consent.

homocysteine measurements
Plasma tHcy was measured in our laboratory by an automated HPLC method with reversed-phase separation and fluorescence detection. The HPLC system consisted of a Gilson 232-401 sample processor, Spectra Physics 8800 solvent delivery system, and LC 304 fluorometer (8).

plasma sample preparation for ADOMET and ADOHCY measurements
Sample cleanup was performed with solid-phase extraction (SPE) columns (Varian Inc.) containing phenylboronic acid, which at pH 7 to 8 selectively binds cis diol groups. The SPE columns were preconditioned by addition of five 1-mL volumes of 0.1 mol/L formic acid and five 1-mL volumes of 20 mmol/L ammonium acetate (pH 7.4). Before SPE, the acidified samples were neutralized with 55 µL of 1 mol/L ammonia to a pH of 7.4 to 7.5, and 110 µL of internal standard [1.5 µmol/L for 2H3-AdoMet (CDN Isotopes) and 0.41 µmol/L for 13C5-AdoHcy (9)] was added. The mixture was then applied to the SPE column for binding of AdoMet, AdoHcy, and their internal standards 2H3-AdoMet and 13C5-AdoHcy. Water-soluble impurities were removed by washing the column twice with 1 mL of 20 mmol/L ammonium acetate (pH 7.4) (10), and AdoHcy and AdoMet were eluted in 1 mL of 0.1 mol/L formic acid. After SPE, AdoMet and AdoHcy were stable for at least 6 months (at –20 °C) because elution from the SPE column by formic acid (pH 2–3) stabilizes AdoMet and AdoHcy. The samples (20 µL) were injected on an equilibrated (0.2 mL/L acetic acid) Symmetry-Shield HPLC C18 column [100 x 2.1 mm (i.d.); Waters Corporation] and eluted in a gradient (0%–0.3%) of methanol in 0.2 mL/L aqueous acetic acid delivered by an HP 1100 binary pump (Agilent Technologies) with the splitter (Acurate; LC Packings) in the 1:4 mode, allowing the injection of 4 µL of sample into the electrospray injection chamber. The retention times were 2.40 and 2.80 min for AdoMet and AdoHcy, respectively. AdoMet and AdoHcy concentrations were measured by LC-ESI-MS/MS with the Micromass Quattro LC (Waters) in the positive-ion (ESI+) mode. Optimal multiple-reaction monitoring conditions were obtained for 4 channels: AdoMet (m/z 399->250), 2H3-AdoMet (m/z 402->250), AdoHcy (m/z 385->136), and 13C5-AdoHcy (m/z 390->136). Data were acquired and processed by Quanlynx for Windows NT software (Micromass).

ADOMET and ADOHCY quantification and ion suppression
Calibrators (AdoMet and AdoHcy) and internal standards (2H3-AdoMet and 13C5-AdoHcy) were included in each analytical run for calibration. Briefly, stock solutions of AdoMet and AdoHcy in deionized water were diluted in ammonium acetate (pH 7.4) to concentrations of 10, 20, 50, 100, 200, 400, and 800 nmol/L for AdoMet and 5, 10, 20, 50, 100, 200, and 400 nmol/L for AdoHcy. We added 110 µL of internal standard to 500 µL of calibration solution and then processed the solution as described above for the samples. Calibration curves were obtained by plotting ratios of the peak area (calibrator/internal standard) against the concentration of the calibrator. We quantified AdoMet and AdoHcy by interpolating the observed peak-area ratio (m/z 399 and 385 peaks for endogenous AdoMet and AdoHcy vs m/z 402 and 390 peaks for the 2H3-AdoMet and 13C5-AdoHcy internal standards) on the linear regression line for the calibration curve. When AdoMet or AdoHcy concentrations were low, the samples were measured again, and an additional low-range calibration curve was prepared (2, 5, 10, 20, and 50 nmol/L AdoMet or 1, 2.5, 5, 10, and 20 nmol/L AdoHcy) as described above.

Ion suppression was calculated from the peak areas of the internal standards added to the calibrator solutions and compared with the peak areas of the internal standard that was added to each plasma sample. The relative change in peak area of the internal standard was attributed to matrix effects.

statistics
Linear regression analysis (Excel) was used to verify the linearity of the calibration curves, and one-way ANOVA (SPSS, Ver. 12.0) was used to assess differences attributable to storage conditions for AdoMet and AdoHcy concentrations in pooled plasma samples.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
chromatography and mass spectra
Shown in Fig. 1 are typical chromatograms of a control plasma prepared and subjected to LC-ESI-MS/MS analysis as described in the Materials and Methods section. Elution times were 2.4 min for AdoMet and 2H3-AdoMet and 2.8 min for AdoHcy and 13C5-AdoHcy. Decomposition MS/MS mass spectra of AdoMet and AdoHcy are shown in Fig. 2 . Optimal multiple-reaction monitoring conditions were obtained in the positive-ion mode: AdoMet, m/z 399->250 (adenosine); AdoHcy, m/z 385->36 (adenine).



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  		Figure 1. Typical MRM chromatograms of control serum.

Panels A and B show the peaks for endogenous AdoMet monitored at m/z 399->250 (A) and the internal standard 2H3-AdoMet monitored at m/z 402->250 (B), both of which elute at ~2.4 min. Panels C and D show the peaks for endogenous AdoHcy monitored at m/z 385->136 (C) and the internal standard 13C5-AdoHcy monitored at m/z 390->136 (D), both of which elute at ~2.8 min.



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  		Figure 2. Mass fragmentograms of AdoMet (A) and AdoHcy (B) generated in the positive-ion mode.

ESI+ MS/MS conditions: capillary voltage, 3.0 kV; cone voltage, 25 V; collision gas, argon at 0.15 Pa; collision energy, 15 eV. (A), the parent ion for AdoMet is at m/z 399, and the main product ion is at m/z 250 (adenosine); (B) the parent ion for AdoHcy is at m/z 385, and the main product ion is at m/z 136 (adenine).

linearity of ADOMET and ADOHCY measurements and quantification limits
The calibration curve was linear over the ranges 10–800 nmol/L for AdoMet and 5–400 nmol/L for AdoHcy, as determined by 3 separate measurements. The coefficient of linear correlation (r2) was >0.999 for the calibration curves of both AdoMet and AdoHcy (Fig. 3 ). For the lower-range calibration curves (2–50 nmol/L for AdoMet and 1–20 nmol/L for AdoHcy), the coefficient of linear correlation was also >0.999 for both curves. The quantification limits, derived from the lower-range calibration curve, were 2.0 nmol/L for AdoMet and 1.0 nmol/L for AdoHcy (mean signal-to-noise ratio >10).



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  		Figure 3. LC-MS/MS calibration curves for AdoMet ({blacksquare}) and AdoHcy ({diamondsuit}).

Calibration curves were linear over a range of 10–800 nmol/L for AdoMet and 5–400 nmol/L for AdoHcy. For AdoMet ({blacksquare}), y = 0.125x + 0.077 nmol/L (r2 = 0.9999); for AdoHcy ({diamondsuit}), y = 0.249x – 0.046 nmol/L (r2 = 0.9997).

quality control, recovery, and precision
Recovery experiments were performed within the physiologic ranges of AdoMet and AdoHcy, as determined in healthy controls (see below), by use of nonacidified pooled plasma samples. The AdoMet concentration of the test pool was 77.3 nmol/L, and for AdoHcy, the concentration was 17.6 nmol/L. Mean recoveries were 94.5% for AdoMet (100 nmol/L added to the test pool) and 96.8% for AdoHcy (20 nmol/L added to the test pool) with CVs of 5.0% and 6.1%, respectively (see Table 1A ). The precision data for the method are presented in Table 1B . For this purpose, a large test pool of plasma was collected and treated according to the standard procedure used by our laboratory to assure metabolite stability over time (also see the next section). The intraassay CVs (n = 9) for AdoMet and AdoHcy were 4.2% and 6.7%, respectively, and the interassay CVs (n = 12) were 3.9% and 8.3%, respectively (Table 1B ). Ion suppression in plasma was 30% and 20% for AdoMet and AdoHcy, respectively. This assay comprises a fast sample preparation step (10 samples in 30 min) and a measurement/column regeneration time of 8.5 min, which enables us to handle 100 samples/day.


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  		Table 1. Recovery (A) and precision (B) of the LC-MS/MS assay for AdoMet and AdoHcy in plasma.

stability of ADOMET and ADOHCY
We observed a decrease in AdoMet over time in nonacidified plasma samples and a simultaneous increase of AdoHcy, suggesting partial degradation of AdoMet to AdoHcy in our plasma samples. We therefore evaluated the AdoMet degradation rate during storage in treated and nontreated EDTA-plasma samples. After only 3 h at room temperature, a marked decrease in AdoMet (~10%) and an increase in AdoHcy (~24%) were observed in the nonacidified plasma samples. This degradation process was not prevented by sample storage at –20 °C, and after 1 month, the AdoMet concentrations had decreased to 43% of the initial value (P = 0.009), and AdoHcy had increased to 150% of the initial value (P = 0.067). Acidification of aliquots of the same plasma samples (with 1 mol/L acetic acid) stabilized both AdoMet and AdoHcy (Table 2 ). A decrease in AdoMet and a parallel increase in AdoHcy were also observed in nontreated plasma when samples were collected in sodium citrate (pH 5.5) or heparin Vacutainer Tubes (BD Vacutainer Systems; data not shown). These observations are in line with earlier results of Stabler and Allen (11), who observed the same phenomenon in plasma and urine sample that were stored at room temperature and in samples stored at 4 °C and below. Even the use of acidic citrate (pH 4.3) may not prevent degradation because the pH increases to ~6.0 after blood sampling (12). We therefore acidified plasma samples for AdoMet and AdoHcy measurements with acetic acid (final concentration, 0.091 mol/L acetic acid; final pH 4.5–5.0) with minimal delay after blood sampling to prevent AdoMet degradation. At this pH, no protein precipitation was observed.


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  		Table 2. AdoMet degradation observed over time in nonacidified but not acidified EDTA-plasma samples.

ADOMET and ADOHCY concentrations in control individuals
As controls, 26 apparently healthy individuals from the Radboud University Nijmegen Medical Centre [mean (SD) age, 28.3 (8.2) years; 69% women] participated in this study to verify our method. Plasma samples were prepared for AdoMet, AdoHcy, and tHcy determination as described. Mean (SD) concentrations were 11.2 (4.8) µmol/L for tHcy (range, 7.0–29.7 µmol/L), 94.5 (15.2) nmol/L for AdoMet (range, 69.4–121.8 nmol/L), and 12.3 (3.7) nmol/L for AdoHcy (range, 6.2–21.9 nmol/L). The resulting mean AdoMet/AdoHcy ratio was 8.5 (3.0). The AdoMet and AdoHcy values we obtained from control individuals are summarized in Table 3 , along with data reported by other groups.


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  		Table 3. Mean (SD) AdoMet, AdoHcy, and tHcy concentrations in plasma samples from healthy controls.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Interest in AdoMet and AdoHcy measurement has increased over the last few years, particularly since increased AdoHcy and decreased cellular methylation capacity have emerged as a mechanism explaining the association between hyperhomocysteinemia and increased risk for cardiovascular and neurologic diseases (1)(4)(7)(13).

In this report we present a highly selective and sensitive high-throughput method for the simultaneous measurement of AdoMet and AdoHcy in plasma samples by stable-isotope dilution LC-ESI-MS/MS. Phenylboronic acid-containing SPE columns were used for AdoMet and AdoHcy extraction, and no metabolite derivatization was needed. Our method meets the criteria of minimal time required for sample preparation (10 samples in 30 min) and measurement/column regeneration (8.5 min), enabling us to process 100 samples per day. The method was linear over a broad range for both AdoMet and AdoHcy (r2 >0.999). Recoveries >94% were obtained at physiologic concentrations, and the inter- and intraassay CVs were <10%. Quantification limits of the assay were 2.0 and 1.0 nmol/L for AdoMet and AdoHcy, respectively, enabling measurement of AdoMet and AdoHcy within the (patho)physiologic ranges.

Several methods to measure AdoMet and AdoHcy have been described, including HPLC with fluorescence detection (14)(15)(16), coulometric electrochemical detection (17), and stable-isotope dilution LC-MS/MS (9)(11) (Table 3Up ). The disadvantages of the published methods include long sample preparation times attributable to the use of inorganic chemicals [e.g., phosphate buffers (9) or perchloric acid for deproteinization (11)], which are less compatible with MS. Other time-consuming steps include derivatization (15), sample analysis, and column regeneration (11)(14)(17). Our method used MS-compatible buffers and solutions, thereby reducing potential ion suppression effects. The use of stable isotopes enables adjustment for ion suppression attributable to matrix effects or early-eluting compounds.

The observation that AdoMet is unstable in untreated samples and partially degrades into AdoHcy, as observed by us (this study) and others (11), may confound the results obtained in previous studies that report relatively low AdoMet and high AdoHcy concentrations. The sensitivity of the assay may be compromised, and the AdoMet/AdoHcy ratio may change because the quantitative decrease in AdoMet is not reflected by the increase in AdoHcy. To prevent this degradation process, we acidified the plasma samples with acetic acid to a pH <5.0 immediately after blood collection and centrifugation. Interassay CVs calculated over a 4-month period indicated that acidification of plasma samples with acetic acid stabilizes AdoMet and AdoHcy for at least 4 months.

We measured AdoMet and AdoHcy in 26 healthy individuals and found AdoMet and AdoHcy concentrations similar to those reported by others (11)(14), and our calculated methylation index approached values observed by Stabler and Allen (11) and Melnyk et al. (17). We found only a slight correlation between tHcy and AdoHcy (data not shown). This result may be attributable to the fact that most of our observations were within the physiologic range for Hcy; we may have observed a stronger correlation if hyperhomocysteinemic individuals had been included in the study.

In conclusion, the presented method provides a fast, reliable method for the routine measurement of AdoMet and AdoHcy in plasma, enabling investigation of disturbed 1-carbon metabolism in various disease states associated with hyperhomocysteinemia.


   Acknowledgments
 
This study was supported in part by The Netherlands Heart Foundation (Grant 2002B68). Martin den Heijer is supported by a VENI grant from the Dutch Organization for Scientific Research (NWO). Henk J. Blom is an Established Investigator of the Netherlands Heart Foundation (D97.021). We greatly acknowledge S.G. Heil for critical evaluation of the manuscript.


   Footnotes
 
1 Nonstandard abbreviations: tHcy, total homocysteine; AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine; LC-ESI-MS/MS, liquid chromatography–electrospray injection tandem mass spectrometry; and SPE, solid-phase extraction.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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C.-W. Hu, C.-J. Wang, L. W. Chang, and M.-R. Chao
Clinical-Scale High-Throughput Analysis of Urinary 8-Oxo-7,8-Dihydro-2'-Deoxyguanosine by Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry with On-Line Solid-Phase Extraction
Clin. Chem., July 1, 2006; 52(7): 1381 - 1388.
[Abstract] [Full Text] [PDF]

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