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Articles |
1
Metabolic Unit, Department of Clinical Chemistry, Free University Hospital, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.
a Author for correspondence. Fax 31-20-4440305; e-mail C.Jakobs{at}AZVU.NL
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Abstract |
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Methods: We used tandem mass spectrometry (MS/MS) for the simultaneous determination of SAM and SAH, with stable-isotope-labeled internal standards. The 13C5-SAH internal standard was enzymatically prepared using SAH-hydrolase and [13C5]adenosine. The method comprises a weak anion-exchange solid-phase extraction procedure serving as clean-up step for the deproteinized plasma and CSF samples. After clean-up, samples were injected on a C18 HPLC column, which was connected directly to the tandem mass spectrometer, operating in MS/MS mode.
Results: In plasma samples, the intraassay CVs for SAM and SAH were 4.2% and 4.0%, respectively, and the interassay CVs were 7.6% and 5.9%, respectively. In CSF, the intraassay CVs for SAM and SAH were 6.8% and 6.9%, respectively, and the interassay CVs were 4.2% and 5.5%, respectively. Mean recovery of SAM and SAH for both matrices at two concentrations was 93%. Detection limits for SAM and SAH in samples were 7.5 and 2.5 nmol/L, respectively. Concentrations of SAM and SAH in plasma from healthy subjects were within the previously reported ranges. In 10 CSF samples, the mean concentrations (range) were 248 (137–385) nmol/L for SAM and 11.3 (8.9–14.1) nmol/L for SAH.
Conclusions: SAM and SAH can be analyzed by MS/MS, taking optimal advantage of the speed and high sensitivity and specificity of this relatively new analytical technique.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-ketoglutarate. SAH is known to be a
competitive inhibitor for several SAM-dependent methyltransferases
(1)(2). In typical situations, SAH
concentrations are relatively low. Increased homocysteine may produce
an increase in intracellular SAH, leading to inhibition of
SAM-dependent methyltransferases. Recent research suggests that alterations in the concentrations of SAM and SAH are associated with several disease states. Altered SAM and SAH concentrations in CSF have been found in HIV-positive patients. The concentration of SAM was decreased and the concentration of SAH was increased, leading to a significantly decreased SAM/SAH ratio (3). Additionally, low SAM concentrations were found in patients suffering from 10-methylenetetrahydrofolate reductase deficiency, cobalamin defects, and methionine adenosyltransferase II deficiency (4). There is evidence that SAM is required for the maintenance of myelin. Patients with significant demyelination, as observed by magnetic resonance imaging, showed complete reversion of the magnetic resonance imaging alterations after oral treatment with SAM (5). Because the interest in plasma and CSF concentrations of SAM and SAH derives from a broad spectrum of research, a fast, sensitive, and precise analytical method for the determination of these important intermediates is required.
Many methods have been designed for the determination of SAM and SAH in body fluids and tissues. Because the concentrations of SAM and SAH in tissue, red blood cells, and lymphocytes are in the micromolar range, HPLC combined with ultraviolet detection is frequently used for these matrices (6)(7)(8)(9). In plasma and CSF, however, the concentrations of SAM and SAH are considerably lower, requiring more sensitive detection. A sensitive HPLC method has been described that is based on the conversion of SAM and SAH to their 6-etheno analogs, highly fluorescent derivatives that enable detection in the nanomolar range (10)(11). More recently, an alternative for this fluorescence method was described in which SAM and SAH were converted to isoindoles by derivatization with naphthalenedialdehyde and cyanide (12). Both fluorescence methods showed adequate sensitivity but had the disadvantage of being very laborious and time-consuming. In this perspective, the method published by Melnyk et al. (13) was a major step forward. In this method, SAM and SAH are measured in trichloroacetate extracts of plasma, tissue, and lymphocytes, using electrochemical detection of nonderivatized SAM and SAH. Although the total analysis time was relatively short, only 20 samples could be processed in 1 day.
Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is a relatively new analytical technique that is establishing itself because of its high selectivity, sensitivity, and sample throughput. Magera et al. (14) showed that LC-MS/MS is a powerful analytical tool for rapid and sensitive determination of total homocysteine in plasma and urine. The possibility of using stable-isotope-labeled internal standards is an additional advantage of LC-MS/MS. In this report, we describe a fast and precise stable-isotope dilution LC-MS/MS method for the combined quantification of SAM and SAH in plasma and CSF samples.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subjects
The applicability of the MS/MS method for SAM and SAH was
investigated by the analyses of 15 plasma samples of premenopausal
women with plasma homocysteine within the reference interval, i.e.,
0–15 µmol/L. Fasting blood samples were collected with EDTA and
immediately centrifuged; plasma was stored at -20 °C until
analysis. CSF values were obtained by the analysis of 10 CSF
discard samples that had been obtained by lumbar puncture from
children with unknown neurological dysfunction and in whom no
disturbance of the methyl transfer pathway was suspected. Blood-free
CSF samples were stored at -20 °C until analysis.
enzymatic synthesis of 13C5-SAH
Because labeled SAH was not commercially available, we prepared it
by enzymatic synthesis. A 300-µL aliquot of 0.5 mmol/L
[13C5]adenosine aqueous
solution was mixed with 300 µL of 25 kU/L SAH hydrolase in 0.1
mmol/L phosphate buffer (pH 7.4) and 100 µL of 1.6 mmol/L
L-homocysteine. The mixture was incubated at 37 °C for
30 min. The progress of the reaction was monitored by timed injections
of 5-µL aliquots on a HPLC system. HPLC was performed with a Symmetry
C18 analytical column (3.9 x 150 mm; 5 µm
bead size; Waters) with H2O-methanol (85:15, by
volume), containing 1 mL/L formic acid as mobile phase at a flow
rate of 1.5 mL/min. Compounds were detected by ultraviolet absorbance
at 260 nm. The conversion of
[13C5]adenosine to
13C5-SAH was quantitative
in 20 min. For clean-up, 100-µL fractions were injected on the HPLC,
and the eluate fractions containing
13C5-SAH were pooled. The
concentration of 13C5-SAH
was determined vs calibration solutions containing unlabeled SAH. The
obtained stock solution was diluted to serve as internal standard
solution. Injection of the stock solution on HPLC with ultraviolet
detection revealed only a single peak with the same retention time as
authentic SAH. Isotopic purity of
13C5-SAH was determined by
LC-MS/MS in scanning and precursor ion mode and was found to be >95%.
sample preparation
Plasma or CSF aliquots of 500 µL were deproteinized by the
addition of 312 µL of 100 mL/L perchloric acid. The mixture
was mixed thoroughly and then centrifuged for 10 min at
2000g at 4 °C. From the clear supernatant, 500 µL was
transferred to a second test tube, and 50 µL of internal standard
mixture containing 0.50 µmol/L
13C5-SAH and 1.0 µmol/L
2H3-SAM was added. The
solution was neutralized by the addition of 140 µL of 1 mol/L
phosphate buffer (pH 11.5) after which 1 mL of
H2O was added. The solution was applied to an
OASIS solid-phase extraction (SPE) column (60 mg, 3 mL; Waters), which
was conditioned with 1 mL of methanol, 750 µL of 10 mmol/L lauric
acid in 0.1 mol/L NaOH, and 1 mL of H2O. After
the sample had passed through the column by gravity, the column was
rinsed with 700 µL of H2O. Analytes were eluted
from the column with 800 µL of H2O-methanol
(85:15, by volume), containing 1 mL/L formic acid. Calibrators
(included in each batch of samples) at concentrations of 0, 10, 25, 50,
and 100 nmol/L for SAH and 0, 20, 50, 100, and 200 nmol/L for SAM were
prepared in 40 mL/L perchloric acid. To 500 µL of calibration
solution was added 50 µL of internal standard mixture, followed by
225 µL of 1 mol/L phosphate buffer (pH 11.5) and 1 mL of
H2O. Calibrators were then processed as described
above. The concentration of the analyte in plasma or CSF was calculated
by interpolation of the observed analyte/internal standard peak-area
ratio into the linear regression line for the calibration curve, which
was obtained by plotting the peak-area ratios vs analyte
concentration.
methods
All analyses were performed on an API 3000 triple quadrupole
tandem mass spectrometer (PE-Biosystems Sciex). Side instrumentation
consisted of a Perkin-Elmer Series 200 HPLC pump, a Perkin-Elmer Series
200 autosampler, and a Harvard Apparatus Pump 11 infusion pump. Liquid
chromatography was performed on a Symmetry C18
analytical column (2.1 x 100 mm; 3.5 µm bead size; Waters)
using H2O-methanol (85:15, by volume) containing
0.2 mL/L butyric acid, pH 4.5, as mobile phase at a flow rate of
175 µL/min. The column was connected directly to the turbo ion
electrospray operating in the positive-ion mode. The temperature of the
turbo ion electrospray was set at 400 °C, turbo ion gas (nitrogen)
was used at a flow rate of 8 L/min, and the ion spray voltage was 6000
V. The mass spectrometric system was automatically optimized by
constant infusion at a flow rate of 5 µL/min of a 20 µmol/L
calibration solution in mobile phase. This optimization included
collision-activated decomposition MS/MS, which was performed in the
second quadrupole using nitrogen at 0.06 kPa as collision gas.
Quadrupole 1 and quadrupole 3 were calibrated monthly by direct
infusion at 10 µL/min of a 0.1 mmol/L polypropyleneglycol solution.
Data were acquired and processed using Analyst for Windows NT software
(Ver. 1.0).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Fragment m/z 136 was found in all MS/MS spectra. In
the MS/MS spectra of both
13C5-SAH and SAH, an
additional fragment of m/z 133.9 was found, which most
likely derives from the homocysteine part of the molecule. To
investigate the origin of the m/z 136 fragment, we obtained
MS/MS spectra of S-adenosylethionine and
[13C5]adenosine (data not
shown). Both compounds also generated the m/z 136 fragment
in the collision quadrupole. The combined information led to the
conclusion that fragment m/z 136 derives from the adenosine
backbone. Because the 13C labeling of
[13C5]adenosine is
localized in the ribose group, fragment m/z 136 must be
protonated adenine. Multiple reaction monitoring (MRM) experiments were
carried out by measuring the transition of protonated
13C5-SAH and SAH
(quadrupole 1) to fragment m/z 136 (quadrupole 3). SAM is
positively charged because of the tertiary sulfur atom; therefore, no
[M + H]+ fragment was observed, but the
[M]+ fragment was observed on quadrupole
1. Therefore, the transition of [SAM]+
to fragment m/z 136 was used for MRM analyses. The
finally used MRM transitions are listed in Table 1
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spe
The main function of the SPE procedure was to eliminate perchloric
acid and other ionization-suppressing compounds from the plasma and CSF
extracts. Perchloric acid strongly interferes with the mass
spectrometric measurements, decreasing sensitivity. OASIS
extraction columns were loaded with 750 µL of 10 mmol/L lauric acid
solution, which enabled weak ion extraction. Elution of SAM and SAH was
achieved by 1 mL/L formic acid in
H2O-methanol (85:15, by volume), which is a
compatible solution for the LC-MS/MS analysis. The efficiency of the
SPE procedure was examined in an experiment in which six identical
pooled plasma samples were used. The samples were split in two groups.
The first group consisted of three plasma samples to which the internal
standard mixture was added before the SPE procedure. The second group
consisted of three plasma samples, and the internal standard mixture
was added to the eluate of the SPE procedure. The average of the
observed calibrators/internal standard peak-areas ratios in group 2 was
divided by the average of the observed calibrators/internal standard
peak-area ratios in group 1. The efficiency found, expressed as the
percentage of recovered analyte, was 65% for SAM and 80% for SAH.
chromatography
Typical MRM chromatograms of SAM and SAH in a plasma extract are
shown in Fig. 2
. Total run time for the combined analyses of SAM and SAH was 3
min. The very polar nature of SAM means that this compound has minimal
retention in reversed-phase HPLC. The addition of 0.2
mL/L butyric acid to the mobile phase induced, possibly because
of ion pair-like interactions, additional retention. This was
beneficial for the final response of SAM in the MS/MS analysis because
suppression of the ionization of SAM in the source, caused by early
eluting compounds, was reduced.
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linearity
The calibration curve was linear over concentrations of 10–100
nmol/L for SAH and 20–200 nmol/L for SAM (Fig. 3
). In all cases, the coefficient of linear correlation
(r2) was >0.99 for the calibration
curves of both SAM and SAH.
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limits of detection, precision, and recovery
Limits of detection for SAM and SAH were estimated in samples by
verifying the peak height of the analyte and the noise in the
chromatographic region of the analyte. The minimal detectable
concentrations of the analytes in samples, at a signal-to-noise ratio
>5, were 7.5 and 2.5 nmol/L for SAM and SAH, respectively. The
validation data of the presented method are listed in Tables
2 and
3. All validation experiments were performed with pooled plasma
and pooled CSF samples. The interassay and intraassay CVs (n = 10)
for SAM and SAH in both matrices were <8%. Recovery experiments were
performed at two different concentrations. Mean recoveries (n = 5)
for SAM and SAH for both matrices were 93% with CVs of 2.7–5.0%.
values in human samples
In 15 plasma samples from premenopausal women with normal plasma
homocysteine, the mean SAM concentration was 74.7 ± 14.5 nmol/L
(range, 49.5–90.7 nmol/L), and the mean SAH concentration was
26.2 ± 6.1 nmol/L (range, 18.6–40.1 nmol/L). The mean SAM
concentration in 10 CSF samples from children was 248 ± 97 nmol/L
(range, 137–385 nmol/L), and the mean SAH concentration was 11.3
± 2.8 nmol/L (range, 8.9–14.1 nmol/L).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We present a rapid and precise LC-MS/MS method that is capable of handling large numbers of samples. The high selectivity of MS/MS does not require extensive HPLC separation, although it is very important to achieve separation between the analytes of interest and early eluting matrix components. The high sensitivity, attributable to the high selectivity, eliminates the need for derivatization. Additionally, MS/MS analysis allows the use of stable-isotope-labeled internal standards, making the method more accurate and precise. The method comprises a SPE procedure as clean-up step for the perchlorate fractions. Other investigators have used C18 or OASIS cartridges loaded with heptane sulfonic acid to enhance strong cation-exchange extraction of SAM and SAH (10)(12). This type of extraction requires either a high modifier concentration in the eluting solvent or a displacer cation to elute the analytes, which makes extracts less compatible with MS/MS. Our extraction procedure on OASIS columns, loaded with lauric acid, was carried out at neutral pH. At this pH, the carboxylic group of lauric acid is negatively charged and the amine groups of SAM and SAH are positively charged. SAM and SAH were eluted at low pH, which protonates the carboxylic group of lauric acid and consequently terminates the electrostatic interaction between the analytes and the extraction column. The use of OASIS extraction columns loaded with lauric acid enabled elution with a mild solvent at low pH, yielding clean extracts with sufficient extraction efficiency. The use of labeled internal standards corrects for sample-to-sample differences in extraction performances.
No interfering peaks were observed in the MRM mass fragmentograms, which was beneficial to automated processing of the data. The validation data show that the LC-MS/MS method is precise and reliable. Known amounts of SAM and SAH that were added to plasma and CSF were almost completely recovered. The fully automated method can run without manual assistance: calibrators are processed, and the concentrations of SAM and SAH in samples are calculated by the Analyst software.
The total analysis time needed for 20 samples is <2.5 h: 1–1.5 h of
sample preparation time plus 1 h of LC-MS/MS measuring time. This
opens perspectives for large-scale SAM and SAH measurements in clinical
studies of the pathophysiology of homocysteine metabolism. In addition,
CSF samples from children and adults with neurological disturbances can
be screened for SAM and SAH to gain more insight into the roles of
these analytes. In Table 4
, the SAM and SAH values described here are compared with values
reported by others. In the literature, there is consensus of the plasma
concentrations of SAH with our data for premenopausal women at the
upper end of the range of reported values. For plasma SAM, however, a
wide range of values [60–156 nmol/L; Refs. (11) and
(13), respectively] has been reported. An explanation might
be technical problems inherent to the HPLC methods used. Other groups,
as well as our laboratory, have encountered difficulties in
chromatographic separation and detection with available HPLC methods.
The resulting coelution of interfering compounds is likely to produce
systematic overestimation of SAM in plasma samples. This problem can
explain the high plasma SAM concentrations in some reported studies
(12)(13). The use of the LC-MS/MS method
overcomes this systematic bias because stable-isotope-labeled internal
standards were used and interferences by other compounds were absent.
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The possibility of measuring isotopically enriched SAM and SAH with minimal sample size might be very useful for researchers using stable-isotope tracer techniques, e.g., [2H3]methyl-[1-13C]methionine (15) or [1-13C]homocysteine (16), to study in vivo and in vitro cellular metabolism of methionine, homocysteine, and related compounds involved in 1-carbon metabolism. Precise and accurate isotopic enrichments and concentrations of SAM and SAH can now be measured using this fast online method.
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Footnotes |
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References |
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The following articles in journals at HighWire Press have cited this article:
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  C. Wagner and M. J Koury S-Adenosylhomocysteine a better indicator of vascular disease than homocysteine? Am. J. Clinical Nutrition, December 1, 2007; 86(6): 1581 - 1585. [Abstract] [Full Text] [PDF]
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 H. Gellekink, D. van Oppenraaij-Emmerzaal, A. van Rooij, E. A. Struys, M. den Heijer, and H. J. Blom Stable-Isotope Dilution Liquid Chromatography-Electrospray Injection Tandem Mass Spectrometry Method for Fast, Selective Measurement of S-Adenosylmethionine and S-Adenosylhomocysteine in Plasma Clin. Chem., August 1, 2005; 51(8): 1487 - 1492. [Abstract] [Full Text] [PDF]
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 J. C. Verdonk, M. A. Haring, A. J. van Tunen, and R. C. Schuurink ODORANT1 Regulates Fragrance Biosynthesis in Petunia Flowers PLANT CELL, May 1, 2005; 17(5): 1612 - 1624. [Abstract] [Full Text] [PDF]
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 R. G. V. Smolders, K. de Meer, P. Kenemans, C. Jakobs, W. Kulik, and M. J. van der Mooren Oral Estradiol Decreases Plasma Homocysteine, Vitamin B6, and Albumin in Postmenopausal Women But Does Not Change the Whole-Body Homocysteine Remethylation and Transmethylation Flux J. Clin. Endocrinol. Metab., April 1, 2005; 90(4): 2218 - 2224. [Abstract] [Full Text] [PDF]
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 L. Anderson Candidate-based proteomics in the search for biomarkers of cardiovascular disease J. Physiol., February 15, 2005; 563(1): 23 - 60. [Abstract] [Full Text] [PDF]
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 N. P. Riksen, G. A. Rongen, G. H.J. Boers, H. J. Blom, P. H.H. van den Broek, and P. Smits Enhanced Cellular Adenosine Uptake Limits Adenosine Receptor Stimulation in Patients With Hyperhomocysteinemia Arterioscler Thromb Vasc Biol, January 1, 2005; 25(1): 109 - 114. [Abstract] [Full Text] [PDF]
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 F. Stam, C. van Guldener, P. M. ter Wee, W. Kulik, D. E. C. Smith, C. Jakobs, C. D. A. Stehouwer, and K. de Meer Homocysteine clearance and methylation flux rates in health and end-stage renal disease: association with S-adenosylhomocysteine Am J Physiol Renal Physiol, August 1, 2004; 287(2): F215 - F223. [Abstract] [Full Text] [PDF]
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S. P. Stabler and R. H. Allen Quantification of Serum and Urinary S-Adenosylmethionine and S-Adenosylhomocysteine by Stable-Isotope-Dilution Liquid Chromatography-Mass Spectrometry Clin. Chem., February 1, 2004; 50(2): 365 - 372. [Abstract] [Full Text] [PDF]
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R. Castro, I. Rivera, E. A. Struys, E. E.W. Jansen, P. Ravasco, M. E. Camilo, H. J. Blom, C. Jakobs, and I. Tavares de Almeida Increased Homocysteine and S-Adenosylhomocysteine Concentrations and DNA Hypomethylation in Vascular Disease Clin. Chem., August 1, 2003; 49(8): 1292 - 1296. [Abstract] [Full Text] [PDF]
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 K. Herbig, E.-P. Chiang, L.-R. Lee, J. Hills, B. Shane, and P. J. Stover Cytoplasmic Serine Hydroxymethyltransferase Mediates Competition between Folate-dependent Deoxyribonucleotide and S-Adenosylmethionine Biosyntheses J. Biol. Chem., October 4, 2002; 277(41): 38381 - 38389. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and SAH (•) concentrations in calibrators. Concentration
ranges: 0–200 nmol/L SAM; 0–100 nmol/L SAH.