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Articles |
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Division of Biochemical Toxicology, National Center for Toxicological Research, 3900 NCTR Rd., Jefferson, AR 72079.
a Author for correspondence. Fax 870-543-7720; e-mail jjames{at}nctr.fda.gov
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We used HPLC with coulometric electrochemical detection for the simultaneous measurement of SAM and SAH in 200 µL of plasma, 106 lymphocytes, or 10 mg of tissue. Filtered trichloroacetic acid extracts were injected directly into the HPLC system without additional processing and were eluted isocratically.
Results: The limits of detection were 200 fmol/L for SAM and 40 fmol/L SAH. In plasma extracts, the interassay CV was 3.4–5.5% and the intraassay CV was 2.8–5.6%. The analytical recoveries were 96.8% and 97.3% for SAM and SAH, respectively. In a cohort of healthy adult women with mean total homocysteine concentrations of 7.3 µmol/L, the mean plasma value was 156 nmol/L for SAM and 20 nmol/L for SAH. In women with increased homocysteine concentrations (mean, 12.1 µmol/L), plasma SAH, but not SAM, was increased (P <0.001), and plasma pyridoxal 5'-phosphate concentrations were reduced (P <0.001). Plasma SAM/SAH ratios were inversely correlated with homocysteine concentrations (r = 0.73; P <0.01), and the SAM/SAH ratio in plasma was directly correlated with the intracellular SAM/SAH ratio in lymphocytes (r = 0.70; P <0.01).
Conclusions: Increased homocysteine in serum is associated with an increase in SAH and a decrease in the SAM/SAH ratio that could negatively affect cellular methylation potential. Accurate and sensitive detection of these essential metabolites in plasma and in specific tissues should provide new insights into the regulation of one-carbon metabolism under different nutritional and pathologic conditions.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An abbreviated overview of one-carbon metabolism with emphasis on
essential cellular methylation reactions is presented in Fig. 1
. Methionine is converted to S-adenosylmethionine
(SAM), the major intracellular methyl donor, by methionine
adenosyltransferase (EC 2.5.1.6) and subsequently to
S-adenosylhomocysteine (SAH) by a variety of cellular
methyltransferases present in all cells. This one-way reaction is
subject to competitive product inhibition by SAH because SAH has a
higher affinity for the methyltransferase active site than does its
precursor, SAM (17). The pathologic accumulation of SAH can
lead to a decrease in the SAM/SAH ratio and inhibition of most cellular
methyltransferases (17)(18)(19). SAH is hydrolyzed to
homocysteine and adenosine by SAH hydrolase (EC 3.3.1.1), a reversible
reaction with thermodynamics that actually favor SAH synthesis
(20)(21). Accumulation of SAH and the associated
inhibition of cellular methyltransferases will therefore occur under
metabolic conditions that interfere with product removal of
homocysteine or adenosine (17)(22)(23)(24)(25).
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Although the regulatory role of SAM on the enzyme activities of MTHFR, CBS, and methionine synthase has been emphasized (21)(22)(26), the regulatory importance of SAH in the maintenance of balanced one-carbon metabolism may be underestimated. Under physiologic conditions, SAH concentrations generally are several-fold lower than SAM concentrations, and SAH concentrations in plasma have only recently become detectable with newer methods (27)(28). Alterations in cytosolic SAH have tissue-specific bioregulatory functions and have been reported to up-regulate CBS activity (18), decrease betaine-homocysteine methyltransferase activity (18), and decrease MTHFR activity (29). Inhibition of the several methyltransferase reactions by SAH would be expected to spare the substrate, SAM. Consistent with this notion, an increase in SAM with increased intracellular SAH has been observed in end stage renal patients (30) and in cultured mouse lymphoma cells (31). Homozygous mutations in the CBS gene (32) or the adenosine deaminase gene (33) lead to substantial increases in homocysteine and SAH without affecting methionine synthase activity. By contrast, drugs or nutritional deficiencies that negatively affect the methionine synthase reaction are generally associated with a decrease in SAM and an increase in homocysteine and SAH (34)(35). The extent of reduction in SAM concentrations with folate or B12 deficiencies, however, does not approach the Km of most methyltransferases (17) and therefore may be less likely to reduce methyltransferase activity than an increase in SAH. An effect of SAH on cellular methylation is most likely in nonhepatic tissues in which the concentration of SAH is more variable than SAM (21).
Although several HPLC methods exist for the simultaneous measurement of SAM and SAH, most of these methods rely on precolumn derivatization and ultraviolet detection, which are more time-consuming and less sensitive than direct injection using coulometric EC detection (36)(37)(38)(39). In addition, these methods often require the use of internal standards to correct for sample losses during preparative procedures (27). In this report, we present a new method in which neutralized trichloroacetic acid (TCA) extracts are injected directly into the HPLC system without any further manipulation. SAM and SAH are separated by isocratic elution and coulometric EC detection to provide femtomolar detection limits with exceptional reproducibility and recovery.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subjects and sample preparation
Subjects were 58 healthy adult females with a mean age of 37.2
years (range, 19–53 years) who had participated in a previous clinical
trial (40). Fasted blood samples were collected into EDTA
Vacutainer Tubes, chilled immediately in an ice-water bath, and
centrifuged at 4100g for 15 min at 4 °C. Aliquots of the
plasma layer were transferred into multiple cryostat tubes and stored
at -20 °C until analysis. Separate aliquots were thawed for
determination of plasma homocysteine, pyridoxal 5'-phosphate (PLP),
SAM, and SAH. In a subset of women, mononuclear cells were isolated by
carefully layering whole blood onto an equal volume of
Histopaque®-1077 (Sigma Diagnostics) and
centrifuged at 400g for 30 min. Mononuclear cells were
recovered from the interface and washed several times as described by
the manufacturer. Samples of liver tissue (10–15 mg wet weight) or
isolated lymphocytes (106 cells) were homogenized
with 200 µL of phosphate-buffered saline. To precipitate
protein, 40 µL of 400 g/L TCA was added to 200 µL of plasma
or cell extract, mixed well, and incubated on ice for 30 min. After
centrifugation for 15 min at 18 000g at 4 °C,
supernatants were filtered through a 0.2 µm filter, and 20
µL was injected into the HPLC system.
chromatography
Separation of SAM and SAH in plasma and cell extracts was
accomplished by HPLC with a Shimadzu solvent delivery system (ESA model
580) and a reversed-phase C18 column (5 µm bead
size; 4.6 x 150 mm; MCM) obtained from ESA. Isocratic elution
with a mobile phase consisting of 50 mmol/L sodium phosphate monobasic,
monohydrate; 10 mmol/L heptanesulfonic acid; and 75 mL/L methanol
adjusted to pH 3.4 with concentrated phosphoric acid, was
performed at ambient temperature at a flow rate of 1.0 mL/min and
a pressure of 100–110 kfg/cm2 (1500–1800 psi).
Extracts were injected directly onto the column using a Beckman
autosampler (model 507E). To assure standardization between sample
runs, calibration and reference plasma samples were interspersed at
intervals during each run. Total homocysteine (tHcy) and cysteine
concentrations were quantified using HPLC and coulometric EC detection
as described previously in detail (15).
coulometric ec detection
After HPLC separation, detection of SAM and SAH was accomplished
using a model 5200A Coulochem II electrochemical detector (ESA)
equipped with a dual analytical cell (model 5010) and a guard cell
(model 5020). A guard cell was placed in line before the injector to
remove oxidizable impurities present in the mobile phase that might
compromise baseline stability. The dual analytical cell contained two
porous graphite electrodes in series. The first electrode (E1) is used
as an oxidative screen and was set at a lower voltage than the second
electrode (E2) to remove interfering compounds that oxidize at lower
potentials than the compounds of interest. For selectivity, E2 was set
at or above the established oxidation potential of the compounds of
interest. For optimum detection of SAM and SAH, the electrode
potentials for the guard cell, electrode E1, and electrode E2 were set
at +1000 mV, +400 mV, and +920 mV, respectively. The current generated
at E2 represents the oxidation of the active species between +400 mV
and +920 mV, which encompasses the peak oxidative range for both SAM
and SAH. These potentials provide peak area response with minimum
background and are the basis for quantification. Peak area analysis was
provided by GOLD Nouveau software (Beckman Instruments) based on
calibration curves generated for each compound. Each day, electrode
sensitivity and baseline stability were confirmed by the application of
-400 mV at each electrode for 30 min with the mobile phase at 1.5
mL/min, followed by a 30-min water rinse at 1 mL/min with the
electrodes turned off, and a final 30-min rinse with 500 mL/L methanol
(electrodes off). Before injection of the first sample, the potential
at the electrodes was increased in a stepwise fashion to the final
working potential and the HPLC-EC system was equilibrated for ~1 h
with the mobile phase at 1 mL/min. The use of an autosampler is highly
recommended and allows continuous sampling overnight. When not running
samples, the mobile phase was set at a rate of 0.2 mL/min with lower
voltages of +50 mV, +100 mV, and +200 mV at E1, E2, and the guard cell,
respectively.
current/voltage curves, calibration curves, and limits of detection
Curves reflecting the current generated by the oxidation of
SAM and SAH with increasing voltage at E2 were used to estimate the
respective voltage requirements for peak sensitivity and
reproducibility. Linear calibration curves for SAM and SAH in 100
µmol/L HCl were generated in the following physiologic ranges
for each compound: 1–600 nmol/L for SAM, and 0.2–100 nmol/L for SAH.
Before each analysis, calibration curves were generated from
aliquots of frozen calibrators and examined for reproducibility.
recovery, precision, and statistical analysis
To determine analytical recovery, known concentrations of
SAM and SAH within the physiologic range were added to plasma and mouse
liver tissue extracts. The concentrations of SAM and SAH in the
supplemented samples were determined in five independent
samples, and the mean quantitative recoveries were calculated. To
determine the intraassay precision, 10 replicates of the same sample
were analyzed in a single analytical run. The interassay precision was
determined by analyzing aliquots from a single sample on 10 different
days over a 1-month period. The CV was calculated as the
standard deviation expressed as a percentage of the mean values.
Statistical differences between means were calculated using the Student
t-test and Sigmastat software (Jandel Scientific).
plp
Concentrations of PLP were determined in fasting plasma by the
radioenzymatic assay described previously in detail (41).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A, the voltage was set at 400 mV and 920 mV for electrodes E1
and E2, respectively, for maximum sensitivity and reproducibility
without damage to the electrodes. In Fig. 2
, B and C, the linearity of
detection within the physiologic range for each compound was confirmed.
Typical chromatograms from calibrators, plasma extracts, and mouse
liver extracts are presented in Fig. 3
, A, B, and C, respectively. The concentration of the
ion-pairing reagent, heptanesulfonic acid, was critical for optimum
separation. The retention times were strongly affected by the pH and
methanol concentration in the mobile phase. Under the conditions
selected, the retention times for SAH and SAM were ~10.5 and 20.5
min, respectively. Excellent separation without interfering peaks was
achieved in both plasma and cell extracts, as shown in Fig. 3
. With EC
detection, the selectivity for SAM and SAH was greatly enhanced at E2
by the oxidation and elimination of interfering compounds at E1.
Biological samples required ~35 min between injections to eliminate a
large unidentified peak that elutes at ~30 min.
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limits of detection, precision, and recovery
The limit of detection for the calibrators, defined as the
concentration that produced a signal-to-noise ratio >5, was 200 fmol/L
for SAM and 40 fmol/L for SAH. These limits of detection are
several-fold lower than those reported previously with methods using
derivatization and ultraviolet detection
(27)(28)(38). The
interassay/intraassay precision and product recovery are presented in
Table 1
. In plasma extracts, the mean within-run (intraassay) CV was
3.4% for SAM and 5.5% for SAH, with mean recoveries of 96.8% for SAM
and 97.3% for SAH. In mouse liver extracts; the mean intraassay CV was
6.1% for SAM and 8.8% for SAH, with mean recoveries of 95.2% for SAM
and 97.1% for SAH. The mean between-run (interassay) CV in plasma
extracts was 2.8% for SAM and 5.6% for SAH; for mouse liver extracts,
the mean interassay CV was 7.3% for SAM and 7.6% for SAH.
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plasma sam, sah, plp, and cysteine concentrations in women
with normal and increased tHcy
In Table 2
, plasma SAM, SAH, PLP, and cysteine are shown as a function of
fasting plasma tHcy in the 58 young women. The women were stratified by
tHcy based on previously published reference values for adult
females (42)(43). In women with normal tHcy
values (mean, 7.3 ± 1.1 µmol/L; range, 5.8–8.7 µmol/L), the
mean SAM and SAH concentrations were 155.9 and 20.0 nmol/L,
respectively, with a mean SAM/SAH ratio of 8.5. In women with increased
tHcy (mean, 12.3 ± 1.8 µmol/L; range, 9.3–16.5 µmol/L), SAM
concentrations were not altered, but SAH concentrations were increased
twofold relative to women with normal tHcy, and the SAM/SAH ratio was
decreased by one-half. Interestingly, increased tHcy was associated
with significantly reduced concentrations of plasma PLP and cysteine in
this cohort of women (P <0.001). A significant negative
correlation was observed when SAM/SAH ratios were plotted as a function
of tHcy concentrations (r = 0.73; P <0.01;
Fig. 4
).
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intracellular sam and sah concentrations and sam/sah ratio in human
lymphocytes and mouse liver extracts
The applicability of the assay for measurement of SAM and SAH in
cell and tissue extracts is presented in Table 3
. Lymphocytes were isolated from whole blood obtained from a
subset of women with a mean plasma tHCy of 7.8 ± 1.1 µmol/L and
a mean plasma SAM/SAH ratio of 8.8 ± 1.6 (n = 7). The mean
intracellular SAM/SAH ratio (9.4 ± 3.1) was not significantly
different from the plasma ratio. Fig. 5
is a scatter plot of the SAM/SAH ratios in plasma and in
lymphocytes obtained from the same individuals. Regression analysis
revealed a significant positive association (r = 0.70;
P <0.01), suggesting that the plasma SAM/SAH ratios may be
a convenient reflection of the intracellular ratio in lymphocytes. In
the mouse liver samples, expression of SAM and SAH concentrations per
milligram of protein produced smaller variation than expression per
gram wet weight. The values obtained for mouse liver TCA extracts were
within previously published ranges obtained with a different
methodology (35).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results presented in Table 2
indicate that increased plasma tHcy
was associated with a twofold increase in SAH, no change in SAM, and a
twofold decrease in the SAM/SAH ratio in this cohort of women. Whether
the decrease in SAM/SAH is of sufficient magnitude to affect
methyltransferase activity and cellular methylation reactions cannot be
determined from the present data. Nonetheless, these observations are
consistent with a homocysteine-mediated reversal of the SAH hydrolase
reaction. The regression analysis in Fig. 4
indicates that plasma tHcy
concentrations are inversely correlated with SAM/SAH ratios and
suggests the possibility that tHcy could have indirect effects on
cellular methylation potential. The decreases in plasma PLP
concentrations in these women further suggest the possibility that
nutritional vitamin B6 deficiency may have
contributed to the increase in tHcy; the decrease in cysteine
concentrations is consistent with this interpretation. Similar
increases in SAH concentrations associated with
B6 deficiency previously have been observed in
rat liver (28). In Fig. 5
, the significant positive
correlation between plasma and lymphocyte SAM/SAH ratios suggests that
the plasma SAM/SAH ratio may be a reasonable reflection of the
intracellular ratio. In lymphocytes, export of SAH has been well
documented and appears to be carrier-mediated and largely
unidirectional (50). The kidney appears to be the only route
for SAH removal from plasma (51). The origin of SAM in
plasma is still an open and interesting question because export from
the hepatocytes does not occur despite the fact that more SAM is used
for creatine synthesis in the liver than in all extrahepatic tissues
combined (52).
The mean plasma concentration of SAM detected with the present HPLC-EC method in women with normal tHcy was 156 nmol/L, which is considerably higher than previously published values of 26.5 nmol/L (38), 60 nmol/L (30), and 102 nmol/L (27) in plasma from healthy individuals obtained with other methods of detection. The higher values with the present method most likely reflect the direct injection of the extract without the need for precolumn processing and derivatization, which can lead to significant losses. The SAM peak was completely eliminated by boiling the extract for 5 min before injection, suggesting that coelution of a contaminating metabolite is unlikely, although this possibility cannot be definitively ruled out. Adenosine elutes before the SAH peak, at ~8 min. The mean plasma concentration of SAH obtained with the present method was 20 nmol/L and is within the ranges reported previously using other methods (27). The limits of detection using the coulometric EC detector were 200 fmol/L for SAM and 40 fmol/L for SAH. These values far exceed previous ultraviolet detection limits of 10 nmol/L (30), 25 pmol (28), and 5 pmol/L (27) for both SAM and SAH. Using the present method, we obtained a SAM/SAH ratio of 8.5 for plasma from healthy individuals, which is considerably higher than previously reported values (38). A disadvantage of the present method is that 40 min is required for complete elution of the accompanying peaks in biological samples; however, the precolumn processing time is minimal, and with the use of an autoinjector, ~20 samples per day can be processed. Because of the relatively long elution times, this method for measurement of SAM and SAH is best suited for research purposes and refinement of diagnoses rather than routine clinical analyses that require high throughput methodology. The ability to sensitively and reproducibly detect these important metabolites in plasma should provide new insights into the differential regulation of one-carbon metabolism with specific nutritional deficiencies and specific genetic polymorphisms.
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Acknowledgments |
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Footnotes |
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References |
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T mutation and unexplained early pregnancy loss. Thromb Haemost 1998;79:1056-1057.
[ISI][Medline]
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, SAM.
