J. Lipid Res.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1194/jlr.M500128-JLR200 on June 1, 2005

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
M500128-JLR200v1
46/9/1974    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lin, Y. H.
Right arrow Articles by Salem, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lin, Y. H.
Right arrow Articles by Salem, N., Jr.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?
Journal of Lipid Research, Vol. 46, 1974-1982, September 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Simultaneous quantitative determination of deuterium- and carbon-13-labeled essential fatty acids in rat plasma

Yu Hong Lin, Robert J. Pawlosky and Norman Salem, Jr.1

Section of Nutritional Neuroscience, Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20892-9410

Published, JLR Papers in Press, June 1, 2005. DOI 10.1194/jlr.M500128-JLR200

1 To whom correspondence should be addressed. e-mail: nsalem{at}niaaa.nih.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
This study reports methods for the quantitative determination of stable isotope-labeled essential fatty acids (EFAs) as well as an experiment in which deuterium-labeled linoleic acid (18:2n-6) and {alpha}-linolenic acid (18:3n-3) were compared with those labeled with carbon-13 in rat plasma in vivo. Standard curves were constructed to compensate for concentration and plasma matrix effects. It was observed that endogenous pools of fatty acids had a greater suppressing effect on the measurements of 13C-U-labeled EFAs relative to those labeled with 2H5. Using these methods, the in vivo metabolism of orally administered deuterated-linolenate, 13C-U-labeled linolenate, deuterated-linoleate, and 13C-U-labeled linoleate was compared in adult rats (n = 11). There were no significant differences in the concentrations of the 2H versus 13C isotopomers of 18:2n-6, 18:3n-3, arachidonic acid (20:4n-6), and docosahexaenoic acid (22:6n-3) in rat plasma samples at 24 h after dosing.

Thus, there appears to be little isotope effect for 2H5- versus 13C-U-labeled EFAs when the data are calculated using the conventional standard curves and corrected for endogenous fatty acid pool size and matrix effects.

Abbreviations: 18:2n-6, linoleic acid; 18:3n-3, {alpha}-linolenic acid; 20:4n-6, arachidonic acid; 20:3n-6, dihomo-{gamma}-linolenic acid; 22:6n-3, docosahexaenoic acid; 20:5n-3, eicosapentaenoic acid; EFA, essential fatty acid; FID, flame ionization detector; ISTD, internal standard; NCI, negative chemical ionization; PFB, pentafluorobenzyl

Supplementary key words deuterated linolenate • deuterated linoleate • carbon-13-labeled linolenate • carbon-13-labeled linoleate • standard curves • concentration effect curves • gas chromatography-mass spectrometry negative chemical ionization analysis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
In essential fatty acid (EFA) metabolic studies, 2H and 13C are the most commonly used stable isotopic atoms in tracer studies (1–6) since their discovery (7, 8) and application to metabolic studies about 70 years ago (9). Compared with 2H, the 13C atom has a relatively small difference in mass with respect to 12C and so is very similar in its physicochemical characteristics. For 13C-labeled fatty acids, a high-precision gas chromatography-combustion isotope ratio mass spectrometric system could detect 13C in fatty acids at very low signal levels, down to 3 pmol per injection (10). 2H-labeled fatty acids were used extensively and measured by GC-MS in electron impact (11), positive chemical ionization (12), and negative chemical ionization (NCI) (13) in biological samples. Other non-mass spectrometry methods, such as conventional GC with a flame ionization detector (FID), could be applied when the deuterated fatty acids had enough mass difference to be chromatographically resolved (13–15) and when sufficient amounts occurred in biological experiments such that they could be adequately detected (15–17). However, for fatty acids, no direct comparisons have been made between the metabolic behavior of 2H and 13C tracers either in vitro or in vivo.

In the course of performing measurements of human (18) and rat (19) plasma samples containing multiple stable isotopes, it became apparent that there were several analytical difficulties. The plasma matrix exerted a suppressing effect on the fatty acid isotopic signals as measured in the NCI mode. This suppression was greater for the 13C signals relative to those of the same fatty acid labeled with 2H. Therefore, standard curves for the various fatty acid isotopes to be used were required. In addition, methods were required for estimating the responses of fatty acid metabolites where isotopic standards were not available. This work describes the standardization and measurement of six stable isotope-labeled EFAs of 18 or 20 carbon length. In addition, the metabolism of 2H- and 13C-labeled linoleic acid (18:2n-6) and {alpha}-linolenic acid (18:3n-3) ethyl esters are compared to evaluate a possible isotope effect, as this information will also be required to perform valid measurements of EFA metabolism.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Stable isotope-labeled fatty acids
Deuterated {alpha}-linolenate (17,17,18,18,18-2H5-18:3n-3 ethyl ester; 2H > 95%), deuterated linoleate (17,17,18,18,18-2H5-18:2n-6 ethyl ester; 2H > 95%), carbon-13-uniformly labeled linoleate (13C-U-18:2n-6 ethyl ester; 13C > 95%), and deuterated dihomo-{gamma}-linolenate (19,19,20,20,20-2H5-20:3n-6 ethyl ester; 2H > 95%) were obtained from Cambridge Isotope Laboratories (Andover, MA); carbon-13-uniformly labeled eicosapentaenate (13C-U-20:5n-3 ethyl ester; 13C > 95%) was obtained from Martek Bioscience Corp. (Columbia, MD). The five isotopes were further purified by HPLC and verified by TLC, GC, NMR and GC-MS. Carbon-13-uniformly labeled linolenate (13C-U-18:3n-3 ethyl ester; 13C > 95%) was purchased from Cambridge Isotope Laboratories and used without further purification. Isotope distributions are as follows: 94.4% for 2H5-18:3n-3 (M+5), 43.1% for 13C-U-18:3n-3 (M+18), and 21.8% for (M+17). For isotope distributions in 18:2n-6 isotopomers, the distributions are as follows: 98.9% for 2H5-18:2n-6 (M+5), 58.8% for 13C18-18:2n-6 (M+18), 23.7% for (M+17), 33.9% for 13C-U-20:5n-3 (M+20), and 89.8% for 2H5-20:3n-6 (M+5). Data were based on three or more determinations. All solvents were of GC or higher grade except acetonitrile, which was HPLC grade.

Preparation of standards
Isotopic standards Stock solutions at concentrations of 2, 2, 10, 10, 2, and 2 mmol/l of 2H5-18:3n-3, 13C-U-18:3n-3, 2H5-18:2n-6, 13C-U-18:2n-6, 13C-U-20:5n-3, and 2H5-20:3n-6 were prepared for standard curve generation for each isotope. All analyses were conducted by serial dilutions of the stock solution into a series of working solutions.

Unlabeled standards Unlabeled commercial standards (reference standard 462 with 20:4n-3 added; Nu-Chek Prep, Elysian, MN) were used to construct standard curves of unlabeled fatty acids. Stock solutions at 5~6 mmol/l were made for unlabeled 18:3n-3, 20:3n-3, 20:4n-3, 20:5n-3, 22:5n-3, and docosahexaenoic acid (22:6n-3) in the n-3 family and 18:2n-6, 18:3n-6, 20:2n-6, 20:3n-6, 20:4n-6, and 22:4n-6 in the n-6 family. The exact concentrations of all standard stock solutions, both the labeled and unlabeled fatty acids, were determined by GC analysis.

Standard curves
Standard curves for stable isotope-labeled fatty acids Three sets of standard curves were constructed for each C18 isotope in various matrices: i) the pure stable isotope; ii) isotopes with unlabeled 18:2n-6 (10 nmol) and 18:3n-3 (2 nmol); and iii) isotopes with 50 µl of plasma from a rat dosed with olive oil vehicle. For C20 isotopes, the standard curves were made only in the plasma matrix. The concentrations of endogenous fatty acids in the plasma matrix were measured in triplicate by GC-FID analysis and are given as µmol/l of plasma (means ± SD): 70.3 ± 2.4 for 18:3n-3, 1,462 ± 51 for 18:2n-6, 58.7 ± 1.8 for 20:5n-3, and 55.5 ± 1.8 for 20:3n-6.

For each C18 isotopic fatty acid stock solution, a series of serial dilutions was made and a constant amount of internal standard (ISTD) 23:0 ethyl ester (0.13 nmol) was added. The total amounts of isotopic fatty acids in each sample are shown in Table 1. However, the amounts injected were much smaller, as one part out of 200 or 2,000 was injected into the GC-MS system. The range of these samples covered the estimated range of the isotope-labeled fatty acids in rat plasma samples. Standard curves were determined using linear regression of the ratio of signal areas between isotopes and ISTD versus the ratio of amounts between isotopes and ISTD.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Working table for standard curves of isotopic fatty acids

 
Standard curves for unlabeled fatty acids Stock solutions of each unlabeled fatty acid were diluted into a series of working solutions within a range of 2–1,000 pmol per sample with ISTD (0.13 nmol) added to each sample. Both the isotope-labeled and unlabeled samples were processed similarly for GC-MS analysis (see below).

To estimate the slopes of the standard curves for n-3 and n-6 metabolites for which stable isotope-labeled compounds were not available, the ratios of the slopes determined from the standard curves for the various 18:3n-3 isotopomers (18:3n-3, 2H5-18:3n-3, 13C-U-18:3n-3), 18:2n-6 isotopomers (18:2n-6, 2H5-18:2n-6, 13C-U-18:2n-6), 20:5n-3 isotopomers (20:5n-3, 13C-U-20:5n-3), and 20:3n-6 isotopomers (20:3n-6, 2H5-20:3n-6) were used. For example, to determine the standard curve for 2H5-20:5n-3, it was assumed that the relationship of the slopes of 18:3n-3 and 2H5-18:3n-3 was the same as that of unlabeled 20:5n-3 and 2H5-20:5n3. The intercepts were generally very close to the origin and so are not specified.

Concentration dependence curves The effects of the varying concentrations of endogenous fatty acids on 2H5 and 13C ions were quantified by adding a known amount of a pure stable isotope standard (92 pmol of 2H5-18:3n-3, 69 pmol of 13C-U-18:3n-3, 476 pmol of 2H5-18:2n-6, and 476 pmol of 13C-U-18:2n-6) to various volumes of rat plasma (0, 10, 20, 30, 40, and 50 µl) with normal saline used to bring the amount up to 50 µl. The concentrations of the four isotopes in the sample were chosen to be similar to those of experimental plasma samples. Concentration curves were constructed for the ratio of 2H5 to 13C fatty acid concentration versus the amount of unlabeled 18:3n-3 or 18:2n-6 in the rat plasma.

Plasma samples from rats dosed with C18 isotopes
This animal study proposal was approved by the Animal Care and Use Committee of the National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health. Eight week old male and female Sprague-Dawley rats (murine pathogen-free; Taconic, Germantown, NY) were purchased and housed in our animal facility under conventional conditions. A custom diet (Dyets, Bethlehem, PA) based on the AIN-93G standard (20) was used with modifications in the fat sources (10 wt%) to achieve a composition of 40% saturated fat, 42% monounsaturates, 15% 18:2n-6, and 3% 18:3n-3 but with no added C20 and C22 polyunsaturated fatty acids. This diet is described in more detail in the accompanying paper (19). After a 4–5 week equilibration on this diet, the animals were fasted for 5 h before dosing with a mixture of the four stable isotopic tracers (in µmol/kg body weight): 2H5-18:3n-3 (45), 13C-U-18:3n-3 (32), 2H5-18:2n-6 (210), and 13C-U-18:2n-6 (210) as the ethyl ester in olive oil (n = 11). The olive oil vehicle was given to only one animal of each gender to generate blank plasma with no stable isotopes for background determinations and matrix effect experiments. Body weights at administration were 477 ± 31 g for males (n = 6) and 263 ± 17 g for females (n = 5). Animals had free access to water during the experimental period and to food at 4 h after dosing. Rats were euthanized by decapitation at 24 h after dosing. Blood samples were collected in heparinized tubes. Plasma was separated from whole blood at 1,670 g for 15 min, frozen on dry ice, and stored at –80°C until analysis. Standard curves of the isotopic fatty acids in the rat plasma matrix were used to quantify the amounts of the various isotopically labeled compounds in rat plasma samples.

Total lipid extraction
Total lipids of the samples for standard curves and isotopic rat plasma samples were extracted using the method described previously (4, 21). Rat plasma (50 µl) spiked with 13 nmol (for GC analysis) or 0.13 nmol (for GC-MS analysis) of 23:0 ISTD was mixed with 1 ml of methanol containing butylated hydroxytoluene (0.2 mM) and then extracted twice with 2 ml of CHCl3. Total lipid extracts acquired from the chloroform phase were evaporated to dryness and then derivatized for instrumental analysis.

GC-FID analysis of unlabeled fatty acids
The total lipid extract was derivatized to the methyl esters using 14% BF3-methanol as described by Morrison and Smith (22) and modified by Salem, Reyzer, and Karanian (23). In brief, a fused-silica, narrow-bored DB-FFAP capillary column (30 m x 0.25 mm inner diameter x 0.25 µm; J&W Scientific, Folsom, CA) coupled with a GC 6890 Plus LAN system (Agilent Technologies, Inc., Wilmington, DE) were used for chromatographic separation of the methyl esters with hydrogen carrier gas at 100 Pka and a linear velocity of 50 cm/s. The inlet and detector temperatures were set at 250°C. Oven temperature was programmed from 130°C to 175°C at 4°C/min, then to 210°C at 1°C/min, and finally increased at 30°C/min to 245°C, with a final hold for 15 min. A 28 component quantitative methyl ester reference standard (GLC-462) was used to identify the retention times of methyl ester peaks and to determine the respective instrumental response factors.

GC-MS analysis of isotope-labeled fatty acids
The total lipid extract was saponified using a 5% KOH-methanol solution, and free fatty acids were derivatized to the pentafluorobenzyl (PFB) esters using the PFB reagent (pentafluorobenzylbromide-diisopropylamine-acetonitrile, 1:100:1,000, v/v/v) followed by heating for 12 min at 60°C for GC-MS analysis in NCI mode as described previously (13, 18). A 1.0 µl portion of the hexane solution was subjected to GC-MS in the splitless mode. The same chromatographic column used in the GC-FID work was used for GC-MS with helium as the carrier gas at a constant column head pressure of 100 kPa. The GC oven temperature was programmed as follows: initially 125°C for 0.5 min, then increased to 245°C at a rate of 8°C/min, followed by an isothermal hold for 39 min. The signal abundances of the anions (M-PFB–) of the various isotopic fatty acid compounds were detected in the selected ion monitoring mode using an interval of 1.0 amu for each ion. For the deuterated compounds, the ion channels with 5 amu above the 12C fatty acid analog were monitored. For the 13C-labeled fatty acids, ion channels that were 17 amu above the 12C analog were monitored, because this resulted in a better signal-to-noise ratio compared with the 18 amu ion channel. The areas under each chromatographic peak were obtained by integration using the ChemStation integrator. Data analysis was performed using MSD ChemStation software (Agilent G1701DA version D.00.00.38).

Calculations
A least-squares linear fitting procedure was used to determine the standard curves of isotopic fatty acids that were obtained by plotting the ratios of peak areas of the fatty acid to the ISTD versus their molar ratios. The slope of this line was then used to calculate the unknown concentrations of the stable isotope-labeled fatty acids in rat plasma. The percent of dosage was determined by dividing the concentration of the labeled fatty acids and their metabolites by the amount of isotopes that each rat received. The criteria used to validate the linearity of the standard curves were based on those described by Green (24). When the regression coefficient R 2 was >=0.98, it was considered to be an acceptable fit of the data to the regression line. The y intercepts were <5% of the dependent values obtained for the analyte at the target level.

To correct for the effect of the endogenous amount of 18:3n-3 and 18:2n-6 in rat plasma on the concentration of 13C-labeled fatty acids in rat plasma, a nonlinear regression was fitted to the concentration curves. Isotopic enrichment was obtained by calculating the ratio of the amount of isotope-labeled fatty acids to unlabeled endogenous fatty acids and then multiplying by 100. The statistical test used was the two-sided, pairwise Student's t-test with P < 0.05 considered significant. Both statistical tests and linear regression fitting were conducted using Microsoft® Excel 2002 SP3 for Windows XP Professional (Microsoft, Seattle, WA).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Response curves for 2H5-18:3n-3, 13C-18:3n-3, 2H5-18:2n-6, and 13C-18:2n-6
As the fatty acids occur in a widely different concentration range in the plasma, varying amounts of each C18 isotope were injected into the mass spectrometer over a range from 0.01 to 6.9 pmol after dilution. It was noted that the addition of unlabeled 18:2n-6 (10 nmol) or 18:3n-3 (2 nmol) depressed the response curves of all four of the isotopes (Fig. 1). This was also true when using the plasma matrix. However, it is possible to obtain linear responses over a wide range of analyte concentrations. The linear range for each isotope was as follows: 0.01–4.6 pmol for 2H5-18:3n-3, 0.01–6.9 pmol for 13C-U-18:3n-3, 0.005–2.4 pmol for 2H5-18:2n-6, and 0.05–4.8 pmol for 13C-U-18:2n-6.



View larger version (36K):
[in this window]
[in a new window]
  		Fig. 1. Plots of MS response (peak areas) of four stable isotope-labeled essential fatty acids versus analyte concentration and effects of unlabeled fatty acid and plasma matrix. Linear ranges were as follows: 0.01–4.6 pmol for 2H5-18:3n-3 (A); 0.01–6.9 pmol for 13C-U-18:3n-3 (M+17) (B); 0.005–2.4 pmol for 2H5-18:2n-6 (C); and 0.05–4.8 pmol for 13C-U-18:2n-6 (M+17) (D) without any matrix (curve i; squares), with unlabeled fatty acids (curve ii; triangles), and with rat plasma matrix (curve iii; circles).

 
Most importantly, these data clearly demonstrate that the different response factors are dependent upon the concentration of the endogenous fatty acids, which vary in different samples. In the worst case, the value of the calculated amount of 13C-U-18:2n-6 decreased to ~50% of its actual value. It follows that enrichment is also linear at least up to the range tested in this experiment: at 0–26% for 2H5-18:3n-3, 0–40% for 13C-U-18:3n-3, 0–6.5% for 2H5-18:2n-6, and 0–13% for 13C-U-18:2n-6; the R 2 for the linear regression fit in all four cases was >=0.99.

Standard curves for isotopic and unlabeled EFAs
Standard curves of four C18 isotopic fatty acids were examined under three conditions: i) no matrix; ii) with a constant amount of unlabeled 18:3n-3 and 18:2n-6 added; and iii) with the rat plasma matrix added. Also, the C20 isotopes were examined in the plasma matrix. The standard curves were constructed by regression fitting for the ratio of the MS signal abundance of the isotope to that of the ISTD versus the ratio of the actual amount of the isotope to that amount of the ISTD. The regression equations for the standard curves for the six isotopically labeled fatty acids in the plasma matrix are given in Fig. 2. It is apparent from this figure that a linear equation provides an excellent fit within the specified ranges, as all had R 2 >= 0.99. The slopes of the standard curves alone, when unlabeled fatty acids were added, and in the plasma matrix were as follows: 0.9783, 0.8956, and 0.7015 for 2H5-18:3n-3 and 0.3115, 0.2174, and 0.1535 for 13C-U-18:3n-3 (M+17), respectively.



View larger version (27K):
[in this window]
[in a new window]
  		Fig. 2. Standard curves for six stable isotope-labeled fatty acids in rat plasma matrix. The ratio of MS response (peak area) of the isotope to internal standard (ISTD) was plotted against the ratio of the quantity of isotope to ISTD. The regression lines fitted to the experimental data are displayed for 2H5-18:3n-3 (A), 13C-U-18:3n-3 (B), 2H5-18:2n-6 (C), 13C-U-18:2n-6 (D), eicosapentaenoic acid [20:5n-3, EPA (13C-U-20:5n-3)] (E), and dihomo-{gamma}-linolenic acid [20:3n-6, DGLA (2H5-20:3n-6)] (F).

 
Standard curves for unlabeled C18-, C20-, and C22 EFAs were constructed in a similar manner as those for the isotopic fatty acids (in the absence of the various matrices). Table 2 shows the slopes and regression coefficients of the linear regression curves for the unlabeled fatty acids, produced from analyses of a quantitative standard containing all of these fatty acids.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Slopes and coefficients of regression equations for standard curves of unlabeled fatty acid pentafluorobenzyl esters

 
Because the majority of the isotopically labeled n-6 and n-3 fatty acids are not available as standards, it was necessary to estimate their slopes. This was done by assuming that the ratio of the slope of the labeled fatty acid to its unlabeled form is the same as that of its precursor. Thus, because three of these four quantities are experimentally determined, the fourth, the labeled metabolite, can be derived. For example, for 22:5n-3, no deuterated or 13C standard was available, so the standard curves for 2H5-22:5n-3 and 13C-22:5n-3 could not be determined experimentally. Therefore, the slopes were determined for the unlabeled 22:5n-3, and the ratio of this to the 2H5-22:5n-3 was calculated by assuming that it was the same as the ratio of 18:3n-3 to 2H5-18:3n-3; similarly, the ratio of the slopes of the 22:5n-3 to 13C-22:5n-3 was assumed to be the same as the ratio of the slopes of 20:5n-3 to 13C-20:5n-3.

For the n-3 fatty acids, the slopes for the deuterated metabolites were calculated similarly using that of 2H5-18:3n-3, and the slopes for the 13C metabolites were calculated using that of 13C-18:3n-3. Similarly, for arachidonic acid (20:4n-6), the isotopically labeled 18:2n-6 was used. In both cases, these determinations were made in the presence of the plasma matrix. However, several minor n-6 fatty acids occur in the plasma at a much lower concentration than 18:2n-6 or 20:4n-6; for these, the slopes were obtained using the labeled 18:2n-6 in the absence of the plasma matrix. In this way, the standard curves for all of the stable isotope metabolites to be measured were estimated (Table 3).


View this table:
[in this window]
[in a new window]
  		TABLE 3. Calculated slopes of the regression curves for isotopic EFA metabolites in rat plasma

 
The slopes for both 13C-U-20:5n-3 and 2H5-20:3n-6 could be compared with estimated values because these labeled isotopes were available. The measured and estimated slopes, when corrected for the respective isotope distribution of the precursors, for 20:5n-3 were virtually identical (0.6483 vs. 0.6480), and the agreement for the 20:3n-6 slopes was excellent (0.884 vs. 0.858). Slightly greater differences in slopes were noted if different ions were used in place of the parent ions (e.g., M+19 ion in place of the M+20 ion for 13C-U-20:5n-3, and M+17 ion in place of the M+18 ion for 13C-U-20:5n-3). There was significant improvement in precision and accuracy using these standard curves compared with using a response factor calculated from a single concentration over a wide range of concentrations. The latter compromised the accuracy of isotopic fatty acid measurements when the unknown was not close to the amount of the ISTD (25).

Concentration-dependent effects of endogenous fatty acids
Greater signal suppression of 13C- relative to 2H5-fatty acid ions by endogenous fatty acids Figure 3 depicts the ion chromatograms of pure stable isotope-labeled fatty acids with and without added rat plasma. There was a greater amount of signal suppression on the 13C-labeled fatty acid ions by the endogenous amounts of 18:3n-3 and 18:2n-6 from the plasma compared with the 2H5-labeled fatty acid ions. The peak area decreased by 16% for 2H5-18:3n-3 (0.5 pmol), 23% for 13C-U-18:3n-3 (0.4 pmol), 26% for 2H5-18:2n-6 (3 pmol), and 65% for 13C-U-18:2n-6 (3 pmol) in the presence of rat plasma that contained 4 pmol of 18:3n-3 and 80 pmol of 18:2n-6. A similar suppressive effect of the endogenous pool on stable isotope tracer measurements has been observed previously by Patterson et al. (11, 26) for electron impact mode GC-MS analyses.



View larger version (25K):
[in this window]
[in a new window]
  		Fig. 3. MS signal suppression attributable to the plasma matrix. Ion chromatograms of 2H5-18:3n-3 (M+5) and 13C-U-18:3n-3 (M+17) without (A) and with (B) endogenous 18:3n-3 and 2H5-18:2n-6 (M+5) and 13C-U-18:2n-6 (M+17) without (C) and with (D) endogenous 18:2n-6 are shown. The peak area was used to compare the signal response of 2H5-labeled ion or 13C17-labeled ion with the absence (peaks A, B, C, D) or presence (corresponding peaks A+, B+, C+, D+) of endogenous 18:3n-3 and 18:2n-6 from rat plasma.

 
Concentration dependence curves The greater suppressive effect of plasma on 13C-labeled fatty acid ions compared with 2H5-labeled fatty acid ions was quantified over a range of plasma fatty acid concentrations by diluting plasma. To demonstrate the relatively greater effect on the 13C-labeled fatty acid ions, the ratios of the 2H5 to the 13C signals were plotted on the ordinate versus the injected amount of the fatty acid in the plasma matrix on the abscissa (Fig. 4). The curves were ranged from 3 to 14 pmol per injection for 18:3n-3 and 60 to 300 pmol per injection for 18:2n-6, as these were relevant ranges for plasma measurements. The plot follows a nonlinear, quadratic regression equation between the concentration ratio and the amount of endogenous unlabeled fatty acid matrix. A substantially greater suppression was found for the 13C-labeled fatty acid, as the ratio was up to 1.8 and 3.4 for 18:3n-3 and 18:2n-6, respectively.



View larger version (12K):
[in this window]
[in a new window]
  		Fig. 4. Concentration dependence curves for 2H- and 13C-labeled 18:3n-3 and 18:2n-6 in which a constant amount of isotopes was added to varying amounts of plasma. The ratios of the concentrations of the two isotopes were plotted as a function of various amounts of endogenous 18:3n-3 (A) and 18:2n-6 (B). A nonlinear, quadratic regression curve was applied for fitting of the experimental data.

 
Matrix effects are well known in biological mass spectrometry. In particular, the suppressing effect of higher concentrations of analytes has been reported, for example, for methyl palmitate in the electron impact mode (11). In our NCI analyses, it is assumed that the plasma matrix effect is attributable to suppressed ionization resulting from competition. This is supported by the results presented here demonstrating that the addition of pure fatty acid standards can suppress the signals from the same compound isotopically labeled. Thus, this effect is expected to be an inherent property of this type of analysis and should occur on every instrument. This effect also indicates that the type of ISTD used is a critical aspect of accurate quantitative analysis. It would be optimal to use a 13C-labeled ISTD for the measurement of 13C metabolites and likewise for 2H-labeled compounds. Also, the chemical structures of the ISTDs should be similar to those of the compounds being measured.

Isotope effect in rat plasma in vivo
An attempt was made to directly compare 2H5- and 13C-labeled 18:2n-6 and 18:3n-3 with respect to the extent of their conversion to longer chain, more unsaturated fatty acids in vivo. Approximately equal doses of each of the forms of each stable isotope-labeled compound were administered orally, and rat plasma was collected 24 h later for analysis. Samples were analyzed by GC-MS, the peak areas were calibrated using the standard curves described above, and the EFA concentration (in nmol/ml plasma) of each isotope was converted to the dosage for each animal, expressed as pmol/ml of plasma over µmol of administered dose. The concentrations of the various endogenous fatty acids in rat plasma were quite different for some of the minor EFA components relative to the more major components 18:2n-6, 20:4n-6, and 22:6n-3 (Table 4). Thus, the secondary correction depicted in Fig. 4 was applied to the precursors and to 20:4n-6 and 22:6n-3. This was done by calculating the amount of endogenous fatty acid injected for each of these fatty acids in the rat plasma sample; then, after interpolation from the graphs shown in Fig. 4, the relevant correction was applied between the 2H5- and 13C-labeled fatty acids. Such corrections were not applied to the fatty acid peaks that occurred at lower concentrations. The accuracy of this approach was verified for the measurement of 13C-18:2n-6 and 13C-20:4n-6. The PFB ester samples was diluted 10-fold before GC-MS analysis and compared with the estimate made at the higher concentration using the secondary correction just described. There were no significant differences between the results with these two approaches in the concentrations (means ± SEM, n = 11) of 13C-18:2n-6 (15.55 ± 0.64 vs. 14.01 ± 1.13 nmol/ml plasma; P > 0.05) or 13C-20:4n-6 (3.64 ± 0.26 vs. 3.70 ± 0.36 nmol/ml plasma; P > 0.05). Similarly, the values obtained for 2H5-18:2n-6 (15.58 ± 0.86 nmol/ml plasma) and 2H5-20:4n-6 (3.71 ± 0.28 nmol/ml plasma) were in close agreement with the corresponding values of the 13C-labeled compound. Thus, sample dilution offers one means to obtain quantitative results for the 13C-labeled fatty acids by decreasing concentration-dependent signal suppression.


View this table:
[in this window]
[in a new window]
  		TABLE 4. Essential fatty acid composition in rat plasma at 24 h after dosing with four isotopic tracers

 
Using the correction factors put forward here, there were no significant differences (P > 0.05) in the amount of 2H5-18:3n-3 and 13C-U-18:3n-3 in rat plasma per dosage (Table 5). There were also no differences in the amounts of principal n-3 metabolites, 2H5-20:5n-3 and 13C18-20:5n-3, or between 2H5-22:6n-3 and 13C18-22:6n-3 (Fig. 5). Similarly, there were no significant differences in the amounts of 2H5-18:2n-6 and 13C18-18:2n-6 or between the isotopomers of the principal n-6 metabolites 2H5-20:4n-6 and 13C18-20:4n-6. The more minor n-3 and n-6 metabolites showed very similar accretion values (Table 5). 20:3n-3 could not be measured because of the relatively high baseline of the M+5 ion and its low signal abundance.


View this table:
[in this window]
[in a new window]
  		TABLE 5. Comparison of concentrations of 2H5-labeled and 13C-labeled fatty acids in rat plasma 24 h after dosing with four isotopic tracers

 


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 5. Comparison of the concentration of isotope-labeled fatty acids per dose (pmol/ml plasma/µmol dose) in rat plasma in vivo. Plotted are the stable isotopic precursors, 18:3n-3 and 18:2n-6, and their major metabolites, 22:6n-3 and 20:4n-6, at 24 h after oral dosing with a mixture of 2H5-18:3n-3, 13C-U-18:3n-3, 2H5-18:2n-6, and 13C-U-18:2n-6 ethyl esters. No significant difference (P > 0.05) was observed in rat plasma between the isotopomers of 18:3n-3, 22:6n-3, 18:2n-6, and 20:4n-6. Values are presented as means ± SEM (n = 11).

 
The results of this study demonstrate that the EFAs used containing 2H and 13C were metabolized in a similar manner in vivo in rats. These results are consistent with those of Bosner et al. (27), who found identical cholesterol absorption for 2H6- and 13C5-cholesterol in human plasma. Also, a pharmacokinetic study showed no differences in the rates of absorption, distribution, and excretion of 2H3-labeled and unlabeled methadone in rats (28). In general, little or no isotope effect has been observed for 13C-labeled compounds (29). Thus, it would follow that the deuterium label used in this study also produced little or no perturbations in fatty acid metabolism relative to the endogenous compound.


   ACKNOWLEDGMENTS
 
The authors thank Drs. Lee Chedester and Raouf Kechrid for their assistance with the animal work. This project was funded by the Intramural Research Program of the National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health.

Manuscript received April 4, 2005 and in revised form May 18, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
  1. Emken, E. A., W. K. Rohwedder, and H. J. Dutton. 1976. Dual-labeled technique for human lipid metabolism studies using deuterated fatty acid isomers. Lipids. 11: 135–142.[Medline]

  2. Sauerwald, T. U., D. L. Hachey, C. L. Jensen, H. Chen, R. E. Anderson, and W. C. Heird. 1996. Effect of dietary alpha-linolenic acid intake on incorporation of docosahexaenoic and arachidonic acids into plasma phospholipids of term infants. Lipids. 31 (Suppl.): 131–135.

  3. Brenna, J. T. 1997. Use of stable isotopes to study fatty acid and lipoprotein metabolism in man. Prostaglandins Leukot. Essent. Fatty Acids. 57: 467–472.[CrossRef][Medline]

  4. Salem, N., Jr., B. Wegher, P. Mena, and R. Uauy. 1996. Arachidonic and docosahexaenoic acids are biosynthesized from their 18-carbon precursors in human infants. Proc. Natl. Acad. Sci. USA. 93: 49–54.[Abstract/Free Full Text]

  5. Demmelmair, H., M. Baumheuer, B. Koletzko, K. Dokoupil, and G. Kratl. 1998. Metabolism of U13C-labeled linoleic acid in lactating women. J. Lipid Res. 39: 1389–1396.[Abstract/Free Full Text]

  6. Vermunt, S. H., R. P. Mensink, M. M. Simonis, and G. Hornstra. 2000. Effects of dietary alpha-linolenic acid on the conversion and oxidation of 13C-alpha-linolenic acid. Lipids. 35: 137–142.[Medline]

  7. King, A. S., and R. T. Birge. 1929. An isotope of carbon, mass 13. Nature. 124: 127.

  8. Lewis, G. N. 1933. The isotope of hydrogen. J. Am. Chem. Soc. 55: 1297–1298.

  9. Schoenheimer, R., and D. Rittenberg. 1936. Deuterium as an indicator in the study of intermediary metabolism. V. The desaturation of fatty acids in the organism. J. Biol. Chem. 113: 505–510.[Free Full Text]

  10. Goodman, K. J., and J. T. Brenna. 1995. High-precision gas chromatography-combustion isotope ratio mass spectrometry at low signal levels. J. Chromatogr. A. 689: 63–68.[CrossRef][Medline]

  11. Patterson, B. W., and R. R. Wolfe. 1993. Concentration dependence of methyl palmitate isotope ratios by electron impact ionization gas chromatography/mass spectrometry. Biol. Mass Spectrom. 22: 481–486.[CrossRef][Medline]

  12. Emken, E. A., R. O. Adlof, S. M. Duval, and G. J. Nelson. 1999. Effect of dietary docosahexaenoic acid on desaturation and uptake in vivo of isotope-labeled oleic, linoleic, and linolenic acids by male subjects. Lipids. 34: 785–791.[CrossRef][Medline]

  13. Pawlosky, R. J., H. W. Sprecher, and N. Salem, Jr. 1992. High sensitivity negative ion GC-MS method for detection of desaturated and chain-elongated products of deuterated linoleic and linolenic acids. J. Lipid Res. 33: 1711–1717.[Abstract]

  14. Patton, G. M., S. Cann, H. Brunengraber, and J. M. Lowenstein. 1981. Separation of methyl esters of fatty acids by gas chromatography on capillary columns, including the separation of deuterated from nondeuterated fatty acids. Methods Enzymol. 72: 8–20.[Medline]

  15. Lefkowitz, W., S. Y. Lim, Y. H. Lin, and N. Salem, Jr. 2005. Where does the developing brain obtain its docosahexaenoic acid? Relative contributions of dietary alpha-linolenic acid, docosahexaenoic acid, and body stores in the developing rat. Pediatr. Res. 57: 157–165.[Medline]

  16. Dinh-Nguyen, N., and A. Raal. 1978. Perdeuterated normal-chain saturated mono- and dicarboxylic acids and methyl esters. Prog. Chem. Fats Other Lipids. 16: 195–206.[CrossRef][Medline]

  17. Colson, C. E., and J. M. Lowenstein. 1981. Separation of partially deuterated methyl palmitate from nondeuterated methyl palmitate by high-pressure liquid chromatography. Methods Enzymol. 72: 53–56.[Medline]

  18. Lin, Y. H., and N. Salem. 2002. A technique for the in vivo study of multiple stable isotope-labeled essential fatty acids. Prostaglandins Leukot. Essent. Fatty Acids. 67: 141–146.[CrossRef][Medline]

  19. Lin, Y. H., and N. Salem. 2005. In vivo conversion of 18- and 20-C essential fatty acids in rats using the multiple simultaneous stable isotope method. J. Lipid Res. 46: 1962–1973.[Abstract/Free Full Text]

  20. Reeves, P. G., F. H. Nielsen, and G. C. Fahey, Jr. 1993. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123: 1939–1951.

  21. Folch, A. C., M. Lees, and G. M. Sloane-Stanley. 1957. A simple method for isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: 497–509.[Free Full Text]

  22. Morrison, W. R., and L. M. Smith. 1964. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoride–methanol. J. Lipid Res. 5: 600–608.[Abstract]

  23. Salem, N., Jr., M. Reyzer, and J. Karanian. 1996. Losses of arachidonic acid in rat liver after alcohol inhalation. Lipids. 31 (Suppl.): 153–156.[CrossRef][Medline]

  24. Green, J. M. 1996. A practical guide to analytical method validation. Anal. Chem. 68: A305–A309.

  25. Troost, J. R., and E. Y. Olavensen. 1996. Gas chromatographic/mass spectrometric calibration bias. Anal. Chem. 68: 708–711.[CrossRef]

  26. Patterson, B. W., G. Zhao, and S. Klein. 1998. Improved accuracy and precision of gas chromatography/mass spectrometry measurements for metabolic tracers. Metabolism. 47: 706–712.[CrossRef][Medline]

  27. Bosner, M. S., R. E. Ostlund, Jr., O. Osofisan, J. Grosklos, C. Fritschle, and L. G. Lange. 1993. Assessment of percent cholesterol absorption in humans with stable isotopes. J. Lipid Res. 34: 1047–1053.[Abstract]

  28. Hsia, J. C., J. C. Tam, H. G. Giles, C. C. Leung, H. Marcus, J. A. Marshman, and A. E. Leblanc. 1976. Markers for detection of supplementation in narcotic programs—deuterium-labeled methadone. Science. 193: 498–500.[Abstract/Free Full Text]

  29. Baillie, T. A. 1981. The use of stable isotopes in pharmacological research. Pharmacol. Rev. 33: 81–132.[Medline]


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Lipid Res.Home page
S. Gagne, S. Crane, Z. Huang, C. S. Li, K. P. Bateman, and J.-F. Levesque
Rapid measurement of deuterium-labeled long-chain fatty acids in plasma by HPLC-ESI-MS
J. Lipid Res., January 1, 2007; 48(1): 252 - 259.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
Y. H. Lin and N. Salem Jr.
In vivo conversion of 18- and 20-C essential fatty acids in rats using the multiple simultaneous stable isotope method
J. Lipid Res., September 1, 2005; 46(9): 1962 - 1973.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
M500128-JLR200v1
46/9/1974    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lin, Y. H.
Right arrow Articles by Salem, N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lin, Y. H.
Right arrow Articles by Salem, N., Jr.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Journal of Biological Chemistry 
 Molecular and Cellular Proteomics   ASBMB Today