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Journal of Lipid Research, Vol. 48, 935-943, April 2007
Compartmental analyses of plasma n-3 essential fatty acids among male and female smokers and nonsmokers
* Laboratory of Metabolic Control, National Institutes on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD Published, JLR Papers in Press, January 17, 2007.
1 To whom correspondence should be addressed. e-mail: bpawl{at}mail.nih.gov
The effects of cigarette smoking on n-3 essential FA metabolism were studied in male and female subjects by fitting the concentration-time curves of the d5-labeled plasma fatty acids (FAs) originating from a dose of d5-18:3n-3 to a compartmental model of n-3 FA metabolism. For 3 weeks, female (smokers, n = 5; nonsmokers, n = 5) and male (smokers, n = 5; nonsmokers, n = 5) subjects subsisted on a beef-based diet. Beginning in the third week, subjects received a dose of d5-18:3n-3 ethyl ester (1 g). Plasma FAs were analyzed using gas chromatography (GC) and GC-mass spectrometry, and the kinetic rate parameters were determined from the concentration-time curves for d5-18:3n-3, d5-20:5n-3, d5-22:5n-3, and d5-22:6n-3. Women smokers had a 2-fold greater percent of dose in plasma (5.8% vs. 2.9%; P < 0.01) and a higher fractional rate constant coefficient for formation of d5-22:6n-3 from d5-22:5n-3 (0.03 h1 vs. 0.01 h1; P < 0.01), compared with nonsmokers. Male smokers had elevated total plasma n-3 FAs, more-rapid turnover of 18:3n-3 (13.3 mg/day1 vs. 4.3 mg/day1; P < 0.001), a disappearance rate of d5-20:5n-3 that was both delayed and slower (0.001 h1 vs. 0.012 h1; P < 0.05), and a percentage of d5-20:5n-3 directed into formation of d5-22:5n-3 (99% vs. 61%; P < 0.03) that was greater compared with nonsmokers. Smoking increased the bioavailability of n-3 FAs from plasma, accelerated the fractional synthetic rates, and heightened the percent formation of some long-chain n-3 PUFAs in men and women.
Supplementary key words linolenic acid docosahexaenoic acid isotope tracer mass spectrometry humans kinetics controlled diet Abbreviations: ANCOVA, analysis of covariance; AUC, area under the concentration-time curve; CV, coefficient of variance; NS, nonsmoking subject; S, smoking subject
Cigarette smoking has been shown to elevate, through a diverse set of free radical-mediated actions, lipid peroxide levels in tissues for which oxidation products of polyunsaturated fatty acids (PUFAs), as measured in the circulation, are indicators of oxidative stress (1, 2). Because PUFAs are highly susceptible to free radical oxidation, habitual smoking may decrease the amount of PUFAs in plasma (3) and red blood cells (4). Because of the constant demand for PUFAs in the replenishing of cell membranes, the making of new cells, and the counteracting of losses that occur through peroxidation, smoking tobacco may indirectly enhance the synthesis of long-chain PUFAs through its additional contribution to the system's oxidative load.
Observational studies that examine the effects of smoking on n-3 FA status without rigorous control for dietary intake can be confounded because of lower intake of n-3 FAs by both men and smokers (4, 8). Smoking's direct effects on n-3 FA status or the in vivo kinetics of n-3 FA metabolism have not been studied using either quantitative tracer methods or compartmental modeling procedures in subjects consuming a diet that is controlled for n-3 FA intake. Previously, we described a compartmental model of n-3 FA metabolism that was used for the determination of several rate constant parameters relative to long-chain PUFA biosynthesis (9). We now extend this analysis to examine the effects of smoking on the kinetics of n-3 FA metabolism in comparing male and female smoking (S) and nonsmoking (NS) subjects on a metabolically controlled diet. Kinetic rate parameters were determined for each subject from the concentration-time curves of the experimentally determined d5-labeled FAs 18:3n-3, 20:5n-3, 22:5n-3, and 22:6n-3 in plasma, which originated from an oral dose of d5-18:3n-3, using compartmental modeling procedures.
Subjects Exclusion criteria included: any major medical problems, including, hepatic, endocrine, and metabolic disorders, a history of head trauma, seizures, or prolonged loss of consciousness, a lifetime history of a major psychiatric diagnosis, or abnormal clinical laboratory findings. Subjects were excluded if they consumed more than the equivalent of two glasses of beer or wine per day, used prescription medications within the last month, including birth control pills or over-the-counter medications including aspirin, ibuprofen, acetaminophen, antihistamines, and topical steroids. Subjects were excluded who had persistent use of vitamins E or C, multivitamins, lipid supplements, herbal or home remedies with unknown composition, home cures, or unusual vitamin use or other unusual dietary habits. The study required eating red meat, turkey, and yogurt, and thus excluded vegetarians; also excluded were patients with allergies to foods making up this diet, and those with lactose intolerance. Subjects were excluded who had donated blood within the previous three months or who were currently pregnant or had imminent plans to become pregnant. All subjects provided written informed consent for all clinical procedures under approval of the NIAAA Institutional Review Board under protocol #92-AA-0194.
Dietary protocol
Administration of the d5-FA ethyl ester and subsequent blood draws occurred during the final week of the study period, and details of the procedures have been reported previously (10). Briefly, subjects were given an overnight fast and then received 1 g of d5- -linolenate ethyl ester (d5-17, 17, 18, 18, 18-18:3n-3; Cambridge Isotope Labs, Woburn, MA) blended into low-fat (1% fat) yogurt prior to a morning meal. Because the overall bioavailability of n-3 FAs as either an ethyl ester or a triglyceride appear to be similar, the isotope was administered as an FA ethyl ester (10). With the exception of the 8 h sample, blood (40 ml) was drawn under fasting conditions from the forearm, at baseline and at 8, 24, 48, 72, 96, and 168 h. The platelet-poor plasma was separated by centrifugation (1,800 g for 10 min) and frozen at 80°C until analysis.
Plasma lipid extraction and gas chromatographic analysis
Sample preparation for mass spectral analysis
Mass spectral analysis
Compartmental model
Fractional transfer rates, flow rates, percents, turnover, and errors
Model illustration and rate equations
Limits, constraints, and statistical analysis Because the FA composition of the diet had been experimentally determined and the caloric intake of the diet had been adjusted to the energy requirements of each subject, the daily n-3 FA intake for each subject could be estimated so that upper and lower n-3 FA limits were assigned. Plasma steady-state FA concentrations were determined for each subject at each blood sampling time point, and because the standardized diet minimized the variability of n-3 FA intake, there was little difference (± 5%) in the concentrations of the individual n-3 FAs within the plasma of a given subject over this period. Therefore, the mean concentration of each plasma n-3 FA from each subject was used to approximate the steady-state mass of the endogenous substrate (MJ) available for biosynthesis. These values were held constant. For statistical analyses, data were imported into StatView 5 (SAS Institute; Cary, NC). Initially, plasma FA concentrations and model-derived rate parameters from male and female subjects were pooled and analyzed for the effects of smoking using unpaired t-tests. A P value of 0.05 or lower was considered significant. The data were also analyzed with respect to gender in unpaired t-tests. A P value of 0.05 or lower was considered significant. Where differences were observed in either plasma FA concentrations or kinetic parameters, the findings were further analyzed in an analysis of covariance (ANCOVA) model for possible confounding influences of age or body mass. Additionally, these data sets were analyzed in multiple linear regression models to assess interactions of age and body mass on plasma FAs or kinetic parameters. A P value of 0.05 or lower was considered significant.
Subject characteristics The n-3 FA intake was estimated using values obtained from a direct determination of the lipid content of the food. These values are reported in amounts that take into consideration energy density differences of the diets for male and female subjects (Table 1). The mean concentrations of plasma n-3 FAs obtained from blood samples drawn over the third week of the dietary period are given in Table 2 . As a group, both male and female smokers had higher concentrations of plasma n-3 FAs compared with nonsmokers. These values, however, were strongly influenced by the higher plasma FA concentrations observed in male smokers as compared with female subjects.
Appearance of d5-18:3n-3 in plasma and area-under-the-curve values Two 18:3n-3 compartments were included in the model, one for isotope administration and absorption through the GI tract and the second for the appearance of the FA in the plasma. To perform proper kinetic analysis, blood collection began 8 h from the time of dosing to assess accurate concentrations of labeled 18:3n-3 entering the plasma as well as quantification of the subsequent labeled FAs appearing there (12). Bioavailability of the precursor in plasma was calculated as percent of dose using isotope values obtained from these two compartments. The model carried with it an assumption that fat absorption was essentially complete for all subjects (98%) and that therefore, variances in the amount of labeled precursor appearing in plasma represent differences in bioavailability among subjects. The P(2,1) parameter (Table 3 ) returns the value for percent of dose of d5-18:3n-3 appearing in plasma over 168 h from the model estimations. Among women smokers, this was significantly greater (5.8%) compared with nonsmokers (2.9%), an outcome that is reflected in the composite time course curves for d5-18:3n-3 (Fig. 2A ). Plasma deuterated FA values may also be expressed in terms of area under the concentration-time curve (AUC) over the time course (here expressed in total amount of label, in mg/hour ± SD of d5-18:3n-3). The mean AUC values for all groups were: 85 ± 17; 89 ± 8; 101 ± 21; and 118 ± 19 mg/hour for male S and NS and female S and NS subjects, respectively. There were no differences in the AUC values; however, the AUC does not inform on either the rate of appearance or disappearance of the isotope plasma, nor is it descriptive of the contour of the concentration-time curve.
n-3 FA plasma concentration-time curves Figure 2 and Figure 3 give illustrations of the composite time course curves of labeled FAs in plasma of both female and male subjects. Figure 3B illustrates the time course curve for d5-20:5n-3 in male smokers and nonsmokers. Male smokers had both a delayed and less rapid rate of disappearance of the labeled FA in plasma compared with nonsmokers. Plasma disappearance rates of d5-20:5n-3 were quantified as the fractional rate constant coefficient, L(0,3), given in Table 4 for nonsmokers (0.012 h1) and smokers (0.0014 h1; P < 0.05). By contrast, the rate constant coefficient for disappearance of d5-18:3n-3, L(0,2), from plasma was greater among male smokers (12.7 h1) compared with nonsmokers (8.8 h1; P < 0.04) (see Fig. 3A, Table 4). The dissimilarity resulted in an accelerated turnover rate of 18:3n-3 among smokers [R(0,1) 1,340 mg/day1] compared with nonsmokers [R(0,1) 429 mg/day1] (Tables 5 and 6 ). The 3-fold greater throughput of 18:3n-3 among smokers strongly suggests that smoking acts to stimulate n-3 FA entrance into the plasma, thereby increasing its bioavailability to tissues. We note that this value was well above estimated dietary intake amounts for 18:3n-3 for male subjects (Table 1) but is consistent with the accelerated turnover rate. The proposition of accelerated throughput of 18:3n-3 is supported by higher plasma concentrations of n-3 FA among both male and female smokers compared with nonsmokers (Table 2) as well as the 2-fold greater percentage of d5-18:3n-3 that appeared in the plasma of female smokers, as discussed above (Fig. 2A).
Among both male and female smokers, the fractional rate constant coefficient for transfer of d5-22:5n-3 to d5-22:6n-3, L(5,4), was greater compared with nonsmokers (Table 4). Among women who smoked, the rate constant coefficient was 3-fold greater compared with nonsmokers (0.028 vs. 0.009; P < 0.03) eliciting a 3-fold greater rate of synthesis of 22:6n-3 from 22:5n-3 (20 vs. 6.2 mg/day1; P < 0.04) (Table 6). The difference in magnitude between these coefficients is reflected in the positive incline of the curves in the composite concentration-time curves for d5-22:6n-3 (Fig. 2D). Notably, among female smokers, the appearance rate of d5-22:6n-3 continued to increase throughout the 168 h (Fig. 2D) compared with that of male smokers (Fig. 3D). Male smokers had a 30% increase in the percent conversion of d5-20:5n-3 to d5-22:5n-3 compared with nonsmokers (Table 3), which was reflected in the greater rate of synthesis of 22:5n-3 from 20:5n-3 among smokers (6.2 vs. 19.9 mg/day1; P < 0.03) (Table 6). Among females, there were no differences in the values of the fractional synthetic rate for conversion of 20:5n-3 to 22:5n-3; however, there was a tendency for an increase in the percent conversion of 20:5n-3 to 22:5n-3. There were no substantial differences noted in the half-lives of n-3 FAs between the groups other than a trend toward a shorter half-life of 18:3n-3 among male smokers and a trend toward a longer half-life of 20:5n-3 among female smokers compared with their NS controls (Table 7 ).
DISCUSSION To our knowledge, no detailed studies examining the effects of smoking on the kinetics of essential FA metabolism in human subjects subsisting on a controlled diet have been reported. We undertook an examination of the effects of cigarette smoking on the in vivo kinetics of n-3 essential FA metabolism among male and female subjects maintained on a rigorously controlled diet that was designed to enhance biosynthesis of long-chain PUFAs using a quantitative isotopic tracer technique in conjunction with a compartmental modeling procedure. Although in this study female smokers were older, they also tended to be leaner than nonsmokers, whereas male smokers had greater adiposity than nonsmokers. To assess whether age and/or adiposity had possible confounding influences on plasma FA concentrations or kinetic parameters, positive findings were subjected to further statistical analyses using an ANCOVA and multiple linear regression models. No direct effects of age or adiposity on plasma FA concentrations or kinetic parameter outcomes were detected. Neither age nor adiposity was found to correlate with those effects of smoking in this group of subjects, and there were no apparent significant interactions between these parameters and cigarette smoking. We did observe that smoking possibly affected an increase in the bioavailability of n-3 essential FAs from plasma and altered several rate parameters that appeared to both accelerate and heighten the synthesis of some long-chain n-3 PUFAs in men and women. The compartmental model of n-3 FA metabolism was previously validated in subjects subsisting on various diets (13). Inasmuch as the model makes use of FA data from the plasma alone, the derived kinetic parameters may only indirectly reflect liver synthesis of long-chain PUFAs. In vivo rate constant coefficients for successive steps of long-chain PUFA biosynthesis were determined for each subject using the concentration-time curves of the d5-labeled FAs, and plasma steady-state FA amounts were used as estimations of total mass of n-3 FA substrate available for transfer through the pathway. Because the number of subjects in this study was small, several perceived differences in rate parameters approached but did not reach significance. Gender-specific differences in the metabolism of n-3 FAs have been previously reported (14, 15), where it has been noted that a greater percent of 22:5n-3 was utilized for synthesis of 22:6n-3 among women compared with men (14). In this study, as well, we observed that women smokers and nonsmokers tended to have greater percent conversions of 22:5n-3 to 22:6n-3 compared with male subjects (Table 3; Figs. 2C, D and 3C, D). Cigarette smoking is well known to increase formation of oxygenated PUFA (1, 2) and because smoking has also been associated with lower amounts of serum PUFA phospholipids, it has been suggested that cigarette smoking may influence processes related to the absorption, synthesis, or metabolism of PUFAs (3). Among men and women subsisting on the control diet, cigarette smoking did not appear to have a negative impact on PUFA absorption, because there were greater concentrations of n-3 FAs in the plasma of male smokers compared with nonsmokers and nonsignificant elevations of plasma n-3 FAs among female smokers compared with nonsmokers. This suggested that cigarette smoking may act to increase transport of PUFA into the plasma, a supposition that was supported by observations among women smokers, where there was a greater influx of the dosed d5-18:3n-3 into the plasma compared with nonsmokers. The difference in plasma influx of d5-18:3n-3 in male smokers was not as great as that in female smokers when compared with their NS controls (Table 3). However, to sustain plasma 18:3n-3 concentrations with their rapid turnover rate in male smokers (L(0,2); Table 4), it was estimated that the 18:3n-3 influx in smokers would need to be three times that of nonsmokers (Table 6). The compartmental model was used to determine the efficiency of individual steps in the biosynthetic processes by estimating the percentage of isotope transferred between the n-3 FA compartments along the pathway. Cigarette smoking was not found to increase the percent utilization of 18:3n-3 for formation of 20:5n-3 (P(3,2); Table 3) in either men or women. However, among both male and female smokers, a higher percentage of 20:5n-3 was utilized for 22:5n-3 synthesis (P(4,3); Table 3), and this difference tended to be greater in men than in women. This is the first detailed study in which the effects of cigarette smoking on n-3 essential FA metabolism were examined in human subjects on a controlled diet. Results from the kinetic analyses (isotope tracer determinations) and steady-state measurements (plasma FA concentrations) in male and female subjects subsisting on the beef-based diet indicated that cigarette smoking increased the amount of n-3 FAs transfered to the plasma. As a consequence, bioavailability of PUFAs to tissues may be enhanced in persons who smoke compared with nonsmokers, in part counteracting losses that occur through peroxidation. Also, among male and female smokers, the percent conversion of 20:5n-3 to 22:5n-3 was enhanced, and the fractional synthetic rate for formation of 22:6n-3 among women smokers was triple that of nonsmokers. The increased synthetic rates and the greater percent transfer of isotope between compartments among smokers indicate that smoking also had a positive effect on the production of some long-chain PUFAs in vivo, possibly as another means to compensate for losses resulting from lipid oxidation (1, 2). Notably, the chronic effects of smoking on the enhancement of long-chain PUFA biosynthesis observed here in humans is in contrast to results obtained from cell culture, where an acute dose of cigarette smoke appeared to inhibit long-chain PUFA formation (16).
The authors wish to thank the clinical and technical support staff of the Laboratory of Clinical Studies, National Institutes on Alcohol Abuse and Alcoholism, National Institutes of Health for their participation in this study. The authors appreciate the work of the staff in the metabolic kitchen at the Clinical Center of the National Institutes of Health, especially that of the dieticians, Ms. Patti Riggs, RD and Ms. Nancy Sebring, RD. The authors especially wish to recognize the excellent work of Mr. Brent Wegher in analyzing and reporting the isotope data and managing the database. Manuscript received July 17, 2006 and in revised form January 16, 2007. REFERENCES
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