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American Journal of Clinical Nutrition, Vol. 84, No. 1, 44-53, July 2006
© 2006 American Society for Nutrition

ORIGINAL RESEARCH COMMUNICATION

Conversion of {alpha}-linolenic acid in humans is influenced by the absolute amounts of {alpha}-linolenic acid and linoleic acid in the diet and not by their ratio1,2,3

Petra LL Goyens, Mary E Spilker, Peter L Zock, Martijn B Katan and Ronald P Mensink

1 From the Department of Human Biology, Maastricht University, Maastricht, Netherlands (PLLG and RPM); the Technical University of Munich, Nuclear Medicine Department, Klinikum rechts der Isar, Germany (MES); the Division of Human Nutrition, Wageningen University, Wageningen, Netherlands (PLZ and MBK); and Wageningen Centre for Food Sciences, Wageningen, Netherlands (PLZ, MBK, and RPM)

2 Supported by the Wageningen Centre for Food Sciences (to PLZ, MBK, and RPM), an alliance of major Dutch food industries, Maastricht University, TNO Nutrition and Food Research, and Wageningen University and Research Centre, with financial support by the Dutch government. The model development was partially supported by NIH grant P41 EB-001975, "Resource Facility for Population Kinetics." The experimental pastries were produced by V.T.I.V.T. Ter Hercke (Herk-de-Stad, Belgium).

3 Reprints not available. Address correspondence to PLL Goyens, Department of Human Biology, Maastricht University, PO Box 616, 6200 MD Maastricht, Netherlands. E-mail: p.goyens{at}hb.unimaas.nl.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Human in vivo data on dietary determinants of {alpha}-linolenic acid (ALA; 18:3n–3) metabolism are scarce.

Objective: We examined whether intakes of ALA or linoleic acid (LA; 18:2n–6) or their ratio influences ALA metabolism.

Design: During 4 wk, 29 subjects received a control diet (7% of energy from LA, 0.4% of energy from ALA, ALA-to-LA ratio = 1:19). For the next 6 wk, a control diet, a low-LA diet (3% of energy from LA, 0.4% of energy from ALA, ratio = 1:7), or a high-ALA diet (7% of energy from LA, 1.1% of energy from ALA, ratio = 1:7) was consumed. Ten days before the end of each dietary period, [U-13C]ALA was administered orally for 9 d. ALA oxidation was determined from breath. Conversion was estimated by using compartmental modeling of [13C]- and [12C]n–3 fatty acid concentrations in fasting plasma phospholipids.

Results: Compared with the control group, ALA incorporation into phospholipids increased by 3.6% in the low-LA group (P = 0.012) and decreased by 8.0% in the high-ALA group (P < 0.001). In absolute amounts, it increased by 34.3 mg (P = 0.020) in the low-LA group but hardly changed in the high-ALA group. Nearly all ALA from the plasma phospholipid pool was converted into eicosapentaenoic acid. Conversion of eicosapentaenoic acid into docosapentaenoic acid and docosahexaenoic acid hardly changed in the 3 groups and was <0.1% of dietary ALA. In absolute amounts, it was unchanged in the low-LA group, but increased from 0.7 to 1.9 mg (P = 0.001) in the high-ALA group. ALA oxidation was unchanged by the dietary interventions.

Conclusion: The amounts of ALA and LA in the diet, but not their ratio, determine ALA conversion.

Key Words: Ratio of {alpha}-linolenic to linoleic acid • conversion • oxidation • stable isotopes • humans


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Eicosapentaenoic acid (EPA; 20:5n–3) and docosahexaenoic acid (DHA; 22:6n–3) play a vital role in many metabolic processes. Although these 2 fatty acids are readily available from fish, these marine-derived fatty acids can also be synthesized by humans from {alpha}-linolenic acid (ALA; 18:3n–3). Humans, however, can obtain ALA only through their diets, because the absence of the required {Delta}12- and {Delta}15-desaturase enzymes makes de novo synthesis from stearic acid impossible. Furthermore, conversion of dietary ALA into EPA is limited. Because the efficacy of n–3 long-chain polyunsaturated fatty acid (LC-PUFA) synthesis decreases down the cascade of ALA conversion, DHA synthesis from ALA is even more restricted than that of EPA (1, 2). It is generally assumed that linoleic acid (LA; 18:2n–6) reduces EPA synthesis because of the competition between ALA and LA for common desaturation and elongation enzymes (1-4). Hence, conversion of {alpha}-linolenic acid into the n–3 LC-PUFAs might be changed by increasing ALA intake or by decreasing LA intake (5-9). In many of these studies, however, the intakes of both ALA and LA changed, and as a result, so did their ratio. In addition, it has been suggested that the ALA-to-LA ratio in the diet determines ALA conversion independent of the absolute intakes of both fatty acids (8). This suggestion does not agree with a recent study in rats (10). Human studies, however, were not able to disentangle the effect of the ALA-to-LA ratio on ALA metabolism from the effects of the absolute intake of these 2 fatty acids. The present nutritional intervention trial was designed to determine whether in vivo conversion of dietary ALA is influenced by the absolute amounts of LA or ALA in the diet or by the ALA-to-LA ratio. To that end, we used compartmental modeling to study ALA conversion at various absolute intakes and ratios of LA and ALA in the diet. In addition, effects of the diets on the oxidation of uniformly labeled 13C-ALA ([U-13C]ALA) were examined.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
The present study was part of a trial that was designed to examine the effects of PUFAs on cardiovascular disease risk markers. The characteristics of the study population have been previously described in detail (11). Thirty healthy subjects, fifteen men and fifteen women, completed the study. During study weeks 3 and 4, one male subject had flu with associated gastrointestinal complaints and weight loss. Therefore, it was decided not to use his data in the analysis of the results. Of the remaining twenty-nine subjects, 2 men and 3 women smoked, 7 women were postmenopausal, and 5 of the 8 premenopausal women used oral contraceptives. The men were on average 53.8 ± 11.6 y old (x± SD), weighed 79.6 ± 9.7 kg and had a body mass index (in kg/m2) of 25.2 ± 3.4. The women were 46.2 ± 14.4 y old, weighed 67.6 ± 8.2 kg, and had a body mass index of 23.5 ± 2.8. The study was approved by the Medical Ethics Committee of Maastricht, and all participants were informed in detail about the purpose and nature of the study before they gave their written informed consent.

Diet and study design
The study consisted of a run-in period of 4 wk that was immediately followed by an experimental period of 6 wk. The study was designed as a double-blind, nutritional intervention trial with 3 parallel arms: a low ratio control group, a high ratio low-LA group, and a high ratio high-ALA group. For each of these 3 intervention groups, diets were formulated at 9 different energy levels. These energy levels varied from 7.5 to 13.4 MJ, whereas the difference between 2 successive energy levels equaled 0.84 MJ. Before the start of the study, the subjects weighed and reported in a 3-d food record all foods and drinks consumed during 1 weekend day and 2 working days. Using the Dutch food-composition table (12), we estimated the energy intakes of the subjects’ habitual diets. At trial entry, the subjects were allocated to the energy level that matched their estimated energy requirements. Body weights were measured once weekly. The subjects remained on the assigned energy level as long as their body weight did not change by >2 kg compared with their weight at the start of the study. Otherwise, they were placed into a more appropriate energy level.

During the run-in period, all participants consumed a control diet with a nutrient composition that approximated the dietary intake of the Dutch population. This control diet had an ALA-to-LA ratio of 1:19 by supplying 7% of energy of LA and 0.4% of energy of ALA. At the end of the run-in period, the subjects were randomly divided over the 3 experimental groups stratified for sex, age, and total cholesterol concentrations as determined at the screening visit. During the experimental period, one group continued to consume the control diet, whereas the other 2 groups consumed either a low-LA or a high-ALA diet. As reported previously (11), the diets of the 3 groups were comparable with respect to protein (15% of energy), carbohydrate (50% of energy), cholesterol (11.5–13.5 mg/MJ), and fiber (30–35 g) intakes. The diets differed, however, with respect to fatty acid composition. The ALA-to-LA ratio of both the low-LA and the high-ALA diets was {approx}1:7, which was higher than the ratio of 1:19 of the control diet. On the low-LA diet, the ALA-to-LA ratio was increased by decreasing the proportion of LA from 7% of energy to 3% of energy, whereas ALA intake did not change. In contrast, the ALA-to-LA ratio of the high-ALA diet was increased by increasing the intake of ALA from 0.4% of energy to 1.1% of energy, whereas LA intake did not change. The modification of the proportions of dietary LA or ALA in the experimental groups was done in exchange for mainly oleic acid and to a small extent saturated fatty acids.

The targeted compositions of the 3 diets were achieved through consumption of nonexperimental and experimental food items that accounted for 10–13% of energy and 22–25% of energy from fat intake, respectively. For each of the 9 energy levels, dietary guidelines were provided, describing the type, daily amounts, and preparation of the nonexperimental food items. These guidelines were identical for the 3 intervention groups and had to be followed precisely. In contrast, each intervention group received experimental products that were made from different experimental fats (NIZO Food Research, Ede, The Netherlands), such as a margarine (NIZO Food Research) and pastries. The fatty acid composition of the experimental fats has been presented before (11).

Once weekly, the subjects had to visit the department to receive a specific amount of experimental products, such as margarines and pastries (cookies, pies, and cake) in which these margarines were incorporated. The margarines, which contained 84% absorbable fats and 16% water, had to be used daily for cooking, baking, or as a spread on bread. The pastries were prepared by a local bakery and were consumed daily (cookies) or weekly (pies, cake). The amount of experimental products that the participants received was determined by their assigned energy level. To estimate their energy and nutrient intakes during the study, the subjects had to weigh and record their food intake for 2 working days and 1 weekend day in the last week of both the run-in and the intervention periods. The subjects were asked not to change their level of physical exercise, smoking habits, use of alcohol, or use of oral contraceptives during their participation in the study. Consumption of fish or marine foods was prohibited.

Each day, the subjects had to record in diaries any signs of illness, including the date and time of occurrence, duration, date of resolution, and medication used. The subjects were also asked to note their menstrual phase and record in detail any deviations from both the protocol and the amounts of products that had to be consumed. Each week, the diaries were checked in the presence of the subjects by a registered dietitian.

Tracer protocol and sampling of blood and breath
An amount of 11.4 g of [U-13C]ALA (isotopic purity of 99%) was bought from Isotec (Isotec Inc, Miamisburg, OH) in 2 batches of 5.7 g each. Each batch was diluted by 27.28 times in olive oil, after which capsules of 0.3 mL were completely filled to obtain 10 mg [U-13C]ALA per capsule.

Ten days before the end of the run-in period and 10 d before the end of the experimental period, on days 19 and 61 respectively, the subjects reported to the department after fasting overnight and abstaining from alcohol for 24 h. The volume of CO2 expiration (VCO2) was measured by using a ventilated-hood system (Omnical, Maastricht University, the Netherlands) (13), and breath was sampled to determine the baseline 13C/12C ratio of expired CO2. For the latter measurement, the subjects had to breathe for 3 min through a mouthpiece with 2-way nonrebreathing valves (2700 series; Hans Rudolph Inc, Kansas City, MO) that were connected to a mixing chamber of 6.75 L. From this mixing chamber, breath was sampled directly into a 10-mL Vacutainer (Becton Dickinson, Meyland, France). Thereafter, a fasting blood sample (t = 0 h) was drawn to measure the background enrichment of 13C-labeled ALA, EPA, docosapentaenoic acid (DPA), and DHA in plasma phospholipids. Subjects then received a single oral bolus of 30 mg [U-13C]ALA that was provided in 3 capsules. Immediately after tracer intake, the subjects had to consume a standardized meal consisting of 200 mL orange juice and 3 slices of bread, each spread with 5 g of the assigned experimental margarine and with 15 g jam. The subjects were allowed to choose between tea and decaffeinated coffee and were free to add milk or sugar. Breath samples were collected at 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, and 9 h after tracer intake and were stored at room temperature for later analysis. VCO2 production was measured before a breath sample was taken. About 5 h after tracer intake, the subjects received a lunch. At day 19 of the run-in period, their meal consisted of bread spread with the experimental control margarine as well as experimental and nonexperimental food items according to their choice. The amount and type of the food items had to meet their individual dietary guidelines. All food items and beverages that were consumed on day 19 were weighed and recorded. At day 61 of the experimental period, the subjects were provided with an identical lunch that included the assigned spread and experimental products.

For the 8 d after the tracer oxidation test, the subjects took 20 mg [U-13C]ALA per day: one capsule at 0800 and one at 2000. Tracer capsules for the next days were provided after blood sampling. Fasting blood samples were further collected at 48, 96, 168, 192, and 216 h, which corresponded with days 21, 23, 26, 27, and 28 of the run-in period and days 63, 65, 68, 69, and 70 of the experimental period.

Blood was sampled by venipuncture with a 0.9 x 38 mm needle (PrecisionGlide; Becton Dickinson Vacutainer System, Plymouth, UK) under minimal stasis with the subject in a recumbent position. Blood was collected in EDTA-containing tubes, which were kept on ice before and after blood sampling. Plasma was obtained within 1 h after blood sampling by centrifugation of the EDTA-coated tubes for 30 min at 4 °C and 2000 x g. Aliquots from the midportion of plasma were taken and snap frozen in liquid nitrogen. Plasma was stored at –80 °C until analyzed for fatty acid composition and the n–3 fatty acid enrichment of the plasma phospholipids.

Sample analysis
Total lipids were first extracted from plasma by means of a modified procedure of the Folch method with 2-dinonadecanoyl phosphatidylcholine (PC[19:0]2) as an internal standard (14). Subsequently, phospholipids were isolated from the total lipid extract on an Extract-Clean NH2-aminopropylsilyl column (500 mg, 4.0 mL; Alltech Associates Inc, Deerfield, IL) and were then hydrolyzed and methylated into their corresponding fatty acid methyl esters (15, 16). The obtained fatty acid methyl esters were then separated and quantified on a gas chromatograph–flame ionization detector (Perkin Elmer Autosystem, Norwalk, CT).

As described earlier in detail (7, 17), the 13C enrichments of the n–3 fatty acid methyl esters from plasma phospholipids were analyzed on a gas chromatograph–combustion-isotope ratio mass spectrometer (Finnigan MAT 252, Bremen, Germany). The 13C/12C ratio of expired CO2 was determined on a chromatograph–isotope ratio mass spectrometer (Finnigan MAT 252) using a coating PoraPLOT Q column (25 m x 0.32 mm; Chrompack International, Varian, CA). Helium was used as the carrier gas, carbon dioxide was used as the reference gas, and the injector and column temperatures were both 40 °C. All plasma and breath samples from one subject were analyzed in a single run at the end of the trial.

Quantification of n–3 fatty acid metabolism
The 13C enrichments of the breath samples as well as the plasma phospholipid–derived fatty acid methyl esters were expressed as tracer-to-tracee ratios (TTRs). The percentage of [U-13C]ALA tracer that was recovered as 13C in expired breath during the first 9 h after tracer intake was calculated from the TTR in breath and VCO2 production (7, 18). The above background concentrations of the tracer and tracee n–3 fatty acids in plasma phospholipids were calculated as described elsewhere (17).

As recently described in detail (17), a compartmental model (Figure 1Go) was derived by using the SAAM II version 1.2 software package (SAAM Institute Inc, Seattle, WA) to quantify the hepatic conversion of n–3 fatty acids. For each individual, this model was applied to the tracer and tracee data from both the run-in and the experimental periods by using POPKINETICS software (SAAM Institute Inc, Seattle, WA; 17). Briefly, the 13C-labeled n–3 fatty acid data were first incorporated into a compartmental tracer model to estimate transfer rate coefficients [k(i,j) and k(0,j)]. These kinetic parameters were then implemented into a compartmental tracee model (Figure 1Go) to estimate simultaneously with dietary intakes of ALA and unlabeled (or 12C-labeled) n–3 fatty acids the fractions of dietary ALA incorporated into plasma phospholipids and subsequently converted into EPA, DPA, and DHA. The compartmental models considered the liver as the principal site for n–3 conversion. Furthermore, it was assumed that de novo–generated n–3 long-chain fatty acids, as well as their dietary precursor ALA, were assembled in plasma phospholipids. Phospholipids were chosen as the modeling framework instead of plasma total lipids, because the latter also consist of lipid fractions that contain only negligible amounts of n–3 LC-PUFAS or do not represent hepatic conversion. The tracee n–3 fatty acid concentrations in plasma phospholipids were stable over time at the end of the run-in period and at the end of the experimental period, as determined by repeated-measures analysis of variance (data not shown). The modeling assumptions, the limitations, and the advantages of the model that was used to estimate the kinetic parameters have already been described and discussed in detail (17).


Figure 1
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  		FIGURE 1. Compartmental tracee model for the conversion of n–3 fatty acids. The circles symbolize the compartments, whereas the rectangular delay boxes represent the delayed passage of n–3 fatty acid between compartments. The first compartment (Q5) represents the stomach, whereas dietary ALA intake is given as U(5). The compartments Q1, Q2, Q3, and Q4 correspond to, respectively, the masses (µmol) of unlabeled {alpha}-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA) in plasma phospholipids. The arrows reflect the flow of fatty acids. The k(i,j) parameters are the transfer rate coefficients, which represent the fraction of fatty acids that is transferred per unit of time (h–1) from compartment j to i. Similarly, k(0,j) equals the fraction of fatty acids that is irreversibly lost per unit of time (h–1) from compartment j. The flux(i,j) denotes the flow of mass per unit of time (µmol/h) from compartment j to i. The flux(i,j) is derived by multiplying the mass of compartment j by the transfer rate coefficient k(i,j). Comparably, the flux(0,j) represents the outflow of mass from compartment j. The parameters U(2), U(3), and U(4) represent the endogenous inflow (µmol/h) of EPA, DPA and DHA, respectively, from endogenous sources other than plasma phospholipids. Reprinted with permission from reference 17.

 
Statistical analysis
Results are expressed as means ± SDs. The possible comparisons that can be made between the 3 study groups to disentangle the effects of the absolute amounts of dietary ALA, LA, and their ratio on the metabolism of ALA are depicted in Figure 2Go. Responses to the experimental diets were calculated for each subject by subtracting the value of a parameter obtained at the end of the run-in period from the value obtained at the end of the experimental period. The effects of the diets were examined with analysis of variance by using the general linear models procedure in SAS (SAS system release 8.2; SAS Institute Inc, Cary, NC). The response to the experimental diet was defined as the dependent variable, and dietary group was a fixed factor. When significant differences were found (P value < 0.05), a Tukey post hoc test was used to make pairwise comparisons of the diets. Although the statistical power may have been too low, additional analyses were performed to see whether dietary effects were modified by sex, use of oral contraceptives, or smoking. For this, the analyses were repeated by including the interaction term between diet and these factors in the model. These interactions terms, however, never reached statistical significance (results not shown).


Figure 2
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  		FIGURE 2. Interpretation of the effects of the absolute amounts of dietary {alpha}-linolenic acid (ALA), linoleic acid (LA), and their ratio in the control group, the low-LA group, and the high-ALA group. The present study was specifically designed to disentangle the effects of the ALA-to-LA ratio from those of the absolute amounts of ALA and LA in the diet on the metabolism of ALA. The following comparisons can be made: 1) Differences between the low-LA group and the control group are due to the decreased intake of LA or the increase in the ALA-to-LA ratio. 2) Differences between the ALA group and the control group are due to the increased intake of ALA or the increase in the ALA-to-LA ratio. 3) If the effects of the low-LA group and the high-ALA group are the same, and at the same time differ from those in the control group, these effects are due to the increase in the ALA-to-LA ratio.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Body weight and fatty acid composition of plasma phospholipids
Changes in body weight during the experimental period were minor: 0.0 ± 0.6 kg for the control group, –0.4 ± 1.1 kg for the low-LA group, and –0.1 ± 1.0 kg for the high-ALA group (P = 0.612 for the difference in changes between the groups). Compliance with the diets was confirmed by the changes in the fatty acid compositions of plasma phospholipids (Table 1Go). As opposed to the control and the high-ALA diets, the proportion of monounsaturated fatty acids (MUFAs) in the low-LA diet was increased at the expense of the proportion of LA (P < 0.001 versus the control group, P < 0.001 versus the high-ALA group). Consumption of the low-LA and the high-ALA diets raised the proportions of both ALA (LA group: P = 0.001, ALA group: P < 0.001) and EPA (LA group: P < 0.001, ALA group: P < 0.001) compared with the control diet. DPA was significantly higher after consumption of the high-ALA diet than after consumption of the low-LA diet (P = 0.016). Changes in DHA were not significantly different between the 3 study groups.


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  		TABLE 1 Effect of the control diet, the low–linoleic acid (LA) diet and the high–{alpha}-linolenic acid (ALA) diet on the fatty acid composition of plasma phospholipids in healthy humans1

 
Oxidation
During the run-in period, the proportion of tracer that was recovered in breath, 9 h after tracer ingestion, was 19% for the control group, 18% for the low-LA group, and 21% for the high-ALA group (Table 2Go). After the experimental period, changes in tracer recovery were not significantly different between the 3 groups.


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  		TABLE 2 Cumulative 13C recovery in breath carbon dioxide as a percentage of [U-13C] {alpha}-linolenic acid (ALA) intake, collected over a period of 9 h after tracer intake, for the control group, the low–linoleic acid (LA) group, and the high-ALA group1

 
Steady-state compartmental masses
The steady-state compartmental masses (Qi) presented in Table 3Go were obtained by multiplying the tracee n–3 fatty acid concentrations (µmol/L), which were measured in plasma phospholipids, by plasma volume. The latter was assumed to be 4.5% of body weight (19). Compared with the control diet, the low-LA diet and the high-ALA diet both raised the amount of unlabeled ALA and EPA in plasma phospholipids (all P < 0.01). The increases in ALA and EPA, however, were not significantly different between these 2 experimental diets. The changes in the masses of unlabeled DPA and DHA did not differ significantly between the 3 study diets.


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  		TABLE 3 Steady-state masses of the n–3 fatty acids in plasma phospholipids for the control group, the low–linoleic acid (LA) group, and the high–{alpha}-linolenic acid (ALA) group1

 
Rate coefficients, endogenous inputs, and fluxes
The effects of the diets on the rate coefficients, endogenous inputs, and fluxes are shown in Table 4Go.


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  		TABLE 4 Kinetic parameters for the control group, the low–linoleic acid (LA) group, and the high–{alpha}-linolenic acid (ALA) group, estimated by compartmental modeling of ALA conversion1

 
Effects of diets on rate coefficients
The transfer rate coefficients k(i,j) represent the fraction of substrate that is transferred per unit of time (h–1) from compartment j to i, whereas k(0,j) equals the fraction of substrate per unit of time (h–1) that is irreversibly lost from compartment j (see Figure 1Go). The latter represents the outflow of n–3 fatty acids that during the time frame of the tracer experiment does not return to plasma phospholipids. Examples of irreversible losses are the oxidation of a fatty acid and the loss of fatty acids into fat tissue or cell membranes.

Compared with the control diet, the fraction of dietary ALA [d(1, 6)] that was incorporated into the plasma phospholipid pool (ALA compartment = Q1) per hour increased after the low-LA diet (P = 0.012), whereas the fraction of dietary ALA that was not incorporated per hour [d(0, 6)] decreased. After consumption of the high-ALA diet, however, the fraction of dietary ALA incorporated into ALA plasma phospholipids per hour decreased significantly compared with both the control diet (P < 0.001) and the low-LA diet (P < 0.001). Once incorporated into the plasma phospholipid pool, the fraction of ALA that was converted into EPA per hour [k(2, 1)] was significantly lower after consumption of the high-ALA diet than after both the control diet (P < 0.001) and the low-LA diet (P < 0.001). Also, the fraction of EPA that entered the delay compartment per hour [k(7, 2)] was significantly lower after the low-LA diet than after the control diet (P = 0.005). In contrast, it was higher after consumption of the high-ALA diet than after both the control diet (P = 0.005) and the low-LA diet (P < 0.001). Furthermore, the fraction of EPA that disappeared from the plasma phospholipid pool per hour [k(0, 2)] declined significantly after consumption of the high-ALA diet (P < 0.001 versus the control diet, P < 0.001 versus the low-LA diet). The other rate coefficients were not significantly changed by the experimental diets.

Effects of diets on endogenous inputs
Except for U(5), which represents the intake of dietary ALA, the endogenous input U(i) corresponds with the inflow of tracee fatty acids into compartment i from endogenous sources other than plasma phospholipids. It represents the inflow of n–3 fatty acids into the plasma phospholipid pool that did not originate from dietary ALA consumed during the time frame of the tracer experiment. Examples of endogenous sources include inputs of n–3 fatty acids into plasma phospholipids that were consumed before the tracer experiment and released from adipose tissue, from lipid bilayers, or from lipid fractions other than plasma phospholipids.

Because of the dietary manipulations, the dietary intake of ALA [U(5)] was not significantly different between the control and the low-LA group, whereas it was {approx}3 times higher in the high-ALA group. The endogenous input into the EPA compartment [U(2)] rose in the high-ALA group (P = 0.006 versus the control group, P < 0.001 versus the low-LA group). The inputs into the DPA and DHA compartment did not differ significantly between the experimental groups.

Effects of diets on fluxes
The flux(i,j), which is derived by multiplying the mass of compartment j by the transfer rate coefficient k(i,j), represents the flow of unlabeled fatty acids per unit of time (µmol/h) from compartment j to i. Likewise, flux(0,j) represents the outflow of unlabeled fatty acids from compartment j.

The flow of dietary ALA that did not enter the plasma phospholipids compartment [flux(0, 6)] was significantly higher with the high-ALA diet (P < 0.001 versus the control diet, P < 0.001 versus the low-LA diet). The flow of dietary ALA into the ALA plasma phospholipid compartment [flux(1, 6)] and the flow from the ALA into the EPA [flux(2, 1)] compartment were higher after consumption of the low-LA diet. The outflow from the EPA compartment [flux(0, 2)] was significantly increased in the low-LA group (P = 0.040 versus the control group), whereas it was significantly decrease in the high-ALA group (P = 0.013 versus the control group, P < 0.001 versus the low-LA group). However, in the high-ALA group, there was a significant rise in the flow from the EPA into the DPA compartment [flux(7, 2); P < 0.001 versus the control group, P < 0.001 versus the low-LA group].

Percentage and absolute amounts of ALA incorporation and n–3 fatty acid conversion
During the run-in period, 7.1%, 6.3%, and 9.7% of the dietary ALA intake was incorporated into plasma phospholipids for the control, low-LA, and high-ALA groups, respectively (Table 5Go). After the low-LA diet, the percentage of dietary ALA incorporated into the ALA plasma phospholipid compartment was significantly increased by 4% compared with the control diet (P = 0.012). In contrast, consumption of the high-ALA diet significantly decreased the incorporation by 8% and 12% compared with the control diet (P < 0.001) and the low-LA diet (P < 0.001), respectively. Expressed in absolute amounts (mg/d), calculated by multiplying the fractional rate constant d(1, 6) by the amount of ALA consumed (mg/d), it was found that incorporation of dietary ALA into plasma phospholipids was higher with the low-LA diet than with both the control and the high-ALA diets (P = 0.020 versus the control group, P = 0.001 versus the high-ALA group). In this respect, no significant differences were found between the control and high-ALA groups.


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  		TABLE 5 Uptake of dietary {alpha}-linolenic acid (ALA) into plasma phospholipids and subsequent conversion into its n–3 fatty acid derivatives expressed as percentages and absolute amounts for the control group, the low–linoleic acid (LA) group, and the high–{alpha}-linolenic acid group1

 
During the run-in period, 99.9% of ALA from the phospholipid pool was converted into EPA in the control group, 99.8% in the low-LA group, and 99.7% in the high-ALA group (data not shown). Thus, 7.1%, 6.3%, and 9.7% of ALA intake was converted into EPA during the run-in period in the control, low-LA, and high-ALA groups, respectively. When compared with the control diet, the change in the percentage of dietary ALA that was converted from ALA into EPA increased significantly after the low-LA diet (P = 0.012), whereas it decreased after the high-ALA diet (P < 0.001). This change was also significantly different between the low-LA and high-ALA diets (P < 0.001). The change in the percentage of dietary ALA intake that was converted from EPA via DPA into DHA was not significantly different after consumption of the 3 diets. The absolute amount of EPA in plasma phospholipids that was derived from dietary ALA increased after the low-LA diet (P = 0.019), whereas it hardly changed after consumption of the high-ALA diet. The change in the absolute amount of newly formed DHA did not differ significantly between the control and the low-LA diet, whereas it increased significantly after the high-ALA diet (P < 0.001 versus the control diet, P < 0.001 versus the low-LA diet).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we showed that conversion of ALA into its longer and more unsaturated fatty acid derivatives is not determined by the ALA-to-LA ratio but by the amounts of ALA or LA in the diet. Decreasing the intake of LA increased the proportion of dietary ALA that was converted into EPA. On the other hand, increasing ALA intake increased the absolute amount of DHA synthesized.

Several human intervention studies have reported that increasing the intake of ALA or decreasing the intake of LA increases the proportions of ALA and EPA, whereas it does not, or only marginally, changes the proportion of DHA in plasma lipid fractions (8, 20-23). In agreement with these observations, we found that the low-LA and the high-ALA diets increased the proportions of ALA and EPA in plasma phospholipids to the same extent, whereas proportions of DHA tended to decrease. The latter finding was also observed by others (23). However, plasma steady-state fatty acid concentrations are actually a dynamic situation that is characterized by a continuous inflow and outflow of fatty acids, processes that can be quantified by the use of stable isotopes.

Emken et al (6) were the first to initiate a tracer study to quantify the effects of a change in dietary LA intake on the conversion of ALA. From measurements of enrichment in the total plasma lipid pool, those authors estimated that conversion of ALA into its LC-PUFAs in young men was 18.5% after a saturated fatty acid–rich diet that provided 4.7% of energy as LA and 0.6% of energy as ALA, with an ALA-to-LA ratio of 1:8. Estimates for the conversion of ALA into EPA from other stable-isotope studies have varied between 0.2% and 8% (4). We found that conversion after a control diet with a low ALA-to-LA ratio (7% of energy as LA, 0.4% of energy as ALA, ratio 1:19) was on average 7.8%. As discussed before (4, 17, 24, 25), a part of the variation in outcomes between various studies is due to differences in experimental approaches, such as the analytic method used to quantify ALA metabolism, the type of lipid fraction chosen to reflect hepatic conversion, and the mode of tracer administration. Another part of this variation, however, is due to differences in the fatty acid composition of the diets. Indeed, Emken et al (6) reported that the conversion of ALA decreased from 18.5% to 11% when LA intake increased from 4.7% of energy to 9.3% of energy and ALA intake decreased from 0.6% of energy to 0.3% of energy and the ALA-to-LA ratio changed from 1:8 to 1:31. This 40% decrease in total n–3 conversion was attributed to the increased intake of LA. However, the simultaneous change in both ALA intake and the ALA-to-LA ratio could have affected n–3 fatty acid biosynthesis as well. In other stable-isotope studies, it was also not possible to disentangle the effects of ALA or LA from those of the ALA-to-LA ratio (5-9). We found that a decrease in the LA content of the diet increased the incorporation of dietary ALA in plasma phospholipids from 6.3% to 11.5% of ALA intake or from 72 mg to 120 mg. Because almost all ALA from the plasma phospholipid pool was converted into EPA, synthesis of EPA increased to a comparable extent. Thus, our results confirm the general view that a high LA intake inhibits conversion of ALA (2, 3). In contrast, when the amount of ALA in the diet increased from 0.4% to 1.1%, ALA incorporation into plasma phospholipids and the successive EPA synthesis decreased from 9% to 3% of ALA intake, whereas no changes were observed when expressed in absolute amounts. Furthermore, the present study confirms that synthesis of DHA in humans is extremely limited. Moreover, our tracer results indicate that conversion from EPA into DPA—a step that appears to be an additional constraint in the n–3 pathway (17)—is affected differently after a low-LA diet than after a high-ALA diet. The change in the percentage of dietary ALA that was converted from EPA into DPA and further into DHA was comparable with the control diet, regardless of whether LA intake decreased or ALA intake increased. In contrast, expressed in absolute amounts of ALA intake, the synthesis of DPA and DHA hardly changed in the low-LA group, whereas it increased significantly in the high-ALA group. Hence, even though EPA synthesis increased after a low-LA diet, most of this marine fatty acid was not available for conversion into longer and more unsaturated fatty acids. An increase in ALA intake had just the opposite effect, because more EPA was converted into DPA and DHA.

It has been postulated that a high LA-to-ALA ratio inhibits conversion of n–3 fatty acids independent of the absolute amounts of these fatty acids in the diet (1, 8). If this were true, then the metabolic parameters of the low-LA and the high-ALA diet should have been the same in the present study, because both diets had the same ALA-to-LA ratio. Clearly, this was not the case. Hence, our findings show that dietary recommendations should not focus on the ALA-to-LA ratio in the diet, but should consider the individual amounts of dietary ALA and LA. Furthermore, our results support the concept presented by Sinclair et al (2) that a reduction in dietary LA together with an increase in ALA intake would be the most appropriate way to enhance EPA and DHA synthesis from their parent fatty acid ALA. However, it is also clear that this approach will not lead to substantial increases in plasma phospholipid DHA contents as can be obtained through a moderate consumption of fish or marine oils (1). This agrees with findings in vegetarians and vegans who have lower proportions of plasma EPA and DHA (26).

In line with other studies (1, 2, 5, 6, 9, 27-31), the present study shows that a major part of the ingested ALA is oxidized within 9 h after consumption. ß-Oxidation in humans ranges between 16% and 33% of the ingested tracer dose, depending on the duration of sampling (31). Our results fall within these ranges. Our data indicate that the percentage of ALA oxidation over a period of 9 h after tracer intake is not affected by the absolute amounts of ALA or LA or by the ALA-to-LA ratio of the diet.

In conclusion, the present study shows that the dietary ALA-to-LA ratio is not a determinant of n–3 fatty acid conversion. An increase in EPA synthesis can be obtained by lowering the amount of LA in the diet, whereas an increase in DHA synthesis is achieved by increasing the amount of dietary ALA. The optimal dietary approach to increase n–3 LC-PUFAs is to consume them preformed. However, in individuals unwilling or unable to eat seafood, exchanging LA for ALA may be the next best approach.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the expert contribution and assistance of David M Foster (SAAM Institute and University of Washington, Seattle), as well as the assistance of Gianna Toffolo and Claudio Cobelli (University of Padua, Italy) during the initial phase of model development. We kindly thank V.T.I.V.T. Ter Hercke (Herk-de-Stad, Belgium), in particular Tony Corthouts and Johny Vanden Dijck, for the production of the experimental pastries. We appreciated the support of the members of our dietary and technical staff and thank all participants for their cooperation and interest.

This study was designed by PLLG, PLZ, MBK and RPM. PLLG and RPM were responsible for the collection, statistical analysis, and interpretation of the data and the writing of the manuscript. PLLG and MES performed the kinetic modeling of the data. The manuscript was reviewed and approved by all authors. None of the authors had a personal or financial conflict of interest.


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 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Received for publication November 15, 2005. Accepted for publication March 17, 2006.




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