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American Journal of Clinical Nutrition, Vol. 80, No. 1, 45-50, July 2004
© 2004 American Society for Clinical Nutrition

ORIGINAL RESEARCH COMMUNICATION

Triacylglycerol-rich lipoprotein margination: a potential surrogate for whole-body lipoprotein lipase activity and effects of eicosapentaenoic and docosahexaenoic acids1,2,3

Yongsoon Park, Philip G Jones and William S Harris

1 From the Lipid and Diabetes Research Center, Saint Luke’s Hospital, and the University of Missouri–Kansas City School of Medicine (YP and WSH), and the Mid America Heart Institute, Saint Luke’s Hospital, Kansas City (PGJ and WSH)

2 Supported by grants from the National Heart, Lung, and Blood Institute (HL-47468), the Saint Luke’s Hospital Foundation, and the Heartland Affiliate of the American Heart Association (postdoctoral fellowship to YP).

3 Address reprint requests to WS Harris, Lipid and Diabetes Research Center, 4320 Wornall Road MP1, Suite 128, Kansas City, MO 64111. E-mail: wharris{at}saint-lukes.org.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Margination occurs when blood borne particles attach to the vessel wall. Triacylglycerol-rich lipoprotein (TRL) particles marginate when they bind to endothelial lipoprotein lipase (LpL).

Objective: This study was undertaken to determine whether TRL margination reflects in vivo LpL activity and whether n–3 fatty acids affect fasting and fed TRL margination.

Design: Healthy subjects (n = 33) began with a 4-wk, placebo (olive oil; 4 g/d) run-in period and were then randomly assigned to 4 wk of treatment with 4 g/d of ethyl esters of either safflower oil (SAF; control), eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA). Margination volume (MV) was calculated by subtracting true from apparent plasma volume.

Results: MVs were 3 times as great during the fasting state as during the fed state (P < 0.0001). In both the fasting and the fed states, MV was significantly correlated with plasma triacylglycerol and TRL half-lives. In the fed state, MV was also correlated with preheparin LpL, whereas in the fasting state it was not. There was no significant correlation between preheparin LpL and postheparin LpL in the fasting state. Relative to SAF, EPA and DHA supplementation resulted in higher MVs by 64% and 53% (both P < 0.001), respectively, in the fasting state, without significantly reducing fasting triacylglycerol concentrations. In the fed state, DHA doubled the MV (P < 0.05), whereas EPA had no significant effect.

Conclusions: The correlations between MV and TRL half-lives and preheparin LpL suggest that MV could be a reflection of whole-body LpL binding capacity. The increases in MVs with EPA and DHA supplementation suggest that these fatty acids may increase the amount of endothelial-bound LpL or its affinity for TRL.

Key Words: Eicosapentaenoic acid • docosahexaenoic acid • margination • chylomicrons • lipoprotein lipase • n–3 fatty acids


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Delayed postprandial clearance of triacylglycerol-rich lipoproteins (TRLs) appears to play a pathogenic role in atherosclerosis (1-4). This may be due in part to a low activity of plasma lipoprotein lipase (LpL; 5), because LpL is the principal enzyme responsible for intraluminal hydrolysis of TRL (6, 7). However, the extent to which LpL activity measured ex vivo reflects activity in vivo is not known. Most investigators have examined activity ex vivo by using plasma drawn without (8-12) or after (12-16) heparin injection or in muscle (17, 18) or adipose tissue (18, 19) biopsy specimens. Unfortunately, correlations among these measures are weak, and no gold standard method for determining whole-body, in vivo enzyme activity exists (8, 10, 11).

Apparent distribution volumes of TRL (in particular, chylomicrons) have been reported to be greater than plasma volumes (20-23). This has been attributed to margination, or sequestration of the TRL during binding to endothelial LpL (24, 25). It is possible that the extent of margination could be a useful marker of LpL activity in vivo: the greater the margination, the greater the LpL binding capacity. In the present study, we measured margination volume, defined as the difference between true plasma volume (calculated from validated equations) and the apparent distribution volume of labeled lipid emulsion particles (a surrogate for chylomicrons calculated from clearance curves). In the absence of margination, these 2 volumes would be the same. If margination is present, the apparent distribution volume would be higher than the plasma volume, and the degree of the discrepancy would reflect LpL binding capacity (Figure 1Go).



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  		FIGURE 1.. Margination volume. If a known number of particles (here, 12) is dissolved in an unknown volume, measurement of the concentration will allow for calculation of the volume. In the case of no binding to the wall (left), the true volume (100 mL) and the apparent distribution volume are the same. However, if some of the particles bind to the wall (right), the particle concentration is reduced, and the apparent distribution volume will be artificially inflated. The margination volume is thus defined as the difference between true and apparent volumes.

 
This report includes new data on margination volumes derived from 33 individuals who were the subject of a previous publication that included other measures of LpL activity (12). Here, we sought to compare the margination volume with these other estimates of LpL activity and to determine whether supplementation with purified eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) would increase margination volumes. These 2 n–3 fatty acids have been shown to reduce postprandial lipemia (26-30) and, in some studies, to enhance LpL activity or LpL-mediated TRL clearance (9, 12, 31). Finding an effect on margination volumes would support the hypothesis that the mechanism by which n–3 fatty acids reduce postprandial lipemia involves the modulation of LpL function.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Volunteers aged 21–70 y with body mass indexes (in kg/m2) of 22–30, fasting serum LDL-cholesterol concentrations <160 mg/dL, HDL-cholesterol concentrations >35 mg/dL, and triacylglycerol concentrations <200 mg/dL were recruited. Individuals with known hepatic, renal, or gastrointestinal disease; those with lactose intolerance; or those taking medications known to affect lipid metabolism or fat absorption were excluded. The subjects’ characteristics are shown in Table 1Go. The Saint Luke’s Hospital Institutional Review Board approved this study, and written informed consent was obtained from all participants.


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  		TABLE 1. Subject characteristics1

 
Protocol
This was a randomized, double-blind, parallel group study that began with a 4-wk placebo run-in period during which 4 g/d of olive oil ethyl esters was administered. This was followed by a 4-wk washout period during which no capsules were taken. (The primary purpose of the washout period was to allow blood volumes to recover.) The subjects then entered the treatment period, during which they were randomly assigned to 4 wk of either safflower oil ethyl esters (SAF; control), EPA ethyl esters, or DHA ethyl esters (4 g/d each; 95% pure). The oils were provided by the Fish Oil Test Material Program of the Department of Commerce and the National Institutes of Health. Four weeks has been shown to be sufficient time for the hypotriacylglycerolemic effects of fish oils to appear (32). Margination volumes were measured during the last week of both the placebo run-in and the treatment periods.

Diets
The subjects were asked to avoid eating fish (or taking fish oil) throughout the study and were counseled to maintain a stable dietary and exercise pattern. They were given low-fat isocaloric meals (<30% of energy from fat) the night before each study visit and were asked to refrain from consuming alcohol and performing strenuous exercise for 48 h before each visit. The supplements were not taken on the days of the margination experiments.

Procedures
This study was designed to measure margination volume in both the fasting and the fed states because the presence of chylomicrons was expected to affect binding capacities. To produce steady-state chylomicronemia, the subjects consumed a priming dose (350 mg fat/kg) of a chocolate-flavored, cream-based drink, and then 2 h later, began sipping the drink every 15 min for the next 5.5 h. We previously found that this technique produces stable chylomicronemia, and that by individually adjusting the fat ingestion rate between 175 and 225 mg · kg–1 · h–1, we could produce steady-state, postprandial serum triacylglycerol concentrations that were similar in the placebo run-in and treatment periods within groups (12, 33).

To determine margination volume, a known amount of a radiolabeled lipid emulsion was injected intravenously, followed immediately by multiple blood samplings over the next 30 min (12). The apparent (or instantaneous) volume of distribution was then determined by back extrapolation of the first exponential disappearance curve to time zero (see the section titled "Calculations" below). The subjects reported to the Metabolic Research Unit in the morning after fasting overnight (12 h). An intravenous sampling cannula was placed in a forearm vein and an injection cannula in a contralateral vein; both were kept patent with infusions of 0.9% NaCl. A commercial lipid emulsion containing 4 µCi [3H]triolein (140 mg triacylglycerol in 2 mL prepared as described below) was injected 4 different times on the study day: once before (fasting state) and 3 times (5, 6, and 7 h) after the ingestion of the first dose of the high-fat drink (12, 33). Blood samples were drawn 0, 1, 3, 5, 7, 9, 13, 17, 20, and 30 min after each of the 4 bolus injections, and plasma was analyzed for tracer concentration by extracting the plasma lipids with chloroform:methanol (2:1, by vol) into a scintillation vial, evaporating the solvents, adding scintillation cocktail (Optifluor; Packard, Meriden, CT), and counting in a Wallac liquid scintillation counter (Pharmacia; Gaithersburg, MD).

We sought to evaluate the utility of the margination volume approach by comparing it with 3 other surrogates of LpL activity: preheparin LpL activity, postheparin LpL activity, and chylomicron triacylglycerol half-life (clearance measured in the chylomicron fraction, not in whole plasma as described for margination volume). The procedures for these 3 tests were recently published (12). Because the method for determining LpL activity (pre- or postheparin) is relatively new, however, it will be described in detail here.

Preheparin lipoprotein lipase assay
LpL activity was measured by incubating plasma with emulsified triolein and then determining the amount of oleic acid liberated. Blood was collected into heparin-coated tubes, and the plasma was separated and stored at –80 °C. Substrate was prepared fresh daily with 200 mg triolein and 5.68 mL of 90 g gum arabic/L in 50 mmol NH4OH-NH4Cl/L (buffer; pH 8.5) by sonication (series 4710; Cole-Parmer Instrument Co, Chicago). Then, 1.375 mL of 200 g bovine serum albumin/L in buffer and 1.375 mL of the internal standard solution were added to the mixture. The internal standard solution was made in advance as follows. Heptadecanoic acid (13.525 mg) was dissolved in 10 mL methanol and 1 mL 10 mol ammonium hydroxide/L and then dried under nitrogen. Bovine serum albumin (20 mL of 200 g/L in buffer) was added and the mixture was sonicated at amplitude 40 for 2 h in an ice bath.

Plasma (100 µL), 880-mmol/L sodium dodecyl sulfate (SDS) solution (20 µL), buffer (180 µL), and substrate (0.5 mL) were added. The blank contained 50 mg NaCl and no SDS to inhibit lipase activity (34, 35). The mixtures were agitated in a vortex mixer and incubated for 2 h at 28 °C. The triacylglycerol hydrolysis reaction was terminated by adding 5.33 mL of methanol:chloroform:heptane solution (38.4%:34.2%:27.4%) and 1.5 mL of 0.1 mol carbonate-bicarbonate buffer/L in 1 mol NaCl/L (pH 10.5). After shaking and centrifugation (1500 x g, 15 min, room temperature), the supernatant fluid (containing nonesterified fatty acids) was transferred, and 0.5 mL of 0.5 mol HCl/L and 3 mL hexane were added. The mixture was shaken vigorously and centrifuged at 1500 x g for 45 min at room temperature. The supernatant fluid was transferred and dried under nitrogen. Samples were methylated by adding 1 mL BF3 and heating at 100 °C for 3 min. The methylated fatty acids were extracted by adding 2 mL distilled water and 2 mL hexane. The supernatant fluid was dried under nitrogen and analyzed by gas chromatography (injection temperature, 200 °C; oven temperature, 210 °C) with a 30-m SP2330 capillary column (Supelco, Bellefonte, PA). The amount of liberated oleic acid was determined (after subtracting appropriate blanks), and activity was expressed as µmol oleic acid released · h–1 · mL plasma–1. This assay was found to be linear with time over 4 h, with substrate and with plasma (enzyme) concentration. Activity was inhibited by known LpL inhibitors such as NaCl (0.5 mol/L), guanidine HCl (0.5 mol/L), paraoxon (12 µg/mL), and tetrahydrolipstatin (3 µg/mL; unpublished data), and unaffected by the freezing and thawing of plasma.

Postheparin plasma lipoprotein lipase assay
Postheparin LpL activity was measured in plasma drawn 15 min after the injection of heparin (100 IU/kg body wt). This injection took place in the morning after the subjects had fasted overnight and 3 d after the tests to determine chylomicron triacylglycerol clearance rates. Postheparin LpL activity was not measured in the fed state because the injection of heparin would have markedly disrupted steady-state chylomicronemia.

The substrate described above was added to 20 µL postheparin plasma mixed with buffer (1:1), 80 µL human serum (as a source of apolipoprotein C-II), and 200 µL buffer. For the blank, 50 mg NaCl, 20 µL 880-mmol/L SDS solution, and 180 µL buffer were used instead of 200 µL buffer. For the hepatic lipase assay, a 3.6-mol/L NaCl solution was used instead of buffer. The rest of the procedure was the same as described above for the preheparin LpL assay.

Calculations
The plasma 3H concentration at the moment of injection (from which apparent distribution volume was calculated) was estimated by back extrapolation of the monoexponential curve to time zero for each of the 4 injections. The following formulas were used (36).

(1)

(2)

(3)

Statistical analysis
The changes from baseline to the end of treatment for the EPA and DHA groups were compared with the change observed in the SAF group by using analysis of covariance with baseline values as covariates (SAS version 8.2; SAS Institute Inc, Cary, NC). Preplanned comparisons with the SAF group were conducted by using Dunnett’s test. Pearson correlations (full and partial) were calculated between margination volume and plasma triacylglycerol concentration, half-life, or LpL (preheparin or heparin-stimulated) activities. A two-tailed P value of <0.05 was required for statistical significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
After all values from both the placebo run-in and the treatment periods were combined, margination volumes were smaller during the fed state than during the fasting state (586 ± 106 compared with 1741 ± 181 mL; P < 0.0001).

With the use of only the data from the placebo run-in period (n = 33), the relation between margination volume and other surrogates of LpL activity was assessed. In the fasting state, margination volume was highly correlated with chylomicron triacylglycerol half-lives and with triacylglycerol concentrations. The latter remained significant even after controlling for fasting triacylglycerol (Table 2Go). No relation of margination volume to pre- or postheparin LpL was found in the fasting state, nor were pre- and postheparin LpL significantly related to each other (data not shown). In the fed state, margination volume remained highly correlated with half-lives and triacylglycerol (although after control for triacylglycerol, the relation between margination volume and half-life was somewhat attenuated). Unlike in the fasting state, in the fed state, margination volume was positively and significantly correlated with preheparin LpL activity (Table 2Go). There was no correlation between fasting and fed preheparin LpL activities (data not shown).


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  		TABLE 2. Correlations between margination volumes (MVs) and chylomicron triacylglycerol (TG) half-lives, TG concentrations, and pre- and postheparin lipoprotein lipase (LpL) activities in the fasting and fed states1

 
The effects of treatment with n–3 fatty acids on plasma triacylglycerol concentrations, on chylomicron triacylglycerol half-lives and preheparin LpL activities during both the fasting and the fed states, and on postheparin LpL activity have been previously reported (12) but are summarized in Table 3Go. There were no significant effects of either on fasting serum triacylglycerol or postheparin LpL activity. Chylomicron triacylglycerol half-lives were shortened with both n–3 fatty acids in the fed state, but neither had any effect on this parameter in the fasting state. Preheparin LpL similarly increased in the fed state with both fatty acids but not in the fasting state.


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  		TABLE 3. Effects of 4 wk of supplementation with 4 g/d of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or safflower oil (SAF) control on serum triacylglycerol (TG), chylomicron TG half-life, and pre- and postheparin lipoprotein lipase (LpL) activity in the fasting and fed states1

 
The effects of EPA and DHA on margination volume are presented in Figure 2Go. Compared with the control group (SAF), supplementation with both EPA and DHA increased fasting margination volumes (by 64% and 53%, respectively; both P < 0.001). As noted above, this occurred without a significant lowering of serum triacylglycerol concentrations. In the fed state, mean margination volumes doubled with DHA supplementation (P < 0.05 versus the change in the control group), but the increase with EPA was not significantly different from that in the control group.



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  		FIGURE 2.. Mean (±SEM) margination volume (MV) during the fasting and fed states after the placebo run-in period ({square}) and the active treatment periods ({blacksquare}). MV was measured once in the fasting state, whereas it was measured 3 times (and averaged) during the fed state. All subjects were tested once at the end of the placebo run-in period and again at the end of the treatment period. Note that the y axes of the 2 graphs are different. n = 11 subjects per treatment group; safflower oil (SAF), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Analysis of covariance (controlled for baseline values) was used to test for differences, followed by Dunnett’s test. *,**The response to EPA or DHA (ie, the changes from the placebo run-in period to the end of the treatment periods) was significantly greater than the corresponding response to SAF: *P < 0.001, **P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Consumption of n–3 fatty acids usually reduces both fasting and postprandial triacylglycerol concentrations (14, 26-28, 30, 37). It has been suggested that the hypotriacylglycerolemic action of n–3 fatty acids may be due, at least in part, to enhanced LpL-mediated lipolysis (9, 12, 30). In the present study, we sought to test this hypothesis by using a novel approach to estimate whole-body LpL binding capacity in vivo: TRL margination volume. Because it is possible that LpL action (the mass present on the endothelium, its affinity for substrate, or the presence of competing TRL) is different in the fasting state than in the fed state, we examined margination volumes under both conditions. As predicted, we found that margination volume was 1) significantly reduced during chylomicronemia and 2) inversely correlated with previously published fasting serum triacylglycerol concentrations and chylomicron triacylglycerol half-lives (12). These findings support the hypothesis that margination volume may be a physiologically meaningful reflection of LpL activity.

The distribution volume of labeled TRL has been reported to be larger than plasma volume in both rats (22, 23) and healthy volunteers (20, 21), which suggests vascular binding of lipoproteins to LpL, ie, TRL margination. The fact that native chylomicrons reduced margination volumes in the present study is further evidence that the particular emulsion used here to assess chylomicron triacylglycerol metabolism is a physiologically meaningful tracer (33, 38).

In our study, margination volume correlated well in the fed state with 3 independent surrogates of LpL activity: preheparin LpL activity, chylomicron triacylglycerol half-lives, and serum triacylglycerol concentrations, and it correlated well in the fasting state with the last 2. It did not, however, correlate with either assessment of LpL enzymatic activity (pre- or postheparin) in the fasting state. Why margination volume would correlate with preheparin activity in the fed state but not in the fasting state is not readily apparent and suggests that further study is needed.

The lack of correlation between preheparin and postheparin LpL was not surprising because this was reported previously (8, 11, 39, 40). Although some have found a positive relation between the two (41, 42), it remains unclear whether preheparin LpL activity actually derives from endothelial turnover or from some other source. Vilella et al (10) reported that plasma contains substantial amounts of LpL mass with low activity, suggesting that the enzyme may circulate catalytically inactive and that heparin not only releases bound LpL but also somehow activates LpL already in the circulation. Even though most preheparin LpL may be inactive, it may nevertheless be physiologically significant; recent studies showed that preheparin LPL is negatively correlated with both coronary atherosclerosis (43) and acute myocardial infarction (44). However, others reported the opposite relation (40) between pre- and postheparin LpL activity. Postheparin LpL activity was associated with coronary artery disease prevalence (45), whereas in another study there was no association with disease (46). The inconsistency in these findings may stem in part from differences in the techniques used to assess LpL activity. Clearly, the precise roles and significance of pre- and postheparin LpL activity (and mass) remain unclear.

The preheparin lipase assay used here showed a marked dependence on the presence of SDS in the incubation cocktail. Without SDS, lipase activity was in the range observed by others (8, 39, 47), but with SDS (final concentration of 22 mmol/L), lipase activity was 8–10 times higher. Baginsky and Brown (35) showed in 1977 that SDS not only inhibits hepatic lipase activity but stimulates LpL activity (at least up to 1 mmol/L). The reason for this enhancement in activity is not clear, but it may relate to the detergent’s ability to remove released fatty acid from the reactive site. A similar stimulatory effect of albumin on LpL activity has been reported (48).

To our knowledge, this was the first attempt to quantify margination volume and to propose that it might serve as a measure of LpL binding capacity. It is also the first study to examine the effects of n–3 fatty acid supplementation (or any other intervention) on TRL margination. We found that both EPA and DHA supplementation significantly increased margination volumes, the former in the fasting state and the latter in both the fasting and the fed states. Previous studies suggested that n–3 fatty acids may accelerate LpL-mediated lipolysis (12, 49), perhaps via increased preheparin LpL activities (9, 12). Postheparin LpL activity has been found by some investigators to be unaffected by n–3 fatty acid supplementation (12, 13, 16), whereas others have reported an increase (31, 50, 51). In 2 studies (31, 52), n–3 fatty acid supplementation increased LpL messenger RNA mass in adipose tissue. The peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) has been shown to modulate LpL activity via a direct transcriptional effect on the LpL promoter (53). Because n–3 fatty acids are known PPAR{gamma} agonists (54), they may increase LpL transcription in vivo. In that light, the recent report by Chambrier et al (55) that PPAR{gamma} messenger RNA concentrations are positively correlated with plasma EPA concentrations is particularly relevant. These studies, combined with our present results, support the hypothesis that n–3 fatty acids increase the endothelial expression or binding capacity of LpL.

In summary, our data support the 2 hypotheses proposed but questions remain. We found that margination volume, especially in the fed state, can potentially be used as a surrogate of whole-body LpL action and that supplementation with long-chain, n–3 fatty acids appears to stimulate LpL action. The lack of correlation between postheparin LpL activity and virtually any other proposed surrogate of LpL action (chylomicron triacylglycerol half-lives, fasting triacylglycerol concentrations, preheparin LpL activities, or margination volumes) does not support the physiologic importance of the postheparin test for assessment of chylomicron clearance capacity. The significant correlations between several of these measures and preheparin LpL activity suggests that this test may be more physiologically meaningful. We have also documented differences in LpL-mediated functions in the fed versus the fasting state that suggest that assessments of LpL activity in one setting may not translate to the other. Finally, we found that both n–3 fatty acids increased margination volumes in the fed state, and that DHA did so in the fasting state. Thus, these fatty acids may increase the expression, intracellular transport, endothelial positioning, or binding affinity of TRL to LpL. Clearly, the mechanisms by which n–3 fatty acids affect LpL metabolism are yet to be fully delineated.


   ACKNOWLEDGMENTS
 
We thank John M Miles for his critical comments on the manuscript and Sheryl Windsor, Bart Damron, and Lan Zhang for their assistance in data collection.

YP was responsible for the day-to-day management of the study, some of the laboratory testing, the data analysis, and the preparation of the first draft of the manuscript. PGJ carried out the statistical analysis of the data. WSH designed the study, obtained funding for it, and wrote the final draft of the manuscript. None of the authors had any conflicts of interest relative to this project.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Received for publication January 23, 2003. Accepted for publication December 29, 2003.




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