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(Circulation. 1996;93:365-371.)
© 1996 American Heart Association, Inc.

Articles

-3 Lipid Infusion in a Heart Allotransplant Model

Shift in Fatty Acid and Lipid Mediator Profiles and Prolongation ofTransplant Survival

F. Grimminger; H. Grimm; D. Führer; C. Papavassilis; G. Lindemann; C. Blecher; K. Mayer; F. Tabesch; H.-J. Krämer; J. Stevens; W. Seeger

From the Departments of Internal Medicine and Surgery, Justus-Liebig University, Giessen, Germany.

Correspondence to F. Grimminger, MD, PhD, Department of Internal Medicine, Justus-Liebig-University Giessen, Klinikstrasse 36, D-35392 Giessen, FRG.

	Abstract

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Background -3 Fatty acids may have a major impact on immune responses involved in heart transplant rejection. We compared the effects of posttransplant intravenous supplementation with -3–rich versus -6–rich lipid emulsions on graft survival, plasma fatty acid profiles, and levels of arachidonic acid versus eicosapentaenoic acid–derived lipid mediators.

Methods and Results Inbred PVG and Wistar-Kyoto rats were used as donors and recipients, respectively, in a model of heterotopic heart transplantation. Animals received 9 g/kg body wt per day of either fish oil–derived (n=8) or soybean oil–derived fat (n=7) in the form of a continuously infused lipid emulsion; controls were sham-infused with saline (n=8). Graft rejection was assessed by loss of activity of the transplant. The fish oil–derived preparation but not that originating from soybean oil caused an increase in total and free plasma fatty acids. Substantial quantities of eicosapentaenoic acid and docosahexaenoic acid appeared in the free fatty acid fraction, surpassing those of arachidonic acid. Ex vivo stimulation of neutrophils with the Ca²⁺ ionophore A23187 demonstrated an increase in 5-series leukotriene (LT) generation in animals undergoing -3 lipid infusion (LTB₅, -oxidation products of LTB₅, LTA₅ secretion), with 5-series/4-series LT ratios ranging between 0.08 and 0.36. Ratios of TX B₃/B₂ liberated from ex vivo stimulated platelets even approached 1:1 in -3 supplemented rats. Graft survival was 7.6±0.3 (mean±SEM) days in saline-infused, 10.4±0.7 in -6 lipid–infused, and 12.9±0.4 in -3 lipid–infused animals.

Conclusions Posttransplant intravenous alimentation with fish oil–derived lipid emulsions prolongs heart transplant survival in excess to -6 lipids. Profound changes in fatty acid profiles and lipid mediator generation may underlie this finding.

Key Words: transplantation • leukotrienes • lipids • fatty acids • platelets

	Introduction

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Acute rejection of the transplanted heart remains one of the major problems in cardiac transplantation. The use of broad-spectrum immunosuppressive agents has dramatically prolonged allograft survival, but at the cost of major deleterious side effects. A variety of alternative approaches has been attempted to interfere with the complex immunological and inflammatory sequelae of events in acute graft rejection.¹ These include manipulations of dietary fat, with focus on polyunsaturated fatty acids^{2 3} Among these, -3 fatty acids, in which the last double bond is located between the third and fourth carbon atoms from the methyl end of the fatty acid chain, have attracted particular attention. EPA and DHA are scarce in normal Western diets but make up an appreciable part of the fat in cold-water fish and seal meat. They serve as alternative lipid precursors for both cyclooxygenase and lipoxygenase pathways, with the formation of trienoic prostanoids (instead of the dienoic ones originating from AA) and 5-series LTs (instead of the 4-series LTs derived from AA).³ Many of the -3 fatty acid–derived metabolites, including 5-series cysteinyl LTs, LTB₅, and TXA₃, possess markedly reduced inflammatory and vasomotor potencies as compared with the AA-derived lipid mediators, and they may even exert antagonistic effects.⁴ Additional immunomodulating efficacy may result from their capability to dampen PMN-related and thrombocyte-related inflammatory events.⁴ Moreover, supplementation with a -3 fatty acid–rich diet has been implicated in a reduction of proinflammatory cytokine synthesis in mononuclear cells.⁵

In a heterotopic rat cardiac transplant model, an increase in allograft survival by 45% was achieved when recipient rats were fed purified diets rich in -3 fatty acids 2 to 4 weeks before and after transplantation.⁶ In both experimental and clinical studies, coapplication of cyclosporine A and a fish oil–enriched diet was recently noted to reduce nephrotoxicity and graft rejection after renal transplantation.^{7 8} However, it might be advantageous to use an intravenous route for -3 lipid administration in order to combine parenteral nutrition and anti-inflammatory intervention: slow kinetics and limited extent of EPA availability for inflammatory processes on enteral supplementation of -3 lipids thus may be overcome. Infusion of fish oil–derived emulsions in rats, rabbits, and humans has been reported to result in a severalfold increase in the membrane contents of EPA and DHA within a few days.⁹ Moreover, plasma content of nonesterified EPA, which in low micromolar concentrations is known to profoundly affect neutrophil leukotriene generation and inflammatory PMN functions,^{10 11} may rise during intravenous application of -3 lipid emulsions via activation of the lipoprotein lipase.^{12 13}

In the present study, we used the heterotopic rat heart allotransplant model to elaborate the efficacy of a fish oil–based lipid emulsion, continuously infused in the posttransplantation period, as an immunosuppressive agent. We focused specifically on the alterations in the plasma fatty acid profiles and related changes in neutrophil and platelet lipid mediator generation, and data were compared with an isocaloric soybean oil–based lipid preparation. Parenteral nutrition with the -3–rich lipids provoked a marked shift toward EPA and EPA-derived products, concomitant with a significant prolongation of graft survival.

	Methods

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Materials
The leukotrienes LTB₄ and 20-OH- and 20-COOH-LTB₄, used as reference substances, and the synthetic LTA₄ methyl ester were generous gifts from Dr J. Rokach (Merck Frosst, Canada). Additional LTs were graciously supplied by Dr Bartmann (Hoechst AG; Frankfurt, Germany). TXB₃ was a gift from C.O. Meese (Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany), and monoclonal antibody against TXB₂ was generously provided by Dr K. Brune (Institute of Pharmacology, Erlangen, Germany). LTA₅ methyl ester, LTB₅, TXB₂, BSA, and all reference fatty acids were obtained from Paesel AG and Sigma Chemie. The nonenzymatic hydrolysis products of LTA as well as omega oxidation products of LTB₅ were prepared for use as reference standards as previously detailed.¹¹ A23187 was from Calbiochem, and the ionic pair reagent tetrabutyl-ammonium-dihydrogenphosphate (TBA, 1 mol/L solution) from Aldrich Chemie. Biotinylated sheep anti-mouse antibody was purchased from Amersham Buchler GmbH. Avidin-biotin-horseradish-peroxidase (AB-complex) was from Dakopatts GmbH. 2,2'-Azinobis (3-ethyl-benzthiazolinesulfonic acid) (ABTS)³ was obtained from Boehringer GmbH. -Tocopherol and Tween 20 were purchased from Sigma Chemie. Microtiter plates (immunoplate Maxisorp F96 with certificate) were supplied by Nunc. Chromatographic supplies included HPLC-grade solvents distilled in glass (Fluka KG) and octadecylsilyl 5 µm (Hypersil) as well as silica gel 5-µm column packing materials (Machery-Nagel), C-18 Sep-pack cartridges (Waters Association), and silica gel 60 F₂₅₄ plates (Merck). RPMI 1640 medium was from Gibco, FCS from Boehringer Mannheim GmbH, and Percoll from Pharmacia Fine Chemicals. All other biochemicals were obtained from Merck.

Animals
The experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals after approval of the local committee of ethics. Inbred PVG (male; weight, 100 to 150 g) and Wistar/Kyoto rats (male; weight, 200 to 250 g, Mollegaard Breeding Center, Skensved, Denmark) served as donors and recipients, respectively. The rats were housed in plastic cages with stainless steel wire bottoms in a laboratory with controlled temperature (20°C), humidity (50%), and a 12-hour light/dark cycle. The animals were allowed to adapt to the environment for at least 1 week before transplantation. They were fed R3-EWOS-ALAB brood stock feed.

Surgical Technique
A spiral-shaped polyethylene catheter (PE 10, 5 cm; Clay Adams), attached to a silicon tube (Silastic 0.012x0.025 inch, No. 602-105 HH 061999, Dow Corning Corp), was heat-united to a 30-cm PE-20 catheter. The silicon part of the catheter was placed in the animal's left jugular vein as described elsewhere¹⁴ and the PE 20 end diverted to the exterior immediately before transplantation. This catheter was connected to a SAGE pump enabling 24-hour continuous infusion. The recipients were anesthetized with phentanylcitrate (0.315 mg/kg body wt IM). The abdomen was opened by a midline incision. The left kidney was removed, and the kidney vessels were cuffed. The donors were anesthetized with pentobarbital (60 mg/kg body wt IP). Heparin 300 IU was injected intravenously before harvesting the heart. The grafts were flushed with cold Ringer's lactate solution containing 50 IU of heparin/mL and anastomized immediately with the cuffed vessels; the cold ischemia time was <5 minutes. Immediately after transplantation of the heart, continuous 24-hour infusion of soybean oil–based (n=7) or fish oil–based (n=8) lipid emulsions (9 g fat/kg body wt per day each) or sham infusion with saline (n=8) was started and continued until rejection was complete. Ad libitum feeding with R3-EWOS-ALAB brood stock feed was continued after surgery.

Fat Emulsions
Emulsions containing 20% oil were prepared using soybean oil or fish oil. The fatty acid composition is given in Table 1. The isotone water phase contained distilled water, purified egg phospholipids, and glycerol. It was heated to 60 to 70°C, and the lipid phase was added in a mixer at high speed. The emulsions contained the antioxidant -tocopherol at a concentration of 1 mg/mL. A fine emulsion was created in a valve homogenizer at high pressure. The emulsion was dispensed in glass vials and heat sterilized.

View this table: [in this window] [in a new window]   Table 1. Fatty Acid Composition of Soybean Oil and Fish Oil (% of Total Fatty Acids)

Assessment of Graft Rejection and Blood Sampling
The transplanted hearts were palpated twice daily. When no pulsation was palpable and the ECG showed no activity of the transplant, rejection was considered to be complete and the grafts were removed. Transverse isotopic 2-mm slices through both the right and left ventricles of the rejected heart were fixed in formalin and paraffin embedded. Sections of 5 µm were cut from the paraffin block using the rotation microtome 2055 AUTOCUT (Reichert-Jung Co), dried on slides for 60 minutes at 60°C, and stained with hematoxylin and eosin. Isocrafts were examined as negative controls. Rejection was evaluated according to the Billingham Score, a standardized cardiac biopsy grading with a high score indicating severe acute rejection.¹⁵ In a second set of experiments, recipients were exsanguinated at day 4 after transplantation via puncture of the abdominal aorta.

Neutrophil Leukotriene Profile
Rat PMNs were isolated by an adaptation of the technique described by Hjorth et al¹⁶ for human PMNs. Blood was immediately mixed with heparin (50 U/mL) and centrifuged in a discontinuous Percoll gradient to yield a fraction of approximately 97% purity. Cell viability, as assessed by trypan blue exclusion, ranged above 96% under all experimental conditions, and LDH release was consistently below 3%. PMNs were incubated in RPMI 1640/10% FCS, washed twice with HBSS/25 mmol/L HEPES sine Ca²⁺ and Mg²⁺, resuspended in HBSS/25 mmol/L HEPES, and stimulated for 10 minutes with 1 µmol/L A23187. After terminating the reaction on ice, cells were removed by centrifugation (3000g, 5 minutes, 4°C), and LTs of the 4- and 5-series were extracted from the supernatant by octadecylsilyl solid phase extraction columns as described by Grimminger et al.¹⁷ Conversion into methyl esters was performed by addition of freshly prepared diazomethan in ice-cold diethylether. RP-HPLC of nonmethylated compounds was carried out on octadecylsilyl columns (Hypersil, 5-µm particles) with a mobile phase of methanol/water/acetic acid (72:28:0.16, pH 4.9; Reference 17). In addition to the conventional UV detection at 270 nm (LTs) and 237 nm (HETES and HEPES), a photodiode array detector (Waters model 990) was used, which provided full UV spectra (190 to 600 nm) of eluting compounds and allowed checking for peak purity and subtraction of possible coeluting material. For additional verification, samples were collected in 15-second fractions in selected experiments and subjected to post–HPLC-RIA with anti-LTB₄ as previously described.¹⁷ RP-HPLC of methylated compounds was performed isocratically (66:34:0.16, pH 4.9) for 5 minutes, followed by a linear gradient to 90:10:0.16 over 10 minutes (Gynkothek gradient former, model 250). SP-HPLC of methylated compounds was carried out using a modification of the method of Nadeau et al.¹⁸ The mobile phase consisted of hexane/isopropanol/acetate (86:14:0.1), and the column was eluated isocratically at a flow rate of 1.0 mL/min. All data obtained by the different analytical techniques were corrected for the respective recoveries of the overall analytical procedure and are given in pmol/10⁶ PMN throughout the experiments. Recovery was determined by separate recovery experiments using different quantities of the individual compounds in the appropriate concentration range. For quantification of the different LTs, correspondence of values calculated from UV absorbance in two different chromatographic procedures was required (deviation <10%). Concerning LTB, quantification was additionally confirmed by the use of post–HPLC-RIA.

Platelet Thromboxane Generation
Blood was collected in 3-mL plastic tubes containing 700 µL of a 7.5% EDTA solution. After centrifugation at 200g for 10 minutes, the platelet-rich plasma was decanted and spun again (1500g, 10 minutes). Pelleted platelets were washed with isotonic PBS (pH 7.4), recentrifuged, and resuspended in Tris buffer (20 mmol/L; pH 7.4; 132.8 mmol/L NaCl, 4.3 mmol/L KCl, 1.1 mmol/L KH₂PO₄, 2.4 mmol/L CaCl₂, and 1.3 mmol/L MgPO₄; 240 mg/100 mL glucose). Platelet count was adjusted to 10⁸/mL, and stimulation was performed with 2 µmol/L A23187 (vehicle DMSO, 0.5% vol/vol, final DMSO concentration). Incubation was terminated after 15 minutes by adding 500 µL TBA (1 mol/L, pH 7.0) and 4 mL ice-cold Tris buffer. After centrifugation at 1500g for 10 minutes, TXA₂ and TXA₃ in the supernatant were measured as their stable hydrolysis products TXB₂ and TXB₃. The analytes were extracted from the buffer solution by solid phase extraction, subjected to RP-HPLC separation, and quantified by post–HPLC-ELISA as detailed recently.¹⁹ To avoid chemical decomposition of the analytes, ionic pair reagent, antioxidant, and high buffer strength were used during critical steps of the analytical procedure. Briefly, buffer samples including zero controls as well as controls with known amounts of thromboxanes were supplied with the ionic pair reagent TBA and submitted to solid phase extraction with preconditioned C18 columns. Elution was performed by addition of acetone/acetonitrile (50/50, vol/vol) into microreaction vessels provided with 10-fold concentrated PBS (pH 7.4) and -tocopherol for protection of prostanoids in the following freeze-drying procedure in vacuum. Dried eluates were extracted with acetonitrile and submitted to RP-HPLC separation (C18 column length, 2x15 cm, 3-µm particles; mobile phase: 28/72 (vol/vol) acetonitrile/water, 0.5 mmol/L TBA, pH 7.3; flow rate, 1 mL/min) to separate 2- and 3-series TX. Eluate fractions of 0.3 mL corresponding to the known retention times of TXB₂ and TXB₃ were collected, freeze-dried, redissolved in water, and subjected to ELISA. A monoclonal mouse antibody against TXB₂ with established cross-reactivity to TXB₃ (90%) was used.

Plasma Fatty Acids
Nonesterified plasma fatty acids were quantified by one-step rapid extractive methylation for gas chromatographic analysis.²⁰ Briefly, citrate plasma was spiked with heptadecanoic acid as internal standard; free fatty acids were converted to methyl esters by mixing with ethereal diazomethane; the ethereal layer was dried, redissolved in chloroform, and transferred to the gas chromatograph. Gas chromatographic analysis was performed on a Chrompack gas chromatograph CP 9000 using a CP-88 fused silica capillary column (50 m x0.25 mm; Chrompack AG). The program used an initial oven temperature of 160°C, lasting 2 minutes. The temperature then was raised at a rate of 4°C per minute to 220°C, where it was held for 3 minutes. Injector and detector were maintained at 250°C and 300°C, respectively. The fatty acid methyl esters were detected by use of a flame ionization detector, and peak area integration was performed. For quantification of total plasma fatty acids, 30 µL of plasma was subjected to hydrolysis and methylation in methanol/2N HCl, 15 hours, 100°C. After evaporation, the residue was redissolved in methanol/water, extracted with hexane, evaporated to dryness, redissolved in chloroform, and subjected to gas chromatographic analysis.

Statistics
Values are given as mean±SEM. One-way ANOVA with Tukey's honestly significant difference post hoc test was used to test for differences between the various groups after a normal distribution had been confirmed by the Kolmogorov-Smirnov test. A probability value of less than .05 was considered to indicate statistical significance.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Plasma Fatty Acids
Linoleic acid (18:2) and AA (20:4) represented the predominant acids within the total plasma fatty acid pool of saline-infused rats (Fig 1 and Table 2). This profile as well as the total sum of plasma fatty acids were not substantially altered in animals undergoing parenteral administration with soybean oil; there was some decrease in AA and a moderate increase in oleic acid under this regimen. However, dramatic changes did occur in response to intravenous fish oil infusion. The -3 fatty acids EPA and DHA became predominant compounds under this regimen, and their sum even surpassed that of 18:2 and 20:4. In addition, there was some change in the profile of other reference fatty acids (16:0, 16:1, 18:0, and 20:1), as given in Table 2. The total amount of plasma fatty acids increased by approximately 50% in fish oil–infused rats.

View larger version (24K): [in this window] [in a new window]   Figure 1. Bar graph shows total plasma fatty acids (TFA) in the different experimental groups (saline infusion [NaCl], soybean oil, and fish oil). Selected fatty acids (20:4, arachidonic acid; 20:5, eicosapentaenoic acid; and 22:6, docosahexaenoic acid) and the total sum of plasma fatty acids (right scale) are given. Values are mean±SEM; error bars are missing when falling into symbol. $P<.05 compared with saline; *P<.05 compared with soybean oil.

View this table: [in this window] [in a new window]   Table 2. Total Plasma Fatty Acids in the Different Experimental Groups

Similar changes were noted in the free plasma fatty acid fraction (Fig 2 and Table 3). Only very small quantities of DHA and virtually no EPA were detected under baseline conditions in saline-infused controls and in rats undergoing parenteral soybean oil supplementation, but a dramatic increase occurred in response to fish oil infusion. This rise in the free -3 fatty acids largely contributed to a nearly 2.5-fold increase in the total sum of free plasma fatty acids, whereas this fraction of plasma lipids was only marginally augmented in response to soybean oil infusion.

View larger version (17K): [in this window] [in a new window]   Figure 2. Bar graph shows free plasma fatty acids (FFA) in the different experimental groups (saline infusion [NaCl], soybean oil, and fish oil). Selected free fatty acids (20:4, arachidonic acid; 20:5, eicosapentaenoic acid; and 22:6, docosahexaenoic acid) and the total sum of free plasma fatty acids (right scale) are given. Values are mean±SEM; error bars are missing when falling into symbol. $P<.05 compared with saline; *P<.05 compared with soybean oil.

View this table: [in this window] [in a new window]   Table 3. Free Plasma Fatty Acids in the Different Experimental Groups

Lipid Mediators
Exclusive liberation of TXA₂ was noted in A23187-challenged platelets from saline-infused controls and from animals subjected to parenteral soybean oil administration. Moreover, the total amount of TXA₂ synthesis was only marginally increased in response to the intravenous -6 fatty acid supplementation (Fig 3). Conversely, in rats undergoing fish oil infusion, total quantities of TXA₂ were reduced by >50%, accompanied by marked TXA₃ synthesis: The TXA₃/TXA₂ ratio approximated 1:1 in these animals.

View larger version (17K): [in this window] [in a new window]   Figure 3. Bar graph shows platelet thromboxane generation. The A23187-induced liberation of TXB2 (stable hydrolysis product of TXA2) and TXB3 is given as mean±SEM.

As anticipated, in vitro stimulation of PMNs isolated from saline-infused controls provoked virtually exclusive generation of 4-series LTs, including LTB₄, 20-COOH- and 20-OH-LTB₄ (summarized as ox-LTB₄ in Fig 4), and LTA₄ (its stable hydrolysis products 6-trans- and 6-trans-epi-LTB₄, summarized as 6t-LTB₄ in Fig 4). The profile and total amount of PMN leukotriene synthesis were virtually unchanged in animals subjected to soybean oil infusion. Intravenous administration of the fish oil–based lipid emulsion did not substantially suppress the 4-series LT generation; however, marked formation of 5-series leukotrienes (LTB₅, its omega oxidation products, and LTA₅ hydrolysis products depicted in Fig 4) was noted. The 5-series/4-series LT ratios approximated 0.08 (LTB₄ and its omega oxidation products) and 0.36 (LTA₄).

View larger version (21K): [in this window] [in a new window]   Figure 4. Bar graph shows neutrophil leukotriene generation. The A23187-induced liberation of 4-series LTs (-6 derivatives) and 5-series LTs (-3 derivatives) is given for the different experimental groups (mean±SEM). Ox-LTB4(5) indicates sum of 20-COOH- and 20-OH-LTB4(5); 6t-LTB4(5), sum of 6-trans- and 6-trans-epi-LTB4(5). *P<.05 (fish oil–infused rats compared with both other groups).

Transplant Survival
As demonstrated in Fig 5, survival of the transplant was significantly increased in response to soybean oil administration as compared with saline-infused controls, and rejection was even more retarded in rats undergoing fish oil infusion (significant difference from the -6 fatty acid–supplemented animals).

View larger version (12K): [in this window] [in a new window]   Figure 5. Bar graph shows heart transplant survival in the different experimental groups (mean±SEM; +P<.05 compared with saline-infused controls; $P<.05 compared with soybean oil infusion).

Seven-day isografts (n=8) had minimal histological changes and no evidence of significant immunological response or rejection. All the allografts examined showed lymphocyte infiltration, myocyte necrosis, and focal hemorrhage, which are the typical histological signs of acute rejection. On the five-point scale according to Billingham et al,¹⁵ the mean cellular rejection was scored 3.8 in the saline-infused group and 3.4 and 2.6 in the soybean oil–infused and fish oil–infused groups, respectively.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present use of a fish oil–derived lipid emulsion for intravenous alimentation and -3 lipid supplementation allows the administration of effective doses of alternative precursor fatty acids within a short time period. A significant prolongation of graft survival was noted, concomitant with a shift in plasma fatty acid and lipid mediator profiles of neutrophils and thrombocytes toward EPA/DHA and EPA-derived products.

The total amount of intravenously provided lipids (9 g/kg body wt per day) is high compared with routine parenteral nutrition in humans. However, it must be considered that rats possess a manifold increased energy turnover per kilogram of body weight in comparison to humans.²¹ Related to the total energy expenditure of these animals, 30% was provided as fat by the current intravenous supplementation regimen, which is well within the range of parenteral nutrition standards.²¹ When infusing 0.3 g EPA ethyl ester into Wistar rats within 20 minutes (which corresponds to approximately one third of the total amount of -3 fatty acids presently infused within 24 hours), a >98% clearance of the infused lipid aggregates from the plasma occurred within 1 hour, and there was no sign of organ lipidosis upon subsequent histological examination.²² Accordingly, no lipidemia occurred in response to the currently used soybean oil supplementation regimen, thus excluding overriding of the natural lipid clearance mechanisms.

A high percentage (75%) of the fatty acids in soybean oil is polyunsaturated, with a 370 to 1 predominance of -6 over -3 lipids. Nevertheless, the profiles of total and free plasma fatty acids differed only marginally between saline-infused and soybean oil–infused rats because of the similarity in lipid composition between the orally provided stock feed and the soybean oil. The low values of EPA and DHA in the plasma lipids of these animals are typical for Western diet alimentation.²³ Infusion of the fish oil–based lipid emulsion, containing polyunsaturated fatty acids at an -3/-6 ratio of 7.6:1, provoked a dramatic shift in both total and free plasma fatty acids toward predominance of EPA over AA, accompanied by large DHA levels. Artificial lipid aggregates are known to activate the endothelial lipoprotein lipase, including a translocation of this enzyme from its cellular binding sites into the vascular compartment.¹² The increasing plasma lipolytic activity then may cause a rise in free plasma fatty acids due to escape from local cellular uptake mechanisms. Interestingly, such increase was presently noted to occur in response to the -3–based (nearly 2.5-fold levels of free fatty acids) but not the -6–based lipid infusion regimen. This finding indicates differential impact of synthetic lipid aggregates on the endothelial hydrolysis and cellular uptake mechanisms in dependence of their fatty acid composition, a feature hitherto not described. Overall, kinetics and extent of plasma esterified and free -3 lipid increase in response to parenteral fish oil supplementation by far exceeded corresponding alterations in response to conventional dietary fish oil uptake.⁴

In addition to being incorporated into membrane phospholipid pools, free extracellular precursor fatty acids may directly affect cell activation and mediator generation. Micromolar concentrations of nonesterified AA have been detected at sites of inflammatory events.²⁴ For organs composed of different cell types with inflammatory potencies, evidence for intercellular exchange of free AA has been presented, which contributes to transcellular eicosanoid synthesis and is susceptible to modulations of extracellular free fatty acid contents.^{25 26} The present finding of plasma-free EPA concentrations of >30 µmol/L, surpassing those of AA approximately twofold, must be anticipated to result in a dramatic shift in LT generation from 4-series to 5-series products upon neutrophil stimulation in this natural environment.^{10 11} Part of this effect was demonstrated by the present technique of in vitro stimulation of neutrophils isolated from their plasmatic environment, which resulted in the formation of appreciable quantities of EPA-derived 5-lipoxygenase products. This finding suggests that some EPA-containing membrane lipid pool(s), providing precursor fatty acids for the neutrophil 5-lipoxygenase pathway, may be rapidly regulated in exchange with plasma EPA concentrations.

The shift in lipid mediator generation toward EPA-derived products was even more prominent for thrombocytes similarly stimulated after isolation from their natural plasmatic environment: The relationship of 1:1 of TXA₃ to TXA₂, measured in response to fish oil infusion, reflects the highest 3-series to 2-series prostanoid ratio hitherto described. For comparison, ex vivo stimulation of thrombocytes obtained from volunteers with dietary -3 fatty acid supplementation for several months resulted in 5% to 15% generation of TXB₃ as related to TXB₂.²⁷

This study did not address the question of which biochemical and cellular changes imposed by infusion of a fish oil–based lipid emulsion significantly contributed to the prolongation of graft survival. Interestingly, some effect was even achieved by parenteral administration of soybean oil. This finding is reminiscent of the moderately reduced allograft rejection occurring upon dietary enrichment with linoleic acid.² Some enhanced generation of prostaglandin E, which is also known to possess immunosuppressive properties in cardiac allografting,²⁸ or direct inhibitory effects of free fatty acids on lymphocyte activation and cytokine generation^{29 30} have been suggested as underlying mechanisms, but direct measurements of these variables were not performed. In line with this reasoning, the presently noted 2.5-fold increase in free plasma fatty acids, occurring in response to intravenous fish oil administration, may exert some suppressive effect on primary immunological events in allograft rejection via modulation of lymphocyte function. In addition, events related to thrombocytes and TX generation as well as neutrophils and leukotriene synthesis may substantially contribute to inflammatory and vasomotor sequelae in transplantation. Increased generation of TXA₂, a powerful vasoconstrictor agent and aggregator of platelets and leukocytes, during acute rejection of heart allografts has been demonstrated clinically and experimentally.³¹ The currently demonstrated major shift from TXA₂ to TXA₃, known to possess reduced or even antagonistic biological properties as compared with the dienoic TX,^{4 32} thus may contribute significantly to the improvement of allograft survival via dampening of vasoconstrictor and thrombotic phenomena in acute rejection. This assumption is supported by previous observations of increased allograft survival upon use of TX synthetase inhibitors and TX antagonists.³³ In line with this reasoning, dietary -3 fatty acids, given in high doses for several weeks in nonhuman primates, reduced both vascular thrombus formation and lesions after mechanical vascular injury.³⁴ The shift in neutrophil leukotriene generation from LTB₄ to LTB₅, which may be anticipated as even more prominent in the natural plasmatic environment with -3 fatty acid predominance, has a variety of implications. LTB₅ possesses a more than 10-fold reduced chemotactic and PMN-activating capacity as compared with LTB₄,⁴ and competition with LTB₄ for receptor occupancy on neutrophils has been demonstrated.³⁵ Because of interference with the LTB₄-based autocrine loop of PMN activation, a marked dampening of neutrophil function is achieved. Additional effects may be exerted via interference with the LTB₄-related stimulation of other inflammatory cells, including mononuclear cells and their cytokine production.⁵

Over the last few years, -3 fatty acid–rich lipid infusions have been developed for use in clinical situations, intending to shift the AA/EPA ratio toward predominance of the latter lipid mediator precursor and thereby combine parenteral nutrition and pharmacological intervention. The presently described prolongation of cardiac allograft survival, accompanied by a shift in plasma fatty acid and lipid mediator profiles under a fish oil infusion regimen, extends the rationale of such an approach. Further studies are warranted to characterize the underlying events in more detail and investigate possible cooperation of -3 lipids with the conventional immunosuppressive agents, corticosteroids, and cyclosporine.

	Selected Abbreviations and Acronyms

 

AA = arachidonic acid DHA = docosahexaenoic acid EPA = eicosapentaenoic acid LT = leukotriene 5-HET(P)E = 5-hydroxy-eicosatetra(penta)enoic acid PMN = polymorphonuclear leukocyte TX = thromboxane HPLC = high-performance liquid chromatography SP-HPLC = straight phase HPLC RP-HPLC = reverse phase HPLC RIA = radioimmunoassay

	Acknowledgments

 
This study was supported by the Deutsche Forschungsgemeinschaft (Klinische Forschergruppe "Respiratorische Insuffizienz").

Received April 4, 1995; revision received July 11, 1995; accepted August 29, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 

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