Metabolomics reveals increased isoleukotoxin diol (12,13-DHOME) in human plasma after acute Intralipid infusion

Intralipid is a fat emulsion that is regularly infused into humans and animals. Despite its routine use, Intralipid infusion can cause serious adverse reactions, including immunosuppression. Intralipid is a complex mix of proteins, lipids, and other small molecules, and the effect of its infusion on the human plasma metabolome is unknown. We hypothesized that untargeted metabolomics of human plasma after an Intralipid infusion would reveal novel insights into its effects. We infused Intralipid and saline into 10 healthy men in a double-blind, placebo-controlled experiment and used GC/MS, LC/MS, and NMR to profile the small-molecule composition of their plasma before and after infusion. Multivariate statistical analysis of the 40 resulting plasma samples revealed that after Intralipid infusion, a less-well-characterized pathway of linoleic acid metabolism had resulted in the appearance of (9Z)-12,13-dihydroxyoctadec-9-enoic acid (12,13-DHOME, P < 10−3), a leukotoxin that has powerful physiological effects and is known to inhibit the neutrophil respiratory burst. Intralipid infusion caused increased plasma 12,13-DHOME. Given that 12,13-DHOME is known to directly affect neutrophil function, we conclude that untargeted metabolomics may have revealed a hitherto-unknown mechanism of intralipid-induced immunosuppression. Intralipid is a fat emulsion that is regularly infused into humans and animals. Despite its routine use, Intralipid infusion can cause serious adverse reactions, including immunosuppression. Intralipid is a complex mix of proteins, lipids, and other small molecules, and the effect of its infusion on the human plasma metabolome is unknown. We hypothesized that untargeted metabolomics of human plasma after an Intralipid infusion would reveal novel insights into its effects. We infused Intralipid and saline into 10 healthy men in a double-blind, placebo-controlled experiment and used GC/MS, LC/MS, and NMR to profile the small-molecule composition of their plasma before and after infusion. Multivariate statistical analysis of the 40 resulting plasma samples revealed that after Intralipid infusion, a less-well-characterized pathway of linoleic acid metabolism had resulted in the appearance of (9Z)-12,13-dihydroxyoctadec-9-enoic acid (12,13-DHOME, P < 10−3), a leukotoxin that has powerful physiological effects and is known to inhibit the neutrophil respiratory burst. Intralipid infusion caused increased plasma 12,13-DHOME. Given that 12,13-DHOME is known to directly affect neutrophil function, we conclude that untargeted metabolomics may have revealed a hitherto-unknown mechanism of intralipid-induced immunosuppression. body mass index intravenous fat emulsion nuclear Overhauser effect spectroscopy projection onto latent spaces discriminant analysis peroxisome proliferator-activated receptor soluble epoxy hydrolase total ion current Intravenous fat emulsions (IVFEs) are regularly infused into both patients and healthy humans in a range of settings. Of these IVFEs, perhaps the most common is Intralipid, a lipid emulsion made from soybean oil and egg yolk phospholipids (1Anon, . 2011. Intralipid official FDA information, side effects and uses. In. Baxter Healthcare Corporation.Google Scholar). In clinical practice, Intralipid is used as a component of parenteral feeding because it contains a rich blend of diet-mimicking triacylglycerols, composed mostly of the 18-carbon unsaturated FAs linoleic (∼46%) and oleic (∼20%) acid (2Hammond, E. G., Johnson, L. A., Su, C., Wang, T., White, P. J., . 2005. Soybean oil. In Bailey's Industrial Oil and Fat Products. F. Shahidi, editor. John Wiley and Sons, Inc. 577–653.Google Scholar). In addition, clinical infusion of Intralipid is an effective therapy for cardiac poisoning by local anesthetic (3Manavi M.V. Lipid infusion as a treatment for local anesthetic toxicity: a literature review.AANA J. 2010; 78: 69-78PubMed Google Scholar) and as a drug delivery vehicle (4Mirtallo J.M. Dasta J.F. Kleinschmidt K.C. Varon J. State of the art review: intravenous fat emulsions: current applications, safety profile, and clinical implications.Ann. Pharmacother. 2010; 44: 688-700Crossref PubMed Scopus (114) Google Scholar). In a research setting, acute Intralipid infusion is often used to study the effects of increased triacylglycerol and nonesterified FA concentrations on metabolism and physiological function (5Minh T.C. Oliver S.R. Flores R.L. Ngo J. Meinardi S. Carlson M.K. Midyett J. Rowland F.S. Blake D.R. Galassetti P.R. Noninvasive measurement of plasma triglycerides and free fatty acids from exhaled breath.J. Diabetes Sci. Technol. 2012; 6: 86-101Crossref PubMed Scopus (11) Google Scholar–7Al-Shayji I.A. Caslake M.J. Gill J.M. Effects of moderate exercise on VLDL and Intralipid kinetics in overweight/obese middle-aged men.Am. J. Physiol. Endocrinol. Metab. 2012; 302: E349-E355Crossref PubMed Google Scholar). Despite its wide application, Intralipid infusion is not without its hazards. Particularly in the very young, it can lead to a number of adverse events, including hypertriglyceridemia, liver disease (8Postuma R. Trevenen C.L. Liver disease in infants receiving total parenteral nutrition.Pediatrics. 1979; 63: 110-115Crossref PubMed Google Scholar), pancreatitis (9Lashner B.A. Kirsner J.B. Hanauer S.B. Acute pancreatitis associated with high-concentration lipid emulsion during total parenteral nutrition therapy for Crohn's disease.Gastroenterology. 1986; 90: 1039-1041Abstract Full Text PDF PubMed Scopus (69) Google Scholar), and immunosuppression that leaves vulnerable patients at an increased risk of infection (4Mirtallo J.M. Dasta J.F. Kleinschmidt K.C. Varon J. State of the art review: intravenous fat emulsions: current applications, safety profile, and clinical implications.Ann. Pharmacother. 2010; 44: 688-700Crossref PubMed Scopus (114) Google Scholar, 10Okada Y. Klein N.J. van Saene H.K. Webb G. Holzel H. Pierro A. Bactericidal activity against coagulase-negative staphylococci is impaired in infants receiving long-term parenteral nutrition.Ann. Surg. 2000; 231: 276-281Crossref PubMed Scopus (49) Google Scholar, 11Okada Y. Papp E. Klein N.J. Pierro A. Total parenteral nutrition directly impairs cytokine production after bacterial challenge.J. Pediatr. Surg. 1999; 34: 277-280Abstract Full Text PDF PubMed Scopus (18) Google Scholar). It has been suggested that Intralipid infusion may inhibit the mononuclear phagocyte system function (4Mirtallo J.M. Dasta J.F. Kleinschmidt K.C. Varon J. State of the art review: intravenous fat emulsions: current applications, safety profile, and clinical implications.Ann. Pharmacother. 2010; 44: 688-700Crossref PubMed Scopus (114) Google Scholar), inasmuch as this system not only contributes to the immune response but is also responsible for the clearance of fat emulsions from plasma (12Waddell W.R. Geyer R.P. Clarke E. Stare F.J. Function of the reticuloendothelial system in removal of emulsified fat from blood.Am. J. Physiol. 1954; 177: 90-94Crossref PubMed Scopus (6) Google Scholar). It has further been suggested that the high linoleic acid content of Intralipid (via metabolic conversion of linoleic to arachidonic acid) may lead to an increase in the eicosanoids (4Mirtallo J.M. Dasta J.F. Kleinschmidt K.C. Varon J. State of the art review: intravenous fat emulsions: current applications, safety profile, and clinical implications.Ann. Pharmacother. 2010; 44: 688-700Crossref PubMed Scopus (114) Google Scholar), 20-carbon lipids that play a critical role in modulating human immune function (13Mancini A.D. Di Battista J.A. The cardinal role of the phospholipase A(2)/cyclooxygenase-2/prostaglandin E synthase/prostaglandin E(2) (PCPP) axis in inflammostasis.Inflamm. Res. 2011; 60: 1083-1092Crossref PubMed Scopus (52) Google Scholar). The chemistry of Intralipid is far from simple. Its soybean and egg yolk constituents are a complex mixture of proteins, lipids, and small molecules, including phospholipids, sterols, tocopherols, hydrocarbons, and flavones (2Hammond, E. G., Johnson, L. A., Su, C., Wang, T., White, P. J., . 2005. Soybean oil. In Bailey's Industrial Oil and Fat Products. F. Shahidi, editor. John Wiley and Sons, Inc. 577–653.Google Scholar), many of which are known to be biologically active. Thus, the reasons for both Intralipid's clinical effectiveness and its occasional toxicity may be more obscure than is currently thought. By extension, the chemical complexity of Intralipid may explain some of the contradictions in the literature with regard to effects of acute increases in circulating lipids (and lipid metabolites); for example, see recent work from this laboratory (14Holland D.J. Erne D. Kostner K. Leano R. Haluska B.A. Marwick T.H. Sharman J.E. Acute elevation of triglycerides increases left ventricular contractility and alters ventricular-vascular interaction.Am. J. Physiol. Heart Circ. Physiol. 2011; 301: H123-H128Crossref PubMed Scopus (10) Google Scholar). Given that untargeted small-molecule profiling of human plasma or serum in response to Intralipid infusion has, to our knowledge, never been carried out, the complex interaction between Intralipid and physiology, both in the clinic and elsewhere, remains unclear. The aim of this study was to measure changes in the human plasma metabolome in response to a standard Intralipid infusion, by using untargeted metabolomic profiling. We used GC/MS, high-resolution LC/MS, and 1H-NMR and aimed to profile novel compounds that were significantly increased in human plasma due either to the infusion or to secondary metabolism of the infusate. Our aim was to shed light on the possible contribution of these small molecules to both the beneficial, as well as the adverse, effects of Intralipid infusion in humans. Ten healthy men were recruited from the community by local advertisement. The subjects’ ages, weights, heights, and body mass index (BMI) were measured during an initial assessment visit. Subjects were deemed eligible for participation if they were aged over 18 years, had a normal BMI (18.5–25), were not on any cardiac or metabolic medications, and had no adverse reaction to egg, nuts, soy, heparin, or Intralipid. They were screened twice for possible complicating conditions (for example coronary heart disease, diabetes mellitus, aortic valve stenosis, and diseases of the liver, pancreas, or kidney). Subjects were informed of the nature and possible risks associated with each experiment and were required to complete both a health screening questionnaire and a statement of informed consent. All protocols were approved by the human research ethics committee of the University of Queensland and conformed to the Declaration of Helsinki. The study was a randomized, double-blind, placebo-controlled experiment. Subjects attended the laboratory on two separate occasions separated by approximately 1 week (alternate randomization was implemented during the second visit). On each visit, the subjects arrived in the morning following an overnight fast. After at least 10 min supine rest in a temperature-controlled room, an intravenous cannula was inserted in the cephalic of each the for infusion and the left to was after which a saline infusion was After a of heparin, subjects were to of two Intralipid or were infused at and with After min of infusion, was a total of 40 were in for clinical and for metabolomic was using a clinical system metabolomics 1 of plasma were separated at using a The and were to in a and at to the was in either of (for mass or NMR acid were in for with a into a a of were the was and to for samples were by on a to a mass using as at in The was a and of 1 were made using a and a in using a The was The was at to at to at The ion was at and the at were using over the range to in ion for the at and with of were also as a in of any were by the of were on the total ion current for each FA with the of which with oleic acid these this the ion at was and this was to the after a of acid to this All FAs were as their of the total FAs were using analysis was using a high-resolution mass The samples were separated using a a at a of using acid in for and acid in for 10 of each was onto the and compounds were over a of over over and over 1 After at for the was in for The was at and the samples were at mass were in the range of at a of to the following of and to and of was used for and was used for were and using G. R. G. mass for profiling using and 78: PubMed Scopus Google Scholar) in the nuclear Overhauser effect spectroscopy were from the using an were with a with a of at total of were into The was to the of the and a of was during the and also during the of the The resulting free were and in NMR All chemical were to at The were and using F. A. G. H. as to for of complex in NMR 78: PubMed Scopus Google Scholar) and D. E. T.C. S. of metabolic in using an PubMed Scopus Google Scholar), both were in due to were from further analysis D. E. Clarke R. The of and to human plasma on from metabolic profiling 2010; 6: PubMed Scopus Google Scholar). experiment with regard to that the small molecules the metabolic profile have from any of human metabolism the soybean metabolism to the egg yolk content of and possible in human from compounds that were of Intralipid (and directly we Intralipid directly to a of it to the and used this as a that were not in the but only in the were to be the of secondary LC/MS, were by by had been we used any that were and for either or increased example, the of acid in the 1H-NMR was used to the of the we used to their mass was with the Our was to not than a number of compounds of were further using mass compounds acid acid acid acid and were and the resulting were with example, the ion that was to to 12,13-DHOME was using at the at both and were with an for 12,13-DHOME in the were not were with using the in S. S. S. In for of mass 2010; PubMed Scopus Google Scholar). no compounds were in this were from a on chemical was using spectroscopy statistical of our standard we an system to that by the and D. P. E. S. A. for metabolic profiling of serum and plasma using and to mass 2011; 6: PubMed Scopus Google Scholar). NMR and were using projection onto latent spaces discriminant analysis using the as implemented in to were and (for of against we F. L. by to Google Scholar) as implemented in was if the P was the due to a of we on each with of P using the and Y. Y. the a and powerful to R. Scholar). were as if the P was and FA were using a as implemented in and analysis were using or the 18 or S. A. for PubMed Scopus Google and S. A. for and mass profile 2010; PubMed Scopus Google or All and are from the on triglycerides and FAs were normal at the infusion of Intralipid resulted in a increase in increase in circulating free FAs was also both and with the increase most due to the of on was no in total in either was a in VLDL and and a in of plasma clinical FAs Total VLDL in a The to increase in circulating free FAs was by a in the FA profile The most was a in the contribution to the total plasma FA from linoleic acid to a of Intralipid. was also a increase in the contribution from from to The contribution of other FAs either or the this be in the of the increase in total free Thus, the contribution from oleic acid was oleic acid have for this to be the by measured by are of total in a are of total total of of in the mass were using from in and a further in Of were significantly by Intralipid infusion < after the and were for (for example, from which were After and were also Of the be the of and Of these were as and and from further is a complete of of the is in the small molecules in human by LC/MS, that were significantly after an Intralipid is for the most if was most P for the most is acid is but was not with is in ion was also ion was also acid is but was not with is in ion was also ion was also ion was also ion was also P is for the most if was most P for the most is is but was not with is in ion was also ion was also in a Of the both and some were FAs that had been from the infused by including linoleic and oleic more were due to of linoleic acid FA in including acid and acid We also a at that may have to 12,13-DHOME The at this mass and was significantly after Intralipid infusion P the and that this was not in our The acid was significantly its as a of by 1H-NMR were a and some FAs as acid and acid was to the 1H-NMR and for of the The of was by as to be due to < The latent between the and and for of the 1 was by a of to a in the NMR at were using spectroscopy E. NMR spectroscopy of the complex metabolic of from patients with of and PubMed Scopus Google Scholar) as Thus, the NMR further of the that was in the samples with the study the effects of Intralipid infusion on the human plasma we the plasma of 10 healthy using GC/MS, high-resolution LC/MS, and before and after infusion with either Intralipid or Intralipid infusion caused an increase in circulating triglycerides and nonesterified we also over novel chemical and that were increased by Intralipid infusion. The of these is have been regularly infused into both patients and healthy humans in a range of for more than they are most used for parenteral either on their or as a component of total parenteral They have also a novel as a drug delivery In this they are used as a for that are soluble in drug in lipid is it 60: PubMed Scopus Google Scholar). infusion of is an effective therapy for cardiac poisoning by local anesthetic (3Manavi M.V. Lipid infusion as a treatment for local anesthetic toxicity: a literature review.AANA J. 2010; 78: 69-78PubMed Google Scholar). also into healthy subjects and as an of increased plasma triacylglycerol and FA Given that plasma lipid concentrations are associated with an increased risk of disease and increased in using have an role in our of the effect of acute of plasma its infusion can and cause adverse liver disease, hypertriglyceridemia, and with infants these adverse are they can be they that the chemical profile of many is complex to their the mechanism adverse remains unclear. we used metabolomics to profile the plasma of 10 healthy before and after infusion with either saline or of the IVFEs, Intralipid. We a range of compounds that were not known to be increased in response to Intralipid infusion, including linoleic acid and their that are to have effects a of these were only their on the metabolic significantly increased our in these an metabolic as a for metabolome has been suggested of mass is at than 1 PubMed Scopus Google Scholar, G. K. R. S. of metabolomic interaction Scopus Google Scholar). Of these 12,13-DHOME J.M. J.F. K.C. of to their by PubMed Scopus Google Scholar) is of 12,13-DHOME is in humans from linoleic the by soluble epoxy hydrolase and of a metabolic pathway that from linoleic acid its to the and the 12,13-DHOME and are with effects. The or peroxisome proliferator-activated are that a of to and lipid metabolism G. S. A. and role of peroxisome proliferator-activated receptor and PubMed Scopus Google Scholar). The effects of the are very G. S. 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Thus, of many of the effects of the and PubMed Scopus Google Scholar), 12,13-DHOME the of including J. of linoleic acid on current in role of PubMed Scopus Google Scholar) and cardiac effects of linoleic acid on cardiac PubMed Scopus Google Scholar). In addition, both and can be to the with the leukotoxin of as in S. A. J. J. and in PubMed Scopus Google Scholar, K. S. A. J. from in and PubMed Scopus Google Scholar). Given that a immune system is a adverse effect of Intralipid infusion, for the very and the both of Intralipid and both of are vulnerable to is on and mechanism that linoleic the FA of Intralipid, is The more of linoleic acid metabolism is the 20-carbon FA arachidonic acid to the a of lipids and that are of and immune function (13Mancini A.D. Di Battista J.A. The cardinal role of the phospholipase A(2)/cyclooxygenase-2/prostaglandin E synthase/prostaglandin E(2) (PCPP) axis in inflammostasis.Inflamm. Res. 2011; 60: 1083-1092Crossref PubMed Scopus (52) Google Scholar). Thus, the is that Intralipid infusion to a in concentrations with effects on and immune this are (for a see J.M. Dasta J.F. Kleinschmidt K.C. Varon J. State of the art review: intravenous fat emulsions: current applications, safety profile, and clinical implications.Ann. Pharmacother. 2010; 44: 688-700Crossref PubMed Scopus (114) Google Scholar). 12,13-DHOME the neutrophil respiratory inhibit the neutrophil respiratory PubMed Scopus Google Scholar), the of to to as In other 12,13-DHOME is significantly after Intralipid infusion, and is known to directly inhibit immune function in a that is with immunosuppression. 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The of the effects of in and Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, acute infusion of into to a increase in work of the heart K. Y. Clarke K. R.L. and J. PubMed Scopus Google Scholar) with very recent work that feeding a both fat S. Y. J. G. Clarke K. R.L. and increased 1 in of a J. 2012; PubMed Scopus Google Scholar) and metabolism Y. R. S. A. Clarke K. R.L. increases and and in the normal 2010; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, the effects of Intralipid infusion that were to acute increases in lipids may in be due to increased In the of our other Intralipid infusion increases the of a number of linoleic acid that have a effect on cardiac an that the effect of Intralipid for example, cardiac function would on the metabolic between the production of as and as leukotoxin work in this is We infused Intralipid into 10 healthy men and the changes in their plasma metabolome using and We that of linoleic acid were significantly after Intralipid infusion. Of 12,13-DHOME is known to have adverse effects on neutrophil Thus, we that of the known adverse effects of Intralipid infusion, may in be by 12,13-DHOME.