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Phagocyte-induced lipid peroxidation of different intravenous fat emulsions and counteractive effect of vitamin E
APPLIED NUTRITIONAL INVESTIGATION
Nutrition Vol. 15, No. 5, 1999
Phagocyte-Induced Lipid Peroxidation of Different Intravenous Fat Emulsions and Counteractive Effect of Vitamin E GUO HAO WU, MD, PHD,*† CONNIE JARSTRAND, MD, PHD,* AND ¨ RGEN NORDENSTRO ¨ M, MD, PHD† JO From the Departments of *Clinical and Oral Bacteriology and †Surgery, Huddinge University Hospital, Karolinska Institute, Huddinge, Sweden Date accepted: 8 July 1998 ABSTRACT
Unsaturated fatty acids, a major component of fat emulsions used in parenteral nutrition, are prone to peroxidation which is an important feature of oxygen-associated tissue damage. We used the nitroblue tetrazolium (NBT) reduction test to measure the production of superoxide radicals by stimulated polymorphonuclear neutrophils (PMN) in the presence of different fat emulsions: Intralipid (containing 100% long-chain triacylglycerols, LCT), Vasolipid (a physical mixture of 50% LCT and 50% mediumchain triacylglycerols, MCT) and Structolipid (structured triacylglycerols containing 63% LCT and 37% MCT). We measured the amount of malonaldehyde (MDA) and 4-hydroxyalkenal to determine the lipid peroxidation of the three fat emulsions in the presence of stimulated neutrophils. Further, we investigated the role of vitamin E (a-tocopherol) in preventing lipid peroxidation in vitro. The results showed that the values of NBT reduction of PMN were significantly decreased in each of the three fat emulsions and that increasing concentrations of fat emulsions were associated with decreased values of NBT reductions, in a dose-dependent way (P , 0.001). There were, however, no statistically significant differences between the values of the three different types of fat emulsions (P . 0.05). Lipid peroxidation increased significantly in the presence of all three types of fat emulsions, and was more pronounced for Intralipid than for Vasolipid and Structolipid after 1 and 2 h of incubation with resting as well as with stimulated phagocytes. The increased lipid peroxidation of the fat emulsions was markedly reduced by vitamin E, and the inhibition was concentration dependent. In conclusion, lipid peroxidation in vitro is more pronounced when PMNs are incubated with fat emulsions. This increase in lipid peroxidation can be reduced by adding vitamin E to the fat emulsions. Nutrition 1999;15:359 –364. ©Elsevier Science Inc. 1999 Key words: free radicals, structured triacylglycerols, lipid peroxidation, vitamin E (a-tocopherol), fat emulsion, neutrophil
INTRODUCTION
Intravenous fat emulsions are used as a source of energy and essential fatty acids. Despite unquestionable nutritional advantages, the infusion of fat emulsions rich in polyunsaturated fatty acids (PUFAs) may become peroxidized in vivo. Lipid peroxidation, generated by the action of free radicals, is enhanced in a variety of clinical conditions such as septic shock, adult respiratory distress syndrome, or ischemia-reperfusion injury.1–3 Earlier studies from our group have shown that the degree of hypertriglyceridemia during Intralipid infusion in patients correlated with the impairment of leucocyte function.4 Furthermore, granulocytes
from patients with type IV hyperlipoproteinemia showed a decreased NBT reduction.5 The hypothesis was that neutrophil-produced oxygen radicals can cause lipid peroxidation of fat emulsions. The purpose of the present study was 1) to measure the production of superoxide anions as the NBT reduction by stimulated neutrophils in the presence of fat emulsions, 2) to measure lipid peroxidation of fat emulsions in the presence of stimulated and unstimulated neutrophils, 3) to evaluate the role of vitamin E in preventing lipid peroxidation in this in vitro system, and 4) to compare three different fat emulsions with respect to production of superoxide anions and lipid peroxidation.
Correspondence to: Jo¨rgen Nordenstro¨m, MD, PhD, Department of Surgery, K53, Huddinge University Hospital, S-141 86 Huddinge, Sweden. E-mail: [email protected]
Nutrition 15:359 –364, 1999 ©Elsevier Science Inc. 1999 Printed in the USA. All rights reserved.
0899-9007/99/$20.00 PII S0899-9007(99)00052-0
360
PEROXIDATION OF IV FAT EMULSIONS AND EFFECT OF VITAMIN E TABLE I. COMPOSITION OF INTRALIPID, VASOLIPID, AND STRUCTOLIPID
Content/1000 mL Soybean oil (g) Structured triacylglycerol (g) Coconut oil (g) Egg phospholipid (g) Glycerol (USP) (g) Water for injection (mL) Mean molecular weight (approx.) Proportion of LCT:MCT (wt:wt) Fatty acids composition (wt:wt) Caprylic acid, 8:0 Capric acid, 10:0 Palmitric acid, 16:0 Stearic acid, 18:0 Oleic acid, 18:1 Linoleic acid, 18:2 a-linolenic acid, 18:3 Other Total
Intralipid
Vasolipid
Structolipid
200 — — 12 22.5 1000 865 100:0
100 — 100 12 25 1000 634 50:50
— 200 — 12 22.5 1000 683 63:37
— — 13 4 22 52 8 1 100
43 18 4 2 9 20 2 2 100
27 10 7 3 13 33 5 2 100
By this reduction, yellow soluble NBT is converted to dark blue formazan that can be measured by a spectrophotometric method. It reflects the oxidative metabolism of the cells known to be enhanced during phagocytosis (respiratory burst). In the present study, we used a modification of the conventional NBT method, using only 10% of the sample volume. All the samples were exposed to three different types of fat emulsions, Intralipid 20% (100% long-chain triacylglycerols, Pharmacia & Upjohn AB, Stockholm, Sweden); Vasolipid 20% (physical mixture of 50% long-chain triacylglycerols [LCT] and 50% medium-chain triacylglycerols [MCT], B. Braun Medical AB, Germany); and Structolipid 20% (containing 63% LCT and 37% MCT, Pharmacia & Upjohn AB). The composition of fat emulsions are presented in Table I. The PMN suspension (0.1 mL) were mixed on a microtite plate with 0.03 mL of each of the fat emulsions in three different concentrations (0.33, 3.33, 33.33 mg/mL). Experiments were performed with or without stimulation of the cells by heat-killed Candida albicans (0.001 mL, 3 3 108 Candida albicans/mL), then 0.05 mL NBT solution was added and the plate was covered with adhesive tape and incubated for 60 min at 37°C in a shaking device. The reaction was then stopped by adding 0.1 mL 0.5 N HCl. The plate was centrifuged at 1500 rpm. for 10 min and the supernatant was removed. A total of 0.2 mL of dimethyl-sulfoxide (DMSO) was added to each well to extract the formazan. The plate was covered again and placed on an agitation table for 15–20 min to accelerate the extraction of the formazan. DMSO alone was added as a blank to the first well of each plate and, finally, the optical densities (OD) were recorded spectrophotometrically at 570 nm.5–7
MATERIALS AND METHODS
PMN Separation Venous heparinized blood (40 –50 mL) was obtained from each of 18 healthy donors (age 18 – 65 y). The polymorphonuclear neutrophils (PMN) were separated on a sodium metrizoate (Sigma Lot 35H-1406) and methylcellulose (Dow Chemical Company, Lot, MN, USA) column by the method of Boyum and suspended in pH-adjusted (7.2–7.4) Eagle’s medium at a density of 5 3 106 cells/mL. NBT Reduction Test Nitroblue tetrazolium (NBT) is an electron acceptor used for indirect detection of the production of superoxide by stimulated PMN, as outlined in the following equation: NBT 1 O1 2
OO 3 formazan 1 O2
(1)
Lipid Peroxidation We used a new colorimetric assay, LPO-586 (OXIS-21012, British Bio-technology Products Ltd., England), to measure the reaction of a chromogenic reagent R1 (10.3 mmol/mL N-methyl2-phenylindole, in acetonitrile) with malonaldehyde (MDA) and 4-hydroxyalkenal at 45°C. MDA and 4-hydroxyalkenal are formed in vitro as breakdown products of peroxidized polyunsaturated fatty acids, and the extent of peroxidation is quantitatively related to the amount of MDA and 4-hydroxyalkenal produced. One molecule of either MDA or 4-hydroxyalkenal reacts with two molecules of reagent R1 to yield a stable chromophore with maximal absorbance at 586 nm.8,9 The detection threshold, based on 10 independent blank measurements, was 0.1 nmol/mL. When the same experiments were performed over a 10-d period under the same experimental conditions, with standard concentrations ranging from 0 –20 mm/mL and stock solutions of 10 mm/mL of
TABLE II. THE VALUES OF NBT REDUCTION (OPTICAL DENSITY) OF NEUTROPHILS AFTER INCUBATION WITH INTRALIPID, VASOLIPID, AND STRUCTOLIPID AT THREE DIFFERENT CONCENTRATIONS (0.33, 3.33, 33.33 MG/ML) Lipid concentration
Resting neutrophils (n 5 18) Stimulated neutrophils (n 5 18)
Intralipid Vasolipid Structolipid Intralipid Vasolipid Structolipid
* Different from control at p , 0.001.
Control (PMN alone)
0.33 (mg/mL)
3.33 (mg/mL)
33.33 (mg/mL)
0.405 6 0.09 0.399 6 0.10 0.401 6 0.10 0.795 6 0.18 0.788 6 0.20 0.779 6 0.21
0.336 6 0.08* 0.340 6 0.08* 0.330 6 0.10* 0.673 6 0.20* 0.665 6 0.21* 0.659 6 0.21*
0.269 6 0.08* 0.273 6 0.07* 0.255 6 0.08* 0.577 6 0.18* 0.564 6 0.17* 0.566 6 0.18*
0.211 6 0.06* 0.218 6 0.08* 0.202 6 0.07* 0.482 6 0.15* 0.467 6 0.12* 0.475 6 0.15*
PEROXIDATION OF IV FAT EMULSIONS AND EFFECT OF VITAMIN E
361
TABLE III. NEUTROPHILS-INDUCED LIPID PEROXIDATION OF THREE FAT EMULSIONS MEASURED AS MDA AND 4-HYDROXYALKENAL (mm/mL) AND ITS PREVENTION BY VITAMIN E (10 mm/mL)
1 h (n 5 10)
Resting neutrophils Stimulated neutrophils
2 h (n 5 10)
Resting neutrophils Stimulated neutrophils
without vitamin with vitamin E without vitamin with vitamin E without vitamin with vitamin E without vitamin with vitamin E
E E E E
Control (PMN alone)
Intralipid (0.33 mg/mL)
Vasolipid (0.33 mg/mL)
Structolipid (0.33 mg/mL)
1.34 6 0.72 0.44 6 0.51† 3.15 6 1.12 1.09 6 0.78† 1.57 6 0.79 0.93 6 0.75† 4.35 6 1.33 3.37 6 1.52†
9.39 6 2.34*‡ 5.78 6 1.91†‡ 14.58 6 3.44*‡ 10.05 6 3.49†‡ 12.20 6 2.15*‡ 7.67 6 1.80†‡ 17.14 6 3.02*‡ 10.52 6 1.64†‡
4.72 6 2.09*‡ 2.89 6 1.86†‡ 8.66 6 2.96*‡ 6.19 6 2.91†‡ 6.29 6 1.73*‡ 3.40 6 1.62†‡ 9.82 6 1.76*‡ 6.69 6 2.01†‡
2.83 6 1.19*‡ 1.58 6 1.67†‡ 6.67 6 2.29*‡ 4.11 6 2.32†‡ 4.12 6 1.90*‡ 2.09 6 1.12†‡ 7.39 6 1.68*‡ 4.93 6 1.51†‡
* Different from control at p , 0.001. † Different from without vitamin E at p , 0.001. ‡ Different between three types of lipid at p , 0.05.
the corresponding acetals stored at 4°C, the SEM values obtained were lower than 5%. Heparinized venous blood was obtained from 16 healthy donors (age 18 – 60 y). The preparation of PMN was similar to that we used for the NBT test, but the cells were suspended in 20 mmol/mL tris-HCl buffer (pH 5 7.4) instead of Eagle’s medium at a density of 5 3 106 cells/mL. The phagocyte suspension (0.5 mL, 0.5 3 106 cells/mL) was mixed on a plate with each of three different types of fat emulsions which were the same as in the NBT test (0.15 mL, 0.33 mg/mL), and the experiments were performed without as well as with vitamin E (a-tocopherol, 0.045 mL, 10 mm/mL). We stimulated the PMN as in the NBT test, and incubated the plate for 60 and 120 min at 37°C in a shaking device. In order to observe the kinetics of lipid peroxidation of fat emulsions, we incubated the
PMN from eight donors with three different types of fat emulsions for 1, 2, 4, 6, 8, and 10 h to measure the content of MDA and 4-hydroxyalkenal at different times. We chose five different concentrations of vitamin E (2.5, 5, 8, 10, and 15 mm/mL) mixed with three different types of fat emulsions, and measured the MDA and 4-hydroxyalkenal after 6 h of incubation. After incubation, 0.2 mL of sample were added to clean glass tubes containing 0.65 mL of R1, and the solutions were mixed thoroughly and then added 0.15 mL of R2 (15.4 mol methanesulfonic acid). The tubes were then closed with tight stoppers, and incubated at 45°C in a water bath for 40 min. After that, the cloudy sample was centrifuged at 15 000 g for 10 min. The clear supernatant was cooled on ice and used for the measurements at 586 nm. We used Tris-HCl buffer as a blank control and PMN alone as the control, and we also measured the MDA and 4-hydroxyalkenal in the three fat emul-
FIG. 1. Kinetics of lipid peroxidation with Intralipid, Vasolipid, and Structolipid after 1, 2, 4, 6, 8, and 10 h of incubation with unstimulated neutrophils from eight healthy donors (Means 6 SD).
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PEROXIDATION OF IV FAT EMULSIONS AND EFFECT OF VITAMIN E
sions to evaluate the lipid peroxide content of these fat emulsions during storage. Statistical Analysis Data were presented as the mean 6 SD and analyzed by analysis of variance (ANOVA) using Sigmastat software (Jandel Scientific, San Rafael, CA, USA). Statistical significance was predetermined as P , 0.05. RESULTS
Effect of Different Fat Emulsions on NBT Reduction The mean values of NBT reduction by PMN incubated with the three types of fat emulsions at three different concentrations are given in Table II. The results showed that the values of NBT reduction by PMN at rest were significantly decreased with three fat emulsions compared with those without fat emulsions (P , 0.001). After adding heat-killed Candida albicans, the decrease in the values of NBT reduction by PMN with fat emulsions also was statistically significant (P , 0.001) (Table II). The lipid-induced decrease in the values of NBT reduction appeared to be dose dependent (r 5 0.78, P , 0.01). The values of NBT reductions by PMN stimulated by Candida albicans were significantly higher than corresponding that with resting PMN (Table II, P , 0.001). There was no statistically significant difference between the effects of the three different types of fat emulsions in the values of NBT reductions of resting or stimulated PMN (Table II, P . 0.05). Lipid Peroxidation Mean values of lipid peroxidation by the three different types of fat emulsions are shown in Table III. We used the PMN alone as a control, and our results showed that lipid peroxidation increased significantly in the presence of each of the three different types of fat emulsions (P , 0.001, Table III). As shown here, there are significantly different values for lipid peroxidation with the three types of fat emulsions after 1 and 2 h of incubation (P , 0.001). The values with Intralipid were higher than with the other lipids. Lipid peroxidation with Structolipid was the lowest. The MDA and 4-hydroxyalkenal levels in each of the three different types of fat emulsions alone (without PMN) were very low and were nearly equal to the values of the blank control (0.072 mm/mL for Intralipid; 0.071 mm/mL for Vasolipid; 0.070 mm/mL for Structolipid versus 0.073 mm/mL for the blank control). The lipid peroxidation was significantly increased when PMN was stimulated by heat-killed Candida albicans (P , 0.001), and the values also increased after 2 h of incubation (Table III). Our results showed that the lipid peroxidation reached the maximum at 6 h of incubation in vitro. The values with Structolipid were higher than with Vasolipid after 4 h of incubation (Fig. 1). Our results also demonstrated that the lipid peroxidation with three different types of fat emulsions was decreased after adding vitamin E. There were statistically significant differences between the values with and without vitamin E (P , 0.001). With vitamin E, the lipid peroxidation of the three types of fat emulsions decreased, and the values were lower at all incubation times (Fig. 2). The lipid peroxidation of the fat emulsions was strongly inhibited by vitamin E, and the inhibition was concentration dependent (Fig. 3). This inhibitory action of vitamin E was similar with the three different types of fat emulsions (Fig. 3). The rate of inhibition was not linear, however, and 70 – 80% inhibition of lipid peroxidation generation was observed at a concentration of vitamin E of about 8 mm/mL (Fig. 3). DISCUSSION
When phagocytes such as neutrophils or macrophages are stimulated by microorganism or other stimuli, they become acti-
FIG. 2. Changes in lipid peroxidation with Intralipid, Vasolipid, and Structolipid after 1, 2, 4, 6, 8, and 10 h of incubation with unstimulated neutrophils from eight healthy donors. The experiments were performed with and without vitamin E (Means 6 SD).
vated with an increased oxygen metabolism as a result. This respiratory burst by neutrophils is characterized by marked changes in oxygen metabolism that result in increased production of superoxide anions (O1 2 ). The chemical reactivity of superoxide anions may be limited, but in the presence of transition metal ions, the partially reduced forms of superoxide, such as hydrogen peroxide (H2O2) and the hydroxyl radical (OH1), might possibly initiate lipid peroxidation under physiological conditions.10 Although neutrophil-generated reactive oxygen metabolites are necessary for the antimicrobial defense system, these free radicals can also cause damage to the neutrophil itself and to surrounding tissues.11,12 The occurrence of the superoxide anions can be mea-
PEROXIDATION OF IV FAT EMULSIONS AND EFFECT OF VITAMIN E
363
FIG. 3. Relationship between lipid peroxidation and different concentrations of vitamin E after 6 h incubation with Intralipid, Vasolipid, and Structolipid with unstimulated neutrophils from eight healthy donors (Means 6 SD).
sured by the NBT test.5–7 Our results indicated that in vitro exposure of human PMN to three different types of fat emulsions decreased NBT reduction. A dose-response pattern was seen when different concentrations of fat emulsions were added to the PMN in vitro. The decreased NBT reduction with the fat emulsions in the present study was similar to that observed in our earlier study.6 The mechanism behind this finding was then explained by the lipid causing alterations in the cell membranes of neutrophils and resulting in decreased production of superoxide anions along with other decreased phagocyte functions. Another explanation for the observed low values of NBT reduction might be that the superoxide anions reacted with lipids instead of with the NBT. Harmful effects of free radical-mediated lipid peroxidation in patients have been previously proposed.13–16 Several different analytic techniques have been investigated for measuring lipid peroxidation and much effort has been devoted to sample preparation and assay validation.8,9,17 In the present study, we have demonstrated that lipid peroxidation increased in the presence of three different fat emulsions. Intralipid is more prone to become peroxidized than the other fat emulsions. This could be related to its higher content of v-6 polyunsaturated fatty acids. Vasolipid, a physical mixture of 50% MCT and 50% LCT, is less peroxidized
than Intralipid. This may be due to its lower content of polyunsaturated fatty acids. Structolipid, incorporating esterification of LCT and MCT on a glycerol backbone, showed the lowest degree of lipid peroxidation at 1 and 2 h of incubation, but after that time, its peroxidation increased and was even higher than that of Vasolipid, and it remains to be determined whether or not this phenomenon is a result of the structural feature of this lipid. Vitamin E, a lipid-soluble vitamin, occurs in nature in eight forms, i.e., a-, b-, d-, and g-tocopherols, and a-, b-, d-, and g-tocotrienols. Of these forms, a-tocopherol is the most biologically active tocopherol isomer.18 The most widely accepted biologic function of vitamin E is its antioxidant property. Vitamin E is a chain-breaking lipid-soluble antioxidant in the biologic membrane, where it contributes to membrane stability. It protects critical cellular structures against damage from oxygen-free radicals and reactive products of lipid peroxidation.19,20 Most of the commercial fat emulsions are made from soybean oil in which tocopherols are natural ingredients. They are supposed to act as antioxidants in the fat emulsions. Several studies have demonstrated that the tocopherol content differs between different commercial fat emulsions, and that intravenous fat emulsions
364
PEROXIDATION OF IV FAT EMULSIONS AND EFFECT OF VITAMIN E
peroxidize during storage.21–23 We measured the MDA and 4hydroxyalkenal in three different types of fat emulsions, and the results indicated that little lipid peroxidation had occurred in them. This means that the amounts of tocopherols in these fat emulsions are sufficient to inhibit peroxidation. However, as shown here, in the presence of PMN, especially PMN stimulated by heat-killed Candida albicans, the lipid peroxidation was significantly increased due to the production of large amounts of free radicals. That Intralipid was found to be more prone to become peroxidized, maybe due to its high levels of g-tocopherol relative to a-tocopherol; it, therefore, has less antioxidant effect.21,23 Hence, from a theoretical point of view, it could be reasonable to increase the content of a-tocopherol for better protection of fat emulsions against lipid peroxidation. Our results demonstrated that the lipid peroxidation by stimulated PMN in the presence of fat emulsions can be effectively inhibited by adding vitamin E in vitro, and the
inhibition was concentration dependent. The optimal concentration of vitamin E was at about 8 –10 mm/mL in vitro. SUMMARY
In conclusion, fat emulsions used for parenteral nutrition increased lipid peroxidation by stimulated PMN in vitro. This increased lipid peroxidation could be overcome by adding vitamin E to these fat emulsions. However, it is not clear whether patients receiving parenteral nutrition, might show increased lipid peroxidation, and further studies are needed before a possible clinical significance of lipid peroxidation can be established. ACKNOWLEDGMENTS
Dr. Wu Guo Hao was, at the time of the study, a postdoctoral research fellow from Zhong Shan Hospital, Shanghai Medical University. He was supported by a grant from Phamacia & Upjohn, Stockholm, Sweden.
REFERENCES 1. Goode HF, Cowley HC, Kalker BE, et al. Decreased antioxidant status and increased lipid peroxidation in patients with septic shock and secondary organ dysfunction. Crit Care Med 1995;23:646 2. McCord JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 1985;312:159 3. Richard C, Lemonnier F, Thibault M, et al. Vitamin E deficiency and lipoperoxidation during adult respiratory distress syndrome. Crit Care Med 1990;18:4 4. Nordenstro¨m J, Jarstrand C, Wiernik A. Decreased chemotactic and random migration of leukocytes during Intralipid infusion. Am J Clin Nutr 1979;32:2416 5. Jarstrand C, Angelin B, Einarsson K. Decreased nitroblue tetrazolium reduction of granulocytes in type IV hyperlipoproteinemia. J Lab Clin Med 1979;94:897 6. Jarstrand C, Berghem L, Lahnborg G. Human granulocyte and reticuloendothelial system function during Intralipid infusion. JPEN 1978; 2:663 7. Urban T, Jarstrand C. Selenium effects on human neutrophilic granulocyte function in vitro. Immunopharmacology 1986;12:167 8. Esterbauer H, Cheeseman KH. Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Meth Enzymol 1990;186:407 9. Janero DR. Malonaldehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic Biol Med 1990;9:515 10. Rosen GM, Pou S, Ramos CL, et al. Free radicals and phagocytic cells. FASEB J 1995;9:200 11. Moslen MT. Reactive oxygen species in normal physiology, cell injury and phagocytosis. In: Armstrong D, ed. Free radicals in dignostic medicine. New York: Plenum Press, 1994:85
12. Fridovich I. Superoxide dismutase: an adaptation to a paramagnetic gas. J Biol Chem 1989;264:7761 13. Gossum AV, Shariff R, Lemoyne M, et al. Increased lipid peroxidation after lipid infusion as measured by breath pentane output. Am J Clin Nutr 1988;48:1394 14. Pitka¨nen O, Hallman M, Andersson S. Generation of free radicals in lipid emulsion used in parenteral nutrition. Pediatr Res 1991;29:56 15. Pitka¨nen O. Peroxidation of lipid emulsions: a hazard for the premature infant receiving parenteral nutrition. Free Radic Biol Med 1992; 13:239 16. Helbock H, Motchnik PA, Ames BN. Toxic hydroperoxides in intravenous lipid emulsions used in preterm infants. Pediatrics 1993;91:83 17. Pitka¨nen OM, Hallman M, Andersson SM. Determination of ethane and pentane in free oxygen radical-induced lipid peroxidation. Lipids 1989;24:157 18. Meydani M. Vitamin E. Lancet 1995;345:170 19. Kanno T, Utsumi T, Kobuchi H, et al. Inhibition of stimulus-specific neutrophil superoxide generation by alpha-tocopherol. Free Radic Res 1994;22:431 20. Kanno T, Utsumi T, Takehara Y, et al. Inhibition of neutrophilsuperoxide generation by a-tocopherol and coenzyme Q. Free Tad Res 1996;24:281 21. Traber MG, Carpentier YA, Kayden HJ, et al. Alternations in plasma and tocopherol concentrations in response to intravenous infusion of lipid emulsions in humans. Metabolism 1993;42:701 22. Steger PJK, Muhlebach SF. In vitro oxidation of IV lipid emulsions in different all-in-one admixture bags assessed by an iodometric assay and gas-liquid chromatography. Nutrition 1997;13:133 23. Steger PJK, Muhlebach SF. Lipid peroxidation of IV lipid emulsions in TPN bags: the influence of tocopherols. Nutrition 1998;14:179
Nutrition Vol. 15, No. 5, 1999
Phagocyte-Induced Lipid Peroxidation of Different Intravenous Fat Emulsions and Counteractive Effect of Vitamin E GUO HAO WU, MD, PHD,*† CONNIE JARSTRAND, MD, PHD,* AND ¨ RGEN NORDENSTRO ¨ M, MD, PHD† JO From the Departments of *Clinical and Oral Bacteriology and †Surgery, Huddinge University Hospital, Karolinska Institute, Huddinge, Sweden Date accepted: 8 July 1998 ABSTRACT
Unsaturated fatty acids, a major component of fat emulsions used in parenteral nutrition, are prone to peroxidation which is an important feature of oxygen-associated tissue damage. We used the nitroblue tetrazolium (NBT) reduction test to measure the production of superoxide radicals by stimulated polymorphonuclear neutrophils (PMN) in the presence of different fat emulsions: Intralipid (containing 100% long-chain triacylglycerols, LCT), Vasolipid (a physical mixture of 50% LCT and 50% mediumchain triacylglycerols, MCT) and Structolipid (structured triacylglycerols containing 63% LCT and 37% MCT). We measured the amount of malonaldehyde (MDA) and 4-hydroxyalkenal to determine the lipid peroxidation of the three fat emulsions in the presence of stimulated neutrophils. Further, we investigated the role of vitamin E (a-tocopherol) in preventing lipid peroxidation in vitro. The results showed that the values of NBT reduction of PMN were significantly decreased in each of the three fat emulsions and that increasing concentrations of fat emulsions were associated with decreased values of NBT reductions, in a dose-dependent way (P , 0.001). There were, however, no statistically significant differences between the values of the three different types of fat emulsions (P . 0.05). Lipid peroxidation increased significantly in the presence of all three types of fat emulsions, and was more pronounced for Intralipid than for Vasolipid and Structolipid after 1 and 2 h of incubation with resting as well as with stimulated phagocytes. The increased lipid peroxidation of the fat emulsions was markedly reduced by vitamin E, and the inhibition was concentration dependent. In conclusion, lipid peroxidation in vitro is more pronounced when PMNs are incubated with fat emulsions. This increase in lipid peroxidation can be reduced by adding vitamin E to the fat emulsions. Nutrition 1999;15:359 –364. ©Elsevier Science Inc. 1999 Key words: free radicals, structured triacylglycerols, lipid peroxidation, vitamin E (a-tocopherol), fat emulsion, neutrophil
INTRODUCTION
Intravenous fat emulsions are used as a source of energy and essential fatty acids. Despite unquestionable nutritional advantages, the infusion of fat emulsions rich in polyunsaturated fatty acids (PUFAs) may become peroxidized in vivo. Lipid peroxidation, generated by the action of free radicals, is enhanced in a variety of clinical conditions such as septic shock, adult respiratory distress syndrome, or ischemia-reperfusion injury.1–3 Earlier studies from our group have shown that the degree of hypertriglyceridemia during Intralipid infusion in patients correlated with the impairment of leucocyte function.4 Furthermore, granulocytes
from patients with type IV hyperlipoproteinemia showed a decreased NBT reduction.5 The hypothesis was that neutrophil-produced oxygen radicals can cause lipid peroxidation of fat emulsions. The purpose of the present study was 1) to measure the production of superoxide anions as the NBT reduction by stimulated neutrophils in the presence of fat emulsions, 2) to measure lipid peroxidation of fat emulsions in the presence of stimulated and unstimulated neutrophils, 3) to evaluate the role of vitamin E in preventing lipid peroxidation in this in vitro system, and 4) to compare three different fat emulsions with respect to production of superoxide anions and lipid peroxidation.
Correspondence to: Jo¨rgen Nordenstro¨m, MD, PhD, Department of Surgery, K53, Huddinge University Hospital, S-141 86 Huddinge, Sweden. E-mail: [email protected]
Nutrition 15:359 –364, 1999 ©Elsevier Science Inc. 1999 Printed in the USA. All rights reserved.
0899-9007/99/$20.00 PII S0899-9007(99)00052-0
360
PEROXIDATION OF IV FAT EMULSIONS AND EFFECT OF VITAMIN E TABLE I. COMPOSITION OF INTRALIPID, VASOLIPID, AND STRUCTOLIPID
Content/1000 mL Soybean oil (g) Structured triacylglycerol (g) Coconut oil (g) Egg phospholipid (g) Glycerol (USP) (g) Water for injection (mL) Mean molecular weight (approx.) Proportion of LCT:MCT (wt:wt) Fatty acids composition (wt:wt) Caprylic acid, 8:0 Capric acid, 10:0 Palmitric acid, 16:0 Stearic acid, 18:0 Oleic acid, 18:1 Linoleic acid, 18:2 a-linolenic acid, 18:3 Other Total
Intralipid
Vasolipid
Structolipid
200 — — 12 22.5 1000 865 100:0
100 — 100 12 25 1000 634 50:50
— 200 — 12 22.5 1000 683 63:37
— — 13 4 22 52 8 1 100
43 18 4 2 9 20 2 2 100
27 10 7 3 13 33 5 2 100
By this reduction, yellow soluble NBT is converted to dark blue formazan that can be measured by a spectrophotometric method. It reflects the oxidative metabolism of the cells known to be enhanced during phagocytosis (respiratory burst). In the present study, we used a modification of the conventional NBT method, using only 10% of the sample volume. All the samples were exposed to three different types of fat emulsions, Intralipid 20% (100% long-chain triacylglycerols, Pharmacia & Upjohn AB, Stockholm, Sweden); Vasolipid 20% (physical mixture of 50% long-chain triacylglycerols [LCT] and 50% medium-chain triacylglycerols [MCT], B. Braun Medical AB, Germany); and Structolipid 20% (containing 63% LCT and 37% MCT, Pharmacia & Upjohn AB). The composition of fat emulsions are presented in Table I. The PMN suspension (0.1 mL) were mixed on a microtite plate with 0.03 mL of each of the fat emulsions in three different concentrations (0.33, 3.33, 33.33 mg/mL). Experiments were performed with or without stimulation of the cells by heat-killed Candida albicans (0.001 mL, 3 3 108 Candida albicans/mL), then 0.05 mL NBT solution was added and the plate was covered with adhesive tape and incubated for 60 min at 37°C in a shaking device. The reaction was then stopped by adding 0.1 mL 0.5 N HCl. The plate was centrifuged at 1500 rpm. for 10 min and the supernatant was removed. A total of 0.2 mL of dimethyl-sulfoxide (DMSO) was added to each well to extract the formazan. The plate was covered again and placed on an agitation table for 15–20 min to accelerate the extraction of the formazan. DMSO alone was added as a blank to the first well of each plate and, finally, the optical densities (OD) were recorded spectrophotometrically at 570 nm.5–7
MATERIALS AND METHODS
PMN Separation Venous heparinized blood (40 –50 mL) was obtained from each of 18 healthy donors (age 18 – 65 y). The polymorphonuclear neutrophils (PMN) were separated on a sodium metrizoate (Sigma Lot 35H-1406) and methylcellulose (Dow Chemical Company, Lot, MN, USA) column by the method of Boyum and suspended in pH-adjusted (7.2–7.4) Eagle’s medium at a density of 5 3 106 cells/mL. NBT Reduction Test Nitroblue tetrazolium (NBT) is an electron acceptor used for indirect detection of the production of superoxide by stimulated PMN, as outlined in the following equation: NBT 1 O1 2
OO 3 formazan 1 O2
(1)
Lipid Peroxidation We used a new colorimetric assay, LPO-586 (OXIS-21012, British Bio-technology Products Ltd., England), to measure the reaction of a chromogenic reagent R1 (10.3 mmol/mL N-methyl2-phenylindole, in acetonitrile) with malonaldehyde (MDA) and 4-hydroxyalkenal at 45°C. MDA and 4-hydroxyalkenal are formed in vitro as breakdown products of peroxidized polyunsaturated fatty acids, and the extent of peroxidation is quantitatively related to the amount of MDA and 4-hydroxyalkenal produced. One molecule of either MDA or 4-hydroxyalkenal reacts with two molecules of reagent R1 to yield a stable chromophore with maximal absorbance at 586 nm.8,9 The detection threshold, based on 10 independent blank measurements, was 0.1 nmol/mL. When the same experiments were performed over a 10-d period under the same experimental conditions, with standard concentrations ranging from 0 –20 mm/mL and stock solutions of 10 mm/mL of
TABLE II. THE VALUES OF NBT REDUCTION (OPTICAL DENSITY) OF NEUTROPHILS AFTER INCUBATION WITH INTRALIPID, VASOLIPID, AND STRUCTOLIPID AT THREE DIFFERENT CONCENTRATIONS (0.33, 3.33, 33.33 MG/ML) Lipid concentration
Resting neutrophils (n 5 18) Stimulated neutrophils (n 5 18)
Intralipid Vasolipid Structolipid Intralipid Vasolipid Structolipid
* Different from control at p , 0.001.
Control (PMN alone)
0.33 (mg/mL)
3.33 (mg/mL)
33.33 (mg/mL)
0.405 6 0.09 0.399 6 0.10 0.401 6 0.10 0.795 6 0.18 0.788 6 0.20 0.779 6 0.21
0.336 6 0.08* 0.340 6 0.08* 0.330 6 0.10* 0.673 6 0.20* 0.665 6 0.21* 0.659 6 0.21*
0.269 6 0.08* 0.273 6 0.07* 0.255 6 0.08* 0.577 6 0.18* 0.564 6 0.17* 0.566 6 0.18*
0.211 6 0.06* 0.218 6 0.08* 0.202 6 0.07* 0.482 6 0.15* 0.467 6 0.12* 0.475 6 0.15*
PEROXIDATION OF IV FAT EMULSIONS AND EFFECT OF VITAMIN E
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TABLE III. NEUTROPHILS-INDUCED LIPID PEROXIDATION OF THREE FAT EMULSIONS MEASURED AS MDA AND 4-HYDROXYALKENAL (mm/mL) AND ITS PREVENTION BY VITAMIN E (10 mm/mL)
1 h (n 5 10)
Resting neutrophils Stimulated neutrophils
2 h (n 5 10)
Resting neutrophils Stimulated neutrophils
without vitamin with vitamin E without vitamin with vitamin E without vitamin with vitamin E without vitamin with vitamin E
E E E E
Control (PMN alone)
Intralipid (0.33 mg/mL)
Vasolipid (0.33 mg/mL)
Structolipid (0.33 mg/mL)
1.34 6 0.72 0.44 6 0.51† 3.15 6 1.12 1.09 6 0.78† 1.57 6 0.79 0.93 6 0.75† 4.35 6 1.33 3.37 6 1.52†
9.39 6 2.34*‡ 5.78 6 1.91†‡ 14.58 6 3.44*‡ 10.05 6 3.49†‡ 12.20 6 2.15*‡ 7.67 6 1.80†‡ 17.14 6 3.02*‡ 10.52 6 1.64†‡
4.72 6 2.09*‡ 2.89 6 1.86†‡ 8.66 6 2.96*‡ 6.19 6 2.91†‡ 6.29 6 1.73*‡ 3.40 6 1.62†‡ 9.82 6 1.76*‡ 6.69 6 2.01†‡
2.83 6 1.19*‡ 1.58 6 1.67†‡ 6.67 6 2.29*‡ 4.11 6 2.32†‡ 4.12 6 1.90*‡ 2.09 6 1.12†‡ 7.39 6 1.68*‡ 4.93 6 1.51†‡
* Different from control at p , 0.001. † Different from without vitamin E at p , 0.001. ‡ Different between three types of lipid at p , 0.05.
the corresponding acetals stored at 4°C, the SEM values obtained were lower than 5%. Heparinized venous blood was obtained from 16 healthy donors (age 18 – 60 y). The preparation of PMN was similar to that we used for the NBT test, but the cells were suspended in 20 mmol/mL tris-HCl buffer (pH 5 7.4) instead of Eagle’s medium at a density of 5 3 106 cells/mL. The phagocyte suspension (0.5 mL, 0.5 3 106 cells/mL) was mixed on a plate with each of three different types of fat emulsions which were the same as in the NBT test (0.15 mL, 0.33 mg/mL), and the experiments were performed without as well as with vitamin E (a-tocopherol, 0.045 mL, 10 mm/mL). We stimulated the PMN as in the NBT test, and incubated the plate for 60 and 120 min at 37°C in a shaking device. In order to observe the kinetics of lipid peroxidation of fat emulsions, we incubated the
PMN from eight donors with three different types of fat emulsions for 1, 2, 4, 6, 8, and 10 h to measure the content of MDA and 4-hydroxyalkenal at different times. We chose five different concentrations of vitamin E (2.5, 5, 8, 10, and 15 mm/mL) mixed with three different types of fat emulsions, and measured the MDA and 4-hydroxyalkenal after 6 h of incubation. After incubation, 0.2 mL of sample were added to clean glass tubes containing 0.65 mL of R1, and the solutions were mixed thoroughly and then added 0.15 mL of R2 (15.4 mol methanesulfonic acid). The tubes were then closed with tight stoppers, and incubated at 45°C in a water bath for 40 min. After that, the cloudy sample was centrifuged at 15 000 g for 10 min. The clear supernatant was cooled on ice and used for the measurements at 586 nm. We used Tris-HCl buffer as a blank control and PMN alone as the control, and we also measured the MDA and 4-hydroxyalkenal in the three fat emul-
FIG. 1. Kinetics of lipid peroxidation with Intralipid, Vasolipid, and Structolipid after 1, 2, 4, 6, 8, and 10 h of incubation with unstimulated neutrophils from eight healthy donors (Means 6 SD).
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PEROXIDATION OF IV FAT EMULSIONS AND EFFECT OF VITAMIN E
sions to evaluate the lipid peroxide content of these fat emulsions during storage. Statistical Analysis Data were presented as the mean 6 SD and analyzed by analysis of variance (ANOVA) using Sigmastat software (Jandel Scientific, San Rafael, CA, USA). Statistical significance was predetermined as P , 0.05. RESULTS
Effect of Different Fat Emulsions on NBT Reduction The mean values of NBT reduction by PMN incubated with the three types of fat emulsions at three different concentrations are given in Table II. The results showed that the values of NBT reduction by PMN at rest were significantly decreased with three fat emulsions compared with those without fat emulsions (P , 0.001). After adding heat-killed Candida albicans, the decrease in the values of NBT reduction by PMN with fat emulsions also was statistically significant (P , 0.001) (Table II). The lipid-induced decrease in the values of NBT reduction appeared to be dose dependent (r 5 0.78, P , 0.01). The values of NBT reductions by PMN stimulated by Candida albicans were significantly higher than corresponding that with resting PMN (Table II, P , 0.001). There was no statistically significant difference between the effects of the three different types of fat emulsions in the values of NBT reductions of resting or stimulated PMN (Table II, P . 0.05). Lipid Peroxidation Mean values of lipid peroxidation by the three different types of fat emulsions are shown in Table III. We used the PMN alone as a control, and our results showed that lipid peroxidation increased significantly in the presence of each of the three different types of fat emulsions (P , 0.001, Table III). As shown here, there are significantly different values for lipid peroxidation with the three types of fat emulsions after 1 and 2 h of incubation (P , 0.001). The values with Intralipid were higher than with the other lipids. Lipid peroxidation with Structolipid was the lowest. The MDA and 4-hydroxyalkenal levels in each of the three different types of fat emulsions alone (without PMN) were very low and were nearly equal to the values of the blank control (0.072 mm/mL for Intralipid; 0.071 mm/mL for Vasolipid; 0.070 mm/mL for Structolipid versus 0.073 mm/mL for the blank control). The lipid peroxidation was significantly increased when PMN was stimulated by heat-killed Candida albicans (P , 0.001), and the values also increased after 2 h of incubation (Table III). Our results showed that the lipid peroxidation reached the maximum at 6 h of incubation in vitro. The values with Structolipid were higher than with Vasolipid after 4 h of incubation (Fig. 1). Our results also demonstrated that the lipid peroxidation with three different types of fat emulsions was decreased after adding vitamin E. There were statistically significant differences between the values with and without vitamin E (P , 0.001). With vitamin E, the lipid peroxidation of the three types of fat emulsions decreased, and the values were lower at all incubation times (Fig. 2). The lipid peroxidation of the fat emulsions was strongly inhibited by vitamin E, and the inhibition was concentration dependent (Fig. 3). This inhibitory action of vitamin E was similar with the three different types of fat emulsions (Fig. 3). The rate of inhibition was not linear, however, and 70 – 80% inhibition of lipid peroxidation generation was observed at a concentration of vitamin E of about 8 mm/mL (Fig. 3). DISCUSSION
When phagocytes such as neutrophils or macrophages are stimulated by microorganism or other stimuli, they become acti-
FIG. 2. Changes in lipid peroxidation with Intralipid, Vasolipid, and Structolipid after 1, 2, 4, 6, 8, and 10 h of incubation with unstimulated neutrophils from eight healthy donors. The experiments were performed with and without vitamin E (Means 6 SD).
vated with an increased oxygen metabolism as a result. This respiratory burst by neutrophils is characterized by marked changes in oxygen metabolism that result in increased production of superoxide anions (O1 2 ). The chemical reactivity of superoxide anions may be limited, but in the presence of transition metal ions, the partially reduced forms of superoxide, such as hydrogen peroxide (H2O2) and the hydroxyl radical (OH1), might possibly initiate lipid peroxidation under physiological conditions.10 Although neutrophil-generated reactive oxygen metabolites are necessary for the antimicrobial defense system, these free radicals can also cause damage to the neutrophil itself and to surrounding tissues.11,12 The occurrence of the superoxide anions can be mea-
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363
FIG. 3. Relationship between lipid peroxidation and different concentrations of vitamin E after 6 h incubation with Intralipid, Vasolipid, and Structolipid with unstimulated neutrophils from eight healthy donors (Means 6 SD).
sured by the NBT test.5–7 Our results indicated that in vitro exposure of human PMN to three different types of fat emulsions decreased NBT reduction. A dose-response pattern was seen when different concentrations of fat emulsions were added to the PMN in vitro. The decreased NBT reduction with the fat emulsions in the present study was similar to that observed in our earlier study.6 The mechanism behind this finding was then explained by the lipid causing alterations in the cell membranes of neutrophils and resulting in decreased production of superoxide anions along with other decreased phagocyte functions. Another explanation for the observed low values of NBT reduction might be that the superoxide anions reacted with lipids instead of with the NBT. Harmful effects of free radical-mediated lipid peroxidation in patients have been previously proposed.13–16 Several different analytic techniques have been investigated for measuring lipid peroxidation and much effort has been devoted to sample preparation and assay validation.8,9,17 In the present study, we have demonstrated that lipid peroxidation increased in the presence of three different fat emulsions. Intralipid is more prone to become peroxidized than the other fat emulsions. This could be related to its higher content of v-6 polyunsaturated fatty acids. Vasolipid, a physical mixture of 50% MCT and 50% LCT, is less peroxidized
than Intralipid. This may be due to its lower content of polyunsaturated fatty acids. Structolipid, incorporating esterification of LCT and MCT on a glycerol backbone, showed the lowest degree of lipid peroxidation at 1 and 2 h of incubation, but after that time, its peroxidation increased and was even higher than that of Vasolipid, and it remains to be determined whether or not this phenomenon is a result of the structural feature of this lipid. Vitamin E, a lipid-soluble vitamin, occurs in nature in eight forms, i.e., a-, b-, d-, and g-tocopherols, and a-, b-, d-, and g-tocotrienols. Of these forms, a-tocopherol is the most biologically active tocopherol isomer.18 The most widely accepted biologic function of vitamin E is its antioxidant property. Vitamin E is a chain-breaking lipid-soluble antioxidant in the biologic membrane, where it contributes to membrane stability. It protects critical cellular structures against damage from oxygen-free radicals and reactive products of lipid peroxidation.19,20 Most of the commercial fat emulsions are made from soybean oil in which tocopherols are natural ingredients. They are supposed to act as antioxidants in the fat emulsions. Several studies have demonstrated that the tocopherol content differs between different commercial fat emulsions, and that intravenous fat emulsions
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PEROXIDATION OF IV FAT EMULSIONS AND EFFECT OF VITAMIN E
peroxidize during storage.21–23 We measured the MDA and 4hydroxyalkenal in three different types of fat emulsions, and the results indicated that little lipid peroxidation had occurred in them. This means that the amounts of tocopherols in these fat emulsions are sufficient to inhibit peroxidation. However, as shown here, in the presence of PMN, especially PMN stimulated by heat-killed Candida albicans, the lipid peroxidation was significantly increased due to the production of large amounts of free radicals. That Intralipid was found to be more prone to become peroxidized, maybe due to its high levels of g-tocopherol relative to a-tocopherol; it, therefore, has less antioxidant effect.21,23 Hence, from a theoretical point of view, it could be reasonable to increase the content of a-tocopherol for better protection of fat emulsions against lipid peroxidation. Our results demonstrated that the lipid peroxidation by stimulated PMN in the presence of fat emulsions can be effectively inhibited by adding vitamin E in vitro, and the
inhibition was concentration dependent. The optimal concentration of vitamin E was at about 8 –10 mm/mL in vitro. SUMMARY
In conclusion, fat emulsions used for parenteral nutrition increased lipid peroxidation by stimulated PMN in vitro. This increased lipid peroxidation could be overcome by adding vitamin E to these fat emulsions. However, it is not clear whether patients receiving parenteral nutrition, might show increased lipid peroxidation, and further studies are needed before a possible clinical significance of lipid peroxidation can be established. ACKNOWLEDGMENTS
Dr. Wu Guo Hao was, at the time of the study, a postdoctoral research fellow from Zhong Shan Hospital, Shanghai Medical University. He was supported by a grant from Phamacia & Upjohn, Stockholm, Sweden.
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