Intralipid

ABSTRACT. The aim of this preliminary study was to assess the possible presence of cholesterol oxidation products in 2 IV lipidic emulsions with different fatty acid compositions (longchain triglyceride, medium-chain triglyceride-long-chain triglyceride). Because these emulsions are currently used in neonatal parenteral nutrition, their direct venous introduction might be potentially dangerous because of the possible atherogenic role of cholesterol oxidation products. The emulsions were analyzed when bottles were opened (ie, under normal condition of administration) and after a 12-hour direct experimental exposure to air and high (90%) oxygen concentrations. 7-Ketocholesterol and 5[alpha]-epoxycholesterol were chosen as markers of cholesterol oxidation and detected by gas chromatographymass spectrometry of their trimethylsilyl ethers. The detected amounts were always very low and in some cases below the detection limit of the analytical method for the 2 cholesterol oxidation products (COPs; 0.1 and 0.3 µg/g of extracted lipids). Immediately after opening the bottles, their concentrations were lower in the emulsions containing the higher amounts of polyunsaturated fatty acids. Experimental hyperoxic exposure generally determined only a mild increase in the content of cholesterol oxidation biomarker, and after exposure to oxygen, the amounts of COPs were slightly higher than after exposure to air. The results of the present study are undoubtedly reassuring for the safety of neonates, although caution is always required when drawing conclusions from in vitro data. (Journal of Parenteral and Enteral Nutrition 28:342-347, 2004)

IV lipid emulsions (ILEs) are routinely used in nutrition management of the critically ill premature newborn. Lipids, which supply the newborn with essential fatty acids and calories, are usually administered as vegetable oil-in-water emulsions, made isotonic with glycerol. Two types of lipid emulsions are currently used: ILEs with a prevalent polyunsaturated fatty acid (PUFAs) content (about 60%), derived from soybean oil, and ILEs composed of 50% medium-chain triglyceride (MCT) and 50% long-chain triglyceride (LCT).

Despite the fact that numerous advantages have been observed, parenteral administration of lipid emulsions has been related to possible health risks in newborns (hypertriglyceridemia, bilirubin toxicity, altered gas exchange and hemodynamics, unpaired antibacterial neutrophil function).1-4

It is likely that preterm newborns undergoing intensive care are exposed to a variety of additional factors and conditions that amplify the oxidative injury mediated by lipid peroxidation. These include hyperoxic exposure, insufficient activity of the protective systems, caused by the reduced supply and activity of cellular antioxidant and free radical scavengers (glutathione peroxidase, catalase, and superoxide dismutase), and increased substrate for lipid peroxidation, particularly PUFAs.5-7

The high amounts of PUFAs in lipid emulsions are exposed to peroxidative stress, which implies hydroperoxide generation.8 Hydroperoxides of PUFAs and their degradation products are reactive species with toxic, mutagenic, and cancerogenic effects.9 Despite the protective effect exerted by the addition of tocopherols, the stability and safety of ILEs is not guaranteed. In fact, several authors have detected the hydroperoxides of linoleic and linolenic acid or their degradation products ethane and pentane in ILEs.10,11

Nevertheless, little attention has been paid to the possible sterol oxidation in ILEs. Both cholesterol and phytosterols are generally present. Cholesterol is mainly the result of the use of egg phospholipids as emulsifying agents. It is subject to oxidation reactions, which may involve either an attack on its molecule by oxygen reactive species (direct oxidation) or an attack on its double bond by PUFA hydroperoxides (indirect oxidation or peroxidation). 7[alpha]-Hydroxycholesterol, 7[beta]-hydroxycholesterol, and 7-ketocholesterol are usually the main products of direct oxidation. As far as indirect oxidation is concerned, the main products of this reaction are cholesterol-5,6-epoxides, even though they may be further transformed into cholestan3[beta],5[alpha],6[beta]-triol by water at low pH values. 7-Ketocholesterol has been extensively used as a marker of the whole oxidation process.12,13 Phytosterols in ILEs result from the presence of soybean oil and might experience similar oxidation pathways, but the study of these processes has not been equally exhaustive.

Several in vitro and in vivo studies have specifically suggested that cholesterol oxidation products (COPs) play an important role in determining cytotoxicity in the vascular wall.14-18 Dietary cholesterol or venous infusion have little atherogenic or citotoxic effects compared with COPs. In arterial lesions COPs are partly derived from dietary sources. It has been demonstrated that they are absorbed in chylomicrons whose remnant fractions easily penetrate into arterial walls and are retained there.1 Although COPs are rapidly cleared from plasma,20,21 at the moment there is a substantial lack of knowledge about the threshold levels above which dietary COPs may play a detrimental role. Nevertheless, direct venous introduction of COPs by infusing ILEs could be potentially more dangerous. As far as phytosterol oxidation products are concerned, few biochemical studies on their biologic activity are available.

The extent of cholesterol oxidation in food depends on the composition of the food itself and on the production technologies that are used. Known factors enhancing COP formation in food processing are the presence of an oxygen-containing environment, high processing temperatures, and long heating times. Unsuitable storage conditions can also play an important role in the development of cholesterol oxidation.22-25

Similarly, the risk of cholesterol oxidation in IV emulsions might be particularly high when their PUFA content is high, especially if the emulsions are stored before infusion at room temperature and without protecting them from light exposure.8,9,26

After a previous randomized investigation in which significant amounts of 7-ketocholesterol had been detected in commercial milk formulas,27 this study is an attempt to investigate the possible presence of COPs in the two kind of ILEs currently used in neonatal parenteral nutrition, namely LCT and MCT/LCT. Thus, 7-ketocholesterol and 5a-epoxycholesterol were respectively chosen as markers of direct and indirect cholesterol oxidation, and their presence in ILEs was investigated by gas chromatography-mass spectrometry (GC-MS) of their trimethylsilyl ethers (TMS). Although ß-epoxycholesterol is often the major product of indirect oxidation pathways and would be a better choice for its monitoring, the lack of its pure standard led us to use its isomer. ILEs were analyzed as soon as bottles were opened, under normal condition of administration, and after a prolonged (12 hours) experimental direct exposure to air and high oxygen concentration (90%), in conditions that reflect administration to preterm newborns.

MATERIALS AND METHODS

Chemicals

Analytical grade solvents were supplied by Merck (NJ), absolute pyridine (over molecular sieves) by Fluka and sylon BFT (N,O-bis(TMS)trifluoroacetamide +1% trimethylchlorosilane) by Supelco Inc. and Diethyl ether was deperoxidized before analysis by molecular sieves (Deperox supplied by Fluka, Sigma-Aldrich, St. Louis, MO).28

Analytical Standards

5,6[alpha]-Epoxy-cholestan-3[beta]-ol(5[alpha]-epoxycholesterol),3[beta]-hydroxycholest-5-ene-7-one (7-ketocholesterol), cholest-5-ene-3[beta],19-diol (19-hydroxycholesterol) were supplied by Sigma Chemicals and 5[alpha]-cholestan-3[beta]-ol ([beta]-cholestanol) by Fluka, Sigma-Aldrich.

Samples

Two ILEs with different fatty acid composition were taken into consideration. The label of one of the emulsions (Intralipid 20% = LCT emulsion) reported that purified soybean oil accounted for 20% of the total formulation, whereas similar amounts (10%) of soybean oil and MCT triglycerides were reported in the second emulsion (Lipofundin 20% = LCT/MCT emulsion). The pH of both emulsions was near pH 7. Three samples of each emulsion were analyzed.

Air and Hyperoxic Environment

One bottle of each emulsion was opened and, in order to avoid possible analytical differences, 1.5 mL of emulsion were withdrawn by syringe from the top, from the middle, and from the bottom of the bottle respectively and transferred onto 2 bacterial culture polystyrene plates.29 Then, these plates were respectively exposed to a stream of air and oxygen at 36°C for 12 hours. Exposure was calculated as the mean time needed for neonatal plasma clearance of infused long-chain fatty acids.30 Air and oxygen acted on the plates at atmospheric pressure. The hyperoxic environment was created using direct oxygen source flow in a Hood cell in which plates and oximeter had been previously set. When an oxygen concentration of 90% was reached, the flow was stabilized.31

Lipid Extraction

CHCl^sub 3^ (5 mL) and 10 mL of CH^sub 3^OH were added to exactly 5 mL of emulsion, and the mixture was homogenized at 8000 rpm for 1 minute. Then, 5 mL more of CHCl^sub 3^ and 5 mL of water were added, carefully stirring with a glass rod for 30 seconds after each addition. The obtained mixture was poured into a 25-mL glass-stoppered cylinder and allowed to separate into 2 clear phases. The lower phase was collected in a 50-mL round-bottomed flask, and the solvent evaporated under vacuum at room temperature.32

Cold Saponification and Extraction of Nonsaponifiable Products

19-Hydroxycholesterol (2.3 µg) and a-cholestanol (56.3 µg) were respectively used as internal standard for COPs and sterol quantitative determination. They were added to 200 ± 20 mg (exactly weighed) of the extracted lipids in a 50-mL round-bottomed flask. Cold saponification was carried out in the dark by 10 mL KOH 1 N in methanol.33 Any warming of the alkaline methanolic solution was carefully avoided.34 Unsaponifiables were then extracted33 and evaporated to dryness in Rotavapor at 20°C.

Enrichment of COPs From Total Unsaponifiables by Solid-Phase Extraction (SPE)

The extracted unsaponifiables were dissolved in 3 mL of diethyl ether (peroxide free). One milliliter of this solution was evaporated to dryness under a stream of nitrogen. Then the residue was dissolved in 1 mL of hexane, and COPs were purified by SPE on a 200-mg silica cartridge.35

Derivatization

The oxysterol-containing acetone fraction was dried under a stream of nitrogen. Then 20 µL of dry pyridine and 80 µL of BFT were added and the mixture was allowed to stand overnight before GC-MS analysis.

GC-MS of COPs

GC-MS was used to verify peak assignment (total ion current mode) and to quantify (single ion monitoring mode) single COPs as TMS ethers. Pulsed splitless injection (injection pulsed pressure 21 psi, injection pulsed time 1 minute, splitless time 1 minute) was performed at 290°C in an HP 6890 GC System coupled with an HP 5973 quadrupole mass spectrometer. A 1 µL sample was injected onto a 30 m × 0.25 mm ID × 0.25 µm film thickness HP5MS fused silica capillary column at an oven temperature of 9O0C and a helium flow rate of 0.8 mL/minute. The oven temperature was kept at 90°C for 1 minute, and then it was programmed at 20°C/minute up to 280°C and at 4°C/minute to 300°C (kept for 16 minutes) under flow-controlled conditions (constant flow 0.8 mL/min). A 5-minute postrun at 310°C was then performed. The mass spectrometer interface temperature was set to 310°C. The temperature of the ion source was 230°C, electron energy 70 eV and quadrupole temperature 150°C. Recoveries were checked by running a solution of 7-ketocholesterol, [alpha]-epoxycholesterol, and 19-hydroxycholesterol standards through all experimental procedures. COPs recoveries were above than 90%.

A 3-level calibration was used for quantitative analysis. The standard solutions were run through all the purification procedures before GC-MS analysis.

Isolation and High-Resolution Gas Chromatography (HRGC) of the Sterol Fraction

Sterols were isolated from 1 mL of the residual unsaponifiable solution by preparative thin layer chromatography according to EC Regulation 2568/91.36 Their TMS derivatives were analyzed by HRGC chromatography and flame ionization detection (HRGC-FID).

Determination of Fatty Acid Composition

Four hundred microliters of the extracted lipids were dissolved in 4 mL of heptane in a stoppered centrifuge glass tube. Then 0.5 mL of 2 N KOH in methanol were added. After vigorous stirring for 30 seconds, the mixture was centrifuged and 1 µL of the upper layer was immediately analyzed by HRGC-FID, according to European Community Regulation 2568/91.

RESULTS

All investigated parameters are summarized in Tables I-III.

Both analyzed ILEs had fatty acid and sterol compositions which were consistent with the emulsion composition reported by the manufacturers (Table IV).

Three sample for each ILEs have been analyzed when the bottle were opened ("baseline" conditions); 3 other samples have been analyzed before and after experimental hyperoxic exposure. Table III shows the amounts of COPs detected in analyzed sample as average value ± SDs of 3 determinations. The detected amounts of cholesterol oxidation markers were always very low and in some cases below the detection limit of the analytical method for the 2 COPs (0.1 and 0.3 µg/g of extracted lipids).

When the bottles were opened ("baseline" conditions), in both emulsions the concentrations of 5[alpha]-epoxycholesterol were higher than the concentrations of 7-ketocholesterol. The concentrations of the detected COPs were lower in LCT than in the MCT/LCT ILE. Moreover, because the total sterol content of the MCT/LCT ILEs was 20 times lower (Table II), the ratio between detected COPs and cholesterol amounts was by far higher in MCT/LCTs than in LCT ILEs.

Experimental hyperoxic exposure generally determined a mild increase in the cholesterol oxidation biomarkers content in ILEs, but after exposure to oxygen the amounts of COPs were higher than after exposure to air (Table III). Nevertheless, in MCT/LCT ILE both 5[alpha]-epoxycholesterol and 7-ketocholesterol contents were slightly lower than in "baseline" conditions. The detected amounts of 5[alpha]-epoxycholesterol were always higher than the amounts of 7-ketocholesterol, even if high oxygen concentrations should favor direct cholesterol oxidation.

DISCUSSION

In the light of previous experimental studies, which took into consideration the involvement of COPs and their LDL-bound acyl esters in the initialization or enhancement of vascular wall damage,15,22,37,38 the results of the present study on the possible presence of COPs in ILEs are undoubtedly reassuring. Because the biologic activity of phytosterols oxides is not still proved, their possible presence in ILEs was not investigated in this study.

Despite the fact that there is a lack of clinical information about the threshold levels above which intake of "preformed" COPs plays negative effects on both adult and newborn, it seems feasible from the present study that the amount of preformed COPs introduced into the bloodstream of infants fed exclusively by IV nutrition is far below the levels of dietary COPs introduced by infants fed with some artificial formula milks.39 It is likely that, in the first days of life, IV infusion "vehiculate" to the newborn amount of 7-ketocholesterol, and thus of COPs, is very small (

However, comparison between the results obtained for the 2 investigated ILEs shows some differences both in "baseline" and in hyperoxic conditions.

The total sterol content in LCT ILEs was remarkably higher than in MCT/LCT ILEs, whereas the "baseline[b]" concentrations of the investigated COPs were lower in LCT than in MCT/LCT ILEs. Thus, both the lower sterol content of MCT/LCT ILE and the more intense oxidation of the remaining cholesterol might have been caused by a more severe heat treatment during the production of these emulsions or of their ingredients.

In both the investigated emulsions, the concentration of 5[alpha]-epoxycholesterol in "baseline" conditions was higher than the concentration of 7-ketocholesterol, whereas that in LCT ILE was below its limit of detection, and this stands for a more intense development of indirect oxidation, probably because of the high amounts of PUFAs. However, the 5[alpha]-epoxycholesterol concentrations in extracted lipids were 3 times higher in MCT/LCT ILE than in LCT ILE. This cholesterol oxidation biomarker, whose formation is favored by PUFA hydroperoxides, was thus paradoxically lower in the emulsions with higher PUFA content. These results appear to contrast the hypothesis that because of their chain-length characteristics, MCT/LCT infusions are exposed to a lesser extent of lipid peroxidation than LCT ILEs.40,41 Nevertheless, some studies have shown that when fatty acids are dispersed in aqueous colloidal dispersion their oxidative stability may surprisingly increase as the degree of their unsaturation increases, and this might explain the higher stability to oxidation of LCT ILE.42

Hyperoxic trials have been carried out in order to establish whether the "baseline" characteristics of the investigated emulsions could be modified in conditions that resembled in vivo conditions. In fact, when lipid emulsions are transported through the newborns' bloodstream before being cleared from the plasma, they may be exposed to oxidation by the prolonged and simultaneously high inspired oxygen concentrations. Ten years ago, Pitkanen et al43 showed that incubation of lipid emulsion with pro-oxidant (H^sub 2^O^sub 2^ and FeCl^sub 2^) alone or combined with human erythrocytes led to increased production of pentane and malonylaldeydes in the experimental setting. In the same study IV lipid (Intralipid) administered to preterm newborns was related to an exponential increase in pentane as a result of peroxidative stress.10,43-45

In our study, both ILEs submitted to high oxygen concentrations showed an increase in the investigated COPs. Nevertheless, although the enhancement of oxidation sometimes determined a doubling of investigated COPs their final concentration was in absolute value very low. The exposure to air caused a slight increase in COPs content only in LCT ILE. Their small decrease in LCT/MCT ILE might be ascribed to the possible decomposition of these COPs favored by gas impurities. The differences between air and oxygen exposure were not particularly significant (Table III), although the content of the detected COPs was higher after oxygen exposure than after air exposure in both MCT/LCT and in LCTILE. Nevertheless, in this experimental environment (air or oxygen exposure) the concentration of 5[alpha]-epoxycholesterol again proved to be higher than the 7-ketocholesterol concentration.

A possible reason for the limited development of cholesterol oxidation in the emulsions we analyzed might be the protective effect of the particular oil-inwater environment.42 Moreover, it should be remembered that oxidation in oil-in water emulsions can be limited by the presence of antioxidants or chelating agents and is highly related to the purity of the ingredients, their possible interaction, the concentration and size of the lipid droplets, and the inside localization of the PUFAs.

As indicated previously, quantitatively definite conclusions cannot be drawn from our data. But it is clear that the amounts of COPs derived from a normal intake of infant formula might be as much as 40-fold higher than the amounts contained in "experimentally oxidized" emulsions.

With regard to the presence and possible intake of preformed amounts of cholesterol oxides in currently used ILEs, the results of the present study are quite reassuring for the safety of neonates,46 although caution is always required when drawing conclusions from in vitro data. Samples of IV preparations seem to be minimally affected up to opening by the possible oxidative stress derived from industrial manufacturing. Moreover, the small increase in COPs content after prolonged oxygen exposure seems to rule out a negative effect of iatrogenic therapy.

This study completes our previous research and, to our knowledge, provides data for the first time on the levels of cholesterol oxides in ILEs. Conclusions are different from our previous studies because the manufacturing technologies used appear to minimize possible risks from the ILEs.

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Fabio Scopesi*; Paola Zunin[dagger]; Carlo Bellini*; Renata Sacchi*; Raffaella Boggia[dagger]; Filippo Evangelisti[dagger]; and Giovanni Serra*

From the *Department of Neonatal Care, Genoa University, G. Gaslini Institute, Genoa, Italy; and the [dagger]Department of Pharmaceutical and Food Chemistry and Technology, Genoa University, Genoa, Italy

Received for publication February 18, 2004.

Accepted for publication June 7, 2004.

Correspondence: Fabio Scopesi, MD, Department of Neonatal Care, Genoa University, "G. Gaslini" Institute, Genoa, Italy. Electronic mail may be sent to fabioscopesi@ospedale-gaslini.ge.it or fabscop@hotmail.com.

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