Intralipid

ABSTRACT. Background: We evaluate the effects of multilayered bags us ethylvinyl-acetate bags on peroxidate formation of various emulsions for all-in-one total parenteral nutrition solutions (TPN)during storage. Methods: Twenty-four parenteral nutritions were prepared with 4 commercial IV lipid emulsions (Soyacal 20%, Grifols; Intralipid 20%, Fresenius-Kabi; Lipofundina 20%, Braun; and Clinoleic 20%, Clintex) and 2 different bags (multilayered [ML] bag, Miramed; and 1 ethylvinyl-acetate [EVA] bag, Miramed). Each kind of TPN was prepared in triplicate. Samples were taken at 3 different times: immediately after preparation (time 0), after 6 days at 4°C and 48 hours at 37°C (time 1), and finally after a total of 14 days at 37°C (time 2). Oxidation of TPN was evaluated by analysis of hydroperoxides by ferrous oxidation-xylenol orange (FOX) reactive, lipoperoxides by thiobarbituric acid reactive species (TEARS), a-tocopherol by high-performance liquid chromatography (HPLC), and ascorbic acid and dehydroascorbic acid by HPLC. Results: TPN admixtures in ML bag showed less oxidation evaluated by peroxide determination using FOX than EVA bag. Lipoperoxides by TEARS did not show significant differences between 2 bags. Ascorbic acid and dehydroascorbic acid disappeared in EVA bags at time 1. No important differences were found in a-tocopherol content. Conclusions: Multilayered bags minimize oxidation. (Journal of Parenteral and Enteral Nutrition 28:85-91, 2004)

Total parenteral nutrition (TPN) admixtures have been studied for physicochemical stability of emulsion, interactions between ingredients, formation of precipitate, and degradation of vitamins and amino acids. Another factor to take into account is the effect of oxygen on the compounds.1 Admixtures of TPN can peroxidize to potentially harmful peroxides.

Toxicity related to peroxides could be particularly significant in the newborn2 because of the lack of maturity of their defensive systems against oxidative processes and also in critically ill patients, whose oxidative metabolism is increased. Hydroperoxides have been associated with hypoxic-ischemic encephalopathy, lung disease, retinopathy, and necrotizing enterocolitis in premature infants.3

Early reports suggested that lipid emulsions used in parenteral feeding are susceptible to peroxidation.4"8 In addition, lipid peroxidation is influenced by the material used in manufacturing the plastic bag. Steger and Muhlebach9 determined the peroxide value of lipid emulsions by iodometric assay and found 450 times more peroxides in emulsions stored in ethylvinyl-acetate (EVA) bags than in glass controls.

Data have been published regarding the influence of vitamins,10-12 trace elements,13 and light10,14 on generation of peroxides in TPN admixtures.

Contact with oxygen is required for generation of peroxides. In TPN admixtures, oxygen from atmospheric air unavoidably enters rigid containers during compounding; the amount of oxygen varies depending on the nature of the filling process.15

The aim of this study was to establish the influence of the bag material on peroxidation of TPN admixtures with varying lipid emulsions. We analyzed formation of peroxides during storage using 2 different methods: ferrous oxidation-xylenol orange reactive (FOX) for hydroperoxides, and thiobarbituric acid reactive substance (TEARS) for lipoperoxides. In addition, the concentration of antioxidant agents, [alpha]-tocopherol, ascorbic acid, and dehydroascorbic acid (DHA), was determined.

MATERIALS AND METHODS

Twenty-four TPN bags were prepared using 4 lipid emulsions available in Europe (Soyacal 20%, Grifols; Intralipid 20%, Fresenius-Kabi; Lipofundina 20%, Braun; and Clinoleic 20%, Baxter) and 2 different bags (multilayered bag, Miramed; and EVA bag, Miramed) all with a capacity of 3 L available in Europe. Composition of the 4 lipid emulsions is shown in Table I. Multilayered bags were composed of: EVA-ethylvinyl alcohol-polyvinylidine combinations. The TPN composition is shown in Table II. Each kind of products used in TPN formulation had the same batch.

All TPN were prepared by the same technician in a laminar flow hood at 21°C without using a pump. Compounds were added in the same order of preparation. Before closing, the air was completely excluded by manually pressing the bag. TPN admixtures were introduced into a photoprotected plastic bag and refrigerated at 4°C for 7 days before transfer to a heater at 37°C for 14 days.

Samples were collected at 3 different times: time 0 immediately after preparation, time 1 after 7 days at 4°C and 48 hours at 37°C, and time 2 after 7 days at 4°C and 14 days at 37°C.

Samples were transferred into closed test tubes under a nitrogenous atmosphere and frozen at -40°C until analysis. Test tubes were codified and numbered. Analysis was performed blind to the identification of the code sample.

Hydroperoxides (by FOX method), lipoperoxides (by TEARS), [alpha]-tocopherol, ascorbic acid, and DHA were determined in all samples. Solutions and emulsions (amino acids, glucose, lipids, and multivitamins) used in preparation of the admixture were also analyzed for hydroperoxide concentration.

Determination of hydroperoxides

Hydroperoxides were measured according to the FOX method modified by Jiang et al.16 The technique had a 2.5% variation coefficient. Linearity, exactitude, and precision of the method were studied before analysis. The maximal volume for conserving linearity was 30 µL. for TPN and 25 µL for lipid emulsion. Frozen samples were tested for stability of peroxides and were found stable for 21 days at -25°C.

Two solutions were prepared for the FOX reactive. Solution A: 38 mg of xylenol orange were dissolved in 50 mL of methanol 90% and solution B: 25 mg of FeCl^sub 2^.4H^sub 2^O were dissolved in 50 mL of methanol 90%.

Concentrated sulfuric acid (138 µL) was added to a solution of 88 mg of tert-butylhydroperoxide in 60 mL of methanol 90%. Immediately after shaking, 5 mL of solution A and then 10 mL of solution B were added. Finally, the solution was completed with methanol 90% to 100 mL. Air was then removed.

A total of 30 µL of sample was incubated for 30 minutes with 970 µL of FOX reactive at room temperature. After centrifugation (10 minutes at 10,000 rpm), absorbance was read at 560 nm. The concentration of peroxide in the sample was calculated by interpolation in a calibration line.

Calibration line was in a 1-L volumetric flask pipet, 113 µL of a 30% (vol/vol) H^sub 2^O^sub 2^ solution previously standardized. The solution obtained (P^sub 0^) was 1000 µM in H^sub 2^O^sub 2^, prepared fresh daily. P^sub 0^ was diluted quantitatively with water to obtain 6 standard solutions (P^sub 1^ to P^sub 6^) of 200 µM, 125 µM, 100 µM, 71.43 µM, 50 µM and 20 µM in H^sub 2^O^sub 2^ respectively. In 1.5-mL Eppendorf tubes, 30 µL of the standard solution and 970 µL of the FOX reagent was pipetted. The tube was closed and mixed in a vortex. A blank solution was prepared using water instead of the standard solution. After 30 minutes' incubation at room temperature, the absorbance at 560 nm was read against the blank.

Validation Procedure

To determine the maximum volume of sample, reactive FOX was added until a total volume of 1000 µL to: 15 µL, 20 µL, 25 µL, 30 µL, and 37 µL, of NPT. After shaking, tubes were incubated for 30 minutes at room temperature and centrifuged for 10 minutes at 10,000 rpm. Absorbances of supernatants measured at 560 nm were 0.417, 0.504, 0.588, 0.671, and 0.752. A small precipitate appeared in samples above 30 µL of NPT, and the absorbance obtained was less than expected.

A standard solution of H^sub 2^O^sub 2^ was used to determine linearity. The correlation coefficient was >0.9996. The method was lineal.

To determine exactitude, 0 µL, 5 µL, 10 µL, 15 µL, and 20 µL, of H^sub 2^O^sub 2^ 200 µmol/L were added to samples of 30 µL of TPN. The same quantities of H^sub 2^O^sub 2^ 200 µmol/L were added to 30 µL of water.

After adding reactive FOX (970 µL), tubes were incubated for 30 minutes and centrifuged. Absorbance of supernatant was read at 560 nm. Recovery was superior to 85% in all cases.

Determination of Lipid Peroxidation

TEARS were measured as an estimation of lipid peroxidation. TEARS are final stable products, such as ketones and aldehydes, formed from polyunsaturated fatty acids during lipid peroxidation.

TEARS was determined as described.17 Briefly, reactive solution was prepared with 375 mg thiobarbituric acid (TEA) dissolved in 100 mL of water containing 15 mL of 100% trichloroacetic acid and 2.5 mL of concentrated HCl. One milliliter of reactive solution was mixed with 0.5 mL of sample and heated at 100°C for 20 minutes. After cooling, lipids were extracted with 2 mL of butanol by intense agitation. After centrifugation at 300 rpm for 5 minutes, the butanol phase was collected and fluorescence was determined with excitation wavelength at 515 nm and emission wavelength at 553 nm. A standard curve was prepared with malondialdehyde-tetramethylacetal (1,1,3,3-tetramethoxypropane). Results were expressed as micromolars of malondialdehyde. Interassay coefficient of variation (CV) of the method was below 10% and was lineal from 0 to 10 µmol/L.

Determination of [alpha]-Tocopherol

A volume of 0.5 mL of samples was mixed with 0.5 mL of internal standard (tocopherol acetate in ethanol at 10 µg/L). Lipids were extracted with 1 mL of heptane by vortexing for 1 minute. After centrifugation, the upper phase (heptane) was separated and evaporated with nitrogen. Dry pellet was dissolved with 100 µL of ethanol, and 20 µL were injected in the chromatograph. [alpha]-Tocopherol was measured by reversephase high-performance liquid chromatography (HPLC) in an Ultrasphere ODS column, 5 µm, 4.5 × 25 mm (Beckman), using acetonitrile:isopropanol:acetic acid (75:24:1) as mobile phase, as described.18 Results were expressed as milligrams per liter. Interassay CV of the method was lower than 5%.

Determination of Vitamin C and DHA

The amount of ascorbic acid was determined by the difference between the total quantity of DHA obtained after treating the sample with iodine solution (conversion of ascorbic acid a DHA), and the initial amount of DHA in the untreated aliquot. DHA was determined by HPLC using reverse-phase column with isocatric system and fluorescence detection.19 Samples were previously treated with o-phenylendiamine in order to obtain a fluorescent derivative. Chromatography was perfomed on a 150 × 3.9 mm (Novapak C^sub 18^, 4 µm) reverse-phase column using a mixture of phosphate buffer 0.07 M (pH 7.5) and methanol (80:20) as mobile phase (flow rate, 2 mL/min).

Analysis Procedure

Metaphosphoric acid solution (4 mL; 0.62M), 150 µL, of a solution of sodium acetate, 4.5 M, and 250 µL of o-phenylendiamine were added to 1.0 mL of sample. The mixture was then homogenized. After 15 minutes at room temperature, a volume of 10 µL was injected into the Chromatographie system:

Detection: fluorescence [lambda]^sub ex^ = 355 nm, [lambda]^sub em^ = 415 nm

Detection limit: 0.1 ppm; and quantification limit: 1 ppm

The within-day and interday reproductions of DHA determinations were 2% and 8% (n = 6), respectively.

Statistical Analysis

All results are given as mean and SD. In all tables, we show results of time 0, 1, and 2, but time 2 has not been used in statistical analysis because conditions of this time are not carried out in clinical practice.

To evaluate relations between commercial IV lipid emulsions, 2 factors of analysis of variance (ANOVA) were used: TPN bag factor (2 levels: ML bag vs EVA bag) and time factor of repeated measures (2 levels: 0 vs 1). We studied the 2 factors and the interaction between them. The statistical significance level was 5% ([alpha] =0.05), and 2-tailed tests were used throughout. SPSS statistical software, Version 10, was used for the statistical analysis.

RESULTS

Determination of Hydroperoxides by FOX

Table III shows concentrations of hydroperoxides found.

Significant differences were found in the interaction of bags and time between ML and EVA bags, considering time 0 and 1 in the 4 lipids evaluated. EVA bags had a higher hydroperoxide content at time 1 (7 days at 4°C and 48 hours at 37°C) in a range of 1.7 to 3.4 times the initial values and at time 2 by 7.5- to 15-fold. A smaller or no increase in hydroperoxide concentration was observed in ML bags compared with EVA bags over time.

All macronutrient solutions and multivitamins used for TPN preparation were also tested for hydroperoxides. Results are shown in Table IV. Amino acid solution is the major contributor to the hydroperoxide content.

Determination of Lipoperoxides by TEARS

Table V shows the concentration of lipoperoxides found.

No differences were found in the interaction of bag and time between ML and EVA bags, evaluating time 0 and time 1.

Determination of [alpha]-Tocopherol by HPLC

Table VI shows concentration of [alpha]-tocopherol (mg/L) and the percentage of loss.

Significant differences were not found in the interaction of bag and time between MLB and EVA bags except in TPN bag with Clinoleic, but the magnitude of the effect is low (p = .028).

Determination of Ascorbic Acid and DHA by HPLC

Tables VII and VIII show the concentrations of vitamin C and its oxidated form. Significant differences were found between 2 bags. At time I, no samples of EVA bags had any vitamin C or DHA. ML bags maintained vitamin C and DHA during storage even at time 2 after extreme conditions.

DISCUSSION

Our analytical data suggested a marked influence of the material from which the bag was made on the peroxide content in the TPN. In the past, polyvinylchloride (PVC) was used, but it is not recommended at present because of leaching of plasticizer into the fat emulsion.20 EVA bags and the more recent multilayered bags are commonly used today.

Because the multilayered bag is at least 100 times less permeable to oxygen than EVA or PVC bags,21 the passage of oxygen during storage is expected to be lower.

Analysis of Peroxides by FOX

This method analyses the amount of hydroperoxide.

In their work about light-dependent reactions in parenteral solutions with multivitaminic contents (MVI Pediatric) including polysorbates, Silvers et al11 reported interference of FOX assay when ascorbic acid concentration was over 40 µmol/L. Other authors consider that polysorbates constitute the major contributor to generation of peroxides.12 In the present work, Cernevit, which does not contain polysorbates, was added.

We found a concentration of peroxides at time O between 38.5 ± 9.5 and 55.3 ± 7.3 µmol/L, and the expected one, taking into account the peroxide content of solutions used in TPN formulation, was between 77.6 and 94.6 µmol/L. We are unable to explain this finding. It could be that peroxides are unstable products that react with other products.

The concentration of ascorbic acid in our samples, considering all times, was between 54 and 305µM. EVA bags at times 1 and 2 did not contain ascorbic acid (Table VII). We cannot discard an interference with ascorbic acid. However, in our validation of the FOX method, different quantities of hydrogen peroxide were added to samples of TPN and to samples of water, and results obtained were as expected. We therefore considered that any interference did not substantially affect the results.

We found that amino acid solution Aminoplasmal L-10 was the solution that provided most peroxides. Other amino acid solutions available on the market that could make different contributions to the hydroperoxide content were not studied.

In EVA bags, the hydroperoxide concentration rose significantly after 1 week of storage in the refrigerator and 48 hours at 37°C protected from light (time 1). This could be the stability time limit, considering an administration of 48 hours. At time 2, after extreme conditions, hydroperoxides in multilayered bags decreased instead of increasing. An explanation for this finding could be the decomposition of peroxides in a closed system. Without oxygen, no further peroxides are formed.

Our results show that the multilayered bags decreased hydroperoxide formation over time compared with EVA bags.

Peroxides are toxic products. European Pharmacopeia gives a maximum peroxide value for parenteral solutions using soybean oil and olive oil of 5 mEq/1000 g by iodometric method, but it does not give any peroxide value for parenteral nutrition. Furthermore, the peroxide value in the Pharmacopeia is an empirical value for pharmaceutical production rather than a toxicological limit. As it is not known whether the peroxide content of TPN has adverse effects, we recommended multilayered bags because of minimizing peroxide formation over time (Table III).

Analysis of Lipoperoxides by TBARS

No significant interaction between time and bag was found, considering time 0 and 1. Adding time 2 to ANOVA, significant interaction was found. Only after keeping TPN admixtures during 14 days at 37°C, conditions that are not carried out in clinical practice, can we see differences between 2 bags.

We did not find studies using TBARS in TPN. TBARS have been used in parenteral fat emulsions to determine peroxability. Arborati et al22 analyzed peroxide content in parenteral fat emulsions before and after oxidation with Cu^sup ++^ . There was a wide dispersion of data. These authors found concentrations between 3.7 ± 6.7 and 16 ± 8 µmol/L (mean ± SD), depending on fat emulsion. We are unable to compare our results with those of Arborati et al because of the wide dispersion of data and because products were not the same (fat emulsions us TPN).

Analysis of [alpha]-Tocopherol

[alpha]-Tocopherol in TPN comes from lipid emulsions and added multivitamins. This vitamin is a natural antioxidant. Braun add [alpha]-tocopherol (200mg/L) to Lipofundina 20% emulsions. The effect of this vitamin in preventing oxidation is controversial. It seems that at high concentrations it acts paradoxically as a pro-oxidant.6,23,24 Banni et al23 reported that at concentrations above than 40 nmol/L, it acted as a pro-oxidant when the ratio between [alpha]-tocopherol and linoleic acid was 1:100. Nevertheless, Manuel-y-Keenoy et al25 found that supplementation of TPN with [alpha]-tocopherol leads to a decrease in the susceptibility of serum LDL and VLDL to peroxidation in vitro.

In our study, concentrations of [alpha]-tocopherol in Lipofundina bags were higher than expected. This could be attributed to the oxidation process natural variability of this vitamin in vegetable oils.

Our results show that the content of [alpha]-tocopherol decreased slightly with storage, but it was not depleted. In the worst case, it was reduced to 66% of the initial amount.

Analysis of ascorbic and dehydroascorbic

Ascorbic acid is a hydrosoluble natural antioxidant. Some reports indicate that vitamin E requires a coantioxidant (vitamin C) in order to eliminate the vitamin E radical.5

Degradation of vitamin C in EVA-TPN and its stability in multilayered bags has already been described.26,27

No vitamin C or its oxidated product DHA was found in any EVA bags after storage. Furthermore, degradation in the multilayered bag was, in the worst case, only 24% of loss in relation to the initial amount of vitamin C and 25% of loss regarding DHA.

Results of the present study suggest that multilayered bags are effective in minimizing peroxidation of TPN and conserving vitamin C.

SUMMARY

Peroxide values were determined by ferrous oxidation-xylenol orange and by thiobarbituric acid reaction in various TPN stored in multilayered bags vs ethylvinyl-acetate bags, [alpha]-tocopherol, ascorbate, and dehydroascorbate were also measured.

Content of peroxides determined by FOX increased significantly in ethylvinyl-acetate bags.

Multilayered bags minimized peroxidation in TPN over time.

ACKNOWLEDGMENTS

The authors thank Baxter Laboratories for their financial support in vitamin C determination.

REFERENCES

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Antonia Balet, PharmD*; Daniel Cardona, PharmD, PhD*; Salvador Jane§; Antoni M. Molins-Pujol, Chem, PhD*; Jose Luis Sanchez Quesada, MD, PhD[dagger]; Ignasi Gich, MD, PhD[double dagger]; and M^sup a^Antonia Mangues, PharmD, PhD*

From the * Pharmacy, [dagger] Biochemistry, and [double dagger] Clinical Epidemiology Services, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; § Pharmaceutical Technology Department, Laboratories Grifols, Barcelona, Spain.

Received for publication May 6, 2003.

Accepted for publication November 11, 2003.

Correspondence: Daniel Cardona, Hospital de la Santa Creu i Sant Pau, Pharmacy Services, Avda. Sant Antoni M^sup a^ Claret, 167.08025-Barcelona, Spain. Electronic mail may be sent to dcardona@hsp. santpau.es.

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