Peroxidation of Intravenous Lipid Emulsions
Stefan Mühlebach, Ph.D.
Chefapotheker, Apotheke Kantonsspital, CH 5001 Aarau (Switzerland)
phone +41-62-838-53 50, fax +41-62-838-42 48, e-mail: stefan.muehlebach@ksa.ch
Objectives
 To list the characteristics of lipid peroxidation (LPO)
 To identify the potential toxic risk of radical-mediated chemical reactions in vivo
 To enumerate factors influencing LPO in parenteral nutrition (PN) admixtures
 To report on the rationale of measures to reduce LPO in all-in-one admixtures in vitro
related to PN compounding, storage, and final administration to the patient
 To identify different methods to measure LPO and its breakdown products
 To characterise the iodometric test method as a cost-effective candidate for
compounding centres to document LPO in ready to use all-in-one bags
Parenteral Nutrition and Lipid Peroxidation
Parenteral nutrition (PN) contains all the nutrients necessary to meet the patient’s needs.
Most patients on PN show severely disturbed organ functions or/and specific changes of
metabolism (e.g. organ failure, post-aggression stress) requiring an adaptation of the
nutrition regimen. The i.v. administration allows nutrients to be administered only in
digested form and readily available for the basal energy and nitrogen metabolism.
To minimise complications, improve tolerance and convenience, and allow home treatment
all-in-one (AIO) admixtures have improved parenteral nutrition considerably. In total
parenteral nutrition these complex AIO admixtures contain more than 50 different
components and show only limited stability. In terms of effectiveness and safety that
stability issue is of special clinical and pharmaceutical interest [1,2].
Triglycerides as main PN lipid source have only reduced water solubility. For their i.v.
administration and for a good biological tolerance they have to be emulsified like
chylomicrons using natural phospholipids. The oil particles of the resulting oil in water
emulsion have to be distributed homogeneously. The typical mean fat droplet diameter is
0.2 –0.4 m. The negatively charged droplets are prone to undergo physical
destabilisation leading eventually to irreversibly deteriorated emulsion systems with
potentially fatal risks for the patient (embolism).
Intravenous long chain triglycerides (LCT) contain high concentrations of essential
polyunsaturated -3 and -6 fatty acids (PUFA) [e.g. soy bean oil: linoleic (-6 C18-2) and
linolenic (-3 C18-3) acid; fish oil: EPA (-3 C20-5) and DHA (-3 C22-6)]. In presence of
oxygen these PUFA show chemical instability forming reactive lipid peroxides (LPO) and
radicals (Figure 1).
In the presence of ambient air LPO is initiated by reactive singlet oxygen in vitro. After
cleaving hydrogen from fatty acids a hydroxyl radical results. Radicals have a reactive
unpaired electron favouring coupling with neighbouring chemicals and initiating chain
reactions. The hydroxyl radical interacts with conjugated double bonds of a PUFA
representing chemically vulnerable sites. The resulting PUFA radical is the initial event of
an autocatalytic chain reaction producing lipid peroxide radicals and lipid hydroperoxides
both potentially harmful chemicals.
LPO is enhanced by increased temperature, exposure to light, or catalytic concentrations
of micronutrients. The latter may also inhibit oxidation as radical scavengers (Figure 2).
The resulting lipid hydroperoxides are still unstable intermediates fragmenting into
products like aldehydes which may polymerise or represent toxic compounds (e.g.
hydroxynoneal or malondialdehyde). LPO as a chemical degradation process is known
since a long time ago for vegetable oils as rancidity. Only in the 1950s LPO was
addressed in biology and medicine.
Lipid Peroxides and Free Radicals in vivo
The balance between formation and inactivation of free radicals in the body is an essential
modulator of cell proliferation and apoptosis and eventually health and disease [3-6].
Therefore, radicals have desirable and noxious effects. Hydroxyl radical, the biologically
most reactive compound, directly attacks lipid-containing membranes, proteins or DNA
and produces tissue injury, organ dysfunction, or cancer.
Nitric oxide is a stable radical with important signalling functions e.g. in the vascular
relaxation of blood vessels (EDRF) and a potent antioxidant quenching agent neutralising
lipid peroxide radicals responsible for membrane damage. Interacting with a lipid peroxide
radical, NO° has a pro-oxidative function and forms a potent oxidising peroxynitrite.
Superoxide O2-°, another physiologic free radical, is involved in inflammation, phagocytosis
and cell growth. It may interact similarly with a lipid peroxide radical with NO° forming
peroxynitrite (ONOO-) too. To preserve the important signalling effect of NO°, superoxide
tissue concentration is kept low by the activity of the selenium-containing superoxide
dismutase.
Different defense systems exist to reduce an inappropriate oxidative attack or stress in
vivo. There is an important distinction to be made between hydrophilic peroxides mainly
inactivated by catalase and the more detrimental lipid peroxides. Vitamins like tocopherol (hydrophobic) and vitamin C (hydrophilic) are able to act as radical scavenger.
Trace elements like selenium, copper, or iron are present as free metal ions or included in
metallo-enzymes forming important bio-catalysts to inactivate or release radicals, e.g. free
Fe2+ or Cu1+ ions, superoxide dismutase (zinc-, copper-, manganese-dependent),
glutathione peroxidase (selenium), catalase etc.
An increased lipid peroxidation may be measured through marker compounds like
malondialdehyde (plasma), pentane (expired air) or a decrease of anti-oxidative
micronutrients [7]. The anti-oxidative capacity of the body depends on presence of specific
micronutrients (nutrition), enzyme maturation (age) but also on the extent of oxidative
stress (pathologies). Pre-term infants, severely ill patients, or long-term home PN patients
may be at specific risks when exposed to in vitro generated lipid peroxides. Therefore,
there is a need to investigate in vitro LPO and the factors affecting its extent.
In vitro Assessment of LPO in i.v. Lipids and AIO Admixtures
LPO represents a complex cascade of unstable and partly volatile intermediate breakdown
products (Figure 1). Hence, analytical assessment is difficult and a variety of analytical
methods are used. The specific advantages and limitations of each method have to be
evaluated. To document chemical stability of lipid containing PN admixtures cost effective
and easy to handle methods are mandatory.
Gas or liquid chromatography (GLC or HPLC) are rather expensive methods. They are
standard in analytical labs but not routinely available in compounding units and hospital
pharmacies. These chromatographic methods allow to determine a changing fatty acid
profile or breakdown products as ultimate effect of lipid deterioration. These effects are not
early manifestation of LPO [8]. The same holds for thiobarbituric assays or oxygensensitive electrodes, both with poor specificity for LPO.
Ferrous oxidation-xylenol orange (FOX) analysis is a peroxide-sensitive
spectrophotometric assay. In its original design using hydrophilic solvents the test detects
peroxides in the water phase but no lipid peroxides [9]. In addition FOX may interact with
vitamins like ascorbic acid or tocopherols present in PN admixtures.
In contrast, a iodometric titration of a lipid extract represents a most direct method to
determine lipid hydroperoxides. The method is a standard in the analysis of vegetable oils
(pharmacopoeia). For parenteral lipids in different pharmacopoeias an upper limit of the
peroxidation value [PV = mmol peroxides / L fat emulsion] of 5 mM is given.
Starting from the Wheeler method [10] we established a lipid hydroperoxide assessment
for i.v. lipid emulsions and AIO admixture [8]. This included a first lipid extraction step by
chloroform-methanol 2:1 (Folch extraction). Test samples from 10 g lipid emulsion were
incubated for 60 seconds with potassium iodide. The iodine formed was reduced by 0.01 N
thiosulfate using an automated voltametric titration system (Dosimat). The reproducibility
expressed by the coefficient of variation CV was 3.2% (n=48; PV < 0.1 mM); the test
conditions had to be strictly defined; light-protection was necessary. The detection limit for
lipid peroxides was 0.02 mM. One single test took about 15 minutes; the cost for
chemicals was 2-3 Euros. These characteristics show the appropriateness of the method
for a compounding unit.
Factors Affecting LPO in vitro
Conclusions for AIO Admixture Handling [8,11,12]
The PV was dependent on the lipid used (PUFA concentration, content of -tocopherol).
Exposure to ambient light is a powerful enhancer of LPO. Starting from the very low PV of
a commercial 20% LCT lipid emulsion in glass containers with air displaced by nitrogen
this value increased over 1 week by a factor of 40 when the lipid was filled into monolayered, air-permeable EVA bags and exposed to ambient daylight at room temperature.
The PV limit given by the pharmacopoeia was reached within a week (Figure 3). The PV
increase was accompanied by a pH drop. Light protection is an efficient measure to
decrease a PV by a factor of 5-10. Cold storage temperature is an even more efficient
LPO stabiliser.
Micronutrients can heavily affect LPO. -Tocopherol is a potent antioxidant when present
in low and usual concentrations of about 10-20 mg/L in parenteral soy bean lipid
emulsions. Increasing the -tocopherol concentration to 160 mg/L showed a pro-oxidative
effect.
Addition of a daily portion of essential trace elements to an i.v. LCT lipid emulsion or a
lipid-containing AIO admixture increased the PV significantly (Figure 3). The effect was
more pronounced in pure lipid with a PV increase factor of 5-10 during a two weeks stress
test at 40°C in a EVA bag.
In conclusion LPO is a complex lipid deterioration reaction occurring in presence of air in
vitro. For safety reasons PV in AIO admixtures should be maximally reduced. This will be
reached when
 eliminating the oxygen content in the bag during compounding and storage (overwrapping with an oxygen absorber)
 storage of the admixture light-protected at 2-8°C
 trace elements are added to the AIO admixture only immediately before infusion and if
compatibility is documented
 multivitamin addition is not allowed in the presence of trace elements. They may even
provoke LPO. There are balanced commercial multivitamin products to be administered
in a small volume infusion separately from the PN admixture.
References
1. Driscoll DF. Clinical issues regarding the use of total parenteral nutrition admixtures. Ann Pharmacother 1990; 24:
296
2. Barnett MI, Cosslett JR, Duffield JR et al. Parenteral nutrition: pharmaceutical problems of compatibility and stability.
Drug Safety 1990; 5(suppl 1): 101
3. Mühlebach S, Steger PJK. Lipid peroxidation of intravenous fat emulsions: a pharmaceutcal issue with clinical
impact? Nutrition 1998; 14: 720
4. Guttrigde JMC, Halliwell B. The measurement and mechanism of lipid peroxidation in biological systems. Trends
Biol Sci 1990; 15: 129
5. Freeman B A. Oxygen: the air-borne nutrient that both sustains and threatens life. Nutrition 2000; 16:478
6. Weisburger JH. Eat to live, not live to eat. Nutrition 2000; 16: 767
7. Neuzil J, Darlow BA. et al. Oxidation of parenteral lipid emulsion by ambient and phototherapy lights: potential
toxicity of routine parenteral feeding. J Pediatr 1995; 123: 785-90
8. Steger PJK, Mühlebach SF. In vitro oxidation of iv lipid emulsions in different all-in-one bags assessed by an
iodometric assay and gas-liquid chromatography. Nutrition 1997; 13: 133
9. Silvers K M, Darlow BA, Winterbourn CC. Lipid peroxides and hydrogen peroxide formation in parenteral nutrition
solutions containing multivitamins. J Parent Enteral Nutr 2001; 26: 14-17
10. Wheeler DH. Peroxide formation as a measure of autooxidative deterioration. Oil Soap 1932; 9: 82
11. Steger PJK, Mühlebach SF. Lipid peroxidation of iv lipid emulsions in TPN bags: the influence of tocopherols.
Nutrition 1998; 14: 179
12. Steger PJK, Mühlebach SF. Lipid peroxidation of iv lipid emulsions and all-in-one admixtures in total parenteral
nutrition bags: the influence of trace elements. J Parent Enteral Nutr 2000; 25: 37