Industrial convenience systems of OliClinomel N6-900 E (Baxter) and Kabiven (FreseniusKabi) were used as all-in-one nutrient mixtures. OliClinomel contains 80% of olive oil (LCT, in 60-80% esterified by C18-MUFA of the ω-9 subgroup) and 20% of soybean oil (LCT, mainly triglyceride esters composed of C-18:2 PUFA of the ω-6 subgroup). Kabiven contains 100% soybean oil (LCT, mainly C18:2 PUFA esters). The three-chamber bags were reconstituted according to the instruction for use by breaking the non-permanent seals between the compartments and then mixing them by inverting the bag .
An overdose of Ca-gluconate (25 ml into 1000 ml out of 100 mg/ml injection = 5.5 mmol/l Ca2+ extra) was added during the preparation process to one of the TPN. Ingredients in g/l and mmol/l after reconstitution and additional Ca-gluconate are as follows:
OliClinomel (samples OA and OC) Macronutrients: 34 g/l Amino acids, 120 g/l Glucose, 40 g/l FatMicronutrient: 32 mmol/l Na+, 24 mmol/l K+, 2.2 mmol/l Mg2+, 2 mmol/l (Sample OA) and 7.5 mmol/l (Sample OC) Ca2+, 10 mmol/l Phosphate, 53 mmol/l Acetate and 46 mmol/l Cl-.
Kabiven (samples KA and KC) Macronutrients: 33 g/l Amino acids, 100 g/l Glucose, 39 g/l FatMicronutrients: 31 mmol/l Na+, 23 mmol/l K+, 3.9 mmol/l Mg2+, 1.9 mmol/l (Sample KA) and 7.4 mmol/l (Sample KC) Ca2+, 9.7 mmol/l Phosphate, 3.9 mmol/l Sulphate, 38 mmol/l Acetate and 45 mmol/l Cl-.
Previously it was found that a moderate difference (up to 20%) in glucose content did not influence the droplet-stability under very similar electrolyte-environment . So we can state that the two TPN systems studied in our present experiments are comparable. Test mixtures signed with C contain ca. 50% more Ca2+ than maximally advised by the manufacturers and it was considered the possible upper limit of Ca2+ content in the comparison of the stability of different mixtures.
After shaking the solution container, it has been connected to an infusion set and hanged up to an infusion stander. Light protection was not used and the storage temperature was hospital ward room-temperature (22–24°C). After a lag time of 5 minutes infusion administration with a speed of 2 ml/min (= 120 ml/hours) has been started. Samples for droplet-size control measurements were taken from the “supernatant” of the infusion bag content (within 3 mm of the surface, later mentioned as “upper level sample” signed as “up”) and from the infusion tube 25 cm after the output port of the container bag (“lower level sample” in short “low”). Samples (5 ml) have been taken just after the start (zero point) and 24 hours later (end point). For zeta-potential measurements we took samples from the middle of the container and sampling was done at start, after 1, 2, 3, 10, 11, 12 hours to simulate the real parenteral nutrition conditions and in the end of the study period (24. hour).
Droplet size measurements were performed by photon-correlation spectroscopy (PCS, made by Zetasizer 4 and Nanosizer ZS, Malvern Instruments, England) and the surface-charge was checked by measurement of electrophoretic movement (Laser Doppler Anemometry by Zetasizer 4, Malvern Instruments, England). Droplet size distributions and zeta-potential figures were made out of 2 or 3 separate samples in each measurement depending on the coherence of measurement results. Nature samples were diluted by purified water in a proportion of 1:300 in order to reach the measurement concentration-range according to the manual for use of the equipment. The measurement limits of the Zetasizer 4 equipment are 3-3000 nm for determination of droplet size and 20-20000 nm in case of LDA . The measuring range of the Zetasizer Nano ZS equipment (Dynamic Light Scattering principle) for particle size measurement is between 0.3 nm-10 μm and for zeta-potential 3.8-100000 nm . We expected the 99.5% of droplets in the range of 10-3000 nm with the main peak between 300-700 nm and the critical point was the size over 5 μm. Surface charge of droplets was expected between −40 and −10 mV. Droplet-size control was performed according to the accepted rules based on the USP [13, 14]. We also controlled the droplet stability by electrokinetic (zeta-potential) measurement as advised .