preparation and examination of tpn systems for the ... · dynamic surface tension measurements ......
TRANSCRIPT
Preparation and examination of TPN systems for the
individual clinical therapy
Ph.D. thesis
Kovácsné Balogh Judit
Semmelweis University Doctoral School of Pharmaceutical and Pharmacological Sciences
Tutor: Dr. Romána Zelkó, Ph.D. Opponents: Dr. Télessy István c. egyetemi docens, kandidátus Dr. Csányi Erzsébet egyetemi docens, Ph.D. President: Dr. Kerpel-Fronius Sándor egyetemi tanár, D.Sc. Committee:Dr. Szökő Éva egyetemi docens, Ph.D. Dr. Simon Kis Gábor ny.egyetemi docens, kandidátus Dr. Takácsné Dr. Novák Krisztina egyetemi tanár, D.Sc.
Budapest, 2007.
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Table of contents
1. Introduction .............................................................................................................4 2. Objectives ................................................................................................................6 3. Literature Review.....................................................................................................8
3.1. Parental Formulas.........................................................................................8 3.1.1. Clinical aspects.........................................................................................8
3.2. Formulation of TPN ...................................................................................12 3.2.1. Nitrogen .................................................................................................12 3.2.2. Choice of amino acid ..............................................................................12 3.2.3. Non-nitrogen energy...............................................................................14 3.2.4. Energy-Macronutrients ...........................................................................14
3.3. Aspects of stability and compatibility .........................................................37 3.3.1. Emulsion design .....................................................................................40 3.3.2. Stability Assessment ...............................................................................43 3.3.3. Parenteral Nutrition Compatibility ..........................................................47 3.3.4. Drug Stability and Compatibility ............................................................48 3.3.5. Labelling ................................................................................................55 3.3.6. Dispensing..............................................................................................56 3.3.7. Storage ...................................................................................................56 3.3.8. Packaging ...............................................................................................56 3.3.9. Costs ......................................................................................................56 3.3.10. Bags .......................................................................................................57 3.3.11. Documentation .......................................................................................60 3.3.12. Manufacturing procedures ......................................................................60
4. Experimental part...................................................................................................61 4.1. Materials ....................................................................................................61
4.1.1. Mixture F35b..........................................................................................61 4.1.2. Mixture F37b..........................................................................................63 4.1.3. Individual TPN mixtures I.(A) ................................................................65 4.1.4. Individual TPN mixtures II .....................................................................68
4.2. Aseptic Production .....................................................................................69 4.2.1. Facility and environment ........................................................................69 4.2.2. Personnel and training ............................................................................69 4.2.3. Receipt of prescription............................................................................69 4.2.4. Collection of materials and preparation...................................................69 4.2.5. Entry into preparation area......................................................................69 4.2.6. First stage preparation.............................................................................70 4.2.7. Second stage preparation ........................................................................70 4.2.8. Positive pressure.....................................................................................70 4.2.9. Inspection ...............................................................................................71
4.3. Preparation of the TPN mixtures.................................................................71 4.4. Storage of the prepared TPN mixtures ........................................................71 4.5. Methods .....................................................................................................71
4.5.1. Photon correlation spectroscopy .............................................................71 4.5.2. Particle size measurement .......................................................................73 4.5.3. Zeta-potential measurements ..................................................................74 4.5.4. Optical microscopy.................................................................................74 4.5.5. pH measurements ...................................................................................74 4.5.6. Dynamic Surface Tension Measurements ...............................................74
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4.5.7. Statistical evaluation...............................................................................75 4.6. Results and discussion ................................................................................76
4.6.1. Comparison of physical stability of two different brands of lipid emulsion for total nutrient ......................................................................................76 4.6.2. Study of the stability of individual of different calcium / glucose-1-phosphate ratios TPN mixtures...............................................................................89
5. New scientific results and conclusion.....................................................................99 6. Summary .............................................................................................................101 7. Acknowledgements..............................................................................................103 8. Publications and lectures......................................................................................104 9. References ...........................................................................................................108
4
1. INTRODUCTION
Parenteral nutrition formulations are designed to provide nutrients in doses
sufficient to meet the patient’s daily requirements. Because parenteral nutrition is an
extremely complex admixture containing amino acids, dextrose, lipids, water,
electrolytes, trace elements, and vitamins–40 or more components–errors in their
formulation and compounding have led to serious and lethal complications. As a result,
parenteral nutrition formulation design must consider the stability, compatibility, which
in some cases limits one’s ability to individualize nutrient doses. Safety issues related to
parenteral nutrition formulations have led to the development of guidelines for safe
practices.
The two major types of parenteral nutrient solutions are the traditional dextrose-
amino acid solution and the TPN. The TPN System involves the addition of dextrose,
amino acids, and lipid emulsion (with electrolytes, vitamins, trace minerals, and other
additives) into a single container. TPN formulations are used frequently because of the
convenience of only one infusion for parenteral nutrition purposes and the improved
tolerance and oxidation of intravenous fatty acids. The stability of these formulations is
a concern, however, because of the destabilization of the emulsion in the presence of an
acidic pH and because of exposure to extremes of temperature. For parenteral nutrition,
these concerns limit the doses of some nutrients such as divalent cations, zinc, and iron
as well as amino acids.
Nutrients are mixed just prior to infusions, by breaking the plastic connectors
between the compartments, then vitamins and trace elements are added
extemporaneously to the bag.
Shelf –life of these bags is at least 12 months, but allow only for standardized
formulas. Due to their easy application „all-in-one” TPN1 systems should save
preparation and handling time on the ward, thus resulting in decreased manpower cost.
The use of three-compartment TPN bags is less expensive in terms of application
costs than separete bottles or hospital-compounded bag systems. TPN application costs
are partly transfered from the pharmacy to the ward in the three-compartment bag
system compared to hospital-compounded bags. Detailed manpower times measured in
1 TPN= Total Parenteral Nutrition
5
the present studies are published, allowing hospitals to calculate their own application
costs using local salaries, product prices and production costs.
6
2. OBJECTIVES
For patients unable to tolerate any form of enteral feeding, the administration of
fluid and nutrients via a parenteral route is necessary. For long-term care a balanced diet
containing all the essential nutrients, including vitamins and trace elements, must be
provided.
Combining all the constituents of a daily TPN feed into one container can result in
the production of very complex pharmaceutical systems including 15-20 individual
crystalline amino acids, hydrated dextrose, multiple electrolytes, vitamins and minerals,
as well as lipid emulsion. The obtained systems are oil-in-water emulsions stabilized by
an emulsifier that imparts a net negative charge to the surface of the globules which
stabilizes the dispersion. These highly complex formulations are subject to an array of
potential interactions, both favorable and unfavorable, thus resulting stability changes of
the system.
When the emulsion becomes unstable, these homogeneously distributed droplets
begin to aggregate and ultimately coalesce into large fat globules. Phase separation
typically occurs when the volume-weighted percent fat greater than 5 μm exceeds 0,4 %
of the total lipid present in a formulation.
Moreover, when the size of the droplets reaches a dimension of 5 μm or larger, the
infused globules may lodge into the pulmonary capillaries and produce a fat embolism
syndrome.
For such systems, product evaluation and quality control are therefore highly
dependent on methods for accurate determination of both average particle size and the
distribution of sizes present in any given sample. To evaluate an emulsion from the
standpoint of its physiological suitability, it may be more important to demonstrate the
presence or absence of droplets above a certain critical size, rather than to accurately
quantify their amount.
Concerning the above reasons the purpose of my thesis was:
• to track the physicochemical stability of TPN mixtures, successfully applied in
treatment of newborns and young children,
• to analyse the possible interactrions between the components,
• to compare the kinetic and chemical stability of TPN admixtures containing two
kinds of triglycerides(structured and exclusively long-chain triglycerides),
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• to monitor the stability of TPNs as a function of storage conditions
(temperature, storage time).
8
3. LITERATURE REVIEW
3.1. Parental Formulas Since parenteral nutrition was introduced in the United States in the late 1960s, new
challenges for providing optimal specialized nutritional support and drug therapy have
arisen. As a result of expanded knowledge about the metabolic consequence of various
illnesses, and increased demand for specialized parenteral nutrition solutions has
resulted in the availability of myriad different types of amino acids, fat emulsions,
carbohydrates, trace elements, vitamins, and electrolytes. Additionally, drug therapy has
become more complex. Because patients who require parenteral nutrition often require
intravenous drug therapy, drugnutrient interactions and medication delivery options are
important considerations in the management of these patients.[1]
The provision of adequate and appropriate nutrition is a necessary part of total care
for any patient. The enteral (gastric) route is preferred whenever possible and, for
patients unable to swallow a normal diet, feeding via a nasogastric tube is the method of
choice.
For patients unable to tolerate any form of enteral feeding, the administration of
fluid and nutrients via a parenteral route is necessary. In the short term e.g. immediately
postoperative administration of fluid dextrose may be adequate, but for long-term car a
balanced diet containing all the essential nutrients, including vitamins and trace
elements, must be provided.
Johnston at al reported in 1978 that an undernourished patient whose
gastrointestinal tract is temporarily or permanently unusable can increase lean body
tissue and also lay down fat if fed a suitable combination of nutrients intravenously.
This chapter gives an overview of TPN therapy its emphasis on the role of the
pharmacist.
3.1.1. Clinical aspects With an understanding of the clinical aspects of TPN, pharmacists can recommend
regimens to fulfil the needs of the patient as diagnosed by the physician. Possible
interactions with concomintant medication may be identified and advice given on
suitable administration systems. [2]
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According to an estimate by the Institute of Medicine in 1999, ≈1 million injuries
and almost 100 000 deaths may be attributed to medical errors annually. Most errors
that occur in he prescription, dispensing, and administration of medications could have
been preventedby redesign of the systems used to deliver medications to patients.
Practical interventions that attempt to change system processes rather than people were
found to be most successful in the prevention of adverse drug events (ADEs).
Unfortunately, the underlying system failures are rarely identified and corrected, so that
physicians, pharmacists, and nurses are often unwitting participants in the recurrence of
a well-known error. The rate for potential ADE is 3 times higher in children than adults
and substantially higher still for neonates in the neonatal intensive care unit (NICU).
Adequate nutritional support for premature or sick neonates requires the daily planning,
calculating, and ordering of parenteral nutrition. The ordering of parenteral nutrition is
associated with a high incidence of medical errors and a significant potential for patient
harm and is very time-consuming (≈10 minutes per patient.). [3][4]
Indications:
The main indications for TPN are as follows:
Adults
• Pre-and postoperative support
• Malignancy
• Inflammatory bowel disease
• Gastrointestinal fistulae
• Pancreatitis
• Severe trauma
• Burns
• Sepsis
• Hepatic failure
• Renal failure
• The intravenous route for nutrition should only be used where the oral or
nasogastric routes are not readily available.
Children
• Protracted infantile diarrhoea
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• Major alimentary tract surgery in newborns
• Prematurity
Harries at al reported in 1978 that in comparison with the adult, the peadiatric
patient has very little reserve of fat and protein to call upon during periods of
malnourish.
As can be seen in Table 1, the expected duration of survival of children and adults
during starvation and shows that the effect of nutritional deprivation is thus quite
dramatic in the neonate as compared to the adult.
As with adults, where a child requires nutritional support, the enteral or nasogastric
route must be used wherever possible. Where this is infeasible, intravenous feeding
should be instituted rapidly.
Table 1: Duration of survival during starvation Source: [2]
Age group Duration of survival (days) Small premature (1 kg) 4 Large premature (2kg) 12 Full-term infant (3.5kg) 32 One-year-old 44 Adult 90
Administration
Powell-Tuck at al reported in 1978 a technique for administration of the total daily
requirement for TPN via on single container. This was a significant advance over the
multiple-bottle method of administration.
Three-litre bag therapy, however, is not ideal for all patients. In an intensive care
unit, for example, requirements for fluids and electrolytes may change rapidly
throughout the day and require the careful titration that can only be obtained with
smaller volume fluids and injections.
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Choice of entry sites
Many of the individual solutions required for TPN, such as high-strength glucose,
together with mixed 3-litre regimens are generally hypertonic. For short-term therapy
rotation of peripheral entry sites may provide a simple means of administering TPN
without the complications of initiating central vein access. Silk at al reported in 1983
that for longer-term therapy a catheter placed into the superior vena cava as being the
safest method of entry providing rapid dilution of the hypertonic solution.
Flow control
Adult TPN patients generally receive 2-3 litres of fluids per day. Rapid,
uncontrolled infusion of this amount of fluid would cause renal overload and would be
of no benefit to the patient. It is thus vital that some form of flow control device is
employed. This may range from simple clamps through to electronic drip controllers.
Patient assessment and monitoring
Once TPN has been initiated on a patient it is essential that routine monitoring is
carried out. The clinical pharmacist shoud have an understanding of the relevance of
these routine tests (particularly those such as 24-hour urine analysis which is the main
determinant of nitrogen requirements) in order to make adjustment to the TPN
formulation.
Home TPN
Patients with severe Crohn’s disease, excessive bowel resection, etc, who may
require long-term, if not permanent, TPN therapy may be ideal candidates for home
TPN provided that their home environment is suitable.
The majority of patients use the 3-litre bag system administered overnight via an
alarmed pump system.
The patient requires a period of training whilst hospitalized which encompases
aseptic technique, product storage and handling, reporting of effects or complications,
use of ancillary items and pumps, before they are capable of treating themselves at
home.
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3.2. Formulation of TPN Parenteral nutrition solutions are complex formulations that generally include
energy supplied as dextrose and fat, as well as protein, electrolytes, trace elements,
vitamins, and water. These components usually need to be individualized for patients
according to their primary diagnosis, chronic diseases, fluid and electrolyte balance,
acid-base status, and specific goals of parenteral nutrition.
The proportions and mix of components of solutions used for intravenous nutrition
can vary considerably depending upon the patient’s nutritional status and underlying
medical or surgical condition. The components available for TPN are detailed below.
3.2.1. Nitrogen The main objective of parenteral nutrition is to supply the undernourished patient
with sufficient utilizable nitrogen to re-establish nitrogen balance, i.e. where the amount
of nitrogen administered is approximately equal to that excreted (mainly as urea).
3.2.2. Choice of amino acid The body’s relative requirements of the individual amino acids is expected as
follows:
Essential, i.e. which cannot be synthesized by man. All the commercially available
solutions contain the eight essential amino acids in varying proportions.
Non-essential, i.e. those amino acids which can normally be synthesized by the
body. These amino acids are used to increase the amount of nitrogen available from the
solutions and the optimum ratio of essential to non-essential amino acids has yet to be
agreed between workers.
Semi-essential, i.e. those amino acids which although they can in theory be
synthesied by the body, may occasionally need to be provided in the TPN solution due
to the patient's age or disease state. [2]
Amino acids provide 4 kcal/g when oxidized for energy. Generally, it is desired to
provide enough total or nonprotein calories that the utilization of the amino acids for
protein synthesis is optimized.
Parenteral amino acid products can be conveniently divided into two groups:
standard amino acid formulations and modified amino acid formulations. The standard
amino acid products are used for patients with normal organ function and nutritional
needs. [1][5]
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Table 2: Neonatal and Pediatric Amino Acid Products Source: [1]
TrophAmine 6% (McGraw)
Aminosyn-PF 7% (Abbott)
L-Amino acid content (g/100 ml) 6 7 Nitrogen (g/100 ml) 0.93 1.1 Essential Amino Acids (mg/100 ml) Isoleucine 490 534 Leucine 840 831 Lysine 490 475 Methionine 200 125 Phenylalanine 290 300 Threonine 250 360 Tryptophan 120 125 Valine 470 452 Nonessential Amino Acids (mg/100 ml) Alanine 320 490 Arginine 730 861 Histidine 290 220 Proline 410 570 Serine 230 347 Tyrosine 140 44 Glycine 220 270 Cysteine <14 - Electrolytes (mEq/ 100 ml) Sodium 5 3.4 Potassium - - Magnesium - - Chloride <3 - Acetate 56 33 Phosphorus (mM/L) - - Osmolarity (mOsm/L) 525 586 Amount Supplied (ml) 500 250 and 500
From financial perspective, a significant cost of parenteral nutrition is the amino
acid source. Modified amino acid products are most costly per gramm of nitrogen
infused.
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3.2.3. Non-nitrogen energy The malnourished patient requires, in order to utilize administered nitrogen in the
form of amino acids, an independent energy source. There are many such individual
sources available which have been utilized historically although many have now fallen
out of favour due to undesirable side-effects. Examples of these are fructose, sorbitol,
xylitol and ethanol. The use of combinations of mixed calorie sources for parenteral
nutrition have been reported but these appear to have no major benefit over and above
the use of glucose alone. [2]
3.2.4. Energy-Macronutrients Dextrose
Dextrose is the primary source of parenteral carbohydrate. Dextrose is needed by
the central nervous system, white blood cells, red blood cells, and renal medulla. Each
gram of hydrated dextrose used in parenteral nutrition yields 3.4 kcal. Important of
formulation design is the maximal rate of dextrose that oxidized by the body: 5
mg/kg/min (=25 kcal/kg/day)
As such, parenteral nutrition solutions suitable for peripheral vein administration
have dextrose concentrations of 10% or less. This method of parenteral nutrition
administration is usually avoided because it depends on a patient’s having satisfactory
veins, tolerating large fluid volumes, having relatively normal nutritional needs, and
requiring therapy for a short period (e.g., <1 week)
Parenteral nutrition solutions with final concentrations of 10% or greater must be
administered by a central vein because of the high osmolarity. [2]
From the literature it would appear that glucose is the carbohydrate of choice in
nutrition, however, it is not without metabolic complications. Glucose handling in the
sick patient becomes complicated due mainly to an inbalance in the normally well
regulated hormonal systems which are in existence.[6]
This can lead to hyperglycaemia, hypoglycaemia, hypophosphataemia etc. Careful
design of TPN regimens plus the use of lipid as a complementary calorie source can
minimise such effects. [2].
15
Lipid
Their administration, whilst providing some benefits to the patient, is not without
clinical difficulties such as interference with diagnostic tests and deposition in tissues
such as the lung.
Administration of lipid emulsion on a daily, twice or three times weekly basis
appears to provide a balanced mixture of nutrients for the patient requiring long-term
feeding.
Intravenous fat emulsions are used in parenteral nutrition as an energy source and to
provide essential fatty acids. Today, the commercially available products in the United
States are manufactured from soybean oil and safflower oil. These oils are
predominantly made up of long-chain fatty acids having a high content of the
polyunsaturated fatty acids linoleic and linolenic acid. They also differ somewhat in
fatty acid composition but are similar in other characteristics important to maintaining
the stability of the emulsion, for example, egg yolk, phosphatide emulsifier, pH, and
osmolarity, resulting in a small particle size. These characteristics are important in
choosing products and formulating parenteral nutrition admixtures. It is essential to
maintain a safe particle size of the emulsion. The metabolism of intravenous fat
emulsions is similar to that of endogenous chylomicrons. In unstable emulsions, larger
particle sizes develop and lead to symptoms of fat embolism when infused. Generally,
acute reactions such as hypotension, pulmonary hypertension, and acidosis may result
when a fat particle larger than 6 µm is infused.
Intravenous fat emulsions are particularly beneficial as an energy source in patients
or conditions that predispose to harmful effects of dextrose. These include diabetes,
stress, and other hyperglycemic conditions; respiratory acidosis; and hepatic steatosis.
When a substantial percentage of energy is administered lipid (e.g., up to 30% of total
calories), less dextrose needs to be given to meet energy requirements.
Amino acids provide 4 kcal/g when oxidized for energy. Generally, it is desired to
provide enough total or nonprotein calories that the utilization of the amino acids for
protein synthesis is optimized. [1]
Intravenous lipid emulsions are utilized clinically both as a calorie source and as a
source of essential fatty acids. Their administration, whilst providing some benefits to
16
the patient, is not without clinical difficulties such as interference with diagnostic tests
and deposition in tissues such as the lung.
Administration of lipid emulsion on a daily, twice or three times weekly basis
appears to provide a balanced mixture of nutrients for the patient requiring long-term
feeding.[2]
The safe use of intravenous lipid emulsion (IVLE)2 has been a therapeutic challenge
to clinicians in the 20th century.
The motivation to develop such a product was the need for a calorically dense
source of energy hat was iso-osmotic and could be given daily by way of a peripheral
vein.
In the early part of the 20th century, the infusion of IVLE in patients met with
variable degrees of success.[1]
Pharmaceutical review of intravenous lipid emulsions
Pharmaceutical-grade IVLE is a complex dispersion of oil droplets that has been
carefully homogenized to produce a high-quality dispersion, safe for intravenous
administration, with particles of a mean dimension approximately 0.3 μm or 300nm in
diameter. Commercial IVLEs are highly concentrated dispersions and are available in
final lipid concentrations of 10, 20, and 30%.
Both the 10% and the 20% IVLEs can be given as separate infusions or as a TPN
formulation. The 30% IVLE is only indicated for pharmaceutical compounding
purposes as a TPN and is not recommended for direct intravenous administration in its
undiluted form. Just how concentrated IVLEs are can be estimated by making some
elementary mathematical assumptions, and then the relative concentrations of lipid
droplets can be calculated in order to illustrate the magnitude of lipid droplets per
milliliter of these undiluted dispersions. [7]
For most commercially available IVLEs, the phospholipid emulsifying agent is held
constant, irrespective of the final lipid concentration. This would suggest that there
exists an amount of emulsifier in excess of that necessary to stabilize the emulsion, at
least in the lower concentrations of IVLE formulations. The proportion of emulsifier to
triglyceride is greatest in the 10% IVLE formulation.
Table 3: Concentration of Lipid Droplets in Intravenous Lipid Emulsions
2 IVLE= intravenous lipid emulsion
17
Source: [16]
Lipid concentration (droplets/ml)
MDD 10% 20% 30%
0.3µm 7.77 x 1014 1.55 x 1013 2.33 x 1013
0.4µm 3.27 x 1012 6.55 x 1012 9.83 x 1012
0.5µm 1.67 x 1012 3.35 x 1012 5.03 x 1012
Droplet Number = Total Mass / Mass of Individual Droplet
Where:
Total Mass = lipid concentration in g/ml
Mass of Individual Droplet = density (of oil) x volume (of sphere)
Droplet Number = Lipid concentration in g/ml/oil density x
4/3 π r3
Example
Where:
Total Mass = 0.1 g/ml, 0.2 g/ml, or 0.3 g/ml
Density of oil = 0.91 (soybean oil)
Volume of Spherer = 4/3 π r3
Radius of Droplet = ½ diameter in centimeters (MDD3 = 0.3 µm or 0.15 x 10-4 cm)
This ratio has been suggested to underlie the hypertriglyceridemia seen with the
separate administration of 10% IVLE to neonates [8] and to adults at very high infusion
rates. [9]
The mean lipid droplet size of 300nm is within the typical range of the dimensions
of endogenous chylomicrons (range, 80 to 500nm) [10] and the formulations are
manufactured in this way so as to behave in a similar manner with respect to their
metabolic fate.[11]
3 MDD = Mean Droplet Diameter
18
Chylomicrons are composed of a core of reconstituted triglycerids surrounded by a
surface-active layer of phospholipids and small amounts of cholesterol and apoprotein
B.
They assist in the digestion of fats, facilitated through the process of emulsification.
Pharmaceutical IVLEs consist of a relatively homogeneous dispersion of lipid
droplets as triglycerides that are similarly surrounded by a phospholipid surfactant in a
continuous aqueous phase, forming an oil-in-water emulsion system.
The most common IVLE emulsifying agent or surfactant used is a purified mixture
of egg yolk phospholipids.
All lipid emulsions intended for intravenous administration must be oil-in-water
mixtures in order to avoid introducing potentially fatal lipid emboli into the
intravascular compartment, which may occur with water-in-oil mixtures or „cracked”
oil-in-water emulsions.
A cracked oil-in-water emulsion is another term for the terminal phase of emulsion
destabilization, in which finely dispersed lipid droplets progress through a stage of
aggregation and coarsening of fat globules, resulting in the separation of the two
immiscible phases oil and water.
The degree to which this occurs in the clinical setting is variable and is not always
visually apparent.
A 1995 study employed a single-particle optical sensing technique using light
extinction to identify the subvisible changes associated with globule size coarsening that
occur in the upper size range of the lipid droplet size distribution of the emulsion (i.e.,
globules >1um).[12]
From this work involving 90 admixtures using a fractional factorial design, it is
clear that when 0.4% or greater of the total fat present exceeds 5 um in diameter using
the single-particle optical sensing technique, the emulsion can be considered
pharmaceutically unstable and therefore unsuitable for intravenous administration.
Although the precise toxic dose of unstable and enlarged fat globules is unknown, a
pharmaceutically unstable lipid emulsion should be considered unsafe and therefore
unfit for human administration.
19
Three principal components affect the final stability and subsequent safety of
parenteral lipid emulsion: the „internal” or dispersed oil phase, the emulsifying agent,
and the „external” or continuous aqueous phase.
(1) Dispersed Oil Phase
The commercial emulsions provide a wide array of lipids used, including the
polyunsaturated fatty acids (PUFAs) as either n-3 or n-6 long-chain triglycerides
(LCTs), such as fish oil and soybean oil, respectively, monounsaturated fatty acids as n-
9 LCT-s, such as medium-chain triglycerides (MCTs).
The commercially available parenteral emulsion products intended for intravenous
administration include these oils either alone or in combination with others as mixtures.
When combinations of these oils are produced, they are prepared by physically
blending or mixing the lipid components (as physical mixtures), or by transesterification
of the MCTs and LCTs to chemical mixtures (as structured lipids).[13]
Combinations of oils can reduce the adverse reaction profile of an individual oil by
decreases in dosage and may even enhance the metabolic clearance, and subsequent
safety, of the IVLE.
For example, from studies assessing the differences in plasma clearance between
certain lipid emulsions, the differences have been explained by changes in the physical
characteristics of lipid droplets from an MCT-LCT physical mixture.
Nuclear magnetic resonance studies performed on the dispersed phase identified
MCT at the surface of the LCT droplets, which might explain the superior clearance
from plasma seen with pure MCT and MCT-LCT mixtures compared with pure LCT
lipid emulsions.
Additional evidence of similar droplet behavior has also been shown in an in vitro
study evaluating the effect of different lipid emulsions on neutrophilic adhesion.[14]
MCT-LCT emulsions acted similarly to pure MCT emulsions in that they
stimulated neutrophilic adhesion, whereas pure LCT did not.
In fact, neither did a structured MCT-LCT mixture, which suggests that the
dominant action of the structured lipid droplet was similar to that of a pure LCT
emulsion.
20
It would appear from these preliminary findings that the pharmaceutical and
physiologic actions of physical mixtures of MCT and LCT behave similarly to those of
pure MCT emulsions, and that structured mixtures of MCT and LCT behave more like
pure LCT dispersions.
These findings might also explain the differences in the physicochemical stability
between IVLEs either alone or as a TPN, [15] yet it will require further study to confirm
and possibly extend the implications of these findings.
Finally, the dispersed oil phase of IVLEs is used as a drug vehicle for compounds
that have poor water solubility and/or stability in aqueous media.
Compared with the unique formulation characteristics of specifically designed drug
liposomes, IVLEs may offer a simpler and cost-effective alternative drug dosage
form.[16]
Since the 1970s, lipid emulsions based on LCTs from soybean or safflower oil have
been used in parenteral nutrition. For many years, lipid supply has been considered as a
means of preventing or correcting essential fatty acid deficiency and of providing an
efficient fuel to many tissues of the body.
Obviously, these effects are related to the dose and infusion rate of lipid emulsions.
The mechanism are not totally clear, but an excessive intake of linoleic acid seems
to be one of the major reasons for interference with immune function.[17]
Therefore, efforts at further developing and optimizing lipid emulsions have
focused on replacing part of the LCTs by MCTs synthesized from coconut oil.
An MCT-LCT –containing lipid emulsion has been available on the European
market since 1984 and later worldwide. Numerous research teams have studied the
parenteral application of this physical MCT-LCT mixture in a clinical environment and
during longterm home parenteral nutrition (HPN).[17]
(2) Structured triglycerids
An alternative to a physical mixture of MCTs and LCTs can be obtained by
interesterifying medium-and long-chain fatty acids to create a mixed triglyceride
21
molecule called a structured triglyceride (STG). One must clearly differentiate between
chemically defined and randomized STGs.
The later can be synthesized by mixing MCT and LCT oils and heating the mixture
in the presence of a catalyst.
During this process, fatty acids of different chain lengths can be esterified into one
triglyceride molecule.
The individual STG molecule within the emulsion is composed of three fatty acids
in which the proportion of medium-and long-chain fatty acids varies randomly, that is,
the structure of the individual triglyceride is heterogeneous and depends on the initial
proportion of LCT and MCT oils.[13] [18]
Chemically defined STGs, in contrast, are made by enzymatic re-esterification. As
a consequence of the positional specificity of the lipase used, these triglycerides contain,
for example, medium-chain fatty acids in the 1.3 positions and long-chain fatty acids un
the 2 position.[19][16]
(3) Oxidative Utilization
For many years, the oxidative utilization of MCTs and LCTs in total parenteral
nutrition of severely injured patients was unknown. [16]
(4) Emulsifier
The principal emulsifying agent used to stabilize IVLEs is a purified mixture of egg
yolk phosphatides. From a physicochemical standpoint, the phospholipid emulsifier
possesses some ideal characteristics for producing a stable dispersion and forms the
basis of the emulsion system.
The amphophilic characteristics of the emulsifier are critical to its ability to adsorb
at the oilwater interface and therefore make the two immiscible phases miscible.
The hydrophilic head occurs at position 3 of the glycerol backbone, which is
esterified with phosphoric acid linked through another ester bond to an alcohol such as
choline.
The head is composed of polar phosphate groups that extend into the continuous
aqueous phase, whereas the hydrophobic tails orient toward the dispersed oil phase of
the emulsion. Ionization of the polar phosphate group in the hydrophilic head produces
a net negative charge on the individual droplets (zeta potential).
22
This electronegative potential, along with its corresponding counterions (required
for electroneutrality), forms a complex electric double layer that provides a formidable
electrostatic barrier against droplet coalescence. The hydrophobic tails comprise
principally nonpolar fatty acid residues, such as palmitic (a saturated fatty acid), which
occupies position 1 of the glycerol moiety: position 2 is generally occupied by an
unsaturated fatty acid, such as oleic acid.
Coupled with the position opposite the hydrophilic head, the closely packed
alignment of the emulsifier along the oil-water interface forms a molecular film around
each submicron lipid droplet that forms an effective mechanical barrier against the
coalescence of fat globules.
23
(5) Continuous Aqueous Phase
The continuous phase of IVLEs is of less importance as a drug vehicle than the
dispersed oil phase.
In terms of IVLEs and TPNs, the composition of the continuous aqueous phase has
major implications for the physicochemical stability of the dispersion. It is the ion-rich
phase of the emulsion. The relative concentrations of prescribed electrolytes will greatly
influence the otherwise stabilizing forces of electrostatic repulsion. (net negative charge
imparted by the emulsifier) between lipid droplets and may adversely affect the
subsequent stability of the emulsion system.
High concentrations of electrolyte salts, especially higher-valence cations such as
calcium and magnesium, tend to make the emulsion less stable by reducing the efficacy
of the electronegative surface charge of the phospholipid emulsifying agent on the lipid
droplets.[12][20]
In addition, low final concentrations of ionic amino acids also decrease
stability.[21]
Although dextrose is a nonelectrolyte, in low final concentrations it too makes for a
less stable emulsion.[22][23]
In essence, formulations with the combination of low final concentrations of both
amino acids and dextrose, as occurs in formulations intended for peripheral vein
administration, tend to be less stable then formulations with higher final concentrations,
such as those intended for central venous alimentation.
Alteration of the dispersion medium, such as a compositional change of the IVLEs
that occurs during the extemporaneous compounding of TPN formulations, influences
the molecular forces that interact at the oil-water interface, as well as the balance of
ionic charges within the bulk, or continuous aqueous phase, of the emulsion. The
variable compositions of TPN formulations, particularly those used in acutely ill
patients to treat severe metabolic disorders, compared with the stable home total
parenteral nutrition (TPN) patient, provide an extraordinary array of physicochemical
challenges to the integrity of the barriers against coalescence and thus the stability and
subsequent safety of the parenteral nutrient admixture.
24
The effects of forming a TPN product may promote destabilization and coalescence,
or a net repulsive energy to stabilize the system may be maintained.
For any parenteral pharmaceutical formulation, destabilization is an expected
outcome of the extemporaneous manipulations of the original container, whether it is an
emulsion or not.
What is important is the assurance of a reasonably assigned „new” shelf life by the
compounding pharmacist that ensures the administration of a stable and compatible
formulation during the infusion period.[16]
(6) Clinically Relevant Glucose Infusion Issues
The rate of glucose infusion has a significant effect on the risk of one’s developing
metabolic complications. Intravenous glucose may follow one of three principal
metabolic pathways.
Oxidation of infused glucose to meet the energy demands of the body is the most
desirable.
The infused glucose may also proceed through nonoxidative pathways. The
nonoxidative disposal to replete glycogen stores is also desirable and occurs at limited
cost and metabolic risk to the host.
However, the glycogen storage capacity of the human body is limited, leaving the
alternative nonoxidative fate of glucose to the production of fat from glucose, a process
known as de novo lipogenesis, to prevail, and this can produce clinically significant
adverse effects.
Finally, the circulating glucose level also increases when excessive infusion rates
are used, and this can produce significant impairments in immune function, particularly
in activated monocytes.[24]
Optimizing the amount of glucose metabolized by the oxidative pathway,
controlling its level in the circulation, and minimizing its metabolism to fat improve the
clinical care of patients receiving nutritional support.
The use of a mixed –fuel system fosters this balance. [16]
(7) Clinically Relevant Long-Chain Triglyceride Infusion Issues
As with glucose, the rate and quantity of LCT emulsions infused in patients has a
significant impact on their tolerance and adverse reaction profile.
25
These effects are magnified in the presence of critical illness and comorbid disease.
Perturbations in reticulo-endothelial system function, hypertriglyceridemia, and adverse
impacts on pulmonary gas diffusion have been associated with the rapid, intermittent
infusion of IVLEs.
However, when given in sufficiently high doses, even continuous administration of
IVLEs can produce pathologic effects.[25]
In an editorial by Klein and Miles in which they reviewed the complication rates
associated with LCT emulsions in humans, they concluded that the adverse effects
reported with LCT administration occured when lipid infusion rates were greater than
0.11 g/kg/h.[26]
Table 4 illustrates variable lipid infusion rates in a series of adult patient
weights.[16]
Table 4: Daily lipid infusions in adults at 15% and 30% and at the Toxic Treshold Source: [16]
Weight (kg) 15% of kcal (g) 30% of kcal (g) 0.11g/kg/h (g) 40 17 34 106 50 21 42 132 60 25 50 158 70 29 58 185 80 33 66 211
(8) Clinical Application of a Mixed-Fuel Regimen
In the acute care setting, IVLEs are generally given as a daily caloric source, often
to reduce the amount of carbohydrate calories and their potential for producing
complications when given in excess to susceptible patients.
As lipids are a preferred source of fuel for skeletal muscle, substitution of a portion
of the glucose calories has resulted in better nitrogen balance.[27]
Given the acute metabolic stress of such patients, excessive administration of either
fat or carbohydrates can be harmful. In this setting, the composition of the TPN
formulation is intended to provide a balance of nonprotein calories.
Generally, 15 to 30% of the daily calories are administered as fat, with the balance
as carbohydrate. Exceeding 30% of calories as fat in the acute care setting has not been
shown to confer additional clinical benefits.[28]
26
IVLEs can be administered either as a TPN formulation or as a separate infusion of
lipid emulsion.
Providing IVLE s as TPN not only simplifies nutritional therapy but affords
additional safety benefits related to better utilization when given continuously,[29]
reduced potential for microbial growth, [30][31] and a reduction in the hyperinsulinemic
state and its attendant complications.[32]
It is clear from the previous discussion that the rate and quantity of energy supplied
are crucial to the safety of either intavenous glucose or intravenous lipids.
As a second principle, once the appropriate feeding weight has been determined, the
rate of infusion should be concordant with the patient’s ability to metabolize the type
and amount of energy supplied. Excessive rates of infusion of either carbohydrate or
lipid can produce serious metabolic disturbances and attendant complications.
Moreover, providing a mixture of glucose and lipids in net quantities that do not
exceed energy needs not only is a more physiologic regimen but also reduces the
likelihood of complications from either substrate.
A daily mixed-fuel parenteral nutrition regimen can be accomplished in a number of
ways, including providing the lipids as a separate infusion, providing lipids, glucose,
and amino acids mixed in one container for immediate use, and adding lipid in one
container but in a separate compartment for later admixing by the clinician, patient, or
caregiver.[16]
(9) Separate Infusion of Lipids
The separate infusion of IVLEs has been the historical means of providing
parenteral lipids and can be done safely, bearing in mind the attendant risks associated
with this method of administration.
Essentially, there are two potential problems with the intermittent administration of
IVLEs.
First, as discussed extensively in previous sections, the discontinuous infusion of
parenteral lipids may increase the risk of metabolic complications if the infusion rate is
excessive. These complications are perhaps viewed by some to be of lesser importance,
since the exposure to IVLEs is limited in time and therefore effectively cleared in the
postinfusion setting.
27
However, it is clear that in susceptible patients receiving large amounts over brief
intervals, such infusions can be harmful.
The IVLEs threshold of 0.11 g/kg/h to produce metabolic complications is
applicable in all infusion conditions, irrespective of whether it is a continuous or a
discontinuous infusion.
A second problem associated with the separate administration of IVLEs is the risk
of inadvertent microbial contamination, especially in prolonged infusion conditions.
The more recent recommendation for administration set changes is in response to
the outbreak of postoperative infections with an anesthetic agent that is in the equivalent
of a 10% IVLEs drug vehicle.[33]
Thus, if IVLEs are given separately, three criteria should be met to ensure their safe
and efficacious administration to acutely ill patients. First, the discontinuous infusion
rate should not exceed 0.11 g/kg/h.
In the 70kg reference man receiving 25kcal/kg and 20% of the total calories as fat, a
200ml bottle of 20% lipids should not be given over an infusion period of less then 6
hours (0.095 g/kg/h) .
Second, no manufacturer’s container of lipids, ready for infusion, should be infused
for a period exceeding 12 hours. [16]
(10) Combined Infusion of Intravenous Lipid Emulsions as Total Nutrient
Admixture
TPNs offer a number of advantages compared with the separate infusion of lipids,
and these have been described.[31]
With respect to the infusion-related issues, like fat emulsion-free TPN admixtures,
TPNs are less able to support the growth of typical nosocomial pathogens than IVLEs
alone.[30]
This is likely due to two major physicochemical differences between native IVLEs
and TPNs.
Specifically, IVLEs are iso-osmotic and have a mean pH of 7.5 whereas TPNs for
central venous alimentation are hypertonic (often exceeding 1500 mOsm/l) and have a
typical final pH of 5.8 to 6 after compounding the all-in-one dosage form.
With regard to their safe hang time, the typical 24- hour infusion applies as it would
for fat emulsion-free TPN.
28
Finally, because TPNs behave like fat emulsion-free TPN with respect to concerns
about microbial growth potential, a study has questioned the need for administration set
changes every 24 hours and suggests that set changes every 72 hours would confer no
additional risks than those of TPN without IVLEs.[34]
Thus, TPNs should be treated in the same manner as fat emulsion-free TPN with the
obvious exception of the size of the in-line filter.
Fat emulsion-free TPN should be filtered through a 0.22 um filter, whereas TPNs
should be administered through a 1.2um filter,[35] which is consistent with the FDA
recommendations.[16][36]
(11) Combined Infusion of Intravenous Lipid Emulsions in
Compartmentalized Infusion Containers
The addition of IVLEs to a compartmentalized infusion container is generally done
when the infusions are intended for later use.
Multicompartmental containers that can accomodate the separation of amino acids,
carbohydrate, and lipids (three-compartment bags) are available in Europe.
A major benefit of this dosage form is in allowing the activation (versus
compounding) of the parenteral nutrition admixture by nonpharmacy personnel without
compromising the pharmaceutical integrity of the formulation. [16]
Clinical Review of pure long-chain triglyceride mixtures
As energy donors, lipid emulsions are an integral element of parenteral nutrition
regimens for critically ill patients.
Lipids are not only structural building blocks of cells and tissues but carriers of
essential fatty acids and fat-soluble vitamins.
In addition, certain fatty acids are precursors of prostaglandins and other
eicosanoids and thereby serve important metabolic function.
Fatty acids can be divided into three groups: saturated, monounsaturated, and
polyunsaturated fatty acids.[37] Each class of fatty acids has a preferential specific role
Saturated fatty acids (medium or long-chain) are more devoted to energy supply,
but one should not forget their specific structural role. The PUFAs of the n-3 and n-6
families have very important structural and functional roles and ideally should not be
extensively used for energy purposes.
29
This section provides a brief overview of the evolution of different types of lipid
emulsions that are already in widespread clinical use or are in the final stages of
development.[16]
(12) Peroxidation in Parenteral Nutrition
Unless prevented by antioxidants, peroxidation reactions in lipid emulsions may
lead to clinical complications. Apart from its implications in chronic diseases, lipid
peroxidation leads to tissue damage and an inflammatory response, together with an
impairment of immune defenses. It may markedly alter the function of several major
organs including the lungs, liver, heart, and kidneys.[16]
(13) Lipid emulsions containing olive oil
As pointed out earlier, soybean oil emulsions provide PUFAs in excess of
requirements, causing the development of abnormal fatty acid profiles, augmentation of
peroxidation, and the creation of eicosanoid imbalances. To reduce these risks of TPN
in critically ill patients, substitution of parts of the soybean oil-based lipid emulsions by
other lipid components is strongly recommended.
Meanwhile, the concept of a physical mixture with MCTs and LCTs has been well
proved.
Another concept is based on the idea of mixing 20% soybean oil with 80% olive oil,
the latter being rich in the monounsaturated fatty acid oleic acid.
Olive oil emulsions are a better source of antioxidants and should contribute to
decreased peroxidation.[16][38]
30
(14) Lipid emulsions containing fish oil
The virtual absence of heart diease or myocardial infarction among Greenland
Inuits was a key epidemiologic observation leading to the focus on n-3 fatty acids.
Studies comparing the diet, blood and tissue lipids, bleeding times, and various
aspects of platelet aggregation among Greenland Inuits, Inuits livingin Denmark , and
Danes have supported the view that diet, in particular the long –chain n-3 fatty acids,
rather than genetics, accounts for the striking advantage Greenland Inuits have over the
other two groups with respect to heart disease.[39][40]
Following these first reports, there was an exponential increase in the number of
publications focusing on the metabolic and clinical effects of fish oils. Preliminary
clinical trials have shown certain beneficial effects of fish oil intakes in diseases
associated with inflammatory reactions such as rheumatoid arthritis or inflammatory
bowel disease, in conditions with impaired immune competence such as burns,
postoperative situations, and cyclosporine treatment after renal transplant.
Lipid emulsions containing fish oil (n-3 fatty acids) are poor substrates for
lipoprotein lipase and triglycerides, and they tend to accumulate in the circulation. In
contrast to most triglyceride fatty acids, triglycerides containing n-3 fatty acids are
taken up by tissues mainly via remnant endocytosis, followed by intracellular
triglyceride hydrolisis.
Even the addition of n-3 containing triglycerides to classic LCT emulsions inhibits
the release of free fatty acids from the soybean oil emulsion.[41]
In contrast, the combination in the same particle of MCTs together with fish oil
triglycerids appears to completely normalize triglyceride hydrolysis by lipoprotein
lipase [42] and to rapidly produce small remnants enriched with n-3 fatty acids.
In healthy subjects, the infusion of an emulsion containing 50% MCT, 40%
soybean, and 10% fish oil triglycerides is associated with a rapid triglyceride
elimination and completely avoids lipid accumulation in plasma.
New preparations seem promising not only for metabolic care but also for
hemodynamic stability in different organ systems of intensive care unit patients. There
is evidence that n-3 fatty acids can also modulate regional blood flow and therefore
prevent intestinal ischemia.
31
Lipid emulsions derived from soybean or safflower oil contain excessive quantities
of PUFAs and insufficient amounts of α-tocopherol. Their parenteral use can rapidly
lead to an unbalanced pattern of eicosanid production and is associated with an
increased production of peroxidative catabolites.
The physical mixture of MCTs and LCTs is a well-proven concept in parenteral
nutrition of critically ill patient.
MCT-containing lipid emulsion do not impair liver function, produce less immune
and no RES function compromise, and do not interfere with pulmonary hemodynamics
or gas exchange.
A promising substrate in the evolution of parenteral lipid emulsions can be seen in
fish oils (n-3 fatty acids). Their fixed combination in a physical mixture of MCT-LCT
emulsion displays a number of interesting aspects. With regard to current literature, n-3
fatty acids have a beneficial influence on the pathophysiologic response to endotoxins
and exert important modulations on eicosanid and cytokine biology. Furthermore, their
intravenous use may improve organ perfusion in different critical situations.[16]
Electrolytes
Electrolytes in maintenance or therapeutic doses need to be added daily to the
parenteral nutrition solution to preserve electrolyte homeostasis. Each patient’s
requirements for individual electrolytes depend on the primary disease state, renal
function, hepatic function, pharmacotherapy, past intake, renal or extrarenal losses, and
nutritional status. Extrarenal electrolyte losses may include those from diarrhea,
ostomies, vomiting, fistulas, or nasogastric suctioning.
32
Table 5: Daily Electrolyte Requirements for Adults4
Electrolyte
Recommended Daily
Supplementation Conventional Dosing Range Calcium 10mEq 10-15 mEq Magnesium 10mEq 8-20 mEq Phosphate 30mmol 20-40 mmol Sodium Variable 1-2 mEq/kg + replacement Potassium Variable 1-2 mEq/kg Acetate Variable As needed to maintain acid-base balance Chloride Variable As needed to maintain acid-base balance
Table 5 shows some of the small volume electrolyte additive solutions available.[1]
Electrolytes may be added to parenteral nutrition solutions using single- or
multipleentity products. The multiple-electrolyte formulations may be used for patients
who have normal organ function and normal serum concentrations of electrolytes. These
products usually lack calcium or phosphorus, or both, so these must be added at the time
of preparation. The obligate electrolyte content of the amino acid product should also be
considered. Most amino acid products contain substantial amounts of chloride and
acetate salts. Some amino acid products are formulated with maintenance electrolytes.
During a pharmacy’s compounding process, inadequate consideration of the phosphorus
content of a manufacturer’s amino acid product resulted in calcium phosphate
precipitation. [2]
(15) Sodium
Sodium is of critical importance in the fluid balance of both of healthy and sick
subjects.
Sodium losses and gains are generally accompanied by similar shifts in chloride
ions and a consequent movement in water.
Severe losses may lead to hypovolaemia, circulatory failure and shock. Generally a
serum concentration of 135-145 mEq/litre is throught to be normal.[2]
4 Assuming patients have normal organ function.
33
(16) Potassium
Potassium is essential for the normal operation of the cell and is an important
determinant of cell membrane resting potential. Thus abnormally high or low levels can
result in poor nerve impulse conduction, fluctuations in heart rhythm and even death
due to heart failure.It also plays a vital role in distribution of body water.
(17) Calcium
Absence of calcium from TPN in the long term may produce symptoms of
hypocalcaemia such as muscle spasm and numbness. The effect of lack of calcium on
the growing child on TPN could understandably have a dramatic effect of growth and
development of bones and teeth. Abnormalities involving both high and low levels of
calcium may be responsible for a wide variety of clinical conditions.
(18) Magnesium
Magnesium has many physiological actions. The most clinically significant effects
of magnesium imbalance are associated with changes in neuromuscular or
cardiovascular function.
(19) Phosphate
By virtue of its buffering action phosphate helps maintain body acid-base balance.
If phosphate is not provided in the TPN solution hypophosphataemia may develop
which can give rise to impaired red blood cell function of many organs.
Hypophosphataemia may also be induced as a result of infusion of high glucose loads.
[2]
(20) Trace elements
The primary use of the individual trace elements which are considered to be
clinically significant:
Zinc, Copper, Selenium, Chromium, Iron, Manganese, Cobalt, Molybdenum.
Trace elements are essential micronutrients that are metabolic cofactors essential for
the proper functioning of several enzyme systems. Most practitioners add these
nutrients to the parenteral nutrition solution daily. The Nutrition Advisory Group of the
American Medical Association has also published guidelines for four trace elements
known to be important to human nutrition. The suggested amounts of zinc, copper,
manganese, and chromium for adults are listed in Table 6. Since the original
recommendations, substantial evidence for the essentiality of selenium has
34
accumulated.[43] Zinc requirements are increased in metabolic stress secondary to
increased unitary losses and in gastrointestinal disease secondary to ostomy or diarrheal
losses. Manganese and copper are excreted through the biliary tract, whereas zinc,
chromium, and selenium are excreted renally. Therefore, copper and manganese should
be used with caution in patients with cholestatic liver disease. Further, evidence
suggests that the amount of manganese in multiple-trace element formulations is too
high, resulting in elevated serum levels that may lead to neurologic symptoms.[44]
Selenium stores have been shown to be depleted in patients receiving long-term
parenteral nutrition [43] or in those with thermal injury,[45] acquired immunodeficiency
syndrome, [46] or liver failure.[47] Therefore, selenium should be added initially to the
parenteral nutrition solution for patients with these disease states or conditions. The
trace elements are available as both single- or multiple-entity products. Parenteral
guidelines for molybdenum and iodine have not been established; however, these trace
elements are available commercially.[1]
Table 6: Suggested intakes for Parental Trace Elements Source: [1]
Trace Elements
Adults Children (µg/kg/day)
Neonates (µg/kg/day)
Zinc 2.5-4 mg/day 100 300 Copper 0.5-1.5 mg/day 20 20 Manganese 150-800 µg/day 2-10 2-10 Chromium 10-15 µg/day 0.14-0.2 0.14-0.2 Selenium 40-80 µg/day 2-3 2-3
35
(21) Vitamins
Patients on long-term TPN therapy will generally require some vitamin
supplementation. The commercial preparations of vitamins available along with
recommended daily requirements which seem to vary according to the current available
recommendations: [2]
• A Retinol
• B1 Thiamines
• B2 Riboflavine
• B6 Pyridoxine
• B12 Cyanocobalamin
• B Nicotinamide
• B Biotin
• B Pantothenic acid
• B Folic acid
• C Ascorbic acid
• D Calciferol
• E Tocopherol acetate
• K Phytomenadione
Vitamins are an essential component of a patient’s daily parenteral nutrition
regimen because they are necessary for normal metabolism and cellular function of the
body. The Nutrition Advisory Group of the American Medical Association has
established guidelines for the 13 essential vitamins (four fat-soluble vitamins and nine
water-soluble vitamins) in adult and pediatric patients.[48][49]
Table 7 shows the 13 essential vitamins in adult and pediatric patients. Individual
parenteral vitamins are recommended when the multivitamins products are not
available. Vitamins that are marketed as single-entity parenteral formulations include
vitamins A, D, E, K, B1 (thiamine), B2 (riboflavin), B3 (niacin), B6 (pyridoxine), B9
(folic acid), B12 (cyanocobalamin) , and C (ascorbic acid). During vitamin shortages,
36
oral multiple vitamins may also be used provided that patient is able to absorbe
adequate amounts orally.[1]
Table 7: Suggested Composition for Parenteral Multivitamin Products
Source: [1]
Vitamin Units of
Measurement Infants and
Children <11yr Adults A RE5 (IU6) 690 (2300) 990 (3300) D µg7 (IU) 10 (400) 5 (200) E mg8 (UI) 4.7 (7) 6.7 (10) K µg 200 - B1 (thiamine) mg 1.2 3.0 B2 (riboflavin) mg 1.4 3.6 Niacin mg 17 40 Folic acid µg 140 400 B6 (pyridoxine) mg 1.0 4.0 Pantothenic acid mg 5.0 15.0 Biotin µg 20 60 B12 (cyanocobalamin) µg 1.0 5.0 C (ascorbic acid) mg 80 100
5 RE: Retinol Equivalents 6 IU: International Units 7 As cholecalciferol 8 As dI-alfa-tocopherol
37
(22) Fluid
In the human body, water is the predominant chemical entity, generally accounting
for more than half of the total body weight. Total body water content varies with age,
sex and obesity.
An inverse relationship exists between the amount of body fat and the amount of
body water present in an individual.
Table 8: Total body water as a percentage of body weight Source: [2]
Constituent Adult male Adult female Infant Body water 60% 50% 77% Fats and fat-free solids 40% 50% 23%
Total water gains and losses in the healthy adult fall within the range of 1500-3000
ml daily.
Thus, where the patient requires TPN, the volume administered will fall into this
range and may need to be supplemented by additional fluids in the special cases of
burns, etc.
Careful patient monitoring is required to ensure that they do not become
dehydrated.
Neonates and growing children may have special requirements for amino acids,
fluid, calcium, etc.[2]
3.3. Aspects of stability and compatibility Parenteral fat emulsions are potentially highly variable products since their raw
materials (soya oil and lecithin) are of biological origin, and undergo considerable
purification prior to use. The precise technology used to produce the emulsions also
differs between manufacturers, and so it is not surprising that fat emulsions from
different sources display different physical characteristics, such as varying droplet
diameter and polydispersity. Given the potential for variation, it is remarkable that the
differences observed in clinical use are so small.
The interproduct variation suggests that there may also be differences between the
stability of total parenteral nutrition (TPN) mixtures made with emulsions from
different manufacturers. Consequently, the introduction of a new product requires that
38
not only its physical stability be evaluated, but also that of TPN mixtures compounded
from it. [50][51]
The stability of a range of total parental nutrition (TPN) mixtures compounded from
Lipofundin S and Aminoplex amino acid solutions was studied. Droplet size was
studied for 180 days using both light scattering and electrical zone sensing techniques;
additionally, the chemical stability was monitored via the pH and osmolality of the
mixtures.
All the mixtures were stable for 90 days, but some irreversible flocculation was
observed in all after 180 days. This appeared to be due to the prolonged storage of bags
in one position, and suggested that TPN mixture stability could be enhanced by
occasional remixing of creamed droplets. The Coulter counter was able to detect the
gradual formation of oil droplets larger than 1 μm in diameter, while the laser
diffraction instrument was less sensitive to these droplets until significant coalescence
had occurred by day 180. The results demonstrate the value of single particle zone
sensing techniques for the study of TPN mixture stability. [50]
Emulsions are heterogeneous system in which one immiscible liquid is dispersed as
droplets in another liquid. Such a system is thermodynamically unstable and is
kinetically stabilized by the addition of one further component or mixture of
components that exhibit emulsifying properties. Depending on the nature of the diverse
components and of the emulsifying agents, various types of emulsions can result from
the mixture of immiscible liquids.
Invariably, one of the two immiscible liquids is water, and the second is an oily
substance, often a long-chain triglyceride. Whether the aqueous or oil phase becomes
the dispersed phase depends primarily on the emulsifying agent used and the relative
amounts of the two liquid phases. Hence, an emulsion in which the oil is dispersed as
droplets throughout the aqueous phase is termed an oil-in-water (O/W) emulsion. When
water is the dispersed phase and oil is the dispersion medium, the emulsion is termed a
water-in oil (W/O) type. All pharmaceutical emulsions designed for parenteral
administration are of the O/W type. [52]
There has been renewed interest in emulsions as a vehicle for delivering drugs to
the human body, especially into the bloodstream through parenteral administration.
Extensive research has been published during the last decade and well reviewed by
numerous authors. As previously by Prankerd and Stella, the reasons for using
39
parenteral emulsions as a drug administration vehicle include solubilization of
hydrolytically susceptible compounds, prevention of drug uptake by infusion sets,
reduction of irritation from of toxicity of intravenously (iv) administered drugs,
potential for sustained-release dosage forms, and possible directed delivery of drugs to
various organs. [50][53][54]
40
3.3.1. Emulsion design Requirements for a Parenteral Emulsion- iv emulsions, like all parenteral products,
are required to meet pharmacopeial requirements. The emulsions must be sterile,
isotonic, nonpyrogenic, nontoxic, biodegradable, and stable, both physically and
chemically. Furthermore, the particle size of the droplets must be < 1μm and generally
ranges from 100-150 nm. With larger particle sizes, a potential oil embolism may occur.
Submicron emulsions intended for parenteral administration are designed for the
incorporation of lipophilic and hydrophobic drugs, which exhibit poor aqueous
solubility. Inclusion of hydrophobic drugs in the inner most oil phase presents special
problems related to the solubilization of the drugs. However, these problems generally
can be overcome by techniques such as the elevation of temperatures and the use of
additives to increase the oil solubility of hydrophobic drugs. [55]
The additional of the other drugs to emulsions for iv application also resulted in
reduced stability or cracking.
It should be emphasized that such a combination of emulsifiers already has been
used in iv fat emulsions and has been found to be free from toxic effects. In view of the
result reported, optimal experimental conditions for stable emulsion formation should
occur when a drug is incorporated into the inner phase of an emulsion. Consequently it
is essential to optimize the emulsion preparation manufacturing process and to make
appropriate choices of a mixture of excipients. Generally, stabilizers that are
cosurfactants in nature should be added to the medicated submicron emulsion
formulation. [50]
Excipient Selection- For complying with the requirements for parenteral emulsions,
careful selection of excipients needs to be performed. Special attention should be given
to two major excipients in the emulsion formulation- the oil and the emulsifier(s). A
detailed description of the excipient specifications for parenteral emulsions was
presented by Hansrani et al.1 Only the major aspects of the physicochemical properties
of the excipients that should be considered are outlined below.
Oil- In previous studies, the oil phase of the emulsion was based mainly on long-
chain triglycerides (LCT) from vegetable sources (soybean, Safflower, and cottonseed
oils). The oils need to be purified and winterized to allow the removal of precipitated
wax materials after prolonged storage at 4˚C. Known contaminants (hydrogenated oils
and saturated fatty materials) should be minimized.
41
The use of medium-chain triglycerides (MCT) in fat emulsion formulations
increased extensively during the 1970s. MCT are obtained from the hydrolysis of
coconut oil and fractionation into free fatty acids that contain between 6 and 12 carbon
atoms. MCT are esterfied with glycerol and are 100 times more soluble in water than
are LCT. MCT have been us mostly in fat emulsion formulations in combination with
LCT. MCT recently were used in medicated emulsions because of their increased ability
to dissolve high concentrations of liposoluble drugs.
Emulsifier(s) – Most of the known synthetic and efficient emulsifiers are toxic upon
parenteral administration because of haemolysis. The emulsifiers most frequently used
in parenteral emulsion formulations are phospholipids (generally from egg yolk
sources), block copolymers of polyoxyethylene- polyoxypropylene (poloxamer) and, to
a lesser extent, acetylated monoglycerides. Other emulsifiers, such as fatty acid ester of
sorbitans (various types of Spans; ICI Americas) and polyoxyethylene sorbitans
(various types of Tweens; ICI, UK), are already approved by the various pharmacopeias
for parenteral administration and can therefore be considered for emulsion formulation
design. However, it should be kept in mind that heat exposure after steam sterilization
can alter the emulsifying ability by reducing the aqueous solubility and result in final
phase separation.
Additives are needed to adjust the emulsion to physiological pH and tonicity.
Glycerol is usually recommended as an isotonic agent and can be found in almost every
parenteral emulsion. The pH is adjust to the desired value with an aqueous solution of
NaOH or HCl, depending on the value that should be reached. The pH of the emulsion
is generally adjusted to 7-8 to allow physiological compatibility and maintain emulsion
physical integrity by minimizing fatty acid ester hydrolysis of MCT-LCT and
phospholipids.
A well-known stabilizer is oleic acid or its sodium salt. Cholic acid, deoxycholic
acid and their respective salts also have been shown to markedly improve drug-
incorporated emulsion stability.[10][49]
Manufacturing process- The mean droplet size of iv emulsions must be smaller than
the finest capillaries likely to be encountered in the vascular system; otherwise, an oil
embolism can occur. Emulsions prepared by use of conventional apparatus, e.g., electric
mixers and mechanical stirrers, etc., show not only large droplet sizes but also a wide
particle size distribution and are often unstable. [52][56][57][58][59][60][61][62]
42
Emulsion Characterization
(1) Droplet Size
Particle size distribution is one of the most important characteristics of an
emulsion. For example, sedimentation and creaming tendencies during long-term and
accelerated stability tests of an emulsion can be conventionally monitored by measuring
the changes in the droplet size distribution. A wide range of particle sizes are found in
emulsion systems, as evidenced by iv fat emulsions that should contain particles in the
range of 50nm- 1 μm and emulsions that are used as contrast media in computerized
tomography and that contain particles 1-3 μm in size. Particles > 5μm in size are
clinically unacceptable because they cause the formation of pulmonary emboli.39 Such
particles are sometimes present because of inefficient homogenization or instability of
the emulsion. Hence, it is necessary to determine their sizes even if they are present in
small numbers. Therefore, two complementary particle size analysis methods, namely,
the photon correlation spectroscopy (PCS) method, which is considered the most
appropriate for studying droplets <1 μm in size, and the computerized laser system,
which can measure droplet sizes >0,6 μm, are needed to effectively cover the measured
size range of 50 nm- 10 μm. The advantage of laser inspection system, e.g., Galai Cis 1
(Galai Co., Migdal Haemek, Israel) over the widely used Coulter Counter system is that
there is no need for an electrolyte solution, which can affect the stability of the
emulsion. [50][63] [64][65]
(2) Droplet Surface Charge
The electrical charge on emulsion droplets is measured by use of either a Zetasizer
(Malvern Instruments, Malvern, England) or the moving-boundary electrophoresis
technique, which has been shown to yield accurate electrophoretic mobility data. The
shape of an electrophoresis cell and the method used to convert the electrophoretic
mobility to the zeta potential have been clearly reviewed.
Emulsifiers can stabilize emulsion droplets, not just through the formation of a
mechanical barrier but also through the production of an electrical (electrostatic) barrier
or surface charge of droplets is produced by the ionization of interfacial film-forming
components. The surface potential (zeta potential) of an emulsion droplet upon the
extent of ionization of the emulsifying agent. The extent of ionization of some
phospholipids present in lecithin is markedly pH dependent.
43
It is believed that emulsions prepared from such highly negatively charged
phospholipids will exhibit high zeta potentials and will be less sensitive to the addition
of small amounts of monovalent and divalent electrolytes.
High zeta potentials (>-30mV) should be achieved in most of a prepared emulsion
to ensure a high-energy barrier, which causes the repulsion of adjacent droplets and
results in the formation of a stable emulsion. [63]
(3) pH
It has already been shown that the main degradation pathway for a fat emulsion
leads to the formation of fatty acids, which gradually reduce the pH of the emulsion.
The initial pH of the emulsion may decrease progressively with time. However, this pH
decrease can be controlled by adjusting the initial pH of the emulsion. Provided that the
initial adjusted pH is satisfactory, the rate of hydrolipids of phospholipids and
triglycerides may be minimized.1 Therefore, the pH of the emulsion should be
monitored continuously over the entire shelf of the emulsion to detect detrimental free
fatty acid formation. [61]
(4) Drug Content
As required for any dosage form, quantitative and sensitive methods of analysis
should be applied to evaluate the chemical fate of the active ingredient in the emulsion
formulation. In a medicated submicron emulsion, the decomposition of the drug can be
accelerated by micellar catalysis. [52][57][58][59][60][61][62][63]
3.3.2. Stability Assessment Accelerated Tests
It should be emphasized that the stability results of accelerated tests based on
elevated temperatures generally do not reflect the actual stability of an emulsion stored
at normal temperatures.
44
Long-Term Tests
It is routine to determine the shelf life of a new product by storing it for various
periods of time at elevated temperatures. The Arrhenius equation is commonly used to
predict the shelf life.
Long-term emulsion stability studies are conducted at temperatures ranging from 4-
50 ˚C. The chemical (drug content) and physical (emulsion droplet size, creaming, and
pH, etc.) changes that might occur in the emulsion during storage are monitored over
long periods of time. However, it must be noted that for emulsions such monitoring can
be erratic, because changes in temperature not only change the rate of the reaction but
also can destroy the physical stability of the emulsion.
During the long testing period, samples stored under various conditions be observed
critically for separation and monitored at reasonable time intervals for changes in the
following characteristic properties: electrical conductivity, viscosity, particle size
distribution, zeta potential, pH, and chemical composition.
In addition to these physical measurements, a shelf life program for an emulsion
should include testing of the emulsion for the establishment of sterility and lack of
pyrogens by validated, recognized microbiological methods.[1]
New Approaches for Emulsion Characterization
Monolayer Studies- Attempts have been made in the last four years to identify the
conditions needed for the formation of stable O/W emulsions by estimating the
interactions occurring between the surfactants at the O/W interfacial film of the
dispensed droplets. For these purposes, surface pressure studies of mixed surfactant
monolayers under dynamic conditions and under equilibrium conditions have been
carried out. Early Detection of Emulsion Instability- Emulsion instability is generally
characterized by a progressive but moderate increase in droplet size that is very difficult
to identify by PCS techniques. Indeed, PCS yield accurate diameter assignments for
monodisperse submicron emulsion but fails to describe the size composition for mixed
samples, especially when the concentration of a particular size population is low
compared with those of other population. Caldwell and Li showed that the combination
of sedimentation field flow fractionation (sedFFF) and PCS was able to characterize the
size distribution for polydisperse samples, for which neither technique alone was
45
capable of providing this information. Therefore, this new combined technique is
recommended for assessing emulsion stability, especially after pertubation of an
emulsion, as suggested by the authors. However, as its name implies, sedFFF is a
sedimentation-based method with elution character. Through the sedFFF process even
highly polydisperse samples can be into fractions of considerable size uniformity, [66]
provided that these fractions are well retained. Such is the case for soybean emulsions,
the oil density of which is 0.9-0.92 g/mL, a density significantly different from the
density of the aqueous mobile fluid. The uniformly sized particle fractions obtained by
this high-resolution separation technique then can be subjected to a PCS analysis for
accurate evaluation of particle diameters at selected elution position. Thus, emulsions
submitted to various accelerated tests can be evaluated by the new combined technique,
which is capable of identifying subtle changes in the particle sizes of emulsions,
changes that forecast later problems.
Stability refers to the degradation of nutritional components over time. The
compounding of parenteral nutrition admixtures accelerates the rate of physicochemical
destabilization, resulting in the recommendation to administer parenteral nutrition as
soon after its preparation as possible. Certain amino acids, lipids, and multivitamins are
most susceptible to instability. [58][59]
Once prepared, dextrose-amino acid solutions without vitamins are chemically
stable for 1 to 2 months if stored in a refrigerator (4°C) are protected from light. At
room temperature, concentrations of tryptophan, arginine, and methionine decrease
significantly. Tryptophan is the least stable of the amino acids when admixed with
dextrose, and its degradation can be initiated by prolonged exposure to light or the
additional of hydrochloric acid. The photoreduction of tryptophan leads to degradation
products that result in an indigo blue discoloration. The clinical significance of
tryptophan degradation products is controversial. Grant and co-workers suggest that
these products may function as hepatotoxins. Therefore, the discoloration should be
prevented by avoiding exposure to extremes of light and temperature.
Characteristics of intravenous fat emulsions are very important to the stability, bio-
availability, and metabolism of the fatty acids. The emulsifier egg yolk phosphatide
maintains a physical barrier and produces electronegative repulsive charges (zeta
potential) to stabilize the oil-in –water dispersion at a particle size of about 0.3 to 0.05
μm. The pH of the fluid significantly influences the participle size of emulsion. Fat
46
emulsions are more stable at an alkaline pH and are buffered to a pH range of from 8 to
8.3. When the fat emulsion is admixed with dextrose and amino acids, the final pH of
the TPN is mostly dependent on the pH of the amino acid product used and usually
ranges from 5.3 to 6.1. This effect is due to the amphoteric nature of amino acids, which
act as a natural buffer. Because of differences in the pH of commercial amino acid
products, the phospholipid content of fat emulsion, and parenteral nutrition preparation,
distribution, storage, and administration, it is recommended that manufactures be
consulted for available stability guidelines. In general, final concentrations of TPN
should be composed of about 2 to 6.7% lipids, 1.75 to 5% crystalline amino acids, and
3.3 to 35% dextrose. Parenteral nutrition solution components outside this range may
also be stable, but this information must be determined for the specific admixture and
not be extrapolated from the literature.
Vitamin stability in parenteral nutrition solution is influenced by many factors,
including solution pH; temperature; the presence of other vitamins, minerals,
preservatives (e.g., bisulfite), and macronutrients; storage time; the type of nutrient
delivery equipment; the flow rate to the patient; and light exposure.[61] Under normal
conditions of light and temperature, most vitamins should maintain their potency for up
to 24 hours from the time of parenteral nutrition admixture. Despite their degradation,
very few vitamin deficiencies have been reported in the acute care setting. Patients who
have marginal body stores and who are dependent on long-term parenteral nutrition
support are most likely to be affected by the short-term stability of vitamins. [1]
47
3.3.3. Parenteral Nutrition Compatibility Parenteral nutrition is considered to be compatible when all the individual
components remain in a form may be safely administered to a patient. Combinations
that form precipitates are considered to be incompatible. Precipitates can be solid and
liquid. The most common solid precipitate in parenteral nutrition is calcium phosphate.
Because both calcium and phosphorus are essential to ensure the proper assimilation of
nutrients into the body, it is desirable to have both included in the parenteral nutrition
formulation. Calcium salts, however, are reactive compounds and readily form insoluble
products with several substances, for example, phosphorous, oxalate, and bicarbonate.
many factors influence the solubility of calcium and phosphorus in parenteral nutrition.
Precipitation is more likely in the presence of high calcium and phosphorus
concentrations, decreased amino acid concentrations, increased environmental
temperature, increased solution pH, or prolonged hanging time beyond 24 hours.
This interaction is prevalent in neonatal parenteral nutrition solutions since this
population requires large doses of calcium and phosphorus, yet fluid intakes are
restricted and amino acid doses are low.
Precipitation can occur in a solution at room temperature even if an identical cold
solution is clear. The effect of body heat on the clinical significance of calcium
phosphate solubility is evident by reports of venous catheter occlusions in parenteral
nutrition solution at the borderline limits of compatibility. As the pH of parenteral
nutrition rises, the more soluble monobasic phosphate salt is converted to dibasic
phosphate, which is more likely to bind with calcium and precipitate. [67]
Calcium gluconate is the preferred salt since it is the least reactive form of calcium.
Parenteral nutrition should be compounded in the proper sequence such that calcium
and phosphorus are added separately and diluted well before mixing together in the final
container. Maximal amounts of calcium and phosphorus doses must not be exceeded,
and “borderline” doses should be avoided by considering separate infusion when
higher-than-normal doses are required. When a base precursor is indicated, only acetate
should not be used. Sodium bicarbonate combines with calcium to form the water-
insoluble salt calcium carbonate. Finally, stability should be ensured by using the most
recent, up-to date information possible, or the information should be verified with the
manufacturer before dispensing the formula to the patient.
48
Phase separation with the liberation of free oil in TPN formulations constitutes a
liquid precipitate. This can occur when an excess of cation is added to a given
admixture. The higher the cation valence, the greater the destabilizing effect on the
emulsion. Monovalent cations such as sodium and potassium have little effect unless
present in very high amounts. Divalent cations, however, can create a bridge when
binding with the anionic component of the emulsifier on two different emulsified fat
particles. This neutralizes the zeta potential, creating repulsive charges, and keeps the
particles near each other. These factors eventually cause the particles to join together to
form larger particles and produce phase separation, the various phases of which are
known as aggregation, coalescence, flocculation and separation, or “oiling out”. In the
terminal stage of emulsion destabilization, small lipid particles form large droplets that
may vary from 5 to 50 μm or more yet may escape visual detection. As the process
continues, coalesced lipid particles in TPN may be seen as yellow-brown oil droplets at
or near the TPN surface. These lipid particles may be either as individual spheric
droplets or as segmented (discontinuous) oil layers. The presence of free oil in TPN is
considered to mean that the formulation is unsafe for parenteral administration. The risk
associated with the infusion of unstable lipid droplets is unclear; however, the existence
of lipid particles greater than 5 μm in diameter comprising more than 0.4% of the total
fat present has been shown to mean that the formulation is pharmaceutically
unstable.[1]
3.3.4. Drug Stability and Compatibility Compounding Considerations for Parenteral Nutrition
Because of the complexity of parenteral nutrition products, safe preparation is a
complicated task. The quality of the final product depends on the facilities, resources,
personnel training and products used in preparation. Since the inception of parenteral
nutrition, pharmacists have developed policies and procedures for parenteral nutrition
compounding based on their training and interpretation of the literature. As a result,
inconsistent practices in parenteral nutrition preparation exist.
Parenteral nutrition is considered a high-risk sterile product. Its compounding
includes complex and/or numerous aseptic manipulations. Specific guidelines for
aseptic processing include media fill validations of both the process and the personnel
carrying out the process. In addition, there are specific requirements for facilities, space,
49
and environmental control similar to those of a Class 100 clean-room environment. The
sterile product release checks require visual inspection against a lighted white and black
background for evidence of visible particulates or other foreign matter. In addition,
compounding accuracy checks of the addition of all drug products or ingredients used to
prepare the parenteral nutrition product are ensured by validating the volume and
quantity used in admixture. Presterilized disposable membrane filtration devices, which
are sensitive in detecting low levels of contamination and easy to use, are commercially
available. The time frame from the preparation of the compound until sterility testing is
important. [1]
50
Table 9: Guidelines for Parenteral Nutrition Compounding, Quality Assurance, and Sterility and Compatibility Source: [1]
Parenteral Nutrition Practice Guidelines Compounding The additive sequence in compounding should be optimized and
validated as a safe and efficacious method. The manual compounding method should be reviewed periodically or when the manufacturer’s brand of nutrient products is about to change. This review should include the most current literature as well as consultation with the manufacturer when necessary. Manufacturers of automated compounding devices should provide an additive sequence that ensures safe parenteral nutrition (PN) compounding. This sequence should also be reviewed by the manufacturer of the nutrient products being used in preparing the PN product. Splitting PN contracts should be avoided unless there is specific stability data concerning the admixture of different brands of amino acids, dextrose, and fat. Each PN product prepared should be visually inspected.
Quality assurance
Gravimetric analysis is suggested as an indirect assessment of the accuracy for PN preparation. Attention should be focused on the most dangerous additives such as potassium chloride. Chemical analysis of the dextrose content may also be used to determine the accuracy of compounding. Refactometric analysis is an alternative as an indirect measurement of the dextrose concentration. This method is limited to PN formulations that do not contain lipids (e.g., neonatal formulations). In-process and end-product testing is recommended daily. Guidelines for aseptic preparation should be followed.
Stability and compatibility
All method used for PN (e.g., dose, admixture, packaging, delivery, storage, and administration) ensure a stable and compatible product. Medication administration in or with PN is safe, stable, and compatible. Stability and compatibility decisions are made with the most reliable information available from the literature or manufacturer. Because of limited stability information, the use of conventional dextrose-amino acid formulas with separate administration of fat is recommended for neonatal and infant patients.
51
Combining all the constituents of a daily TPN feed into one container can result in
the production of very complex pharmaceutical systems, particularly where lipid is
present.
With so many chemical entities present there is much opportunity for interactions
and incompatibilities which may affect the therapeutic value of preparation or increase
its toxicity.
Basic principles of chemistry indicate that changes in temperature, pH, light,
oxygen levels, etc, can have an impact on the potential for interactions to take place.
This potential continues even after all product mixing within the pharmacy aseptic
suite has been completed. Adverse temperature conditions, during storage or within the
ward environment, administering product in direct sunlight, may lead to a disturbance
of product stability.
Stability refers to the degradation of nutritional components over time.
The compounding of parenteral nutrition admixtures accelerates the rate of
physicochemical destabilization, resulting in the recommendation to administer
parenteral nutrition as soon after its preparation as possible.certain amino acids, lipids,
and multivitamins are most susceptible to instability.
Pharmacist involved in TPN should have a thorough understanding of the potential
stability issues in these mixtures and be able to advise physicians accordingly.
[67][68][69][70] [71][72]
Maillard reaction
The Maillard reaction involves the reaction of carbohydrate with certain amino
acids (e.g. glycine), causing the carbohydrate to decompose. This reaction is enhanced
by the high temperatures used in sterilization.Thus, dextrose and amino acids combined
in the same container are not available commercially but must be prepared by a
pharmacist. [1][2]
Amino acids and glucose can participate in a number of chemical reactions. The
interaction between amino acids and glucose may not only reduce the therapeutic effect
of the components but also present a risk of toxicity to the patient.
Once prepared, dextrose-amino acid solutions without vitamins are chemically
stable for 1 to 2 months if stored in a refrigerator (4˚C) and protected from light.At
room temperature, concentrations of tryptophan, arginine, and methionine decrease
52
significantly. Tryptophan is the least stable of the amino acids when admixed with
dextrose, and its degradation can be initiated by prolonged exposure to light or the
addition of hydrochloric acid. The photoreduction of tryptophan leads to degradation
products that result in an indigo blue discoloration. The clinical significance of
tryptophan degradation products is controversial. Grant and coworkers suggest that
these products may function as hepatotoxins. Therefore, the discoloration should be
prevented by avoiding exposure to extremes of light and temperature.
Characteistics of intravenous fat emulsions are very important to the stability,
bioavailability, and metabolism of the fatty acids. The emulsifier egg yolk phosphatide
maintains a physical barrier and produces electronegative repulsive charges (zeta
potential) to stabilize the oil-in-water dispersion at a particle size of about 0.3 to 0.05
μm. The pH of the fluid significantly influences the particle size of the emulsion. Fat
emulsions are more stable at an alkaline pH and are buffered to a pH range of from 8 to
8.3. When the fat emulsion is admixed with dextrose and amino acids, the final pH of
the TPN is mostly dependent on the pH of the amino acid product used and usually
ranges from 5.3 to 6.1. This effect is due to the amphoteric nature of amino acids, which
act as a natural buffer. Because of differences in the pH of commercial amino acid
products, the phospholipid content of fat emulsion, and parenteral nutrition preparation,
distribution, storage, and administration, it is recommended that manufacturers be
consulted for available stability giudelines.
In general, final concentrations of TPN should be composed of about 2 to 6.7%
lipids, 1.75 to 5 % crystalline amino acids, and 3.3 to 35 % dextrose. Parenteral
nutrition solution components outside this range may also be stable, but this information
must be determined for the specific admixture and not be extrapolated from the
literature. [1]
Calcium and other electrolytes
Calcium appears to be the electrolyte with the most potential to challenge mixed
TPN systems, particularly where lipid is employed as a calorie source.
Niemiec & Vanderveen reported in 1984 that the requirement for concomitant
administration of both calcium and phosphate in the same solution introduces the
potential for the precipitation of calcium phosphate. This can be influenced by many
53
parameters, of which pH is by far the most significant since it influences the equilibrium
of phosphate ions.
Modification of pH in TPN mixtures is somewhat difficult due to the buffering
capacity of amino acids. Amino acids also have the ability under suitable conditions to
combine with calcium and phosphate to form soluble complexes.
Other electrolytes and trace elements
Monovalent cations in therapeutic quantities do not appear to give problems with
stability, even with lipid-containing mixed TPN systems.
As trace elements are added to TPN solutions in very small amounts there is
generally not a great potential for interaction. Available literature should be consulted
for specific issues, e.g. the effect of copper on vitamin C degradation.[2]
Vitamins
Vitamin stability in parenteral nutrition solutions is influenced by many factors,
including solution pH. temperature, the presence of other vitamins, minerals,
preservatives (e.g. bisulfite) and macronutrients, storage time, the type of nutrient
delivery equipment, the flow rate to the patient and light exposure.
Under normal conditions of light and temperature, most vitamins should maintain
their potency for up to 24 hours from the time of parenteral nutrition admixture. Despite
their degradation, very few vitamin deficiencies have been reported in the acute care
setting. Patients who have marginal body stores and who are dependent on long-term
parenteral nutrition support are most likely to be affected by the short-term stability of
vitamins.
Similarly, ascorbic acid added in batch fashion to parenteral nutrition degraded and
resulted in calcium oxalate precipitation. Because of these short-term stability
considerations, it is suggested that vitamins be added to parenteral nutrition
formulations shortly before their administration. Parenteral nutrition with vitamins
added should be given an expiration date and time of approximately 24 hours. [1]
54
Niemiec & Vanderveen highlight in 1984 that vitamin stability in TPN systems may
be affected by solution pH, presence of electrolytes, trace elements and other vitamins,
environmental temperature, light and storage time.
Thus, vitamins, if required, shoud be added to TPN mixtures immediately prior to
administration and should not be stored in excess of 24 hours from the thime of mixing.
[2].
Lipid
In order to minimize risks of instability, attention must be paid to the following
points during formulation and manufacture of TPN mixtures containing lipid:
1. Level of cations, particularly di-and trivalent
2. pH of resultant mixture
3. Order of mixing of constituents
4. Choice of plastic container
5. Conditions of storage and administration
6. Manufacturers recommendations
Instability of lipid emulsion systems progresses through “creaming”to “cracking”or
separation of the oil and water phase. Administration of such unstable mixtures can give
rise to fatty deposits in the lung and other tissues. Thus comprehensive testing should be
carried out on potential mixtures to ascertain their suitability for administration to the
patient. This should include direct microscopic examination as well as particle size
measurements. [2]
55
3.3.5. Labelling In general the following information will be required on the label:
• Patient name/number
• Ward
• Product constituents
• Batch (dispensing number)
• Expiry date/time
• Storage conditions
• Other instructions such as guidance on administration rate or technique,
limitations on further additions etc., may also be required.
Table 10 shows an example of label format which takes these factors into
account.[2]
Table 10: Labelling example (after Allwood 1984) Source: [2]
Parental Nutrition Mixture
Total energy supplied in 24 hours is………..kcal is ……….ml.
Contents: Nitrogen g Carbohydrate kcal Sodium mmol Potassium mmol Phosphate mmol Magnesium mmol Trace elements Cu: Zn: Cr: Mn: F: I: Fe. Vitamins A: B Co: C: E: Folate: Biotin: Final volume ml Patient Ward
Expiry date Date: Prepared by: Batch
N°
Warning: Protect from light. Contains approx. 20% w/v dextrose, do not infuse too rapidly. Refrigerate until ready for use. Do not make any further additions to this container.
56
3.3.6. Dispensing Once the product has been filled and labelled a pharmacist should perform a final
check against the prescription prior to sending the product to the ward. This check
should include the patient’s name, ward, etc. and should once again compare the
constituents requested against the final label. Details of further additions, storage
conditions, expiry date, etc. should also be confirmed and the batch number or other
reference allocated should be checked to facilitate traceability in the event of any
difficulties arising subsequent to dispensing, e.g. precipitation, discoloration, etc.
The hospital pharmacist may be involved in development of nursing care guidelines
with particular reference to further additions, storage, etc. It may also be useful for the
ward pharmacist to check that TPN is being correctly administered to the patient, i.e.
with correct flow control device, away from direct sunlight, etc.
3.3.7. Storage Allwood at al recommends in 1984 that compounded TPN solutions should be
stored at 2-8˚C in light of both microbiological and chemical considerations. The
pharmacy/ ward/ home patient – refrigerators should be calibrated to ensure that they
are able to maintain this level of temperature, as bags, particularly those containing
lipid, should not be allowed to freeze and should not be stored at room temperature for
periods in excess of the 12-24 hours required for administration.
3.3.8. Packaging Where supplies of compounded product are to be made to hospitals or home
patients away from the site of manufacture, the quality of the packaging system to
maintain product temperature during transit should be validated to the satisfaction of
local quality control standards. Insulated polystyrene containers may be useful for this
purpose.
3.3.9. Costs Providing a TPN compounding service within a hospital may be a costly venture for
the pharmacy department. Amino acid and lipid presentations are, by their specialist
nature, expensive items to purchase. The materials cost of compounding is easy to
identify, however, dispending into a 3-litre bag requires other, sometimes not so
apparent, costs such as labour input, overheads, consumables, etc. All these factors must
be considered when developing true service costs and deciding whether to produce
inhouse or obtain product from a regional hospital or commercial source. [2]
57
3.3.10. Bags Bags made of poly-ethylene and poly-vinylchloride and of the copolymer ethylene-
vinylacetate were used as containers of perfusion solutions for total parenteral nutrition.
The bags were characterised by tensyometry (free energy and its polar and dispersed
components) and atomic force microscopy (AFM) before and after various periods of
storage of solutions for total parenteral nutrition containing L-aminoacids, electrolytes
or glucose. In most of the cases, after storage of these solutions, tensiometric
characterisation and atomic force microscopy analysis of the internal surface of bags
showed deep modifications which highlight the adsorption of the solutes. The changes
of surface characteristics were found to depend on the time of contact, the wettability of
the polymer and the compounds present into the solutions, while their concentration has
a negligible effect. Generally, the aminoacid solutions produced a higher increase in the
polar component even after short storage times.Poly-ethylene and the copolymer
ethylene-vinylacetate showed a greater inertia if compared with the poly-vinylchloride
bags.
Injectable solutions for Total Parenteral Nutrition containing L-aminoacids,
electrolytes and glucose, are commonly sold as medicinal specialities in glass
containers. Many studies have been carried out to evaluate the possibility of
commercialising these solutions and/or their mixtures, packaged directly in plastic bags.
Pignato in 1996, Morra and Cassinelli in 1997 as a result of these studies bags made of
plastic materials such as copolymer ethylene-vinylacetate (EVA), poly-ethylene (PE)
and poly-vinylchloride (PVC) are being used more and more often in the manufacture
of containers of perfusion solutions. A problem, associated with the use of these plastic
bags, is the loss of solution components through adsorption on the inner surface of the
container. This phenomenon has been reported to occur for several drugs, such as
diazepam, insulin and organic nitrates for which significant losses have been noted after
storage of perfusion liquid in TPN bags, [69] the adsorption of solution components
being dependent on contact time and wettability of the polymers.
Hasma and Tersoff 1987, McPherson et al. 2000 from these premises and the
technological needs, by the use of tensiometry and atomic force microscopy (AFM) an
experimental protocol was planned to evaluate the effects of storage of TPN solutions
58
on the characteristics of the inner wall of bags made by plastic materials widely used in
the medical field. [73][74][75]
In times of budget constraints, it is essential to choose a total parenteral nutrition
(TPN) system that is clinically effective and economically efficient, because TPN is a
potent but relatively expensive nutrition therapy.
TPN is indicated in patients with a non-functioning digestive tract to correct or
maintain their nutritional status.
TPN regimens contain more than 40 different components, including
macronutrients (carbo-hydrates lipids, amino acids) and micronutrients (electrolytes,
trace elements, vitamins). They can be administered in either of two ways:
• The classic separate bottles (SB) system: nutrients are stored in separate bottles
or bags and infused through separate i.v. lines. This system requires numerous
i.v. line manipulations associated with increased risk of administration errors, as
well as septic and metabolic complications.
• The „all-in-one” system: all nutrients are mixed in one bag and infused
simultaneously. This system requires only one i.v. line, and contributes to
decrease manipulation related and metabolic risks. Two major all-in-one
systems exist: hospital-compounded bags and industrial three-compartment
bags. Both have their respective advantages and disadvantages.
Hospital-compounded bags must be prepared almost daily by the hospital pharmacy
because of limited stability (a few days under refrigeration). Their compounding
requires special, expensive equipment and infrastuctures.
TPN formulas can be either „á la carte” to exactly match the patients specific
needs, but are standardized in most hospitals. Three-compartment bags contain
macronutrients and electrolytes in three separate compartments
Nutrients are mixed just prior to infusions, by breaking the plastic connectors
between the compartments, then vitamins and trace elements are added
extemporaneously to the bag. Shelf-life of these bags is at least 12 months, but allow
only for standardized formulas. Thanks to their easy application, „all-in-one” TPN
systems should save preparation and handling time on the ward, thus resulting in
decreased manpower cost.
59
The use of three-compartment TPN bags is less expensive in terms of application
costs than separete bottles or hospital-compounded bag systems. TPN application costs
are partly transfered from the pharmacy to the ward in the three-compartment bag
system compared to hospital-compounded bags. Detailed manpower times measured in
the present studies are published, allowing hospitals to calculate their own application
costs using local salaries, product prices and production costs.
[76][77][78][79][80][81][82][83][84][85].
60
3.3.11. Documentation A work sheet should be generated for each TPN-dispensing activity to be carried
out for recording materials, patient name, label details, etc. The format for such a work
sheet should be agreed between the production and quality control departments of the
hospital in accordance with local policy.
This is by no means the only documentation which is required to control the overall
aseptic porcess. Raw material testing, environmental monitoring records, cleaning
records, operator training records, patient records should all form part of the
documentation packages which are developed and retained to best fit the requirements
of the hospital or industrial environment and the standards laid down in BS 5295 and
recommended in the ‘Guide to good pharmaceutical manufacturing practice’ (DHSS
1983).
3.3.12. Manufacturing procedures Manufacturing procedures or guidelines should be drawn up jointly by production
aud quality control staff depending upon the manufacturing environment. These should
be adhered to by all personnel involved in the process, updated regularly and audited
periodically to ensure conformance. This is essentail to the quality assurance of the
operation.
These procedures should cover all the activities in the department down to specific
tasks, such as use of syringes, etc. [2]
61
4. EXPERIMENTAL PART
4.1. Materials 4.1.1. Mixture F35b
Table 11 summarizes the composition of the examined TPN mixture F35b. Table 11
comprises the total ionic concentrations of the corresponding TPN mixture F35b.
Table 11: Composition of TPN mixture
Quantity(ml) Composition
TPN mixture
1
TPN mixture
2 Rindex 10 % (TEVA) 500.0 ml Magnesium Chloride hexahydreate 0.051 g Calcium Chloride 0.09 g Potassium Chloride 0.13 g Sodium Chloride 1.985 g Glucose monohydrate 55.0 g
1500 1500
Electrolite C (University Pharmacy of the Semmelweis University, Hungary) 100.0 ml Natrium chloratum 2.337 g Kalium chloratum 3.727 g Magnesium sulfuricum cryst. 2.0 g Aqua destillata pro inj. ad 100.0 ml
100 100
Aminoven 10 % (Fresenius Kabi, Uppsala, Sweden) 500.0 ml 500 500
Intralipid 20% inf. (Fresenius Kabi, Uppsala, Sweden) 500.0ml Soybean oil: 200 g Purified egg phospholipids: 12 g Glycerol (anhydrous) (Ph Eur): 22.0 g Water for injection to 1000 ml
500 -
Structolipid 20% inf. (Fresenius Kabi, Uppsala, Sweden) 500.0ml Structured triglycerides: 200 g Purified egg phospholipids: 12 g Glycerol (anhydrous) (Ph Eur): 22.0 g Water for injection to 1000 ml
- 500
62
Table 12 summarizes the ionic concentrations of the prepared TPN mixture.
Table 12: ionic concentrations of the prepared TPN mixture F35b
Compounds Concentration (mol/dm3) in the TPN mixture
Na+ 0.0545
K+ 0.0212
Mg²+ 0.0067
Clˉ 0.0772
SO42- 0.0064
Ca²+ 0.0009
63
4.1.2. Mixture F37b Table 13 summarizes the composition of the examined TPN emulsion F37b. Table
13 comprises the total ionic concentrations of the corresponding F37b mixture.
Table 13: Composition of the TPN mixture F37b
Quantity (ml) Compounds TPN mixture 1 TPN mixture 2 Infusio glucosi 40% (University Pharmacy of the Semmelweis University, Budapest) Glucose anhydrate 400 g Hydrochloric acid 0.1N 1.000 ml per 1000 ml solution
500
500
Electrolite A (University Pharmacy of the Semmelweis University, Budapest) Sodium chloride 4.675 g Potassium chloride 3.727 g Magnesium sulfate cryst 2.00 g Aqua destillata pro inj. ad 100.0 ml
100
100
Aminoven 10% 500ml inf. (Fresenius Kabi AB Sweden) L-isoleucine 5.00 g, L-leucine 7.40 g, L-methionine 4.30 g, L-lysine-acetate 9.31 g (=6.6 g L-lysine), L-phenylalanine 5.10 g, L-threonine 4.4 g, L- tryptophane 2.00 g, L-valine 6.20 g, L-arginine 12.0g, L-hystidine 3.00 g, L-alanine 14.0 g, Glycine 11.0 g, L-proline 11.2 g, L-serine 6.50 g, L-tyrosine 0.40 g, Taurine 1.00 g per 1000 ml solution Total amino acid content 100.0 g/l
1000
1000
Intralipid 20% inf. (Fresenius Kabi, Germany GmbH) Soybean oil: 200 g Purified egg phospholipids: 12 g Glycerol (anhydrous) (Ph Eur): 22.0 g Water for injection to 1000 ml
500
-
Structolipid 20% inf. (Fresenius Kabi, Germany GmbH) Structured triglycerides: 200 g Purified egg phospholipids: 12 g Glycerol (anhydrous) (Ph Eur): 22.0 g Water for injection to 1000 ml
-
500
64
Table 14 summarizes the ionic concentrations of the prepared F37b TPN mixture.
Table 14: ionic concentrations of the prepared F37b TPN mixture
Compounds Concentration (mol/dm3) in the TPN mixture
Na+ 0.0380 K+ 0.0238 Mg2+ 0.0039 Clˉ 0.0618 SO4
2- 0.0039
65
4.1.3. Individual TPN mixtures I.(A) Table 15 summarizes the composition of complex emulsions prepared for the
individual therapy of neonates and Table 15 shows the corresponding ionic
concentrations. The two mixtures differ from each other in the ionic concentrations.
Table 15: Composition of the individual TPN mixtures
Quantity (ml) Compounds
TPN mixture 1
TPN mixture 2
Infusio glucosi 20% (University Pharmacy of the Semmelweis University, Hungary) Glucose anhydrate: 200 g Hydrochloric acid 0.1N 1000 ml per 1000 ml solution
2000 940
Glucose 20% inf. (Human Ltd, Hungary) Glucose monohydrate: 220 g Hydrochloric acid9 per 1000 ml solution
200 -
Aminoven infant 10% inf. (Fresenius Kabi, Germany GmbH) L-arginine: 7.500 g, L-leucine:13.000 g, L-isoleucine:8.000 g, L-methionine: 3.120 g, L-phenylalanine: 3.750 g, L-alanine: 9.300 g, L-proline: 9.710 g, L-valine: 9.000 g, L-threonine: 4.400 g, L-lysine-acetate: 12.000 g (=8.510 g L-lysine), Glycine: 4.150 g, L-histidine: 4.760 g, L-serine: 7.670 g, N-acetyl-tyrosine: 5.176 g (=4.200 g L-tyrosine), L-tryptophane: 2.010 g, N-acetyil-cysteine. 0.770 g (=0.520 g L-cysteine), L-malic acid: 2.620 g, Taurine: 0.400 g per 1000 ml solution Total amino acid content: 100 g/l
600 400
Intralipid 20% inf. (Fresenius Kabi, Germany GmbH) Soy oil: 200 g Egg phospholipids9 Glycerine9 Sodium hydroxide9 per 1000 ml solution
400 200
NaCl 10 % inj. (Pharmamagist Ltd, Hungary) 34 14
Panangin inj.(Richter Gedeon Ltd, Hungary
Magnesium aspartate anhydrate 400 mg(=33.7mg magnesium)
Potassium aspartate anhydrate 452 mg(=103.3 mg potassium)
per 10 ml solution
80 40
66
Calcimusc 10% inj. (Richter Gedeon Ltd, Hungary) Calcium gluconate 1000 mg Boric acid9 per 10 ml solution
60 14.8
Glucose-1-phosphate concentrate for inf. (Fresenius Kabi, Austria GmbH) Glucose-1-phosphate disodium tetrahydrate 3.762 g Sodium hydroxide9 per 10 ml solution
30 12
KCl 10% inj. (Pharmamagist Ltd, Hungary) 24 10
Peditrace concentrate for inf. (Fresenius Kabi Norge AS) Potassium iodide 0.0131 mg Magnesium chloride tetrahydrate 0.036 mg Sodium selenose pentahydrate 0.0666 mg Copper chloride 0.537 mg Sodium fluoride 1.26 mg Zinc chloride 5.21 mg Hydrochloric acid9 per 10 ml solution
30 10
Vitalipid-N infant emulsion for inf. (Fresenius Kabi, Sweden) Retinolpalmitate 135.3 μg Retinol (Vitamin A) 69 μg Ergocalciferol (Vitamin D2) 1 μg α-Tocopherol (Vitamin E) 0.64 mg Phytomenadione (Vitamin K1) 20 μg Soy oil 1000 mg Egg lecithin 120 mg Glycerine 225 mg Sodium hydroxide9 per 10 ml solution
30 10
Humaqua solvent for parenteral use (Water for injection, Human Ltd, Hungary)
- 200
9 The exact quantity of these excipients was not given.
67
Table 16 summarizes the ionic concentrations of the prepared TPN mixtures.
Table 16: ionic concentrations of the prepared TPN mixtures
Compounds Concentration (mol/dm3)
TPN 1 mixture Concentration (mol/dm3)
TPN 2 mixture Na+ 0.017 0.013 K+ 0.015 0.013 Ca2+ 0.004 0.002 Mg2+ 0.003 0.003 Cl- 0.026 0.020 Glucose-1-phosphate 0.009 0.006 Aspartate 0.012 0.012
68
4.1.4. Individual TPN mixtures II Table 17 summarizes the composition of complex emulsions prepared for the
individual therapy of neonates.
Table 17: Compositions of TPN mixtures
Compounds TPN mixture 1 Quantity
(ml)
TPN mixture 2 Quantity
(ml) Glucose inf. 20 % (Human Ltd, Hungary) - 700
Glucose inf. 40% (Human Ltd, Hungary) 500 -
Aminoven infant inf. 10 % (Fresenius Kabi, Germany GmbH) 1000 200
Intralipid inf. 20 % (Fresenius Kabi, Germany GmbH) 500 150
NaCl 10 % inj. (Pharmamagist Ltd, Hungary) 80 20
Panangin inj. (Richter Gedeon Ltd, Hungary) - 40
Calcimusc inj. 10 % (Richter Gedeon Ltd Hungary) - 20
Glucose-1-phosphate inj. (Fresenius Kabi, Austria GmbH) - 10
Magnesium phosphate 10% (Pharmamagist Ltd, Hungary) 20 -
KCl inj. 10% (Pharmamagist Ltd, Hungary) 50 10
Addamel N concentrate for inj. (Fresenius Kabi Norge AS) - 10
Vitalipid-N infant. inj. (Fresenius Kabi, Sweden) - 10
69
4.2. Aseptic Production
This section looks at the parameters of control for aseptic TPN compounding.
The steps in the dispensing of TPN solutions in the hospital facility are detailed
below.
4.2.1. Facility and environment As with all aseptic processes, the environment used for manufacturing can
contribute considerably to product quality and must thus be designed, cleaned,
maintained and monitored to the highest achievable standards.
4.2.2. Personnel and training Aseptic preparation of TPN solutions should only be carried out by personnel who
have undergone a suitable documented training programme. This should cover not only
aseptic technique and validation but also theoretical aspects such as patient
requirements and use of products.
4.2.3. Receipt of prescription The documents used for the prescribing of TPN solutions at ward level may very
from adaptation of a basix fluids chart to a custom-made TPN chart often developed
jointly by pharmacy and medical staff.
On receipt of the request for TPN the pharmacist should check that the requested
combination is feasible, stable and within normal clinical limits. Information can then
be transferred to the dispensing worksheet.
4.2.4. Collection of materials and preparation Once the documentation for a bag or number of bags has been generated and
checked the manufacturing process proper may commence.
The first stage in this process will be the identification and collection together of all
materials required to be taken into the aseptic suite.
The components assembled are the checked against the work sheet by the
pharmacist who should initial the sheet. At this stage either the work sheet or label
containg a copy of the formulation should be passed through with the ingredients,
utilizing a transparent pocket which can be swabbed.
4.2.5. Entry into preparation area
70
This is best effected suing a controlled pass-through hatch or chamber with
interlockiing doors in order to prevent thie hingress of dirty air from the surrounding
environment. Specially designed trays, trolleys or plastic containers may be utilized for
this purpose providid that these are sanitizable and effect bag or batch control and
segregation during handling and transit throughout the manufacturing process.
4.2.6. First stage preparation In order to minimize the microbiological bioburden on the final aseptic process all
excess packaging should be removed and discarded at this stage and all surfaces should
be cleaned.
The cleaning process is normally effected using a 70% alcoholic solution (e.g.
industrial meghylated spirit or isopropyl alcohol) together with sterile lint-free swabs or
cloths utilizing systematic and thorough wiping routine.
Where more than one bag is being processed in the preparation room, care should
be taken to avoid cross-contamination of source materials, labels, etc., and another
reconciliation should be carried out prior to the passage of materials into the aseptic
(Class I) room.
4.2.7. Second stage preparation Entry of materials into the Class I area should again be effected by means of a pass-
through system with inter-locking doors.
Following a defined and through decontamination of the laminar air flow cabinet,
materials passed into the area from the Class II room should then be subjected to a
repeat of the surface cleaning process before being passed directly into the laminar air
flow cabinet.
As with all aseptic operations, materials should be placed well within the laminar
air flow cabinet making use of all the available space and organized in a manner which
will facilitate the pre-defined systematic steps in the dispensing process and cause
minimum discruption of air flow.
4.2.8. Positive pressure Several methods now exist for positive-pressure bag filling. Some pharmacist may
choose to utilize the standard pumps used for a variety of hospital manufacturing
activities, however, specialist systems for TPN do exist (Automix (Baxter), Fillmat
71
2000 (Miramed)), which may also be combined with volumetric measuring systems
providing a useful means of dispensing paediatric TPN solutions.
4.2.9. Inspection The completed nutrition bag should be inspected to check for integrity of all ports,
leaks, splits and particulates for which TPN solutions should conform to the BP 1988
criteria together with the limit test for particulate matter
4.3. Preparation of the TPN mixtures The blending of the compounds of various TPN systems was carried out in a
laminar airflow box (Relatec, Germany) under vacuum. The final preparations consisted
of 4 different types of basic ingredients: amino-acids, carbohydrates, electrolytes and
lipids. The blending of the compounds was carried out under vacuum in a completely
closed system. First of all, half of the volume of the Glucose inf. was sucked into the
plastic bag through one of the plastic tubes which was connected to the bag. The
electrolytes were added to the remained volume of Glucose infusion and then sucked
into the plastic bag. Next, amino-acids were blended to the obtained solution. The last
step was the addition of lipids to the solution by sucking the lipid emulsions into the
plastic bag. The right order of the blending assured the homogeneity of the TPN
mixtures.
4.4. Storage of the prepared TPN mixtures The TPN mixtures were stored at 2-8 °C and 37 ± 0.5°C temperatures for 10 days.
4.5. Methods 4.5.1. Photon correlation spectroscopy
The particle size distribution of TPN emulsions of different compositions was
examined before storage and after 4, 7 and 10 days. Dynamic light scattering
measurements were carried out for checking the kinetic stability of the TPN emulsions.
The apparatus (Brookhaven Instruments Corporation) used consisted of a BI-200SM
goniometer and a BI-9000BO Correlator. An Argon-Ion Laser (Omnichrome 543 AP)
set to the wavelength of 488 nm was applied as a light source. The homodyne
autocorrelation function in channel 238 was determined at real time mode using
logarithmic timescale with a range of 1-200000 μs. Detector angle was set to 90.0 deg.,
and the gap was 100 μm. Before the measurements the emulsions were diluted to reach
the appropriate count rate value. The time of measurement was 180s. 6 parallel
72
examinations were carried out on each sample (four different samples – according to the
temperature of storage and the type of lipid emulsion used for the preparation). Data
were evaluated assuming an exponential distribution of the emulsion particles. The
results were plotted as intensity vs. particle size of the emulsion droplets.
73
4.5.2. Particle size measurement Mean size, size distribution and polydispersity of the emulsion droplets were
measured at 25ºC by an advanced technique of photon correlation spectroscopy (PCS)
using a Malvern Zetasizer 4 apparatus (Malvern Instruments, UK) with autosizing
mode and auto sample time. Analysis of the fluctuations in the intensity of light
scattered from particles undergoing random Brownian motion enables the determination
of an autocorrelation function G(τ) that, in effect, is measure of the probability of a
particle moving a given distance in a τ time (τ is the correlation delay time).
( ) ( )[ ]iic,ii aτ/tkτG −∑∝ exp (1)
The relaxation time (tc) of fluctuations is related to the diffusion coefficient (D) of
particles:
2c DKt /1= (2)
from which the particle size can be calculated via the Stokes – Einstein equation,
(K) is the wave vector.
By determining the autocorrelation function for the dispersions stored at 2-8 °C and
37 ± 0.5°C for various times, the diffusion coefficient and the hydrodynamic radii (ai)
of emulsion droplets have been evaluated.
74
4.5.3. Zeta-potential measurements Laser Doppler-electrophoresis (LDE) was used for investigating the surface-electric
properties of the emulsion droplets. Measurements were carried out before storage and
after 4, 7 and 10 days. For electrically charged particles moving in response to an
applied electric field, a correlation function of laser Doppler-shift was measured with a
Malvern Zetasizer 4 apparatus at 25 ± 1°C (Malvern Instruments, UK), and the resulting
frequency spectrum was translated to electrophoretic mobility. Using an AZ 104 type
cell, 5 mobility measurements were ordinarily done on each sample (four different
samples – according to the temperature of storage and the type of lipid emulsion used
for the preparation) in cross beam mode. The zeta potential (η) of the particles was
calculated from the mobility measurements, using the Smoluchowsky formula.
4.5.4. Optical microscopy The emulsions were observed under a Carl Zeiss optical microscope (Carl Zeiss
Axiostar plus T 0,8A Germany) which was equipped with a video camera. The size and
arrangements of the droplets were studied at 400x magnification..
4.5.5. pH measurements pH values of the TPN mixtures were measured right after preparation and after 1, 4,
7 and 10 days of storage with a Radelkis OP-300 electroanalytical analyser.
4.5.6. Dynamic Surface Tension Measurements The examinations were carried out on the day of preparation and after 1, 4, 7 and 10
days. The surface tension of emulsions was determined by dynamic method, applying
Du-Noüy ring and Wilhelmy plate operations of a computer-controlled KSV Sigma 70
tensiometer (KSV Sigma 70, RBM-R. Braumann GmbH, Germany) at 25°C ± 0.5 °C.
The method determines the maximum mass of liquid pulled from the surface by lifting
the specified solid (e.g. ring or plate). The force (f) measured on the electric balance is
necessary for lifting out and pushing down the solid measuring device from the surface
of the liquid.
The contact angle can be calculated from the extrapolated buoyancy slope:
cos θ = f/pγLV
where θ is the contact angle, f is the force measured on the balance, p is the
measured plate perimeter and γLV is the surface tension (interfacial free energy between
the liquid and vapour) of the examined liquid. 3 parallel measurements were carried out
on all four kinds of samples.
75
4.5.7. Statistical evaluation Zeta-potential values of the two kinds of mixtures at different temperatures and
storage intervals were compared using the two-sample t-test assuming equal variances.
In this case, the comparison was made between Intralipid-containing infusions and
Structolipid-containing ones. Surface tension values measured after different storage
intervals were compared via the paired two-sample t-test for both kinds of mixtures.
The comparison was made between data obtained right after preparation and after 1, 4, 7
and 10 days, respectively.
The statistics were calculated using Microsoft Excel 2002.
76
4.6. Results and discussion 4.6.1. Comparison of physical stability of two different brands of lipid
emulsion for total nutrient Table 18 summarizes the ionic concentrations of the prepared F37b and F35b.
The ionic concentrations of the prepared F35b is higher, than F37b.
Table 18: ionic concentrtions of the TPN mixtures
F37b (electrolyte A) mol/dm3 F35b (electrolyte C+ Rindex10) mol/dm3 Na + 0.038 0.0545 K+ 0.0238 0.0212 Mg 2+ 0.0039 0.0067 Cl - 0.0618 0.0772 SO4
2- 0.0039 0.0064 Ca 2+ 0.0009
Table 19 summarizes the pH of the prepared F35b. There isn’t significant
difference between the two different TPN emulsions.
Table 19: The values of pH of the prepared F35b
Table 20 summarizes the density of the prepared mixtures F35b. There was no
significant difference between the two different TPN emulsions.
Table 20: The values of density of the prepared F35b
pH Intralipid (TPN 1) Structolipid (TPN 2)
Storage (Days) 2-8˚C 37˚C 2-8˚C 37˚C
0 5.95 - 5.9 - 1 5.98 5.92 5.97 5.92 4 5.85 5.84 5.86 5.83 7 5.90 5.78 5.89 5.86
10 6.05 5.94 6.01 5.93
Density(g/cm3) Intralipid (TPN 1) Structolipid (TPN 2)
Storage (Days) 2-8˚C 37˚C 2-8˚C 37˚C
0 1.0203 1.0203 1.0281 1.0281 1 1.0253 1.0249 1.0232 1.0241 4 1.0186 1.0230 1.0225 1.0219 7 1.0281 0.9841 1.0251 1.0199 10 1.0243 1.0241 1.0180 1.0239
77
Table 21 summarizes the conductivity of the prepared mixtures F35b
There isn’t significant difference between the two different TPN emulsions.
Table 21: The values of conductivity of the prepared mixtures F35b
Conductivity (mS/cm) Intralipid (TPN 1) Structolipid (TPN 2)
Storage (days) 2-8˚C 37˚C 2-8˚C 37˚C 1 0.238 - 0.135 -
4 0.142 0.345 0.125 0.319
7 0.213 0.387 0.221 0.263
10 0.250 0.294 0.280 0.274
Table 22 summarizes the surface tension values of different TPN emulsions stored
under different conditions (average of three parallels, ± S.D.)
Table 22: Surface tension values of different TPN emulsions F35b stored under different conditions (average of three parallels, ± S.D.) p refers to the comparison of the surface tension values with the
corresponding values obtained after 1 day (α = 0.01). Surface tension (mN/m)
Intralipid (TPN 1) Structolipid (TPN 2) Storage time
(days) 2-8°C p 37°C p 2-8°C p 37°C p
1 30.43±0.42 - 30.43±0.42 - 31.44±0.45 - 31.44±0.45 - 4 30.95±0.65 <0.01 28.98±0.86 >0.01 30.18±0.73 >0.01 29.24±0.17 <0.01 7 27.64±0.16 <0.01 25.99±0.06 <0.01 31.18±0.15 >0.01 29.38±0.23 <0.01 10 27.76±1.06 <0.01 27.13±0.41 <0.01 31.19±0.19 >0.01 28.29±0.51 >0.01
78
Dynamic surface tension of TPN liquid mixtures containing Structolipid is more
stable, than that of the ones containing Intralipid at storage on 2-8°C and 37°C.
Figure 1: The dynamic surface tension at storage on 2-8°C
Figure 2: The dynamic surface tension at storage on 37°C
Storage on 37oC
25
26
27
28
29
30
31
32
33
0 2 4 6 8 10 12
Storage time (days)
Sur
face
tens
ion
(mN
/m)
Intralipid
Structolipid
Storage on 2-8oC
26
27
28
29
30
31
32
33
0 2 4 6 8 10 12Storage time (days)
Sur
face
tens
ion
(mN
/m)
Intralipid
Structolipid
upper limit
lower limit
79
The particle size distribution of TPN mixtures F35b containing Structolipid seems
to be more stable as a function of temperature and time than that of the ones containing
Intralipid.
Figure 3: Size distribution functions by volume of TPN 1 emulsions stored for various times at (a) 2-8 °C and (b) 37 ± 0.5°C
80
Figure 4: Size distribution functions by volume of TPN 2 emulsions stored for various times at (a) 2-8 °C and (b) 37 ± 0.5°C
81
Figure 5: Zeta potential of the droplets in TPN 1 emulsions F35b stored for various times at 2-8 °C and 37 ± 0.5°C
Figure 6: Zeta potential of the droplets in TPN 2 emulsions F35b stored for various times at 2-8 °C and 37 ± 0.5°C
82
Figure 3 and Figure 4 illustrate the size distribution functions by volume and the
mean droplet sizes determined after various times for the two TPN F35b emulsions
stored at 2-8 °C and 37 ± 0.5°C, respectively. These results clearly show that the peaks
of the distribution functions obtained after longer storage times shift towards larger
values, indicating that during storage, the size of the droplets of both emulsions increase
and in the meantime, the emulsions become more polydisperse.
By comparing the corresponding size distributions of the two emulsions, it can be
seen that the TPN 2 emulsion containing structured triglycerides exhibit higher kinetic
stability. In addition, the rate of droplet coalescence in the emulsions stored at lower
temperature definitely slowed, especially in the emulsions containing only long-chain
triglycerides. In Figure 5 and Figure 6, the electrokinetic properties of the emulsions are
illustrated. The emulsion droplets are negatively charged. The zeta potential of the
droplets in the emulsions of original composition is fairly low, mainly because of the
high ionic concentration in their media. In a previous study, we found a significant
difference between the zeta potential values of LCT- and ST-containing mixtures,
respectively. In the present emulsions, however, notable differences in the zeta potential
of the droplets of the two compositions could not be detected even after longer storage
times and at both temperatures (peaks of the individual samples overlap). This might be
attributed to the higher Na+ (0.0545 M vs. 0.0380 M), Mg2+ (0.0067 M vs. 0.0039 M)
and Ca2+ (0.0009 M vs. 0.0000 M) content compared to the earlier examined mixtures.
The higher electrolyte concentrations could have reduced the advantageous effects of
the structured lipids on the zeta potential values.
Nevertheless, considerably larger (minus 17 – 19 mV) zeta potentials were obtained
for the droplets in the emulsions diluted 10-fold by distilled water, and the (slightly)
higher values were measured in the TPN 2 emulsions.
Since the ionic concentration of the two TPN emulsions was equal, and pH and
conductivity values measured in the course of storage (Table 19 and Table 21) did not
change markedly, the lower physicochemical stability of emulsions prepared with LCTs
cannot be ascribed to electrostatic effects or chemical decomposition. Very likely, the
formation of a “mixed” interfacial layer formed from the medium and the long chain
fatty acids in the case of structured triglycerides is responsible for the more efficient
stabilization [86]. The latter is responsible for the efficient stabilization, which could be
tracked by the different interfacial surface structure of the dispersed droplets.
83
The surface tension values measured by the Wilhelmy plate operations are
summarized in Table 22. The surface tension values determined with Du-Noüy ring
correlated well to values measured by the plate method, but the latter resulted in higher
accuracy. As it can be seen in Table 22, in the case of admixtures containing the
structured lipid component, the obtained surface tension values did not show significant
changes at 2-8°C – indicating a more stable interfacial surface structure. In contrast, the
surface tension of emulsions containing exclusively long-chain triglycerides stored at 2-
8°C, significantly decreased during storage, indicating that there were interfacial
structural changes. It is important to note, that this method is appropriate for the
determination of the tension on the surface of the Wilhelmy plate, not the droplets.
Normally, a decrease in surface tension is linked to stabilization, but in this case it
means that structural changes (e.g. leaking of surfactants from the droplets) occurred
during storage. The samples kept at 37°C presented significant changes (surface tension
decrease) in both cases, which suggests that relatively high temperature storage affects
the stability of admixtures containing structured lipids, as well. In the earlier work, we
found that the surface tension values of ST-containing mixtures remained constant even
at 37°C [86]. The difference between study findings can again be explained by the
higher electrolyte content of the emulsions in the present study, because the higher ionic
concentrations could counteract with the stabilizing effect of structured lipids.
The results of the droplet size distribution and surface tension measurements are in
good correlation with the results of Driscoll et al. concerning the stability of all-in-one
admixtures containing MCTs and LCTs previously mixed in a single emulsion or added
separately to the mixtures [87] . As it was reported, separate droplets of MCTs and
LCTs had poorer physicochemical stability than did the droplets containing both kinds
of triglycerides. In the case of structured lipids, both medium and long chain fatty acids
can be found in the starting lipid emulsion, leading to a favorable interfacial location of
structured triglycerides.
An important finding of the study is that the favorable stabilizing effect of
structured lipids can be deteriorated by the ionic concentration of the media of the
emulsions. [88][89][90][91][92][93][94]
84
Figure 7 and Figure 8 illustrate the average droplet size of the two different TPN
F37b emulsions at different storage temperatures.
Figure 7:Effect of storage time on the average droplet size of the prepared TPN systems; Storage temperature: 2- 8°C
Figure 8: Effect of storage time on the average droplet size of the prepared TPN systems Storage temperature: 37 ± 0.5ºC
85
The mean droplet size of Structolipid 20% before mixing with the other components
was reported to be 276 nm [95] and proved to be between 300-400 nm in the admixtures
at zero time. The results unambiguously indicate that the average droplet size of
emulsions containing structured triglycerides did not significantly change during the
examined storage period. In contrast, the droplet size of emulsions prepared with lipids
containing exclusively long-chain triglycerides, showed remarkable increase even after
4 days of storage. As commercially available lipid emulsions can be stored for 24
months, these findings confirm the fact that the additives mixed to these systems
negatively influence their stability. [105]
The possible explanation of the observed tendency could be the different interfacial
surface structure of the dispersed droplets. The structured lipid component presumably
decreases the surface tension at the droplet/solution interfaces in a greater extent than
the long-chain triglycerides, resulting in a long-term physical stability of the system.
Since the ionic concentration of the two TPN emulsions was equal, and significant
differences in the zeta potential of the droplets could not be detected (Table 23), the
higher physical stability of emulsions prepared with structured triglycerides can not be
ascribed to electrostatic effects. Very likely, the formation of a “mixed” interfacial layer
formed from the medium and long chain fatty acids in case of structured triglycerides is
responsible for the more efficient stabilization. Nevertheless, further studies are still
needed to elucidate the mechanism of the (steric) stabilization. Besides the
advantageous metabolic effects of structured triglycerides, their application is
recommended to improve the physical stability of TPN admixtures, as well.
Table 23 shows the zeta-potential values of the two mixtures after storing at
different temperatures for 10 days. Such values of intravenous lipid emulsions can be
found in the literature and are in the range of -40 to -50 mV [55], which shows
remarkable increase (i.e. weaker repulsive forces between the droplets) in the
admixtures. No significant difference could be observed between the two kinds of
compositions at zero time, which suggests that their initial stability can be considered
equivalent. p values indicate significant differences between the two compositions after
4 and 7 days of storage. The more negative zeta-potential values of the mixture
containing structured lipids confirm the results of the particle-size analysis, i.e. the
enhanced stability of the system prepared with Structolipid. After 10 days, the zeta-
86
potential values can be considered equivalent again, which is probably the result of the
starting destabilization process of the composition containing structured lipids.
Since the ionic concentration of the two TPN emulsions was equal and pH values
measured in the course of storage Table 24 did not present remarkable changes, the
lower physicochemical stability of emulsions prepared with LCTs can not be ascribed to
electrostatic effects or chemical decomposition. [51][55][64][65][66][50]
Table 23: Electrokinetic characteristics of different TPN F37b emulsions (average of 5 parallel measurements, ± S.D.; α = 0.05)
Zeta potential (mV) Storage time (days)
Temperature (°C±0.5°C ) Intralipid Structolipid
p
0 25 -2.2 ± 0.10 -1.9 ± 0.35 > 0.05 4 2-8 -2.4 ± 0.15 -2.9 ± 0.15 < 0.05 4 37 -3.0 ± 0.40 -4.1 ± 0.40 < 0.05 7 2-8 -1.7 ± 0.05 -2.9 ± 0.60 < 0.05 7 37 -2.7 ± 0.60 -3.9 ± 0.15 < 0.05
10 2-8 -2.0 ± 0.20 - 2.9 ± 0.90 > 0.05 10 37 -3.3 ± 0.05 - 2.9 ± 0.40 > 0.05
Table 24: ph values of the mixtures before and after storage under different conditions (average of 3 parallels, ± S.D.)
pH Storage time (days) Temperature (°C±0.5°C) Intralipid Structolipid
0 25 5.8 ± 0.1 5.8 ± 0.2 1 2-8 5.7 ± 0.1 5.9 ± 0.1 1 37 5.7 ± 0.2 5.7 ± 0.1 4 2-8 5.9 ± 0.2 6.0 ± 0.1 4 37 5.8 ± 0.1 5.8 ± 0.2 7 2-8 5.9 ± 0.1 5.9 ± 0.2 7 37 5.7 ± 0.1 5.7 ± 0.3 10 2-8 5.9 ± 0.2 5.9 ± 0.1 10 37 5.7 ± 0.1 5.7 ± 0.2
87
Very likely, the formation of a “mixed” interfacial layer formed from the medium
and long chain fatty acids in case of structured triglycerides is responsible for the more
efficient stabilization. The latter could be tracked by the different interfacial surface
structure of the dispersed droplets. The surface tension values measured by the
Wilhelmy plate operations are summarized inTable 25. The measured surface tension of
purified water was 58.81 ±0.113 mN/m. The surface tension values determined with
Du-Noüy ring correlated well to values measured by the plate method, but the latter
resulted in higher accuracy. As it can be seen in Table 25, the obtained surface tension
remained almost constant within the examined storage intervals in the case of
admixtures containing the structured lipid component – indicating a more stable
interfacial surface structure. p values indicate significant difference compared to zero
time only after 10 days of storage at 2-8°C. In contrast, the surface tension of emulsions
containing exclusively long-chain triglycerides remarkably decreased during storage
referring to the interfacial structural changes. In the case of the sample stored at 37°C, a
significant change could be observed after 4 days. Although further studies are needed
to elucidate the mechanism of the (steric) stabilization, dynamic surface tension
measurements can be recommended as sensitive means for the stability tests of
intravenous lipid emulsions.
Table 25: Surface tension values of different TPN emulsions stored under different conditions (average of 3 parallels, ± S.D.). p refers to the comparison of the surface tension values with the corresponding values
at zero time (α = 0.05).
Surface tension (mN/m) Structolipid Intralipid Storage
time (days) 2-8°C P 37°C P 2-8°C P 37°C P
0 30.49
±0.384 - 30.49 ±0.326 - 33.48 ±0.620 - 33.48 ±0.408 -
1 30.90
±0.846 >0.05 30.28 ±0.846 >0.05 33.06 ±0.887 >0.05 31.53 ±0.725 <0.05
4 30.39
±0.164 >0.05 30.47 ±0.095 >0.05 28.12 ±0.867 <0.05 24.33 ±0.826 <0.05
7 30.19
±0.503 >0.05 30.47 ±0.437 >0.05 26.16 ±0.584 <0.05 26.36 ±0.500 <0.05
10 32.17
±0.342 <0.05 31.50 ±0.425 >0.05 27.58 ±0.872 <0.05 27.06 ±0.537 <0.05
The findings of this study are in good correlation with the results of Driscoll et al.
concerning the stability of all-in-one admixtures containing MCTs and LCTs previously
mixed in a single emulsion or added separately to the mixtures [96]. As it was reported,
88
separate droplets of MCTs and LCTs resulted in impaired physicochemical stability
compared to the ones containing both kinds of triglycerides. In the case of structured
lipids, both medium and long chain fatty acids can be found in the starting lipid
emulsion, leading to a favourable interfacial location of structured triglycerides.
The clinical significance of the present study lies in the recognition that with the
application of total nutrient admixtures containing structured lipids, the incidence of
fatal consequences of parenteral nutrition (e.g. fat embolism) could be decreased
[87][95][96][97][98][99][100][101][102][103][104][105][106].
89
4.6.2. Study of the stability of individual of different calcium / glucose-1-phosphate ratios TPN mixtures
Individual TPN mixtures I.(A)
Figure 9 and Figure 10 illustrate the particle size distribution of TPN mixture I.A.
The results indicate that the mixture was homogeneous after the preparation and
aggregation could not be observed after 3 days storage without remixing.
Figure 9 B, C and Figure 10 B, C refer to the sedimentation of the system with the
two characteristic particle size distributions.
The major destabilizing factors in TPN mixtures are the ionic strength and presence
of specifically binding electrolytes, which control the non-specific and specific
adsorption processes on the droplet surface. [50][51]
In contrast to TPN mixture 1 (Ca2+/phosphate ratio =0.44), TPN mixture 2 contains
less specifically binding calcium ions compared to the glucose-1-phosphate
(Ca2+/phosphate ratio =0.33) thus increasing the possibility of the formation of bridges
between the adjacent complexes.
If there are divalent cations (such as calciums) present in the mixture, they become
associated with the lecithin-stabilized globules, thus further stabilizing the emulsion as
the oil globule-lecithin-Ca²+ complex repels other similar complexes [97]. However, the
anionic electrolyte (glucose-1-phosphate) could form a bridge between adjacent
complexes. It could cause flocculation, leading to coalescence and, ultimately, to
breaking of the emulsion. Besides the destabilizing ionic effect, the presence of larger
initial sizes (Figure 10A) means that very large, unstable globules can be formed fairly
quickly owing to aggregation (Figure 10C). The wide size distribution allows closer
packing of the globules since small globules fit into the spaces between large globules.
Concerning the differences in the kinetic stability of these two emulsions, the
results of the zeta potential measurements confirm the above statements. At these high
ionic strengths the particles of both emulsions exhibit slightly negative zeta potentials
indicating that other (steric) effects may also play a role in maintaining the kinetic
90
stability of the emulsions. The low zeta potentials of the droplets of the more stable
TPN mixture 1 are practically constant even after a 7 days strorage. The changes in the
zeta potential of the droplets during the storage of the less stable TPN emulsion 2 are
shown in Table 26.
These data clearly demonstrate that during storage, the negative surface charge of
the droplets of the TPN mixture 2 definitely increases. This effect is more pronounced
at the higher temperatures. The increase of the zeta potential (see Figure 11) without
considerably changing the ionic strength in the aqueous medium may presumably be
attributed to a slow chemical decomposition of the droplets. Some changes in the
chemical composition of the TPN emulsion 2 could be visually followed, as well. On
the storage at the higher temperatures, a transition from white to yellow colouring of the
emulsion could be observed.
Table 26: Electrokinetic characteristics of TPN emulsions
Storage time Temperature Zeta Potencial (mV) (day) (oC) TPN 1 TPN 2 P
1 2-8 -2(±1) -1(±1) >0,05 1 37 -2(±1) -2(±1) <0,05 4 2-8 -3(±1) -1(±1) <0,05 4 37 -3(±1) -6(±1) <0,05 7 2-8 -3(±1) -9(±1) >0,05 7 37 -3(±1) -11(±1) >0,05
91
Figure 9: Characteristic Photon Correlation Spectra of TPN mixtures type 1 A: without storage, B: 4 days storage, C: 7 days Storage
100 10000
20
40
60
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100
d (nm )
Inte
nsity
(%)
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nsity
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nsity
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92
Figure 10: Characteristic Photon Correlation Spectra of TPN mixtures type 2 A: without storage, B: 4 days storage, C: 7 days Storage
100 10000
20
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nsity
(%)
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nsity
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nsity
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93
Figure 11: The changes of the value of Zeta-potential plotted
-12
-10
-8
-6
-4
-2
0
TPN18°C
TPN137°C
TPN28°C
TPN237°C
1st Day4th Day7th Day
94
Individual TPN mixtures II.
Figure 12 and Figure 13 illustrate the particle size distribution of TPN mixture 1 and
2, while Figure 14 and Figure 15 demonstrate the related microscopic image. The
results indicate that the mixture was homogeneous after the preparation and coalescence
could not be observed after 4 days storage without remixing. Figure 12B and C refer to
the sedimentation of the system with the two characteristic particle size distributions,
and the corresponding microscopic photo (Figure 14C) demonstrates that the
sedimentation is not associated with flocculation.
The major destabilizing factors in TPN mixtures are the ionic strength and presence
of specifically binding electrolytes, which control the non-specific and specific
adsorption processes on the droplet surface [51][102][103].
In contrast to TPN mixture 1, TPN mixture 2 contains more divalent specifically
binding ions from the Panangin, Calcimusc and Addamel components thus increasing
the ionic strength of the emulsion. If there are divalent cations (such as calciums)
present in the mixture, they become associated with the lecithin-stabilized globules, thus
further stabilizing the emulsion as the oil globule-lecithin-Ca2+ complex repels other
similar complexes[97]. However, the anionic electrolyte (glucose-1-phosphate) could
form a bridge between adjacent complexes. This causes flocculation, leading to
coalescence and, ultimately, to breaking of the emulsion. Besides the destabilizing ionic
effect, the presence of larger initial sizes (Figure Figure 13A) means that very large,
unstable globules can be formed fairly quickly owing to coalescence (Figure 15B and C,
and Figure 13C). The wide size distribution allows closer packing of the globules since
small globules fit into the spaces between large globules (Figure 15C).
The studied mixtures showed coalescence within relatively short storage time,
depending on their initial particle size and ionic content. The combination of photon
correlation spectroscopy and optical microscopy enabled the study of the dynamics of
droplet formation during storage. Since the coalescence of the droplets is reversible,
remixing is recommended preceding the application to avoid the presence of particles
larger then 1 μm.
95
Figure 12: Characteristic Photon Correlation Spectra of TPN mixtures type 1 A: without storage, B: 4 days storage; C: 7 days storage
100 10000
20
40
60
80
100
d (nm)
Inte
nsity
(%)
100 10000
20
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60
80
100
d (nm)
Inte
nsity
(%)
100 10000
20
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60
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nsity
(%)
96
Figure 13: Characteristic Photon Correlation Spectra of TPN mixtures type 2
A: without storage, B: 4 days storage; C: 7 days storage
100 10000
20
40
60
80
100
d (nm)
Inte
nsity
(%)
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nsity
(%)
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nsity
(%)
97
Figure 14: Optical Microscopic photos of TPN mixtures type 1 A: without storage, B: 4 days storage; C: 7 days storage; Magnification: 400x
98
Figure 15: Optical Microscopic photos of TPN mixtures type 2 A: without storage, B: 4 days storage; C: 7 days storage
Magnification: 400x
99
5. NEW SCIENTIFIC RESULTS AND CONCLUSION
• Kinetic stability of two total nutrient admixtures prepared with different lipid
emulsions (Intralipid and Structolipid, respectively) was tracked for 10 days
with an array of several physicochemical methods, including particle size
analysis via photon correlation spectroscopy, light obscuration, laser diffraction
or microscopy . While these methods can follow the physical changes, zeta-
potential and pH measurements are able to indicate chemical processes that take
place along with storage. Dynamic surface tension measurements could provide
additional information concerning the physicochemical processes that take place
on the surface of the lipid droplets, therefore the method enabled the tracking of
the destabilizing interaction during the storage of TPN mixtures.
• Electrolytes play an especially important role from stability aspects, as they are
present in all admixtures and have a major effect on the zeta potential of the
emulsions. In the case of nonspecific adsorption, they physically adhere to the
surface of the lipid droplets, and above the Critical Flocculation Concentration
(CFC) cause the disappearance of repulsive forces. Specific adsorption occurs;
when besides the physical ones, chemical interactions also arise (e.g. Ca2+ and
phospholipids). In this case, further adsorption is possible above the CFC, and
repulsive forces arise again. In the case of the examined emulsions, however,
notable differences in the zeta potential of the droplets of the two compositions
of different lipid components could not be detected even after longer storage
times and at either temperature. This might be attributed to the higher Na+, Mg2+
and Ca2+ content. The higher electrolyte concentrations could have deteriorated
the advantageous effects of the structured lipids on the zeta potential values.
• The formation of a “mixed” interfacial layer formed from the medium and the
long chain fatty acids in case of structured triglycerides is responsible for the
more efficient stabilization.
• The favorable stabilizing effect of structured lipids can be deteriorated by the
ionic concentration of the media of the emulsions.
• Droplet size distribution and surface tension data showed that the emulsions
containing structured lipids proved to be more stable, especially at lower storage
temperatures.
100
• The practical usability of our results is that in addition to the advantageous
metabolic effects of structured triglycerides, their application is recommended
also to improve the physical stability of TPN admixtures, which could decrease
the risk of fat embolism in the clinical practice.
101
6. SUMMARY
Lipid emulsions have been used in routine clinical practice for more than 40 years. Intralipid, the first well tolerated lipid emulsion, is still the most commonly used lipid emulsion worldwide containing long-chain triglycerides (LCT) with a fatty acid chain length of 16-20 carbon atoms (long-chain fatty acids, LCFA). Structured triglycerides, in which both medium-chain fatty acids and long-chain fatty acids are esterified to the same glycerol molecule, have positive metabolic effects, which make them competitive or even more efficient as an energy source compared with conventional fat emulsions.
The purpose of my thesis was to compare the kinetic stability of two admixtures containing different lipid components. A further aim was to collect more evidence for the stabilizing effect of structured triglycerides, with special concern to the ionic concentration of the mixtures.
Kinetic stability of two total nutrient admixtures prepared with different lipid emulsions (Intralipid and Structolipid, respectively) was tracked under different storage conditions with an array of physicochemical methods. Several methods were applied for the assessment of physical stability of lipid emulsions, including particle size analysis via photon correlation spectroscopy, and microscopy. While these methods can follow physical changes, zeta-potential and pH measurements are able to indicate chemical processes that take place along with storage. Dynamic surface tension measurements could provide additional information concerning the physicochemical processes that take place on the surface of the lipid droplets.
Electrolytes play an especially important role from this point of view, as they are present in all admixtures and have a major effect on the zeta potential of the emulsions. Very likely, the formation of a “mixed” interfacial layer formed from the medium and the long chain fatty acids in case of structured triglycerides is responsible for the more efficient stabilization.
Droplet size distribution and surface tension data showed that the emulsions containing structured lipids proved to be more stable, especially at lower storage temperatures. Higher electrolyte concentrations of the mixtures can adversely influence this stabilizing effect.
The obtained results indicate that besides the advantageous metabolic effects of structured triglycerides, their application is recommended to improve the physical stability of TPN mixtures.
• J. Balogh, J. Bubenik, J. Dredán, F. Csempesz, D. Kiss, R. Zelkó: The effect of structured triglycerides on the kinetic stability of total nutrient admixtures. J. Pharm. Pharmaceut..Sci. 8(3):552-557, 2005.
• J. Balogh, D. Kiss, J. Dredán, I. Puskás, F. Csempesz, R. Zelkó: Tracking of the Kinetic Stability of Two Types of Total Nutrient Admixtures Containing Different Lipid Emulsions. AAPS Pharm.Sci.Tech. 2006;7 (4) Article 98
102
Összefoglalás Individuális terápiában alkalmazott TPN emulziók előállítása és vizsgálata
A zsíremulziók közül az első, az emberi szervezet számára jól tolerálható iv. lipidemulzió az Intralipid volt, amely hosszú szénláncú triglicerideket (LCT) és 16-20 C atomos zsírsavat tartalmaz (LCFA). A strukturált lipidek, amelyekben mind a közepes, mind a hosszú szénláncú zsírsavak ugyanazzal a glicerin molekulával létesítenek észterkötést, pozitív metabolikus hatással rendelkeznek, amely hatékonyabb energiaforrássá teszi őket, mint a konvencionális, csak hosszú szénláncú zsírsavat tartalmazó zsíremulziók. Nagyobb oxidációs sebességgel, gyorsabb clearance-szel, megnövelt N-megtartó képességgel rendelkeznek a kizárólag hosszú szénláncú zsírsavat tartalmazó emulziókhoz viszonyítva, valamint a retikuloendoteliális rendszerben kevésbé akkumulálódnak.
Doktori értekezésem célja volt, hogy a strukturált trigliceridek hatását vizsgáljam a teljes parenterális táplálásra szánt emulziók (TPN) kinetikai stabilitására, összehasonlítva a kizárólag hosszú szénláncú zsírsavat tartalmazókéval.
Az Intralipid és Structolipid tartalmú különböző összetételű TPN oldatkeverékek kinetikai stabilitásának változását a tárolási körülmények (idő, hőmérséklet) függvényében különböző fizikai-kémiai módszerekkel (foton-korrelációs spektroszkópia, zéta-potenciál-mérés, dinamikus felületi feszültség meghatározása, pH-és vezetőképesség-mérés) vizsgáltam.
Az elvégzett vizsgálatok szerint a kizárólag hosszú szénláncú triglicerideket tartalmazó (Intralipid) emulziók cseppmérete 4 nap után jelentős növekedést mutatott a strukturált lipid-komponenst (Structolipid) tartalmazóval szemben.
Megállapítható, hogy a magasabb elektrolit-koncentráció csökkenti a struktúrált lipid-komponens előnyös hatását a zéta-potenciálra. Amennyiben TPN emulziók ionkoncentrációja azonos, és a zéta potenciál, valamint a pH értékek a tárolás során nem mutattak érzékelhető változást az idő függvényében, a kisebb fizikai–kémiai stabilitás az LCT tartalmú emulziók esetében nem tulajdonítható elektrokémiai vagy kémiai bomlásnak.
A strukturált trigliceridet tartalmazó hosszú- és közepes szénláncú zsírsavak keveréke felületi réteget képez a zsírcsepp felszínén, amely feltételezéseim szerint a nagyobb stabilitásért felelős, ugyanakkor a strukturált lipidek előnyös stabilizáló hatását a magasabb ionkoncentráció csökkentheti. A részecskeméret-analízis és a felületi feszültség mérés adatai alapján megállapítható, hogy a strukturált trigliceridet tartalmazó zsíremulziók stabilabbak, különösen az alacsonyabb tárolási hőmérsékleten.
A magasabb elektrolit koncentráció kedvezőtlen hatást fejt ki a stabilizáló hatással szemben.
A kapott eredmények alapján megállapítható, hogy a strukturált trigliceridek kedvező metabolikus hatásuk mellett a TPN emulziók fizikai stabilitását is növelik, így alkalmazásuk ajánlott a teljes parenterális táplálásra szánt emulziók előállításánál.
• J. Balogh, J. Bubenik, J. Dredán, F. Csempesz, D. Kiss, R. Zelkó: The effect of structured triglycerides on the kinetic stability of total nutrient admixtures. J. Pharm. Pharmaceut..Sci. 8(3):552-557, 2005.
• J. Balogh, D. Kiss, J. Dredán, I. Puskás, F. Csempesz, R. Zelkó: Tracking of the Kinetic Stability of Two Types of Total Nutrient Admixtures Containing Different Lipid Emulsions. AAPS Pharm.Sci.Tech. 2006;7 (4) Article 98
103
7. ACKNOWLEDGEMENTS
First of all I wish to express my sincere thanks to Dr. Romána Zelkó, Director of the
University Pharmacy Department of Pharmacy Administration of the Semmelweis
University, for guiding me with her valuable advices and inspiring me in my work.
Furthermore, I express my special thanks to Professor Ferenc Csempesz (Eötvös
Loránd University, Institute of Chemistry, Department of Physical Chemistry) for his
interesting discussions and collaboration in the Experimental part.
I am very thankful to Dr.Judit Dredán (Semmelweis University, Department of
Pharmaceutics) for her advices and help with the measurements in the Experimental
part.
104
8. PUBLICATIONS AND LECTURES
Publications:
1. Kovácsné Balogh Judit, Zelkó Romána, Vincze Zoltán: A parenterális táplálás
gyógyszerészi vonatkozásai I. Gyógyszerészet 48. 666-671, 2004.
2. Kovácsné Balogh Judit, Zelkó Romána, Vincze Zoltán: A parenterális táplálás
gyógyszerészi vonatkozásai II. Egyedi parenterális oldatkeverékek.
Gyógyszerészet 49. 92-97, 2005.
3. J. Balogh, J. Bubenik, J. Dredán, F. Csempesz, D. Kiss, R. Zelkó: The effect of
structured triglycerides on the kinetic stability of total nutrient admixtures. J.
Pharm. Pharmaceut..Sci.(www.cspsCanada.org) 8(3):552-557, 2005.
4. J. Balogh, D. Kiss, J. Dredán, I. Puskás, F. Csempesz, R. Zelkó: Tracking of the
Kinetic Stability of Two Types of Total Nutrient Admixtures Containing
Different Lipid Emulsions. AAPS Pharm.Sci.Tech. 2006;7 (4) Article 98
(http://www.aapspharmscitech.org).
Other publications:
1. Kovácsné Balogh Judit, Dr. Szász Györgyné: Atropin tabletta és szemcsepp
kvantitatív ellenőrzése indikátorszinezék módszerrel.
Semmelweis OTE Egyetemi Gyógyszertár Gyógyszerügyi Szervezési Intézet
Acta Pharmaceutica Hungarica 53. 150-153. 1983.
2. Kovácsné Dr. Balogh Judit, Dr. Zalai Károly: Gyógyszerésznők
Magyarországon.
Gyógyszerészet 42. 468-474. 1998.
3. Balpataki Rita, Kovácsné Dr. Balogh Judit, Dr. Zelkó Romána, Dr. Vincze
Zoltán: Antibiotikum-felhasználás költségeinek elemzése.
Acta Pharmaceutica Hungarica 71. 108-113. 2000.
4. Kovácsné Balogh Judit, Zelkó Romána, Vincze Zoltán: Minőségbiztosítás-
minőségügyi definíciók és tevékenységek
Gyógyszerészet 45.418-420, 2001.
105
5. Reszkető Zsuzsa, Dr. Szlávik J., Kovácsné Dr. Balogh Judit, Dr. Zelkó
Romána,
Dr. Vincze Zoltán: HIV/AIDS betegek gyógyszeres kezelésének lehetőségei és a
terápiás módszerek értékelése.
Acta Pharmaceutica Hungarica 71. 428-432. 2001.
6. Jelinekné Nikolics Mária, Stampf György, Kovácsné Balogh Judit, Zelkó
Romána, Turmezeiné Horváth Judit: Glukóz infúziók technológiai, stabilitási és
additív-képzési problémái. Gyógyszerészet 47.725-728, 2003.
7. Major Csilla, Vincze Zoltán, Meskó Attiláné, Balogh Judit, Zelkó Romána,
Németh Erzsébet: Gyógyszerelés a rendelőn kívül, Orvosi Hetilap 148.7.291-X
2007
8. Major Csilla, Vincze Zoltán, Meskó Attiláné, Balogh Judit, Németh Erzsébet:
Az öngyógyszerezés helyzete Magyarországon - szakmai szemmel.
Gyógyszerészet 51. 2007. március
Lectures:
1. Kovácsné Dr. Balogh Judit, Dr. Buday Tamásné, Dr. Meskó Attiláné, Dr. Soós
Gyöngyvér: Klinikai laboratóriumi vizsgálatokhoz használt kémszerek és
festékoldatok előállításának klinikai és gazdaságossági jelentősége.
Congressus Pharmaceuticus Hungaricus XI.
Gyógyszerészet 1999. október 6-10. 36.oldal
2. Kovácsné Dr. Balogh Judit, Dr. Buday Tamásné, Dr. Rókusfalvy Andrea, Dr.
Rixer András, Gável Mónika: „Párhuzamos” klinikák
gyógyszerfelhasználásának elemzése a Semmelweis Egyetemen I.
Magyar Kórházi Gyógyszerészek XII. Kongresszusa Budapest, 2000.
3. Jelinekné Nikolics Mária, Stampf György, Kovácsné Balogh Judit, Zelkó
Romána: Glukóz infúziók, terápiás igények, technológiai problémák.
XIV. Országos Gyógyszertechnológiai Konferencia Hévíz, 2002. nov. 8-10.
4. Balpataki Rita, Kovácsné Dr. Balogh Judit, Dr. Buday Tamásné: „Párhuzamos”
klinikák antibiotikum felhasználásának elemzése a Semmelweis Egyetemen II.
Kórházi gyógyszerészek XIII. Kongresszusa Szeged 2002.
106
5. Kovácsné Balogh Judit, Jelinekné Nikolics Mária, Komlódi Tibor, Turmezeiné
Horváth Judit, Vincze Zoltán: A parenterális táplálás gyógyszerészi
szempontból.
Congressus Pharmaceuticus Hungaricus XII. Budapest, 2003. május 8-10.
6. Turmezeiné Horváth J. Kincs J. Bókay J. Kovácsné Balogh J. Zelkó R., Vincze
Z.: Keverékinfúzió sikeres alkalmazása súlyosan atrophiás csecsemőnél.
Congressus Pharmaceuticus Hungaricus XII. Budapest, 2003. május 8-10.
7. Balpataki Rita, Buday Tamásné, Kovácsné Balogh Judit, Vincze Zoltán:
Szisztémás antibiotikum felhasználás elemzése a Semmelweis Egyetemen.
Congressus Pharmaceuticus Hungaricus XII., Budapest 2003. május 8-10.
8. Kovácsné Balogh Judit, Jelinekné Nikolics Mária, Túrmezeiné Horváth Judit,
Vincze Zoltán: A parenterális táplálás gyógyszerészi vonatkozásai.I.
Magyar Mesterséges Táplálási Társaság 2003. évi Kongresszusa, Budapest
2003. november 21-22.
9. Kovácsné Balogh J., Farkas E., Vincze Z., Zelkó R.:A parenterális táplálás
gyógyszerészi vonatkozásai II. A TPN emulziók stabilitási vizsgálata.
Magyar Mesterséges Táplálási Társaság 2003. évi Kongresszusa Budapest,
2003. november 21-22.
10. Kovácsné Balogh Judit: Teljes parenterális táplálás a Semmelweis Egyetem
Gyermekgyógyászati Klinikáin. Magyar Kórházi Gyógyszerészek XIV.
Kongresszusa Debrecen, 2004. május 13-15.
11. Kovácsné Balogh Judit, Jelinekné Nikolics Mária, Farkas Edit, Kiss Dorottya,
Vincze Zoltán, Zelkó Romána: Egyedi összetételű „all in one” oldatok
stabilitásával, inkompatibilitásával kapcsolatos összefüggések vizsgálata.
Magyar Kórházi Gyógyszerészek XIV. Kongresszusa Debrecen, 2004. május
13-15.
12. Kovácsné Balogh Judit, Jelinekné Nikolics Mária, Farkas Edit, Vincze Zoltán,
Zelkó Romána: Egyedi összetételű all in one oldatkeverékek összehasonlítása
energiatartalom és stabilitás szempontjából. Gyógyszer az ezredfordulón V.
Sopron, 2004. március 25-27.
107
13. Kovácsné Balogh Judit, Dredán Judit, Csempesz Ferenc, Kiss Dorottya,
Jelinekné Nikolics Mária, Zelkó Romána: Strukturált trigliceridek hatása teljes
parenterális táplálásra szánt oldatkeverékek fizikai stabilitására.
Magyar Mesterséges Táplálási Társaság 2005. évi Kongresszusa Budapest,
2005. november 18-19.
14. Judit Balogh, Mária Nikolics, Judit Dredán, Ferenc Csempesz, Romána Zelkó:
Comparison of the physical stability of two different brands of lipid emulsion
for total nutrient admixtures. BBBB Conference on Pharmaceutical Sciences
September 26-28, 2005. Siófok, Hungary
15. Judit Balogh, Mária Nikolics, Judit Dredán, Ferenc Csempesz, Romána Zelkó:
Comparison of the physical stability of two different brands of lipid emulsion
for total nutrient admixtures.
Pharmacy: Smart Molecules for Therapy. Semi-centennial conference of
Semmelweis University, Faculty of Pharmacy. Hungarian Academy of Sciences
October 12-14, 2005. Budapest, Hungary
16. Kovácsné Balogh Judit: Egyedi öszetételű teljes parenterális tápláló oldatok
előállítása és minőségellenőrzésük szempontjai. Congressus Pharmaceuticus
Hungaricus XIII. Budapest, 2006. május 25-27.
17. Rácz Bernadett, Kovácsné Balogh Judit, Zelkó Romána: Teljes parenterális
táplálás gyógyszerészi szempontból. Magyar Mesterséges Táplálási Társaság
2006.évi Kongresszusa Galyatető 2006. október 27-28.
18. Szép Ágnes, Kovácsné Balogh Judit, Zelkó Romána: Lipid-tartalmú teljes
parenterális táplálásra szánt emulziós oldatkeverékek fizikai-kémiai
stabilitásának vizsgálata. Magyar Mesterséges Táplálási Társaság 2006. évi
Kongresszusa Galyatető, 2006. október 27-28.
108
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