Phytosterolaemia associated with parenteral nutrition administration in adult patients.

Vegetable lipid emulsions (LE) contain non-declared phytosterols (PS). We aimed to determine PS content depending on the brand and LE batch, and in adult hospitalised patients treated with parenteral nutrition (PN), to establish the association between plasma and administered PS. Part I was the LE study: totals and fractions of PS in three to four non-consecutive batches from six LE were analysed. Part II was the patient study: patients with at least 7 previous days of PN with 0·8 g/kg per d of an olive/soyabean (O/S) LE were randomised (day 0) 1:1 to O/S or 100 % fish oil (FO) at a dose of 0·4 g/kg per d for 7 d (day 7). Plasma PS, its fractions, total cholesterol on days 0 and 7, their clearance and their association with PS administered by LE were studied. In part I, LE study: differences were found in the total PS, their fractions and cholesterol among different LE brands and batches. Exclusive soyabean LE had the highest content of PS (422·36 (sd 130·46) µg/ml). In part II, patient study: nineteen patients were included. In the O/S group, PS levels were maintained (1·11 (sd 6·98) µg/ml) from day 0 to 7, while in the FO group, significant decreases were seen in total PS (-6·21 (sd 4·73) µg/ml) and their fractions, except for campesterol and stigmasterol. Plasma PS on day 7 were significantly associated with PS administered (R2 0·443). PS content in different LE brands had great variability. PS administered during PN resulted in accumulation and could be prevented with the exclusive administration of FO LE.

Oxylipins and Free Fatty Acids in Parenteral Lipid Emulsions Currently Used in Preterm Infant Care: An In Vitro Study.

Lipid emulsions used to support nutrition in preterm infants contain long-chain polyunsaturated fatty acids (LCPUFAs) as a source of essential fatty acids; these LCPUFAs and their parent polyunsaturated fatty acid (PUFA) can be oxidized by a variety of mechanisms to bioactive molecules called oxylipins, which are signaling molecules that initiate and/or resolve inflammation. The aim of this study was to explore levels of free LCPUFA and their related oxylipins in 3 commercially available lipid emulsions (Intralipid, SMOFlipid, and ClinOleic) using ultra high-performance liquid chromatography mass spectroscopy. Free LCPUFA were detected in all lipid emulsions tested. Seven, 8, and 9 different oxylipin compounds were detected in the 3 emulsions, respectively. The oxylipins detected were mainly derived from omega-6 PUFAs; these included 13-hydroxyoctadecadienoic acid from linoleic acid and 5-hydroxyeicosatetraenoic acid derived from arachidonic acid. It may be clinically important to know that oxylipins exist in lipid emulsions and to evaluate their potential effects on preterm infants.

[Fat emulsion tolerance in preterm infants of different gestational ages in the early stage after birth].

OBJECTIVE: To investigate the fat emulsion tolerance in preterm infants of different gestational ages in the early stage after birth. METHODS: A total of 98 preterm infants were enrolled and divided into extremely preterm infant group (n=17), early preterm infant group (n=48), and moderate-to-late preterm infant group (n=33). According to the dose of fat emulsion, they were further divided into low- and high-dose subgroups. The umbilical cord blood and dried blood filter papers within 3 days after birth were collected. Tandem mass spectrometry was used to measure the content of short-, medium-, and long-chain acylcarnitines. RESULTS: The extremely preterm infant and early preterm infant groups had a significantly lower content of long-chain acylcarnitines in the umbilical cord blood and dried blood filter papers within 3 days after birth than the moderate-to-late preterm infant group (P<0.05), and the content was positively correlated with gestational age (P<0.01). On the second day after birth, the low-dose fat emulsion subgroup had a significantly higher content of short-, medium-, and long-chain acylcarnitines than the high-dose fat emulsion subgroup among the extremely preterm infants (P<0.05). In the early preterm infant and moderate-to-late preterm infant groups, there were no significant differences in the content of short-, medium-, and long-chain acylcarnitines between the low- and high-dose fat emulsion subgroups within 3 days after birth. CONCLUSIONS: Compared with moderate-to-late preterm infants, extremely preterm infants and early preterm infants have a lower capacity to metabolize long-chain fatty acids within 3 days after birth. Early preterm infants and moderate-to-late preterm infants may tolerate high-dose fat emulsion in the early stage after birth, but extremely preterm infants may have an insufficient capacity to metabolize high-dose fat emulsion.

Vitamin K1 distribution following intravenous vitamin K1-fat emulsion administration in rats.

This study investigated vitamin K1 (VK1 ) distribution following intravenous vitamin K1-fat emulsion (VK1 -FE) administration and compared it with that after VK1 injection. Rats were intravenously injected with VK1-FE or VK1 . The organ and tissue VK1 concentrations were determined using high-performance liquid chromatography method at 0.5, 2 and 4 h to determine distribution, equilibrium and elimination phases, respectively. In the VK1-FE group, the plasma, heart and spleen VK1 concentrations decreased over time. However, other organs like liver, lung, kidney, muscle and testis, reached peak VK1 concentrations at 2 h. In the VK1 injection group, the liver VK1 concentrations were significantly higher than those in other organs at the three time points. However, VK1 concentrations in the other organs peaked at 2 h. In addition, in VK1-FE group, the heart, spleen and lung VK1 concentrations were significantly higher than those in the VK1 injection group at the three time points, and the liver VK1 concentration was significantly higher than that in the VK1 injection group at 4 h. The VK1 amount was greatest in the liver compared with the other organs. Thus, the liver is the primary organ for VK1 distribution. The distribution of VK1 is more rapid when injected as VK1-FE than as VK1 .

Evolution of lipid profile, liver function, and pattern of plasma fatty acids according to the type of lipid emulsion administered in parenteral nutrition in the early postoperative period after digestive surgery.

BACKGROUND: The metabolic effects of intravenous lipid emulsions (ILEs) used in parenteral nutrition (PN) depend on their fatty acid composition. METHODS: Subjects in this prospective and randomized double-blind study were 28 adult patients post digestive surgery. PN was started after surgery and lasts for 5 days. Randomly, patients receive 1 of 4 different ILEs: medium-chain triglycerides/long-chain triglycerides (soybean oil; MCT/LCT), olive/soybean oil (oleic), long-chain triglycerides (soybean oil; LCT), and structured lipid. On days 0 and 6, serum liver function tests were analyzed for cholesterol, triglycerides, lipoproteins, and serum fatty acids. RESULTS: No differences were found in the 4 groups according to their gender, age, body mass index, diagnosis, baseline white blood cell, C-reactive protein, glucose levels, and other study parameters. Differential significant changes were not observed in any of the hepatic function parameters or plasmatic lipid levels between the groups. A significant decrease was observed in cis monounsaturated fatty acids (MUFAs) and a significant increase in omega-6 polyunsaturated fatty acids (PUFAs) and omega-3 PUFA values in LCT and structured groups compared with MCT/LCT and oleic groups, and a tendency for a decrease in trans fatty acids in the oleic and structured groups was found. CONCLUSIONS: All ILEs administered were safe and well tolerated. The changes in serum fatty acids reflected the pattern of fatty acids administered with different ILEs. The group receiving the olive oil emulsion achieved a fatty acid composition of serum lipids that could offer major therapeutic or biological advantages.

Performance evaluation of a particle-enhanced turbidimetric cystatin C assay on the Hitachi 917 analyzer.

BACKGROUND: Human cystatin C is a low molecular weight protein that has been proposed as a new endogenous marker of glomerular filtration rate. We investigated the performance of the Genzyme cystatin C assay on the Hitachi 917 analyzer. METHODS: Imprecision, linearity, recovery, and interference studies were performed on the Hitachi 917 analyzer. For method comparison, split sample aliquots were assayed using the described method and 2 other commercially available cystatin C assays. RESULTS: The assay was linear from 0.24 to 6.36 mg/l. Within-run coefficient of variation (CV) was 4.2 and 0.8% at cystatin C concentrations of 0.50 and 2.00 mg/l, respectively. Between-run CV was 4.3 and 2.7% at the same concentrations. The average analytical recovery was 99%. Bilirubin (< or =30 mg/dl), triglycerides (< or =1000 mg/dl), intralipid (L index < or =1000), and rheumatoid factor (< or =1000 IU/ml) did not interfere with the assay. A >10% change in cystatin C level was observed when hemoglobin concentration was >800 mg/dl. The assay compared well with the Dade Behring immunonephelometric assay and the Dako immunoturbidimetric assay. CONCLUSION: The Genzyme cystatin C immunoassay is an acceptable method for the determination of cystatin C on the Hitachi 917 analyzer.

Extraction of diethylhexylphthalate by home total parenteral nutrition from polyvinyl chloride infusion lines commonly used in the home.

OBJECTIVES: Recently, our group detected that polyvinyl chloride (PVC) perfusion lines leach large amounts of the toxic plasticizer diethylhexylphthalate (DEHP) under conditions typical of intensive care units. In the present study, we investigated the extraction of DEHP from PVC connecting tubes that are commonly used for total parenteral nutrition (TPN) solutions. The aim of the study was to estimate the amount of DEHP to which children receiving home TPN are exposed for months and years. MATERIALS AND METHODS: 1000 mL of TPN, identical in constitution and amount to the home TPN of 1 of our patients, were perfused through 5 different connecting tube systems and collected in hexane-rinsed glass bottles. The concentration of DEHP in the TPN was analyzed before and after perfusion. RESULTS: Before perfusion of the lines, the solution had a DEHP concentration of 0.05 to 0.69 microg/mL (baseline value). After perfusion of the lines, the load of DEHP in the solution varied between 1.41 and 2.07 microg/mL. This TPN was established for children weighing 20 kg. The daily dosage is between 71 and 104 microg x kg(-1) x day(-1). TPN is administered at home for many months and years. The monthly charge of DEHP is between 42.3 and 62.1 mg. Children weighing 20 kg therefore receive a dosage between 2.1 and 3.1 mg x kg(-1) x month(-1). CONCLUSIONS: Diethylhexylphthalate and its metabolite monoethylhexylphthalate have been demonstrated to be carcinogenic, embryotoxic, hepatotoxic, pneumotoxic, and cardiotoxic and are known to disrupt endocrine pathways and liver detoxifying capacity in animals. They are suspected of having multiple effects in humans as well. The doses presented above should therefore be avoided in children receiving home TPN by the use of tubing systems that are completely free of DEHP. Such systems are available.

Shortened hemofilter survival time due to lipid infusion in continuous renal replacement therapy.

BACKGROUND: Continuous renal replacement therapy is widely used for the treatment of critically ill patients with acute renal failure in critical care units. The survival time of the extracorporeal circuit is an important factor in providing renal replacement therapy. Despite rigorous efforts to maintain hemofilter patency, clinicians are occasionally faced with an unexplained short circuit survival time. METHODS: We present a critically ill patient undergoing continuous venovenous hemofiltration with regional citrate anticoagulation for management of acute renal failure in the context of sepsis. Once the patient was started on lipid infusion as part of total parenteral nutrition, we observed a shortened circuit survival due to premature hemofilter failure necessitating frequent changes of the hemofilter. The known potential causes for this phenomenon were ruled out. RESULTS: Evaluation revealed grossly lipemic serum associated with severe hypertriglyceridemia. Discontinuation of the lipid infusion was followed by a rapid return of circuit survival time to its baseline. Evaluation of the hemofilter by electron microscopy revealed that the rapid blockage of the hollow fibers was caused by lipid microparticles and fibrin deposits. CONCLUSION: Since total parenteral nutrition is commonly administered to malnourished and hypercatabolic critically ill patients on continuous renal replacement therapy, we suggest that intravenous lipid therapy might be a previously unreported and unappreciated remediable cause of premature hemofilter failure.

Phytosterol determination in lipid emulsions for parenteral nutrition.

OBJECTIVE: The presence of phytosterols in vegetal lipid emulsions has been  associated with alterations of liver function tests. Determination of phytosterols  content, currently undeclared, would allow the development of strategies to  prevent or treat these alterations. METHOD: 3-4 non-consecutive batches of 6 lipid emulsions from different providers (Clinoleic, Intralipid, Lipofundina, Lipoplus, Omegaven and Smoflipid) were analyzed. Differences in total phytosterol assay between providers and batches were statistically studied by a one-way ANOVA and Kruskal-Wallis non-parametric approximation and post hoc Scheffé test (p < 0.05)Results: The absence of phytosterols was confirmed in Omegaven, emulsion  based on fish oil. The highest assay of phytosterols (422.4 ± 130.5 µg/mL) has  been related with the highest percentage of soya bean oil in Intralipid. In the  remaining emulsions, concentrations were from 120 to 210 µg/mL related to the  percentage of soya bean oil. Statistically significant differences of phytosterol  content in lipid emulsions were observed among different providers (F = 23.59;  p = 0.000) as well as among non-consecutive batches. Clinolenic (F = 23.59; p  = 0.000), Intralipid (F = 978.25; p = 0.000), Lipofundina TCL/TCM (F = 5.43; p  = 0.045), Lipoplus (F = 123.53; p = 0.000) and Smoflipid (16.78; p = 0.000).  Except for Lipofundina TCL/TCM, the differences between batches were marked. CONCLUSIONS: Lipid emulsions, registered on Spanish pharmaceutical market,  contain variable quantities of phytosterols dependent on commercial brand and  batch.

Objetivo: La presencia de fitoesteroles en emulsiones lipídicas de origen vegetal se ha relacionado con la aparición de alteraciones de los parámetros de la  función hepática. El objetivo es determinar la presencia de fitoesteroles en las  emulsiones registradas en el mercado farmacéutico.Método: Se analizaron tres-cuatro lotes no consecutivos de seis marcas  distintas de emulsiones lipídicas (Clinoleic®, Intralipid®, Lipofundina®,  Lipoplus®, Omegaven® y Smoflipid®) y las diferencias en contenido de  fitoesteroles totales entre marcas y entre lotes se estudiaron estadísticamente  (ANOVA de un factor, aproximación no paramétrica de Kruskal-Wallis y análisis  post hoc Scheffé; p < 0,05).Resultados: Se encontró ausencia de fitoesteroles en el preparado Omegaven® con aceite de pescado. El contenido más alto de fitoesteroles (422,4 ± 130,5  µg/mL) coincidió con el porcentaje más alto de aceite de soja (Intralipid®). En el resto de las emulsiones se detectaron concentraciones de fitoesteroles entre 120  y 210 µg/mL, relacionadas con el contenido de aceite de soja. Se  observaron diferencias estadísticamente significativas entre todas las marcas de  emulsiones lipídicas (F = 42,97; p = 0,000) y entre lotes no consecutivos.  Clinolenic® (F = 23,59; p = 0,000); Intralipid® (F = 978,25; p = 0,000);  Lipofundina® TCL/TCM (F = 5,43; p = 0,045); Lipoplus ® (F = 123,53; p =  0,000),; y Smoflipid® (16,78; p = 0,000). Excepto en el caso de la  Lipofundina® TCL/TCM las diferencias entre lotes fueron marcadas.Conclusiones: Las emulsiones lipídicas registradas en el mercado farmacéutico español contienen cantidades variables de fitoesteroles en función  e la marca comercial y el lote. La determinación del contenido de fitoesteroles, actualmente no declarados, permitiría desarrollar estrategias para prevenir o tratar la aparición de estas alteraciones.

A HPLC method to monitor the occurrence of lipid peroxidation in intravenous lipid emulsions used in parenteral nutrition using in-line UV and charged aerosol detection.

Parenteral Nutrition (PN) provides life sustaining support where gastrointestinal nutrition is inadequate due to disease or prematurity. Intravenous lipid emulsions (IVLEs) form a staple part of PN. Whilst the physical stability of IVLE's is relatively well known and quantified, chemical stability is an area where little testing has occurred. We report a new sensitive method for the monitoring of selected triglycerides present within two IVLEs and the detection and quantification of the peroxidation product 4-hydroxynonenal (HNE) using HPLC with in-line UV and charged aerosol detection (CAD). IVLEs used included the soy-bean oil based emulsion Intralipid® 20% and SMOFlipid® 20% (Fresenius Kabi UK), based on soy-bean, olive, fish oil and medium chain triglycerides. Assay validation gave R2 values of ≥0.99 for all selected triglyceride peaks and 4-hydroxynonenal. Inter and intra-day repeatability gave RSD values < 7.2% for CAD detection, achieving a precise and repeatable method. HNE was confirmed through internal standardisation of the HPLC method. Selected triglycerides were identified using ESI-MS with MicroTOF. This novel method permits the chemical stability of IVLEs to be quantified and monitored in respect to lipid peroxidation during storage prior to delivery to the patient, ensuring the optimal safety of IVLEs in a clinical setting.

An Institutional Change in Continuous Renal Replacement Therapy.

BACKGROUND: Critically ill patients with acute kidney injury may require parenteral nutrition (PN) and continuous renal replacement therapy (CRRT). Introduction of a phosphate-free premixed renal replacement fluid without system-wide education in May 2011 resulted in increased incidence of hypophosphatemia, necessitating change in practice. Changes included (1) maximizing phosphate in PN, (2) modifying the CRRT order set, and (3) developing a CRRT competency evaluation for nutrition support team members. This study evaluates the effect of these changes on the incidence of hypophosphatemia. METHODS: Phosphate levels and predicated probability of hypophosphatemia were evaluated for patients receiving PN and CRRT over 3 time periods: prior to implementing the changes (preimplementation), during change implementation (intermediate), and following implementation (postimplementation). Hypophosphatemia was defined as a serum phosphate level <2.5 mg/dL. Generalized linear mixed models were applied for statistical analysis. RESULTS: The retrospective study includes 336 measures from 49 patients. Patients in the intermediate and postimplementation periods were not significantly different from each other and had significantly higher mean phosphate levels than patients in the preimplementation period ( P < .0001). They were also less likely to develop hypophosphatemia compared with preimplementation patients (intermediate: odds ratio [OR], 0.07; 95% confidence interval [CI], 0.03-0.18, P < .0001; postimplementation: OR, 0.09; 95% CI, 0.03-0.27, P < .0001). CONCLUSIONS: Modifications in phosphate dosing together with CRRT education reduced the incidence of hypophosphatemia in PN patients receiving CRRT. Communication of significant changes in clinical care should be shared with all services prior to implementation. Communication and planning between services caring for complex patients are necessary to prevent systems-based problems.

Physicochemical stability and compatibility testing of levetiracetam in all-in-one parenteral nutrition admixtures in daily practice.

BACKGROUND: Parenteral antiepileptic drugs are frequently used in critically ill patients for seizure control therapy or prevention. Many of these patients require additional parenteral nutrition (PN). Therefore, a parallel infusion of the frequently used antiepileptic drug levetiracetam (LEV) is interesting in terms of the restricted i.v. lines (e.g., neonates). The potential interactions of the complex PN admixture with the drug product and the appropriate admixing of a drug at effective dosages require physicochemical lab assessments to obtain specific and reliable pharmaceutical documentation for the intended admixing. AIM: To assess the of compatibility and stability of LEV, a neutral and hydrophilic drug, in commercial all-in-one (AiO) PN admixtures using simple validated tests to provide necessary data in a timely manner and to allow convenient, documented and safe treatment with PN as the drug vehicle. METHODS: Different concentrations of LEV were injected into two different AiO PN admixtures with no further additives. Stability and compatibility tests for the drug and the PN admixtures were performed over seven days at +4°C, +23±1°C and +37°C without light protection. Stability and sample characteristics were observed by visual inspection and the validated light microscope method. Moreover, the pH level of the admixture was checked, as were the concentrations of LEV over time in the PN admixtures, using an established LC-MS/MS method. RESULTS: The stability controls of LEV at different temperatures were within absolute ±20% of the theoretical value in a concentration range of 98.91-117.84% of the initial value. No changes in pH occurred (5.55±0.04) and no microscopic out of specification data or visual changes were observed. The mean value of the largest lipid droplet in each visual field over seven days was 2.4±0.08µm, comparable to that of the drug-free AiO admixture. Samples stored at +37°C showed yellowish discolorations after 96h of storage. CONCLUSION: LEV showed compatibility and stability over seven days in the selected PN admixtures, and the described methods represented a valuable and timely approach to determine the stability and compatibility of the highly hydrophilic, not dissociated LEV in AiO admixtures under conditions of use. Further studies with clinically relevant and representative examples of physicochemically different drug classes are needed.

Metabolic effects of parenteral nutrition enriched with n-3 polyunsaturated fatty acids in critically ill patients.

BACKGROUND & AIMS: n-3 fatty acids are expected to downregulate the inflammatory responses, and hence may decrease insulin resistance. On the other hand, n-3 fatty acid supplementation has been reported to increase glycemia in type 2 diabetes. We therefore assessed the effect of n-3 fatty acids delivered with parenteral nutrition on glucose metabolism in surgical intensive care patients. METHODS: Twenty-four surgical intensive care patients were randomized to receive parenteral nutrition providing 1.25 times their fasting energy expenditure, with 0.25 g of either an n-3 fatty acid enriched-or a soy bean-lipid emulsion. Energy metabolism, glucose production, gluconeogenesis and hepatic de novo lipogenesis were evaluated after 4 days. RESULTS: Total energy expenditure was significantly lower in patients receiving n-3 fatty acids (0.015+/-0.001 vs. 0.019+/-0.001 kcal/kg/min with soy bean lipids (P<0.05)). Glucose oxidation, lipid oxidation, glucose production, gluconeogenesis, hepatic de novo lipogenesis, plasma glucose, insulin and glucagon concentrations did not differ (all P>0.05) in the 2 groups. CONCLUSIONS: n-3 fatty acids were well tolerated in this group of severely ill patients. They decreased total energy expenditure without adverse metabolic effects.

New Strategy to Reduce Hypertriglyceridemia During Parenteral Nutrition While Maintaining Energy Intake.

BACKGROUND: Hypertriglyceridemia is a frequent metabolic complication associated with fat administration in parenteral nutrition (PN). No clear guidelines have been published on how to proceed once hypertriglyceridemia has been detected. A new strategy could be to substitute the initial fat emulsion with another emulsion with faster clearance. Our objective was to determine the effectiveness in reducing triglyceridemia values, maintaining the caloric intake, and improving nutrition parameters in patients who had moderate hypertriglyceridemia during PN when an olive oil-based fat emulsion (OOFE) was substituted with a multiple-source oil fat emulsion (MOFE). We also assessed the safety of this substitution in hepatic and glycemic profiles. MATERIALS AND METHODS: We performed a retrospective, observational study that included 38 adult patients to whom OOFE in PN was substituted with MOFE when moderate hypertriglyceridemia (≥250-400 mg/dL) was detected. RESULTS: Triglyceridemia values decreased in 36 (94.7%) patients. The mean reduction was 71 (88-22) mg/dL. Fat load was slightly reduced after substitution (-0.14 [-0.23 to 0] g/kg/d; P < .001), but total caloric intake increased from 22.5 (19.7-25.1) to 23.1 (19.8-26.8) kcal/kg/d (P = .053). After substitution, nutrition parameters improved, liver parameters remained unchanged, and insulin requirements increased. CONCLUSION: The substitution of OOFE with MOFE in patients with moderate hypertriglyceridemia during PN resulted in a reduction in triglyceridemia values of about 70 mg/dL. That allowed maintaining the caloric intake and improved nutrition parameters without affecting the hepatic profile. For some patients, insulin requirements increased moderately.

Distribution of Tocopherols and Tocotrienols in Guinea Pig Tissues Following Parenteral Lipid Emulsion Infusion.

BACKGROUND: Tocopherols and tocotrienols possess vitamin E activity and function as the major lipid-soluble antioxidants in the human body. Commercial lipid emulsions are composed of different oils and supply different amounts of vitamin E. The objective of this study was to measure all 8 vitamin E homologs within 4 different commercial lipid emulsions and evaluate their distribution in guinea pig tissues. MATERIALS AND METHODS: The distribution of vitamin E homologs within plasma and guinea pig tissues was determined using a high-performance liquid chromatography (HPLC) system. Lipid hydroperoxides in lipid emulsions were determined using a commercial kit (Cayman Chemical Company, Ann Arbor, MI), and malondialdehyde tissue levels were determined using an HPLC system. RESULTS: The lipid emulsions contained variable amounts of tocopherols, which were significantly different between emulsions. Tocotrienols were present at very low concentrations (≤0.3%). We found no correlation between the amount of vitamin E present in the lipid emulsions and lipid peroxidation. Hydroperoxides were the lowest with an olive oil-based emulsion and highest with a fish oil emulsion. The predominant vitamin E homolog in guinea pig tissues was α-tocopherol. No tissues had detectable levels of tocotrienols. Vitamin E levels (primarily α-tocopherol and γ-tocopherol) were highly variable among organ tissues. Plasma levels were a poor reflection of most tissue levels. CONCLUSION: Vitamin E levels within different lipid emulsions and plasma/tissues are highly variable, and no one tissue or plasma sample serves as a good proxy for levels in other tissues. All study emulsions were well tolerated and did not significantly increase systemic lipid peroxidation.

Development of a validated LC-APCI-MS/MS method to study the plasma and tumor distribution of CHO-PTX intravenous lipid emulsion.

Conjugation of a cholesterol moiety to active compounds for cancer treatment or diagnosis is an attractive approach for increasing lipophilicity and improving loading into lipid carriers. We developed a highly sensitive and specific liquid chromatography atmospheric-pressure chemical ionization tandem mass spectrometry (LC-APCI-MS/MS) analytical method to investigate the in vivo plasma and tumor distribution characteristic of a cholesterol-paclitaxel conjugate (CHO-PTX) in nude mice with MDA-MB-231 human breast cancer xenografts. The samples were analyzed in positive ion, multiple reaction monitoring mode. The plasma and tumor tissue samples were processed by liquid-liquid extraction with methyl tert-butyl ether (MTBE). Docetaxel was used as the internal standard (IS) for sample processing and analysis. MS/MS detection was carried out by monitoring the transitions of m/z 1266.7→369.4 and 330.3 for CHO-PTX, and m/z 808.7→226.4 and 509.1 for IS. The calibration curves were linear over 100-25,000 ng/mL in mouse plasma and tumor homogenate samples. The limit of quantitation of CHO-PTX was 100 ng/mL in both matrices. The intra-day and inter-day precisions were less than 15%, and the accuracy was between -8.0% and 8.6% for both matrices. The developed method was successfully applied to measure CHO-PTX levels in plasma and tumor tissues in nude mice. The mean tumor concentrations in mice tumor tissues after intravenous administration of CHO-PTX emulsion at a dose equivalent to 20 mg/kg paclitaxel were 2022±630 ng/mL ng/mL, 2516±982 ng/mL, 3056±1438 ng/mL, and 2367±1029 ng/mL at 0.25, 3, 24, and 120 h, respectively. The accumulation of CHO-PTX in the tumor suggests that cholesteryl drug conjugates are a promising approach for medical treatment of various human cancers.

In Vitro Studies Indicate Intravenous Lipid Emulsion Acts as Lipid Sink in Verapamil Poisoning.

Intravenous lipid emulsion (ILE), a component of parenteral nutrition, consists of a fat emulsion of soy bean oil, egg phospholipids, and glycerin. Case reports suggest that ILE may reverse hypotension caused by acute poisoning with lipophilic drugs such as verapamil, but the mechanism remains unclear. The methods used are the following: (1) measurement of ILE concentration in serum samples from a patient with verapamil poisoning treated with ILE, (2) measurement of free verapamil concentrations in human serum mixed in vitro with increasing concentrations of ILE, and (3) measurement of murine ventricular cardiomyocyte L-type Ca(2+) currents, intracellular Ca(2+), and contractility in response to verapamil and/or ILE. Maximum patient serum ILE concentration after infusion of 1 L ILE over 1 h was approximately 1.6 vol%. In vitro GC/MS verapamil assays showed that addition of ILE (0.03-5.0 vol%) dose-dependently decreased the free verapamil concentration in human serum. In voltage-clamped myocytes, adding ILE to Tyrode's solution containing 5 µM verapamil recovered L-type Ca(2+) currents (ICa). Recovery was concentration dependent, with significant ICa recovery at ILE concentrations as low as 0.03 vol%. ILE had no effect on ICa in the absence of verapamil. In field-stimulated intact ventricular myocytes exposed to verapamil, adding ILE (0.5 %) resulted in a rapid and nearly complete recovery of myocyte contractility and intracellular Ca(2+). Our in vitro studies indicate that ILE acts as a lipid sink that rapidly reverses impaired cardiomyocyte contractility in the continued presence of verapamil.

Lipid composition and structure of commercial parenteral emulsions.

In order to study the influence of the phospholipid/triacylglycerol (PL/TG) ratio of parenteral emulsions on the distribution and the physico-chemical properties of their fat particles, commercial 10, 20 or 30% fat formulas were fractionated by centrifugation into an upper lipid cake (resuspended in aqueous glycerol) and a subnatant or mesophase, from which a PL-rich subfraction (d = 1.010-1.030 g/l) was purified by density gradient ultracentrifugation. Chemical and 31P-NMR analyses of these fractions indicated that at least two types of fat particles coexist in parenteral emulsions: (i) TG-rich particles (mean diameter: 330, 400, 470 nm in the 10, 20, 30% emulsion) which contain practically all the TG and esterified phytosterols of native emulsions, but only a fraction of their PL, unesterified cholesterol and phytosterols, and other minor lipids; (ii) PL-bilayer particles or liposomes (mean diameter: 80-100 nm) which are constituted with the remaining PL and relatively very small amounts of TG and other lipids. The higher the oil content of the emulsion, the lower the amount of these PL-rich particles, which represent the major particle population of the mesophase. Indeed, minute amounts of TG-rich particles (probably the smallest ones) are also present in the mesophase, even in the PL-rich subfraction which contains the bulk of liposomal PL. Since the PL-rich particles of the infused emulsion generate lipoprotein X-like particles, only the large TG-rich particles can be considered as true chylomicron counterparts.

Effects of intravenous infusions of commercial fat emulsions (Intralipid 10 or 20%) on rat plasma lipoproteins: phospholipids in excess are the main precursors of lipoprotein-X-like particles.

Like most commercial parenteral emulsions, Intralipid contains the same amount of phospholipids (12 mg/ml) to stabilize 100 or 200 mg of soybean oil (10 or 20% formula, respectively). By centrifugation, 10 or 20% Intralipid was separated into a supernatant, fat particles containing the bulk of triacylglycerols stabilized by a fraction of phospholipids and an infranatant--called mesophase--consisting mainly of phospholipids used in excess as emulsifier. We observed that the initial triacylglycerol/phospholipid ratio of the emulsion (100/12 and 200/12, respectively) determines the size of the triacylglycerol-rich particles (260 and 350 nm) as well as the phospholipid content of the mesophase (6.02 and 4.67 mg/ml). To understand the mechanism of the lipoprotein-X (LPX) accumulation generally reported after intravenous fat infusions, plasma lipid levels and lipoprotein profiles were first compared in the rats after infusion (at a constant rate of 0.5 or 1 ml/h for 43 h) of Intralipid 10 or 20%. For the same intravenous triacylglycerol load (100 mg/h), rats infused with Intralipid 10% at 1 ml/h displayed higher triacylglycerol levels than rats infused with the 20% emulsion at 0.5 ml/h, suggesting that the size of exogenous fat particles modulated the catabolic rate of their triacylglycerols. The plasma levels of LPX varied according to the infusion rate of phospholipids not associated with triacylglycerol-rich particles of the emulsion. Moreover, an apo E and apo B enrichment of plasma and an elevation of the apo B48/apo B100 ratio was always observed after Intralipid infusions. In order to confirm that phospholipids of the mesophase are the main LPX precursors, lipoprotein profiles were then compared in the rats after intravenous infusion, at a constant rate of 1 ml/h, of either the mesophase or a suspension of triacylglycerol-rich particles isolated from Intralipid 20%. As expected, significant LPX amounts were only detected in rats infused with the pure mesophase of the emulsion. It was concluded that products of the lipolysis of exogenous fat particles play only a minor role in the formation of LPX. In fact these abnormal lipoproteins are generated by phospholipids of the mesophase which, like infused liposomes, actively mobilize endogenous free cholesterol. Consequently, in order to be considered as true chylomicron models for safe fat delivery in parenteral nutrition and in order to prevent some detrimental effects on cholesterol metabolism, commercial emulsions should be cleared of phospholipid excess.

Effects of triacylglycerol-saturated acyl chains on the clearance of chylomicron-like emulsions from the plasma of the rat.

We previously found that a single saturated acyl chain at the glycerol 2-position affected the metabolism of chylomicrons. The explanation for the effect is not clear, but could be reproduced by saturated monoacylglycerols. In the present work we have extended our measurements to several different triacylglycerols containing one or two saturated chains in specific locations in an attempt to define structural features that affect chylomicron clearance. Lipid emulsions containing triacylglycerol, egg yolk phosphatidylcholine, free cholesterol, cholesteryl oleate (CO) and labelled with 3H-CO and [14C]triolein (OOO) were prepared as models of lymph chylomicrons. When injected intravenously into rats, the metabolism of the emulsions was influenced by the acyl chains of the constituent triacylglycerols. Compared with emulsions containing OOO as the only triacylglycerol, plasma clearances of emulsion [3H]CO were extremely slow in emulsions containing either 1,2-dioleoyl-3-stearoylglycerol (OOS) or 1-stearoyl-2,3-dioleoylglycerol (SOO). As little as 10% of SOO in mixture with OOO slowed the clearance, and increasing proportions of SOO in OOO emulsions progressively slowed the removal of OOO and CO labels from plasma. With 50% and 100% SOO in the emulsions clearance was negligible. In emulsions containing the triacyl-sn-glycerols, 1,3-dimyristoyl-2-oleoylglycerol (MOM), 1,3-dipalmitoyl-2-oleoylglycerol (POP), 1-oleoyl-2,3-distearoylglycerol (OSS) or 1-palmitoyl-2-oleoyl-3-stearoylglycerol (POS), clearance rates of CO and OOO labels from plasma were significantly decreased compared with control OOO emulsions. With emulsions prepared with the triacylglycerols, 1-oleoyl-2,3-dimyristoylglycerol (OMM) and 1-oleoyl-2,3-dipalmitoylglycerol (OPP), clearances of CO label were significantly slower than with control OOO emulsions, while the removal of OOO label was not significantly affected. The uptake of CO label in the liver was decreased in conjunction with the lower rates of clearance of emulsion CO from the plasma. The clearance from plasma of 1,3-distearoyl-2-oleoylglycerol (SOS) emulsions was similar to the control OOO emulsions, but significantly more emulsion OOO label was taken up by the liver. Emulsions made with the triacylglycerols extracted from natural cocoa butter, which contained a high proportion of saturated acyl chains, were cleared similarly to the control OOO emulsions. Our findings indicate that the plasma clearance of triacylglycerol-rich lipoprotein particles depends upon the specific arrangements of the acyl chains of the constituent triacylglycerols, and not necessarily on the overall saturation of the triacylglycerols.(ABSTRACT TRUNCATED AT 400 WORDS)