Interaction Between a Perfluorocarbon Emulsion and Radiographic Contrast Media

ABSTRACT: This study evaluated specially designed perfluorocarbon (PFC) emulsions as blood substitutes in case of induced ischemia of the left heart ventricle in healthy farm pigs. Two hundred milliliters of perfluorocarbon emulsion were infused while 200 ml of blood were simultaneously drawn. Radiographic contrast media were given to aid placement of balloon catheters in the left coronary artery. Histopathological analysis showed that right heart failure caused the deaths of both pigs. Particles (up to > 3 µm) of foreign body materials obstructed capillaries of all organs analyzed (heart, lung, liver, kidneys and spleen). Laboratory investigation showed severe interference between the PFC emulsion and radiographic contrast media, resulting in the deterioration of the PFC emulsion. The strongest interference occurred when PFC emulsion and Accupaque® interacted; particle size started at an initial 311 nm and went up to > 3 µm within seconds. Great care must be taken when PFC emulsions are used in combination with x-ray contrast media. None of the described radiographic contrast media should be used within 48 hours prior to the use of this PFC emulsion. Also, the use of these contrast media should be avoided for a certain period of time after using PFC emulsion. The mechanisms of elimination of PFC emulsions from the circulation are not completely understood and has yet to be evaluated.

Key words: interaction, right heart failure

The aim of this study was to evaluate the potential of a specially designed perfluorocarbon (PFC) emulsion as blood substitute in case of experimentally-induced ischemia of the left heart ventricle. PFC emulsions were used because they were well-known, high-volume carriers of oxygen and carbon dioxide and revealed a high organ-preserving capability in transplant organs.¹ Myocardial ischemia was induced in pigs via balloon catheter in the left coronary artery. At the same time, a decrease in hematocrit was induced by an isovolumetric hemodilution using PFC emulsions as blood substitute. The balloon catheter was placed under X-ray control. Radiographic contrast media were given to control the positioning of the balloon catheter. We report on previously unobserved interactions between a radiographic contrast medium and the PFC emulsions. Methods The study was performed on healthy farm pigs, 3–4 months old, with a body weight between 20–30 kg. The study was approved by the official animal care committee of the Technical University Dresden according to the Declaration of Animal Rights and Welfare. The pigs were first anesthetized with stresnil (azaperon; 3.5 mg/50 kg-bw), followed by a mixture of ketamine (6–8 ml/50 kg-bw) and xylazine (2 ml/50 kg-bw) and then by a mixture of diazepam (4 ml/50 kg-bw) and atropine (1 ml/50 kg-bw). Anesthesia was continued intravenously with methohexital (2–3 mg/kg-bw/h) and fentanyl (5–10 µg/kg-bw/h). For muscle relaxation, 4 mg of intravenous pancuronium were applied. The experimental protocol was as follows: premedication, endotracheal intubation, anesthesia, catheter positioning for the measurement of blood pressure and heart rate (carotid artery), sternotomia, thoracotomia, opening of the pericardium, positioning and fixation of the pO2 and temperature probes in the myocardium and the skeletal muscle, and positioning of the balloon catheter under x-ray control. Two hundred milliliters of PFC emulsion were infused while 200 ml of blood were drawn at the same time. The blood was taken from the femoral artery while the PFC emulsion was injected — via catheter in the jugular vein — into the left coronary artery. Perioperatively, arterial pressure, heart-minute volume and central venous pressure were assessed. Tissue oxygen partial pressures were monitored in the myocardium of the left and right ventricles and in the skeletal muscle (m. gluteus max.). Tissue temperature was measured in the right and left ventricles, m. gluteus max. and the rectum. After the animals were sacrificed, the liver, the lung, parts of the heart and other organs were explanted, fixated and processed for histopathological analysis. PFC emulsion. For the production of the PFC emulsion, 962.5 g perfluorodecaline (F2-Chemicals, Springfield, Great Britain), 25 g perfluorodimorpholinopropane (Prof. Gambaretto, University of Padova, Italy), 75 g Pluronic PE6800 (BASF, Ludwigshafen, Germany) and 25 g soybean oil (Fresenius, Germany) were diluted in lactated Ringer’s solution onto 2.5 L.2 Afterward, the emulsion was homogenized at 400 bar over 20 minutes in the high-pressure homogenizer LAB 60 (APV Gaulin, Lübeck, Germany). The emulsion was then filtered by a sterile and pyrogenic-free filter with a pore size of 4 µm in order to remove contaminants.3 The emulsion was then sterilized in an autoclave (KSG 113; Ohling, Germany) for 20 minutes at 121 °C and 2 bar water vapor atmosphere. Radiographic contrast medium. Iohexol (Accupaque®, Amersham Buchler, Germany), a non-ionic tri-iodated, water-soluble contrast medium with an iodine concentration of 350 mg/ml, was used for radiographic contrast medium. Particle size analysis. Particles were sized with the Autosizer IIe (Malvern, Great Britain).⁴ To assess the diameter of bigger particles, they were measured microscopically in a Neubauer chamber at a primary magnification of 1:1000. Histopathology. Specimens were studied with transmission electron microscopy (TEM). Tissue specimens were fixed in 3% cacodylate-buffered glutaraldehyde for 48 hours. After postfixation in osmium and buffering in 0.1 M cacodylate, they were dehydrated in ethanol and embedded in epon. Semi-thin sections (1.0 µm) were stained with methylene blue-azure II. Ultra-thin sections were mounted on copper grids, stained with uranyl acetate and lead citrate and examined with TEM (Philips, EM400T). Results In the first animal, the coloration of the skin took on a bluish note after 9 minutes. The pig died under the clinical aspects of a right heart failure 20 minutes later. The same procedure was performed in a second pig and gave the same results. After these unexpected outcomes, the study was interrupted. The clinical signs attributed the right heart failure to an obstruction of the lung capillaries with particles of foreign body materials. A laboratory analysis revealed that an interaction between the PFC emulsion and the x-ray contrast medium led to the formation of big particles of spherically-shaped foreign body materials. Histopathological findings. As the morphological correlate of an acute right heart failure, massive dilatations in the myocardium of the right ventricle as well as a distinct interstitial edema were found. There were also indirect signs; blood congestion occurred in the liver, in both kidneys and especially in the spleen. The majority of lung capillaries were completely obstructed by particle-containing emboli. TEM evaluation of PFC particles in the lung showed that particles were carried in the blood and already endocytosed to a very high percentage in granulocytes after only this short time. The size distribution ranged from 300 nm to more than 3 µm. Coalescence of PFC particles can be seen in all intermediate stages (Figure 1). Arrow 1 shows the beginning of coalescence between 2 big-sized particles. Arrow 2 visualizes the end-stage of coalescence between one small and one big PFC particle. Asterisks demonstrate a PFC-particle loaded granulocyte. Figure 2 shows PFC particles in liver sinusoids and in granulocytes. Particle size analysis. In order to determine the interaction of the radiographic contrast media with the PFC emulsion in-vitro, one drop was mixed with 1 drop of the contrast medium. The mixture was diluted with distilled and sterile filtered water to analyze the particle size distribution. We found that the particle size increased so strongly that the measuring range of the instrument (3 nm to 3 µm) was surpassed within seconds. Therefore, the development of particles was assessed in a Neubauer chamber via light microscopy. The PFC emulsion was diluted 1:10 in PBS (phosphate-buffered saline) and then the radiographic contrast medium was added (0.1 ml contrast medium per 0.1 ml PFC emulsion). One hundred twenty seconds after addition of the contrast medium, microphotographs were taken and the diameters of the particles were measured offline. In addition to the radiographic contrast medium used in this study, three other contrast media were analyzed (Table 1): Xenetix® (Iobitridol, Guerbet, Germany), Imeron® (Iomeprol, Byk Gulden, Germany) and Ultravist® (Iopromid, Schering, Germany). All four of the contrast media contained tris(hydroxymethyl)-amino-methane and salts of the ethylenediamine-N,N,N’,N’-tetraacetic acid. Additional examinations with saturated aqueous solutions of these compounds mixed with the same volume of PFC emulsion were carried out. It was found that in the presence of these salts of the etylenediamine-tetraacetic acid, the particle size increased dramatically up to > 20 µm within seconds. Discussion Perfluorocarbons (PFCs) as a class of chemicals are biologically inert, have no toxicity and are not broken down by a biological system into other products. Liquid-liquid systems employing aqueous and perfluorocarbon phases have a very high surface area available for mass transfer. PFCs have been shown to carry very high loads of physically dissolved oxygen and carbon dioxide and to load and unload oxygen two times faster than loading and unloading of oxygen from hemoglobin, where the oxygen is chemically bound. With all of the oxygen carried by PFCs in a dissolved state, the partial pressure of oxygen (pO2) is very high in blood vessels providing additional gradients for diffusion of dissolved oxygen into the tissues. With a median particle diameter of emulsified PFCs of 0.15–0.3 µm, PFC particles easily perfuse the available capillaries in tissues. Even when vasoconstriction or ischemia prevent normal perfusion with red cells, some plasma flow will transport the PFC emulsion through tissues. That is why perfluorocarbon emulsions as intravascular oxygen carriers were designed to temporarily augment oxygen delivery in patients at risk of acute tissue oxygen deficit due to either transient anemia, blood loss or ischemia (inadequate oxygenation resulting from blood flow abnormalities). A PFC named FLUOSOL was approved by the Food and Drug Administration in 1989 for use as an adjunct to high-risk coronary balloon angioplasty. A further PFC named OXYGENT was developed by ALLIANCE Pharmaceutical Corporation and underwent nonclinical and clinical development. In canine studies, a single dose of OXYGENT (1.8 gm PFC/kg) was as effective as autologous blood transfusion in maintaining better myocardial function. In clinical Phase 1 and Phase 2 studies, three hundred forty subjects received OXYGENT. In Phase 2a studies, patients (n = 81) in cardiac surgery (CABG) received OXYGENT. In January 2001, ALLIANCE suspended the Phase 3 studies with OXYGENT due to an imbalance in adverse events, primarily the incidence of stroke. At that time, OXYGENT had been evaluated in 19 clinical studies involving more than 1,400 subjects. Among other reasons, the adverse events might be correlated to increased levels of hemodilution and to higher degrees of blood loss (Internet Release from Alliance Corporation, January 08, 2001: https://www.allp.com/press/press.cgi?@B0108). The histopathological analysis gave clear evidence that a right heart failure caused the deaths of both pigs. Light (not shown here) and electron microscopic examinations showed that big particles of foreign body material obstructed capillaries of all organs analyzed, namely the heart, lung, liver, kidneys and spleen. In the microphotographs taken from TEM, spherically shaped particles with sizes ranging from 300 nm to more than 3 µm showed the phenomenon of coalescence, which is typical of PFC emulsion particles when the emulsion gets deteriorated.⁵ A laboratory investigation was started to show whether the PFC emulsion in these cases and other pharmacological agents used might interfere and result in the deterioration of the PFC emulsion. All 4 of the x-ray contrast media examined interfered with the PFC emulsion; the strongest interference occurred when PFC emulsion and Accupaque interacted. In this case, the particle size started from an initial 311 nm up to more than 3 µm within seconds. This was an unexpected result, because PFC emulsions of this type were tested in multiple animals.^6,7 Moreover, similar PFC emulsions were used in clinical tests in more than 1,400 subjects.⁸ Evidently, greatest care has to be taken when PFC emulsions are used in combination with x-ray contrast media. For the type of PFC emulsion and the x-ray contrast media used here, a pre-, intra- and post-operative contrast medium enhanced x-ray diagnostic must be carefully reflected. Therefore, the day before the use of this PFC emulsion, no radiographic contrast media should be used, because the elimination of these contrast media takes from 24–48 hours.^9–12 Equally, after use of these PFC emulsions, the use of contrast media should be avoided for a certain period of time, depending on the type of PFC medium used. The reason is that the type of PFC medium determines the time of their elimination from the blood. However, the mechanisms of elimination of PFC emulsions from the blood are not completely understood, so the precise day when radiographic contrast media may be used again must be evaluated.