Effects of Lipemia and Trauma on Experimental Fat Embolism Franz X. Hausberger, MD
According to some investigators, trauma is thought to bring about physical changes which produce a coalescence of normally dispersed blood lipids into fat droplets and pulmonary fat embolisms. Lipemia is thought to increase the extent of this embolization. If this theory is correct, intravenously infused labeled oil which is retained in the lungs should be mixed with and diluted byv the fat emboli originating from the blood. Radioactive olive oil of known specific activity was slowly administered to a) control rats, b) rats made lipemic by dietary measures, c) severely traumatized rats, and d) traumatized lipemic rats. There was no difference in the specific activity of the oil recovered from the lungs of rats of any group, indicating that no recognizable amount of blood lipids had been added to the experimentally produced emboli. (Am J Pathol 84:515-520, 1976)
THERE IS N\O GREE\1E\T on the etiology of pulmonary fat embolism. In the classic theorx-, it is chiefly caused bx- intravasation of fat from the site of injurx%, most often the marrow caxity- of fractured bones. Several observations are in variance with this concept, these include the occurrence of fat embolism in poisoning and other conditions not associated X ith bone injury. It has been pointed out, however, that the degree of embolization in these conditions is always minor and without clinical significance."2 Opponents of the classic theory emphasize that the amount of fat in the marrom.- cavity of even the largest bones, like femur or tibia, is not suffient to produce fatal embolization. This opinion is based on experiments by Lehman and Moore.3 wvho found that an intravenous dose of fat of 1.5 to 1. ml kg is lethal for dogs. These authors calculated that the lethal dose for a man of 75 kg is 112 to 113 ml, m-hile the fat content of the marrow cavity of the human femur is 65 to 70 ml. Several other theories about the origin of the embolic fat have been proposed. According to the physicomechanical theory, the embolic fat originates chiefly from blood lipids and only to a minor degree from marrow fat. Trauma is thought to produce instability of normally dispersed lipids such as chvlomicrons, which coalesce into larger fat droplets -hich are retained by From the Department of Anatomy. Jefferson \Medical College of Thomas Jefferson U ni\ ersit\. arnd the Department of Xnatomic Sciences. Temple L nis ersit\ School of Dentistrx. Philadelphia. Penns% 1vania.
Suipported by Grant HL-14322 from the National Heart and Loine Instituite. National Instituites of Health.
Xecepted for publication Mlay 7. 1976 Address reprint requests to Dr Franz X. Hausbercer. Department of Anatomic Sciences. Temple U niversity. School of Dentistry. :3223 North Broad Street. Philadelphia. PA 19140 515
516
HAUSBERGER
American Journal of Pathology
the pulmonary capillaries. Sexeral investigators have reported that lipemia 4-7 or soft tissue injury 8 produce pulmonary fat embolism. and that lipemia greatly increases its severity after fracture.5'7 Other investigators could not confirm these reports. Although lipemia increases the normally low- number of fat droplets in blood.9 it does not significantly affect the number of pulmonary emboli after fracture.9",0 nor does soft tissue injury produce fat embolism.1' The physicomechanical theory was greatly supported by LeQuire and co-workers.'2 wvho found that pulmonary emboli from human patients had a composition that was similar to blood lipids and different from adipose tissue fat. Ellis and WNatson 13 and Holczabek 14,15 could not confirm these findings on their o-n material from human patients. The strongest evidence against the physicomechanical theory was presented by Kerstell 16 and Kerstell and co-workers.'7 Pulmonary emboli washed from the lungs of dogs after femoral fracture showed the chemical composition of marrosw fat. and neither plasma lipids nor free fatty acids contributed to the formation of the emboli. Despite these findings. LeQuire and co-workers 18.19 are "convinced that the histologically massive pulmonary fat embolism seen in human patients" is caused by demulsification of plasma lipoproteins. 19 It had been sho-n earlier that intravenously infused olive oil, labeled with 'l'I, is quantitatively retained in the lungs in the form of fat emboli.20'2' According to the physicomechanical theory, lipemia and or trauma taking place at or near the time of the infusion should produce a coalescence of blood lipids and additional emboli. Furthermore. these emboli should be retained in the lungs and mix with and dilute the externally introduced labeled fat. The resulting subsequent reduction of the specific activitv. i.e., the activity per milligram of olive oil. can be measured. and it would provide an estimate of the degree of formation of emboli from blood fat. This approach wvas used to investigate the validity of the physico-
mechanical theory. Material and Methods Male Wistar rats %seighing betxseen :350 and 380 g 'vere used. The animals "ere anesthetized bv an initial intraperiteoneal injection of 2. to 30 mg kg of pentobarbital Anesthesia or heavy'- sedation wvas maintained throughout the experiment bx- repeated injections of small amounts of the barbiturate In addition. ethy l ether X-as used during the surgical procedures. Approximately 0.3 g kg of olive oil, labeled Nvith 1311 triolein \lal-
linckrodt X -as infused mvith constant speed into the femoral -ein during a period of 1 hour The amount infused \vas determined by %veighing of the sy ringe assembl!- before and after the injection. One to three hours later. the animals %vere decapitated. and their blood "as collected. One piece of plastic tubing wsas tied into the left auricle and a second one into the pulmonarx- trunk. Warm physiologic saline solution "as gentlx forced into the pulmonary veins. and the effluent from the pulmonarx- trunk \-.as collected into one or txN-o 15-
Vol. 84, No. 3 September 1976
LIPEMIA AND FAT EMBOLISMS
517
millimeter test tubes. After the vials w-ere let stand for not more than one-half hour, and they w-ere occasionally tapped to dislodge fat droplets. 1 to 2 ml of petroleum ether %vas added to the clearly recognizable top lav er of fat. The lipid-solvent lav er w as gentlx- stirred with a thin w-ire without disturbing the aqueous laver. After the solvent mixture was let stand for about 3 to 5 minutes, most of the mixture w-as transferred X ith the aid of a sy ringe and needle to a w-eighed glass scintillation v ial. This extraction procedure w-as repeated tw-ice: the solvent was then evaporated, and the vial mvas weighed again. In most experiments. 20 to 4c0 of the administered oil was recovered. and no attempts were made to increase this amount. The activitv of the recovered oil and the activ itv of the oil used for the infusion w-ere determined in a scintillation well detector. The specific activ.ities of the two samples were calculated and compared. The actisity of the thyroid gland of each animal w-as also counted. The preparation of the oil used for infusion has been described earlier." The experiments w-ere carried out in four groups of rats with 10 to 1, animals in each group. Group A consisted of 14 normally fed controls. The 17 rats of Group B wsere starxed for 2 and 3 dav s and refed for 20 to 12 hours w-ith a high fat diet consisting of Smviss cheese and Purina Laboratorx Chow- pellets soaked in olise oil. The 10 rats of Group C %vere traumatized according to the method of Whiteley," by placing a tourniquet around one low-er limb and around both upper limbs for 2 to 4 hours. Infusion X"as started 3 hours after release of the tourniquet. Group D consisted of 1:3 rats u-hich had been both starved and refed like the animals of Group B and traumatized like the animals of Group C.
Results and Discussion The intestinal tract of all animals of Groups B and D contained large amounts of the fat-rich diet; the intestinal lacteals were clearly xvisible as white thread-like structures, the c-sterna chvli contained milky fluid, and the serum was cloudy. All traumatized rats developed extremely large limb edema soon after release of the tourniquet. The first 3 rats of Groups C and D each died 20 to 40 minutes after the start of the infusion, after 0.1 to 0.2 g, kg of the oil had been administered. The lethal dose for healthy rats is 1 g, kg.' Lipemia was minor or absent in the 3 rats of Group D. although the intestines were full of food. The remaining traumatized rats repeatedly received water by mouth with the aid of a pipette and or subcutaneous injections of 10 to 20 ml of physiologic saline solution soon after release of the tourniquet. One rat of Group C and 2 rats of Group D died about 30 minutes after the end of the infusion period. Only animals which survived the completed infusion for 1 hour are included in the results. Rats of Group D showed lipemia, although to a lesser degree than those in Group B. The finding that the intravenous dose of fat necessary to produce death is considerably reduced by trauma confirms many similar experimental and clinical observations which have been reviess ed elsewhere.21 The amounts show n to be fatal during trauma are compatible wvith the amounts w-hich can be mobilized from the marroxv of fractured bones. Text-figure 1 show-s no difference in the specific acti-ities of the oil recovered from the lungs of the four groups, nor w%as there any consistent
Ameican Joumal of Pathology
HAUSBERGER
518
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1-SpeCific of "1-1triolein labeled activit%_TL'xr-F1GueE
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olive oil recovered from lungs
controls (Group A), lipemic ~~~~~~~~~~~~~~~~~of (Group B). traumatized ~~~~~~~~~~~~~~~rats
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_
According to some investigators, trauma is thought to bring about physical changes which produce a coalescence of normally dispersed blood lipids into fat droplets and pulmonary fat embolisms. Lipemia is thought to increase the extent of this embolization. If this theory is correct, intravenously infused labeled oil which is retained in the lungs should be mixed with and diluted byv the fat emboli originating from the blood. Radioactive olive oil of known specific activity was slowly administered to a) control rats, b) rats made lipemic by dietary measures, c) severely traumatized rats, and d) traumatized lipemic rats. There was no difference in the specific activity of the oil recovered from the lungs of rats of any group, indicating that no recognizable amount of blood lipids had been added to the experimentally produced emboli. (Am J Pathol 84:515-520, 1976)
THERE IS N\O GREE\1E\T on the etiology of pulmonary fat embolism. In the classic theorx-, it is chiefly caused bx- intravasation of fat from the site of injurx%, most often the marrow caxity- of fractured bones. Several observations are in variance with this concept, these include the occurrence of fat embolism in poisoning and other conditions not associated X ith bone injury. It has been pointed out, however, that the degree of embolization in these conditions is always minor and without clinical significance."2 Opponents of the classic theory emphasize that the amount of fat in the marrom.- cavity of even the largest bones, like femur or tibia, is not suffient to produce fatal embolization. This opinion is based on experiments by Lehman and Moore.3 wvho found that an intravenous dose of fat of 1.5 to 1. ml kg is lethal for dogs. These authors calculated that the lethal dose for a man of 75 kg is 112 to 113 ml, m-hile the fat content of the marrow cavity of the human femur is 65 to 70 ml. Several other theories about the origin of the embolic fat have been proposed. According to the physicomechanical theory, the embolic fat originates chiefly from blood lipids and only to a minor degree from marrow fat. Trauma is thought to produce instability of normally dispersed lipids such as chvlomicrons, which coalesce into larger fat droplets -hich are retained by From the Department of Anatomy. Jefferson \Medical College of Thomas Jefferson U ni\ ersit\. arnd the Department of Xnatomic Sciences. Temple L nis ersit\ School of Dentistrx. Philadelphia. Penns% 1vania.
Suipported by Grant HL-14322 from the National Heart and Loine Instituite. National Instituites of Health.
Xecepted for publication Mlay 7. 1976 Address reprint requests to Dr Franz X. Hausbercer. Department of Anatomic Sciences. Temple U niversity. School of Dentistry. :3223 North Broad Street. Philadelphia. PA 19140 515
516
HAUSBERGER
American Journal of Pathology
the pulmonary capillaries. Sexeral investigators have reported that lipemia 4-7 or soft tissue injury 8 produce pulmonary fat embolism. and that lipemia greatly increases its severity after fracture.5'7 Other investigators could not confirm these reports. Although lipemia increases the normally low- number of fat droplets in blood.9 it does not significantly affect the number of pulmonary emboli after fracture.9",0 nor does soft tissue injury produce fat embolism.1' The physicomechanical theory was greatly supported by LeQuire and co-workers.'2 wvho found that pulmonary emboli from human patients had a composition that was similar to blood lipids and different from adipose tissue fat. Ellis and WNatson 13 and Holczabek 14,15 could not confirm these findings on their o-n material from human patients. The strongest evidence against the physicomechanical theory was presented by Kerstell 16 and Kerstell and co-workers.'7 Pulmonary emboli washed from the lungs of dogs after femoral fracture showed the chemical composition of marrosw fat. and neither plasma lipids nor free fatty acids contributed to the formation of the emboli. Despite these findings. LeQuire and co-workers 18.19 are "convinced that the histologically massive pulmonary fat embolism seen in human patients" is caused by demulsification of plasma lipoproteins. 19 It had been sho-n earlier that intravenously infused olive oil, labeled with 'l'I, is quantitatively retained in the lungs in the form of fat emboli.20'2' According to the physicomechanical theory, lipemia and or trauma taking place at or near the time of the infusion should produce a coalescence of blood lipids and additional emboli. Furthermore. these emboli should be retained in the lungs and mix with and dilute the externally introduced labeled fat. The resulting subsequent reduction of the specific activitv. i.e., the activity per milligram of olive oil. can be measured. and it would provide an estimate of the degree of formation of emboli from blood fat. This approach wvas used to investigate the validity of the physico-
mechanical theory. Material and Methods Male Wistar rats %seighing betxseen :350 and 380 g 'vere used. The animals "ere anesthetized bv an initial intraperiteoneal injection of 2. to 30 mg kg of pentobarbital Anesthesia or heavy'- sedation wvas maintained throughout the experiment bx- repeated injections of small amounts of the barbiturate In addition. ethy l ether X-as used during the surgical procedures. Approximately 0.3 g kg of olive oil, labeled Nvith 1311 triolein \lal-
linckrodt X -as infused mvith constant speed into the femoral -ein during a period of 1 hour The amount infused \vas determined by %veighing of the sy ringe assembl!- before and after the injection. One to three hours later. the animals %vere decapitated. and their blood "as collected. One piece of plastic tubing wsas tied into the left auricle and a second one into the pulmonarx- trunk. Warm physiologic saline solution "as gentlx forced into the pulmonary veins. and the effluent from the pulmonarx- trunk \-.as collected into one or txN-o 15-
Vol. 84, No. 3 September 1976
LIPEMIA AND FAT EMBOLISMS
517
millimeter test tubes. After the vials w-ere let stand for not more than one-half hour, and they w-ere occasionally tapped to dislodge fat droplets. 1 to 2 ml of petroleum ether %vas added to the clearly recognizable top lav er of fat. The lipid-solvent lav er w as gentlx- stirred with a thin w-ire without disturbing the aqueous laver. After the solvent mixture was let stand for about 3 to 5 minutes, most of the mixture w-as transferred X ith the aid of a sy ringe and needle to a w-eighed glass scintillation v ial. This extraction procedure w-as repeated tw-ice: the solvent was then evaporated, and the vial mvas weighed again. In most experiments. 20 to 4c0 of the administered oil was recovered. and no attempts were made to increase this amount. The activitv of the recovered oil and the activ itv of the oil used for the infusion w-ere determined in a scintillation well detector. The specific activ.ities of the two samples were calculated and compared. The actisity of the thyroid gland of each animal w-as also counted. The preparation of the oil used for infusion has been described earlier." The experiments w-ere carried out in four groups of rats with 10 to 1, animals in each group. Group A consisted of 14 normally fed controls. The 17 rats of Group B wsere starxed for 2 and 3 dav s and refed for 20 to 12 hours w-ith a high fat diet consisting of Smviss cheese and Purina Laboratorx Chow- pellets soaked in olise oil. The 10 rats of Group C %vere traumatized according to the method of Whiteley," by placing a tourniquet around one low-er limb and around both upper limbs for 2 to 4 hours. Infusion X"as started 3 hours after release of the tourniquet. Group D consisted of 1:3 rats u-hich had been both starved and refed like the animals of Group B and traumatized like the animals of Group C.
Results and Discussion The intestinal tract of all animals of Groups B and D contained large amounts of the fat-rich diet; the intestinal lacteals were clearly xvisible as white thread-like structures, the c-sterna chvli contained milky fluid, and the serum was cloudy. All traumatized rats developed extremely large limb edema soon after release of the tourniquet. The first 3 rats of Groups C and D each died 20 to 40 minutes after the start of the infusion, after 0.1 to 0.2 g, kg of the oil had been administered. The lethal dose for healthy rats is 1 g, kg.' Lipemia was minor or absent in the 3 rats of Group D. although the intestines were full of food. The remaining traumatized rats repeatedly received water by mouth with the aid of a pipette and or subcutaneous injections of 10 to 20 ml of physiologic saline solution soon after release of the tourniquet. One rat of Group C and 2 rats of Group D died about 30 minutes after the end of the infusion period. Only animals which survived the completed infusion for 1 hour are included in the results. Rats of Group D showed lipemia, although to a lesser degree than those in Group B. The finding that the intravenous dose of fat necessary to produce death is considerably reduced by trauma confirms many similar experimental and clinical observations which have been reviess ed elsewhere.21 The amounts show n to be fatal during trauma are compatible wvith the amounts w-hich can be mobilized from the marroxv of fractured bones. Text-figure 1 show-s no difference in the specific acti-ities of the oil recovered from the lungs of the four groups, nor w%as there any consistent
Ameican Joumal of Pathology
HAUSBERGER
518
0
0
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w
o O
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00
*
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pdi
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~~~~~~0
80>
1-SpeCific of "1-1triolein labeled activit%_TL'xr-F1GueE
Wr
olive oil recovered from lungs
controls (Group A), lipemic ~~~~~~~~~~~~~~~~~of (Group B). traumatized ~~~~~~~~~~~~~~~rats
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