(Received for publication, May 28, 1946)

REMOVAL OF PLASMA PHOSPHOLIPIDES AS A FUNCTION OF THE LIVER: THE EFFECT OF EXCLUSION OF THE LIVER ON THE TURNOVER RATE OF PLASMA PHOSPHOLIPIDES AS MEASURED WITH RADIOACTIVE PHOSPHORUS BY C. ENTENMAN, I. L. CHAIKOFF, AND D. B. ZILVERSMIT (From the Division of Physiology, University of California Medical School, Berkeley) (Received for publication, May 28, 1946) Although the reaction involving the incorporation of inorganic phosphate into phospholipides is not restricted to any one tissue (surviving slices of liver, kidney, brain, spinal cord, small intestine, and muscle (1) have been shown to effect this reaction), the phospholipides contained in plasma are synthesized mainly by the liver (2). This function of the liver in the formation of plasma phospholipides was demonstrated by comparing the rates at which labeled inorganic phosphate introduced into the blood stream is converted to labeled plasma phospholipides by the normal and the liverless dogs; negligible amounts of labeled phospholipides were recovered in the plasma of the latter as late as 6 hours after excision of the liver (2). The present investigation deals with another phase of the metabolism of plasma phospholipides; namely, their removal as measured by the rate at which intravenously introduced, labeled (such as radioactive) phospholipides disappear from the blood stream. EXPERIMENTAL Preparation of Labeled Plasma PhospholipidesThe method used here for obtaining radioactive plasma phospholipides has been described elsewhere (3). Each of the donor dogs received by stomach tube 2 to 3 millicuries of radioactive inorganic phosphate. Blood was removed from the dogs 20 to 30 hours later, treated with heparin, and the plasma separated by centrifugation. This plasma, which contained the radioactive phospholipides, was injected intravenously into dogs and the rate of disappearance of the labeled phospholipides from their plasma measured. Preparation of AnimalsThe dogs used in this study were maintained for about 3 weeks on a stock diet, the composition of which has been described elsewhere (4). After being fasted for 24 hours they were eviscerated in the following manner: Each dog was anesthetized with ether and injected with 1 mg. of atropine sulfate to inhibit salivation. The abdominal cavity was opened and the inferior mesenteric artery ligated and severed. Two clamps were applied to the lower end of the pelvic colon and the gut severed between them. The celiac axis was exposed, its arteries clamped, and their 15

16 PLASMA PHOSPHOLIPIDES IN LIVER connections with the gastrointestinal tract severed. The portal vein was cut in a similar manner. The esophagus was clamped just below the diaphragm and the entire gastrointestinal tract removed from the abtdominal cavity, but the liver was left in situ. A towel wet with saline was placed in the cavity and the abdominal cavity closed. The entire procedure as a rule occupied less than 15 minutes. After recovery from the anesthetic the dogs behaved normally and were in no apparent pain. Blood was removed for analyses at various intervals throughout the experiment. It should be noted that the eviscerated animals showed no symptoms of shock. They walked around and behaved in a normal manner. Hematocrit readings, obtained each time that blood samples were taken, did not deviate from the normal. Such observations are in agreement with those of other investigators (5). Analytical Proc4zduresThe methods employed for determination of phospholipide P31 and phospholipide Ps2 have been described elsewhere (3). Total fatty acids and cholesterol were determined by oxidative methods (6). Results The liver of the eviscerated dog, although it received no blood by way of the portal vein or hepatic artery, is left in situ. Several investigators have pointed out that in such a preparation the liver is not completely excluded from. the circulation (7). In order to test the functional significance of the residual circulatory connections of the liver of the eviscerated dog, its capacity to contribute phospholipides to plasma was investigated. Test of EviscerationEight dogs were eviscerated and immediately thereafter injected intravenously with radioactive inorganic phosphate. Each dog received 10 cc. of an isotonic solution of Na2HP04 containing 2 millicuries of radioactive phosphorus. Blood was removed from the animals at intervals of 4 to 9 hours after the radiophosphorus had been introduced into the circulation, and radiophospholipides of the plasma determined. It is apparent from Table I that the quantity of radiophospholipides in the plasma of the eviscerated dog is negligible as compared with that previously observed in normal dogs (8). Since plasma phospholipides are synthesized in the liver, it may be concluded that any circulation of the liver remaining after the portal vein and hepatic artery are severed is of little significance so far as the synthesis of plasma phospholipides is concerned. E$ect of Evisceration on Lipide Content of PlasmaThe effect of evisceration upon the concentration of phospholipides, total fatty acids, and cholesterol in plasma is recorded in Table I. In each dog the level of each lipide constituent was measured immediately before and at one or more intervals after evisceration.

ENTENMAN, CHAIKOFF, AND ZILVERSMIT 17 No appreciable decrease in the concentration of phospholipides occurred in any of the eight dogs examined. The maximum difference between the values found before and after evisceration did not exceed 12 per cent. The levels of total fatty acids in the plasma were also unaffected by evisceration. In seven of the eight dogs the values for total fatty acids TABLE Plasma Lipides of Eviscerated Dog (with Hepatic Artery Severed) The lipides are expressed as mg; per 100 cc. of plasma. Dog No. EV3t BUY weight kg. 12.5 EV5t 8.5 EV6 8.8 EV7 11.7 EVlOt 8.0 346 18.2 350A 10.0 350B 9.0 Hn. after visceration I Cholesterol TOtd I Ester I TOti fatty acids Total lipides Before 206 72 134 535 741 4.5 204 81 123 542 746 9 202 77 125 580 782 Before 173 80 93 515 688 5 152 80 72 535 687 7.5 165 81 84 535 700 Before 271 67 204 440 711 4.5 262 63 199 480 742 8 223 67 156 525 748 Before 115 38 77 306 421 3.5 113 53 60 440 553 4.75 103 46 57 295 398 Before 188 63 125 417 605 10 196 63 133 397 593 Before 108 48 60 298 406 5 93 25 68 324 417 Before 154 45 109 424 578 5 146 44 102 427 573 Before 174 65 109 408 582 4 169 81 88 354 523 _ P. hosjhliplde 385 368 388 334 314 319 375 345 363 206 187 180 281 253 172 158 270 250 300 284 _ Spxific activity of PLP, x 10 * PLP refers to phospholipide phosphorus, r. u. to radioactive units as determined by the GeigerMiiller counter. Specific activity is calculated as i. u. PLP3*:mg. PLPar, the PLP*Z being expressed as a percentage of the injected Ps2. t Received intravenously 2 gm. of glucose in 25 cc. of isotonic saline every hour following evisceration. 0.20 0.45 0.35 0.21 0.13 0.18 0.16 0.17 observed after evisceration were either the same as, or slightly higher than, the corresponding preoperative values (Table I). The concentration of total fatty acids found in the plasma of Dog 350B 4 hours after evisceration is about 15 per cent lower than the preoperative value. No demonstrable change in the level of either free or ester cholesterol was produced by evisceration.

18 PLASMA PHOSPHOLIPIDES IN LIVER TABLE II Disappearance of Injected Radiophospholipides from Plasma of Eviscerated Dogs (wifh Hepatic Artery Severed) 5 6 Body weight kg. 9.0 8.7 6.8 6.5 7.8 8.7 MipCP;;ier R. u.t PLPS Mg. PLPS injection g2.ie~ $sz (Eviscerated) 45 795 180 597 300 521 360 475 55 1156 60 (Eviscerated) 90 1140 185 1028 270 1030 345 lboo 55 1540 60 (Eviscerated) 75 1540 175 1270 255 1188 330 1010 90 584 100 (Eviscerated) 110 577 170 562 230 537 290 527 95 592 105 (Eviscerated) 115 585 175 547 250 530 40 517 95 367 180 347 185 (Eviscerated) 210 380 255 348 315 273 375 268. 0.122 0.106 0.104 0.108 0.073 0.073 0.073 0.073 0.071 0.167 0.173 0.176 0.173 0.176 0.117 0.117 0.116 0.116 0.115 0.110 0.107 0.105 0.105 0.074 0.062 0.072 0.078 0.078 0.067 0.065 Specific actwity of LP, x lo~ 6.50 5.62 5.00 4.40 15.8 15.7 14.1 14.1 14.1 9.21 8.90 7.22 6.85 5.74 5.00 4.92 4.85 4.65 4.58 5.40 5.47 5.22 5.07 7.03 5.95 4.86 4.44 4.35 4.12 4.18 I Turnover time of plasma phospholipide before or after evisceration (min.)$ After 920 After 10,000 After 640 After 2500 After 2000 Before 380 After 2250

ENTENMAN, CHAIROFF, AND ZILVERSMIT 19 TABLE IIConcluded El: Body weight Specific activity of LP, x10a Turnover time of plasma phospholipide before or after evisceration (min.)t 7 8 9 ks. 15.0 18.0 7.0 60 1080 0.131 8.78 125 940 0.123 7.64 210 775 0.123 6.30 215 (E seerated: 265 733 305 686 370 667 430 665 50 870 0.120 145 734 0.126 215 630 0.131 220 (Eviscerated 275 600 340 585 415 540 0.119 6.16 0.112 6.12 0.112 5.95 0.113 5.89 0.123 0.123 0.125 7.25 5.82 4.81 Before 420 After 3400 Before 410 4.87 4.75 4.31 After 4750 60 2575 0.124 20.8 Before 580 120 2180 0.126 17.2 180 I 2100 0.128 16.4 185 (Eviscerated 265 2090 0.128 16.3 325 2050 0.127 16.2 385 2030 0.127 16.0 After 9500 * PLP refers to phospholipide phosphorus. t Radioactive units as determined by the GeigerMtiller counter. The specific activity is calculated as the ratio of r. u. PLP32:mg. PLP3*. $ The turnover time is defined as the time required for the disappearance of an amount of plasma phospholipide equal to that present in the plasma (3). Phospholipide Turnover TimeSince in the absence of the liver labeled plasma phospholipides were not synthesized from injected y, the finding that the level of this plasma lipide remained constant in the liverless dog suggests that in the latter the removal of plasma phospholipides had practically ceased. To test this point, evisceration and severance of the hepatic artery were carried out in nine dogs (Table II). Before evisceration all nine dogs received intravenously plasma containing radioactive phospholipide. The changes that occurred in phospholipide P32 and phospholipide P31 with time are recorded in Table II. The turnover time for plasma phospholipide, i.e. the time required for the disappearance of an amount of plasma phospholipide equal to that present in the plasma, was

20 PLASMA PHOSPHOLIPIDES IN LIVER calculated after the manner described elsewhere (3), and the values shown in Table II. In the first five dogs (Table II) the radiophospholipide was injected just before evisceration; it was therefore possible to obtain measures of turnover TABLE Disappearance of Injected Radiophospholipides from Plasma of Eviscerated Dogs (with Hepatic Artery Intact) %z 10 11 12 Body weight kg. 19.0 12.5 15.0 45 690 75 620 107 585 130 (Eviscerated) 135 604 195 502 263 416 315 409 43 713 74 705 105 632 135 (Eviscerated) 140 205 260 335 42 75 135 150 (Eviscerated) 157 630 217 490 280 402 337 364 L _, III Mg. PLP l ~afs~ 0.102 0.103 0.101 0.133 0.133 0.127 0.140 0.140 0.140 0.145 0.100 0.103 0.104 0.107 0.106 0.103 Specific activity of I LPJ Xlo 6.76 6.08 5.73 4.54 3.78 3.12 3.07 5.48 5.41 4.85 4.65 4.29 3.17 2.88 7.98 7.25 6.11 6.00 4.66 3.82 3.47 T I Turnover time of plasma phospholipide before and after evisceration (min.) Before 280 After 280 Before 430 After 430 Before 350 After 350 * PLP refers to phospholipide phosphorus. t Radioactive units as determined by the GeigerMiiller counter. $ Specific activity is calculated as the ratio of r. u. PLP?mg. PLPSI. times only for the interval after evisceration. The values obtained, even though much higher than normal, varied considerably. This variability made it desirable to compare the turnover time of plasma phospholipides in the same dog before and after evisceration. This was done in the last four dogs in Table II. The values found for turnover time in these four

ENTENMAN, CHAIKOFF, AND ZILVERSMIT 21 dogs before evisceration fell within the range reported earlier (8); they were respectively 380, 420, 410, and 580 minutes. Following evisceration the values increased to 2250, 3400, 4750, and 9500 minutes, respectively. It is evident from the above data that in the eviscerated dog the turnover of plasma phospholipides practically stops. In this type of preparation, the entire gastrointestinal tract is excised and the liver excluded from the circulation. In order to determine whether this impaired turnover results from the removal of the liver or from the removal of the gastrointestinal. REMOVAL GC G.LTRACTHEPATK ARTERY SEVERED 0 REMOVAL ff G.LTRACTHEPATK ARTERY IHTACT UdRMAL CONTROL 3.2. I I I I I I I,@k] 200 I50 lee &a 0 50 100 150 200 250 TIME IN MINUTES FIG. 1. Semilogarithmic plot of the specific activities of phospholipide phosphorus against time. 0 = Dog 7 (Table II) ; 0 = Dog 10 (Table III) ; = the average of three normal dogs (3). tract or from both, the following experiment was carried out: Three dogs were injected with plasma containing radioactive phospholipides, and 130 to 150 minutes later were eviscerated in the manner previously described, with the exception that the blood supply to the liver via the hepatic artery was not interfered with. The changes in the phospholipide P32 and phospholipide P31 content of plasma in these dogs and the turnover times are presented in Table III. The data demonstrate clearly that, if the arterial blood supply to the liver is intact, the complete removal of the gastrointestinal tract, pancreas, spleen, etc., has no effect on the rate of disappearance of plasma phospholipides. Fig. 1 shows a plot of the specific activities of plasma phospholipide

22 PLASMA PHOSPHOLIPIDES IN LIVER phosphorus on a logarithmic scale against time. The curves were drawn from data obtained for (1) the average of three normal dogs (3) ; (2) an eviscerated dog (Dog 7, Table II) in which the liver was deprived of blood supply from portal vein and hepatic artery; (3) an eviscerated dog (Dog 10, Table III) in which the blood supply t.o the liver via the hepatic artery was left intact. The disappearance of labeled phospholipides from the plasma of the normal dog proceeds at a constant rate throughout the 5 hour experi ment. So long as the blood supply to the liver by way of the hepatic artery is left intact, removal of the gastrointestinal tract has no apparent effect on the disappearance of labeled phospholipide from the plasma. When, however, the blood su,pply to the liver via the hepatic artery was eliminated in Ihe eviscerated dog, there occurred an abrupt decrease in the rate at which labeled phospholipides disappeared from the plasma. DISCUSSION 6 hours after the injection of radioactive inorganic phosphate, negligible amounts of radiophospholipide can be recovered from liverless dogs made so either by complete excision of the liver (2) or, as in the present study, by excluding this organ from the circulation. Although such findings have been offered as evidence for the view that the liver is the main site for synthesis of plasma phospholipide, the possibility that the extrahepatic tissues contribute to plasma appreciable amounts of phospholipide of low specific activity was not ruled out. That this does not occur, however, is shown by the data contained in Column 6 of Table II and by Fig. 1. The specific activities of plasma phospholipide phosphorus of dogs that had received intravenously radioactive plasma phospholipides did not decrease significantly after the liver had been excluded from the circulation. If, in the eviscerated dog with the liver excluded from the circulation, new plasma phospholipide had been formed, the specific activities of plasma phospholipide phosphorus could not have remained so constant. Since plasma phospholipides are not synthesized in the absence of the liver, the finding that the level of plasma phospholipides remains constant or nearly so in the liverless dog means that in the latter the utilization of plasma phospholipides has practically ceased. This suggests that the liver is also the main tissue in which their utilization occurs. Further evidence for this concept is provided by a comparison of the rates at which the injected labeled plasma phospholipides disappeared from the plasma of the normal and the liverless dogs. The fact that these labeled plasma phospholipides disappeared at a normal rate when the gastrointestinal tract was removed but not when the liver was excluded can leave little doubt as to the significance of the liver in the utilization of plasma phospholipides. The turnover of plasma phospholipides did not completely stop in all

t of the eviscerated dogs. This may be due to small amounts of blood reaching the liver by routes other than the portal vein and hepatic artery. On t he other hand, the small decrease in labeled plasma phospholipide in the eviscerated dogs m.a,y be indicative of some utilization of plasma phospholipide by extrahepatic tissues, a utilization, however, t hat is negligible as compared with that observed in dogs in which the liver remains intact. The results presented here raise the interesting question as to what function plasma phospholipides serve in the animal body. The finding that the liver alone is so much more important than the total mass of extrahepatic tissues in their utilization lends little support to the view that the main function of plasma phospholipides is to transport fatty acids from liver to other tissues. SUMMARY 1. The concentration of phospholipides, total fatty acids, and cholesterol in plasma is not appreciably decreased by excluding the liver from the circulation. 2. The rate at which intravenously injected radiophospholipide disappears from the plasma was used as a measure of the turnover time of plasma phospholipides. In confirmation of earlier work, it was found that plasma phospholipides are completely turned over in 6 to 10 hours in normal dogs weighing from 7 to 18 kilos. By depriving these dogs of their livers, the time required for complete turnover was prolonged to 33 to 160 hours. 3. It is concluded that the liver is t,he principal tissue in the body concerned not only with the synthesis and supply of plasma phospholipides but also with t,heir removal. BIBLIOGRAPHY 1. Chaikoff, I. L., Physiol. Rev., 22, 291 (1942). 2. Fishler, M. C., Entenman, C., Montgomery, M. L., and Chaikoff, I. L., J. Biol. Chem., 160, 47 (1943). 3. Zilversmit, D. B., Entenman, C., Fishler, M. C., and Chaikoff, I. L., J. Gen. Physiol., 26, 333 (1943). 4. Entenman, C., and Chaikoff, I. L., J. Biol. Chem., 142,129 (1942). 5. Markowitz, J., Textbook of experimental surgery, Baltimore, 493 (1937). 6. Chaikoff, I. L., and Kaplan, A., J. BioZ. Chem., 106, 267 (1934). 7. Peterson, J. M., Physiol. Rev., 14, 586 (1934). 8. Friedlander, H. D., Chaikoff, 1. L., and Entenman, C., J. BioZ. Chem., 166.231 (1945).

REMOVAL OF PLASMA PHOSPHOLIPIDES AS A FUNCTION OF THE LIVER: THE EFFECT OF EXCLUSION OF THE LIVER ON THE TURNOVER RATE OF PLASMA PHOSPHOLIPIDES AS MEASURED WITH RADIOACTIVE PHOSPHORUS C. Entenman, I. L. Chaikoff and D. B. Zilversmit J. Biol. Chem. 1946, 166:1523. Access the most updated version of this article at http://www.jbc.org/content/166/1/15.citation Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's email alerts This article cites 0 references, 0 of which can be accessed free at http://www.jbc.org/content/166/1/15.citation.full.ht ml#reflist1