THE REMOVAL OF LIPOPROTEIN LIPASE FROM THE BLOOD BY THE NORMAL AND DISEASED LIVER * t By WILLIAM E. CONNOR t AND JOHN W. ECKSTEIN From the Cardiovascular Research Laboratories, Department of Internal Medicine, State University of Iowa College of Medicine, Iowa City, Ia.) (Submitted for publication November 25, 1958; accepted June 19, 1959) Lipoprotein lipase is an enzyme found in many organs and tissues. The highest concentration is in heart and adipose tissue (2, 3). Its unique physiological substrate consists of the triglyceride component of lipoproteins which it hydrolyzes to fatty acids and glycerol (4). Thus, it differs from pancreatic lipase which will act upon triglycerides not bound in a lipoprotein complex. Hahn first noted that this enzyme appeared in the blood in a highly active form following parenteral heparin administration. It had the property of reducing the turbidity of lipemic plasma (5) and became universally known as "clearing factor." Heparin or a heparin-like substance is now thought to be a part of the lipoprotein lipase molecule (6, 7). Although the precise role of lipoprotein lipase in the metabolism of triglycerides has not yet been defined, it has excited great interest. It may play a role in the hydrolysis of chylomicron triglyceride formed after the ingestion of dietary fats. It may be involved in the pathogenesis of atherosclerosis. It may be important in the outflow of the unesteri- fied fatty acids which provide a significant and readily mobilizable source of energy during the fasting state (8, 9). Jeffries ( 10) and later Spitzer and Spitzer ( 11 ) showed that the isolated, perfused rat liver de- stroyed lipoprotein lipase. Jeffries also found that hepatic venous blood of the heparinized rat had less clearing activity than did aortic blood. Morris and French, however, were unable to dem- onstrate loss of clearing factor by perfusing the rat liver (12). Hepatectomized and cirrhotic rats had higher plasma lipemia clearing activity * Presented at the annual meeting of the American So- ciety for the Study of Arteriosclerosis in San Francisco, Calif. October 24, 1958 (1). t Supported by research grants from the Iowa and American Heart Associations. : This work was performed during the tenure of an American Heart Association Research Fellowship. than control rats (13). Baker, Levine, Turner and Dubin found that patients with hepatic cir- rhosis had higher post-heparin blood levels of clearing factor than normal subjects (14). It was suggested that this increased activity might result from failure of the liver to inactivate the enzyme. The present investigation was undertaken to define the role of the liver in the removal of lipo- protein lipase from the blood in dogs and humans. Lipoprotein lipase levels were determined in vari- ous vascular beds. In these species the normal liver removed lipoprotein lipase from the blood; liver disease prevented the usual removal. METHODS Animal experiments. Dogs in the fasting state, weigh- ing from 17 to 25 Kg., were anesthetized with ether or sodium pentobarbital. Ten normal dogs were studied. Hepatic vein catheterization was performed via the ex- ternal jugular vein. The catheter was placed in a branch of the hepatic vein draining the left lobe of the liver. A polyethylene catheter was introduced into a surgically exposed femoral vein for peripheral venous sampling. At the beginning of the experiment a preheparin sample was drawn from the femoral vein. Ten mg. of heparin 1 was then given intravenously. The hepatic vein catheter was kept patent by a slow saline-heparin drip. Five mg. of additional heparin was given slowly by this route during the experiment. Simultaneous femoral and hepatic venous samples were drawn at 10, 60 and 90 minutes post-heparin. Blood in 9 ml. amounts was added to 1 ml. of 1.85 per cent potassium oxalate solution in tubes chilled in an ice bath. Specimens were centrifuged at 4,000 rpm and 40 C. for 10 minutes. Subsequently, the plasma specimens were kept at 40 C. for a period not longer than two hours before enzymatic activity was de- termined. The enzyme remains stable in plasma for at least 24 hours at 40 C. Hepatic necrosis was induced in three dogs by carbon tetrachloride or chloroform poisoning (15, 16). The same studies were performed as in the normal dogs. In Dogs 1 and 2 studies were performed before and after liver poisoning. All three dogs developed severe icterus, 1 Sodium heparin, Abbott Laboratories. 1746
Transcript
THE REMOVALOF LIPOPROTEIN LIPASE FROMTHE BLOODBY THE NORMALANDDISEASED LIVER * tBy WILLIAM E. CONNORt AND JOHN W. ECKSTEIN
From the Cardiovascular Research Laboratories, Department of Internal Medicine, StateUniversity of Iowa College of Medicine, Iowa City, Ia.)
(Submitted for publication November 25, 1958; accepted June 19, 1959)
Lipoprotein lipase is an enzyme found in manyorgans and tissues. The highest concentration isin heart and adipose tissue (2, 3). Its uniquephysiological substrate consists of the triglyceridecomponent of lipoproteins which it hydrolyzes tofatty acids and glycerol (4). Thus, it differs frompancreatic lipase which will act upon triglyceridesnot bound in a lipoprotein complex. Hahn firstnoted that this enzyme appeared in the blood in ahighly active form following parenteral heparinadministration. It had the property of reducingthe turbidity of lipemic plasma (5) and becameuniversally known as "clearing factor." Heparinor a heparin-like substance is now thought to be apart of the lipoprotein lipase molecule (6, 7).
Although the precise role of lipoprotein lipasein the metabolism of triglycerides has not yet beendefined, it has excited great interest. It may playa role in the hydrolysis of chylomicron triglycerideformed after the ingestion of dietary fats. It maybe involved in the pathogenesis of atherosclerosis.It may be important in the outflow of the unesteri-fied fatty acids which provide a significant andreadily mobilizable source of energy during thefasting state (8, 9).
Jeffries ( 10) and later Spitzer and Spitzer ( 11 )showed that the isolated, perfused rat liver de-stroyed lipoprotein lipase. Jeffries also foundthat hepatic venous blood of the heparinized rathad less clearing activity than did aortic blood.Morris and French, however, were unable to dem-onstrate loss of clearing factor by perfusing therat liver (12). Hepatectomized and cirrhoticrats had higher plasma lipemia clearing activity
* Presented at the annual meeting of the American So-ciety for the Study of Arteriosclerosis in San Francisco,Calif. October 24, 1958 (1).
t Supported by research grants from the Iowa andAmerican Heart Associations.
: This work was performed during the tenure of an
American Heart Association Research Fellowship.
than control rats (13). Baker, Levine, Turnerand Dubin found that patients with hepatic cir-rhosis had higher post-heparin blood levels ofclearing factor than normal subjects (14). Itwas suggested that this increased activity mightresult from failure of the liver to inactivate theenzyme.
The present investigation was undertaken todefine the role of the liver in the removal of lipo-protein lipase from the blood in dogs and humans.Lipoprotein lipase levels were determined in vari-ous vascular beds. In these species the normalliver removed lipoprotein lipase from the blood;liver disease prevented the usual removal.
METHODS
Animal experiments. Dogs in the fasting state, weigh-ing from 17 to 25 Kg., were anesthetized with ether orsodium pentobarbital. Ten normal dogs were studied.Hepatic vein catheterization was performed via the ex-ternal jugular vein. The catheter was placed in a branchof the hepatic vein draining the left lobe of the liver.A polyethylene catheter was introduced into a surgicallyexposed femoral vein for peripheral venous sampling.At the beginning of the experiment a preheparin samplewas drawn from the femoral vein. Ten mg. of heparin 1
was then given intravenously. The hepatic vein catheterwas kept patent by a slow saline-heparin drip. Five mg.of additional heparin was given slowly by this routeduring the experiment. Simultaneous femoral and hepaticvenous samples were drawn at 10, 60 and 90 minutespost-heparin. Blood in 9 ml. amounts was added to 1 ml.of 1.85 per cent potassium oxalate solution in tubeschilled in an ice bath. Specimens were centrifuged at4,000 rpm and 40 C. for 10 minutes. Subsequently, theplasma specimens were kept at 40 C. for a period notlonger than two hours before enzymatic activity was de-termined. The enzyme remains stable in plasma for atleast 24 hours at 40 C.
Hepatic necrosis was induced in three dogs by carbontetrachloride or chloroform poisoning (15, 16). Thesame studies were performed as in the normal dogs. InDogs 1 and 2 studies were performed before and afterliver poisoning. All three dogs developed severe icterus,
anorexia and vomiting. The experiments were per-formed on the third day of illness. Hepatic necrosis wasconfirmed by gross and microscopic examination in twodogs which died later in hepatic coma.
Wehave assumed in these studies that the lipoproteinlipase level in peripheral venous blood is similar to thelevel in blood entering the liver (i.e., in portal vein andhepatic artery blood). In four normal dogs this hy-pothesis was checked by obtaining post-heparin bloodsamples simultaneously from the portal vein, the femoralvein, the hepatic vein and the femoral artery. The portalvein was exposed after laparotomy. A Cournand needlewas inserted into the femoral artery of these dogs forarterial blood sampling.
Human experiments. Ten human subjects were stud-ied in the fasting state. All had been receiving the gen-eral hospital diet with regard to fat content and wereeating well. Five, to be termed normals, had no evi-dence of liver disease. Four had mild hypertension andone had valvular heart disease. None was in cardiacfailure. Five other subjects had long-standing Laennec'scirrhosis documented by physical examination, very ab-normal liver function tests, and liver biopsy. Ascites,present in three patients, did not influence the resultsof this study. The cirrhotic patients were in good nu-tritional state. All had hepatic vein wedge pressures ele-vated to at least 20 mm. of mercury. Hepatic veincatheterization was performed by introducing a catheterinto an antecubital vein and under fluoroscopic controlpassing it through the right atrium and inferior vena cavainto a branch of the hepatic vein draining the right lobeof the liver. A Cournand needle was inserted into theother antecubital vein and in some experiments into thefemoral artery. At the beginning of the experiment apreheparin sample was drawn from the antecubital vein.After the catheter was put into position in the free he-patic vein, 10 mg. of heparin was given intravenously.Simultaneous antecubital and hepatic venous sampleswere drawn at 10, 60, and occasionally 90 minutes post-heparin. Femoral arterial samples were also obtainedin several subjects. Blood was handled as in the dogexperiments.
Lipoprotein lipase activity. Plasma lipoprotein lipasewas quantified in two ways: (a) by the reduction in op-tical density or "clearing" of a plasma-coconut oil emul-sion and (b) by the measurement of glycerol produced bylipolytic action. These techniques were modified fromdescriptions by Grossman (17), Korn (2) and Baker(18). Coconut oil provides a suitable substrate forlipoprotein lipase despite the inability of the enzymeto act upon purified triglycerides. As Korn has dem-onstrated (4), the incubation of coconut oil with plasmalipoproteins results in the formation of a complex analo-gous to chylomicrons with respect to lipoprotein lipaseaction.
Plasma was warmed at 370 C. for three minutes. Then2.5 ml. was mixed with 0.3 ml. of a coconut oil emulsion.2
2Ediol, Schendley Laboratories; kindly provided byDr. B. M. Lamman.
The concentration of the emulsion was adjusted so thatthe mixture of plasma and coconut oil gave an initialoptical density reading of approximately 0.60 unit. Theoptical density and glycerol content of the plasma-coconut oil emulsion were determined in duplicate atzero time and after 120 minutes of incubation at 370 C.The 120 minute incubation period was selected as asuitable time for the measurement of both optical densitychange and glycerol production in the same incubationtube. We had found glycerol production still linear at120 minutes. The change in optical density was linearfor the lower levels of lipoprotein lipase usually foundin 60 and 90 minute post-heparin specimens. The 120minute period was long enough so that the low levels oflipoprotein lipase present in some plasma specimenscould be determined. Bovine albumin was not addedto the incubation system because we did not wish to maska possible effect of the altered plasma proteins of liverdisease upon lipoprotein lipase activity. Since oxalatedplasma was employed, the formation of turbid calciumsoaps was not a problem. In no instance did the incu-bation tube develop more turbidity after maximal clear-ing had occurred. Anfinsen's data had indicated a similarlack of returbidification after 180 minutes of incubation(19). Korn had used a 120 minute incubation periodfor many of his experiments employing lipoprotein lipasefrom rat heart (2).
The optical density was read in 10 mm. cuvettes at awave length of 650 mAt in a Coleman Junior Spectropho-tometer. For glycerol determinations 0.5 ml. aliquotsof the plasma-coconut oil emulsion were added to chilledtubes containing 0.1 ml. of 0.1 N sulfuric acid. Theplasma proteins were precipitated by 2 ml. of 20 per centtrichloroacetic acid. Glycerol was determined on 0.25 ml.of the filtrate by the method of Lambert and Neish (20)and expressed in ,uMole per ml. The results were ana-lyzed statistically by the methods of Fisher (21).
RESULTS
Normal dogsTen healthy mongrel dogs had much higher
levels of lipoprotein lipase in peripheral vein bloodthan in hepatic vein blood 60 and 90 minutes post-heparin. The data for each dog at 10, 60 and 90minutes post-heparin are tabulated in Table I.No significant differences occurred at the 10 min-ute period for the group, but Dog 9 had consid-erable 10 minute peripheral vein-hepatic veindifferences. These were 0.230 optical density unitand 0.54 ,uMole of glycerol.
At 60 minutes the optical density change forall normal dogs averaged 0.304 unit in the periph-eral vein samples and 0.213 unit in the hepaticvein samples. The mean difference was 0.091(p < 0.001). Glycerol production in these ani-
Means for each 10 0.421 0.403 0.414 0.357 0.74 0.65 0.63 0.57vascular bed 60 0.306 0.251 0.289 0.191 0.32 0.31 0.28 0.19
90 0.273 0.239 0.263 0.169 0.41 0.41 0.37 0.31
mals was found to be 0.40 ,uMole in peripheralvein and 0.28,uMole in hepatic vein samples. Themean difference was 0.12 ,uMole, also significant(p < 0.01).
Similar results occurred at 90 minutes post-heparin. The optical density difference was 0.109unit (p < 0.001) and the glycerol difference be-tween peripheral and hepatic vein samples was0.16 pMole (p < 0.001). In one dog 10 mg. ofintravenous protamine 3 caused a prompt reduc-tion of lipoprotein lipase activity in both vascularbeds. The experimental results with the use ofeither Nembutal®D or ether were similar.
Comparison of lipoprotein lipase in the femoralvein, femoral artery, portal vein and hepaticvein in normal dogs. The question arose: Doesthe significant difference in the lipoprotein lipaselevels of peripheral and hepatic vein blood indicatethat the liver removes this enzyme from the blood?The answer could only be in the affirmative iflipoprotein lipase in peripheral vein blood wasshown to be equivalent to the level in the bloodentering the liver, i.e., by the portal vein andhepatic artery. Such an assumption has appearedtrue for Bromsulphalein®D levels (22). Peripheralvein-hepatic vein differences could mean removalof the enzyme by the lungs, intestinal tract, spleenor kidneys as well as by the liver. These possibili-
3 Protamine sulfate, Eli Lilly and Co.
ties were investigated in four normal dogs by de-termining simultaneous lipoprotein lipase levelsin the femoral, portal and hepatic veins and thefemoral artery. Since hepatic and femoral arterialblood are identical, this experiment permitted acomparison of all blood entering the liver with theblood leaving the liver. Enzymatic activity inblood from these vascular beds and from periph-eral venous blood can also be related. As shownin Table II both the optical density change andglycerol production values were much lower in thehepatic vein than in any of the three other vascularbeds. At 60 minutes post-heparin, hepatic veinoptical density change averaged 0.191 unit ascompared with 0.306 unit in the femoral vein,0.251 unit in the femoral artery and 0.289 unit inthe portal vein. The glycerol values from the fe-moral artery, the femoral vein and the portal veinwere grouped together, from 0.32 to 0.28 ,uMole.These were clearly different from the hepatic veinglycerol production of 0.19 ,uMole. At 90 minutespost-heparin the data show the same grouping.At 10 minutes post-heparin, as also occurred in thegroup of normal dogs (Table I), no great differ-ences occurred between any of the vascular bedsexcept for Dog 9 which did have marked differ-ences. In this instance, too, femoral vein, fe-moral artery and portal vein values were groupedtogether in contrast to much lower hepatic vein
1749
WILLIAM E. CONNORANDJOHN W. ECKSTEIN
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The peripheral vein-hepatic vein differences for opticaldensity change and glycerol production are given for 60minute post-heparin specimens.
values for both optical density change and glycerolproduction. These data fortify the assumptionthat the lipoprotein lipase level in peripheral veinblood (femoral or antecubital) is representative ofthe level in the blood entering the liver.
While the lipoprotein lipase levels in the fe-moral vein and femoral artery are similar, slight
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Each vertical bar represents the peripheral vein-hepatic vein difference in lipoprotein lipase activity as
measured by optical density change and glycerol produc-tion. The normal liver is contrasted with the diseasedliver in humans having Laennec's cirrhosis and in dogswith acute hepatic necrosis. All values are those ob-tained 60 minutes post-heparin.
differences are apparent in Dogs 8, 9 and 10, butnot in Dog 11. Dog 8 at 60 minutes post-heparinhad a femoral vein-femoral artery difference of0.107 optical density unit and 0.03 ,pMole ofglycerol; at 90 minutes differences were 0.065optical density unit and 0.10 fAMole of glycerol.Dog 9 had greatest peripheral venous-arterial dif-ferences at the 10 minute time. Dog 10 had dif-ferences at 60 and 90 minutes.
Dogs 'with hepatic necrosis
Acute liver poisoning with chloroform or carbontetrachloride greatly reduced the hepatic removalof lipoprotein lipase. At the 10 minute post-heparin time there was a negative peripheral vein-hepatic vein difference of both optical densitychange and glycerol production (Table I). At60 minutes post-heparin, the optical density changeof the peripheral vein plasma averaged 0.234 op-tical density unit as compared with the 0.225 unitof hepatic vein plasma. The two vascular bedshad also similar glycerol values. At 90 minutespost-heparin there was again little lipoproteinlipase removal by the necrotic liver.
Twq dogs, 1 and 2, were studied before andafter the induction of acute hepatic necrosis. Fig-ure 1 shows that the normal liver of each dog re-moved lipoprotein lipase as indicated by peripheralvein-hepatic vein differences in optical densitychange. and glycerol production. After hepaticnecrosis there was a great diminution in lipopro-tein lipase removal.
A comparison of lipoprotein lipase removal forall dogs indicates greater removal by the normalthan by the necrotic liver. Figure 2 comparesthe mean lipoprotein lipase removal for normaldogs and dogs with hepatic necrosis at 60 minutespost-heparin. The normal dogs had a peripheralvein-hepatic vein difference of 0.091 optical den-sity unit; the dogs with hepatic necrosis had adifference of only 0.009 (one-tenth as much).The normal dogs had a difference of 0.12 /AMole ofglycerol as compared with only a difference of0.01 1uMole in the dogs with hepatic necrosis. At90 minutes post-heparin the normal dogs removed0.109 optical density unit versus a removal ofonly 0.042 unit for dogs with hepatic necrosis.In terms of glycerol the normal liver removed 0.16,uMole and the necrotic liver only 0.05 juMole.
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Human subjects
Results in five normal human subjects are tabu-lated individually in Table III. Subjects 2 and 5had the greatest peripheral vein-hepatic vein dif-ferences for both optical density and glycerol at10 minutes post-heparin, while Subjects 1, 3 and4 developed greatest differences at 60 minutespost-heparin. These peak differences ranged be-tween 0.140 to 0.237 optical density unit and be-tween 0.28 to 0.47 1AMole of glycerol. When thedata from all five normal subjects are consideredfor the 60 minute post-heparin time, the meandifferences were 0.120 optical density unit and0.31 /uMole of glycerol. These were both sig-nificant (p < 0.05). The mean differences forall subjects at 10 minutes post-heparin were 0.087optical density unit and 0.30 .uMole of glycerol.The two subjects, 2 and 4, studied at 90 minutespost-heparin both showed lipoprotein lipase re-moval by the liver. Their mean differences were0.096 optical density unit and 0.23 ,LMole ofglycerol.
The five patients with Laennec's cirrhosis hadless clear cut peripheral vein-hepatic vein differ-ences (Table III). Differences were especiallyminimal as regards glycerol production. Thepeak difference for each patient occurred at 60minutes post-heparin and ranged from 0.003 to0.08 optical density unit and from 0 to 0.24,LMole of glycerol. The mean differences were0.042 optical density unit and 0.06 MuMole ofglycerol. The differences at 10 and 90 minutespost-heparin were much less. The one patientstudied at 90 minutes had negative differences.The intravenous injection of 10 mg. of protaminein two patients caused the lipoprotein lipase levelsin peripheral and hepatic veins to fall to the pre-heparin levels within 10 minutes.
When the hepatic removal of lipoprotein lipasewas compared in normal and cirrhotic subjects,the normal liver removed more lipoprotein lipase(Figure 2). At 10 minutes post-heparin thestandard error of the mean differences (normalsas opposed to cirrhotics) was 0.0565 optical den-sity unit (p > 0.05) and 0.309 MMole of glycerol(p > 0.05). At 60 minutes the standard errorwas 0.075 optical density unit (p > 0.1) and0.152 JMole of glycerol (p < 0.05).
In two normal humans and in two cirrhotic pa-
tients the lipoprotein lipase level was higher at 60minutes post-heparin in the perhipheral vein thanin the peripheral artery. The two normal sub-jects had 15 and 12 per cent higher optical den-sity values in venous samples; the two cirrhoticshad 9 and 7 per cent higher values in venous sam-ples. At 10 minutes post-heparin the normal sub-jects had 5 and 25 per cent higher venous values,but the cirrhotics had no peripheral venous-arterialdifference at this time. In terms of glycerol,there were similar venous-arterial differences forthe four subjects.
DISCUSSION
These results indicate that the normal liver re-moves lipoprotein lipase from the blood in bothdogs and humans. Another interpretation of thedata might be that the liver adds a substance tohepatic vein blood which inhibits the action oflipoprotein lipase. No information is available onthis question. The hepatic removal may be re-garded as an extraction or denaturation phe-nomenon in which the lipoprotein lipase contentof blood entering the liver is related to the contentin blood leaving the liver. The close similarityof the levels of enzyme in peripheral vein bloodand in blood entering the liver has been demon-strated. Lipoprotein lipase has been quantifiedby the change in the optical density of the plasma-coconut oil emulsion which occurred as particlesize was reduced and by the production of glycerolfrom hydrolysis of lipoprotein triglycerides. Bothmethods of measurement, the optical and thechemical, yielded data supporting the same conclu-sion about hepatic removal of lipoprotein lipase.A double check on the data was thus obtained. Athird technique of measurement, used by others(23), has been the determination of fatty acidsformed by enzymatic activity.
If the normal liver removes lipoprotein lipase,then the diseased liver might well have a reduc-tion of this function.. Such has been the case inhuman cirrhosis and in dogs with acute hepaticnecrosis. The differences across the liver foroptical density change and glycerol productionwere reduced strikingly. The failure of the dis-eased liver to perform as did the normal liverprovided further evidence of hepatic removal oflipoprotein lipase from the blood. Two reasons
may be advanced for the reduced removal of theenzyme in liver disease. First, hepatocellulardamage might prevent a normal function of theliver; second, it is known that anastomoses de-velop between the portal and hepatic veins incirrhosis and following hepatic necrosis (24).Such shunts would greatly reduce the usual flowof blood reaching hepatic cells and thus cause anoverall impairment of hepatic function.
Previous work on this subject has been doneonly with the rat, an animal whose liver is saidnot to contain heparin or mast cells (25). Jef-fries (10) and Spitzer and Spitzer (11) foundthat the isolated perfused rat liver destroyedclearing factor. This was not confirmed by Mor-ris and French who employed a similar prepara-tion (12). Wesuggest a possible explanation forthis one divergent experimental report. Theamount of plasma lipoprotein lipase with whichthe liver is presented may be a critical factor inthe determination of hepatic removal by perfusionor sampling techniques. The time of sampling maybe another factor. Weroutinely sampled at severaldifferent times after the administration of hepa-rin-at 10, 60 and usually 90 minutes. Heparineffect was maximal at 10 minutes, as indicated byprolonged whole blood clotting times and highlevels of lipoprotein lipase. Differences betweenperipheral vein and hepatic vein levels were thenless significant. We might ascribe this lack ofdifference to the possible overloading of the liverby large amounts of the enzyme. This effectmight be expected soon after heparin injectionwhen circulating blood levels of heparin were highand continuing to stimulate the entry of lipo-protein lipase into the blood. For most of thehuman subjects and for nine dogs the sample timesgiving the greatest differences were at 60 and 90minutes when the whole blood clotting times werelower or even normal and overall lipoprotein lipaselevels were somewhat lower.
Recently, Robinson and Harris have shownthat after a single passage of blood containingheparin through the rabbit hind limb that lipo-protein lipase activity can be detected in the ve-nous return (26). Such a rapid release suggestedto them that a lipolytic enzyme was probably de-rived from the inner lining of blood vessel walls.This work substantiates another report, that the
peripheral tissues in the rat release lipoproteinlipase into the blood (10). In humans and indogs this same enzymatic release by the peripheraltissues appears to occur. Wefound higher levelsof lipoprotein lipase in peripheral venous than inperipheral arterial blood. The high content oflipoprotein lipase in adipose tissue (3) may meanthat this tissue or its capillaries supplied at leastsome of the lipoprotein lipase appearing in theblood after heparin.
The physiological significance of hepatic re-moval of lipoprotein lipase is uncertain. The en-zyme may possibly be destroyed in the liver byheparinase, known to be present in the livers ofsome species (27). The incubation of lipopro-tein lipase with heparinase from liver (11), witha bacterial heparinase (6) and with a liver tissuehomogenate (28) all caused a great reduction ofenzymatic activity. A recent study demonstratedthat injected heparin is inactivated by the liver(29). Heparin or a heparin-like substance ap-pears to be an integral part of the lipoproteinlipase molecule. Whenheparin was removed fromit by passage through an anion exchange resin,enzymatic activity disappeared (30). It could berestored by the addition of heparin. Other evi-dence indicates that lipoprotein lipase represents acombination of heparin and apo-enzyme fromwhich heparin is dissociated readily (31). Add-ing further to the idea of the close relationship ofheparin and lipoprotein lipase is the often re-peated observation that heparin antagonists blockthe action of the enzyme both in vitro and in vivo.The prompt decline of lipoprotein lipase levels inthe blood following intravenous protamine in ourexperiments illustrates this antagonism.
Much of the current interest in lipoproteinlipase relates to its possible role in atherosclerosis.It has been suggested that alimentary hyperlipemiaor chylomicronemia may be an etiological factorin the development of this disease (32). The de-layed clearing of dietary triglycerides in patientswith coronary atherosclerosis (33, 34) providesevidence of a possible lipoprotein lipase deficiencyin such individuals. Block, Mann and Barkerhave shown that a small dose of intravenous hepa-rin produced less clearing of the lipemia inducedby a fatty meal in coronary patients than in nor-mal persons (35). Others have found that plasma
1753
WILLIAM E. CONNORANDJOHN W. ECKSTEIN
from atherosclerotic individuals was inhibitory toclearing activity (36, 37). It has been shown,also, that the cofactor activity in the lipoproteinlipase system is reduced in atherosclerotic pa-tients (38).
Patients with cirrhosis of the liver appear tohave accelerated clearing of triglycerides from theblood (39) and exhibit very high levels of clearingfactor after the injection of heparin (14). Ourstudies in cirrhotic patients and in dogs with he-patic necrosis suggest that such phenomena mayoccur because of the reduced removal of lipo-protein lipase from the blood in liver disease.The pathological and clinical evidence indicatesthat patients with cirrhosis of the liver have lessatherosclerosis and myocardial infarction thancomparable groups of patients dying from othercauses (40-42). One may then pose the ques-tion: Might the decreased hepatic removal oflipoprotein lipase in cirrhosis contribute to alessened incidence of atherosclerosis? The hyper-estrogenism, absence of hypertension, hypocho-lesterolemia, poor nutrition, hypoprothrombinemia,and increased fibrinolysis, all occurring at timesin advanced cirrhosis, might be additional con-tributing factors.
SUMMARY
1. The lipoprotein lipase content of plasma afterthe administration of heparin was compared inblood collected simultaneously from the peripheraland hepatic veins. In five normal human subjectsand in 10 normal dogs the liver removed or in-activated significant amounts of the lipoproteinlipase with which it was presented.
2. Impairment of liver function greatly reducedthe hepatic removal of the enzyme. This wasshown in five human patients with Laennec's cir-rhosis of the liver and in three dogs with acutehepatic necrosis.
3. In four normal dogs the lipoprotein lipaselevel in the blood entering the liver was similar tothe peripheral venous level and different from thehepatic venous level. On the basis of these datahepatic removal of the enzyme could be deter-mined from peripheral and hepatic venous blood.
4. Lipoprotein lipase was measured in periph-eral venous and arterial blood in four normal dogsand in four humans. The differences between
peripheral venous and arterial blood were con-sistent with the release of lipoprotein lipase intothe peripheral tissues.
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