- Email: [email protected]
In vitro lipid transformations in serum
468
BIOCHIMICA ET BIOPHYSICA ACTA
I N V I T R O L I P I D TRANSFORMATIONS IN SERUM G. V. MARINETTI Department o/Biochemistry, University o/Rochester School o] Medicine and Dentistry, Rochester, N. Y. (U.S.A.)
(Received July 23rd, 196o)
SUMMARY Snake venom phospholipase A effects a rapid hydrolysis of serum lecithin to yield lysolecithin which has predominantly saturated fatty acids. The fatty acids which are released are highly unsaturated. The venom also hydrolyzes serum phosphatidylethanolamine to its lyso-compound. The activity of the venom is abolished byVersene, citrate, and oxalate and reactivated by calcium ions. Pancreatin effects a rapid hydrolysis of serum triglycerides to yield diglycerides. The triglycerides are resynthesized within 1-3 h with a concomitant disappearance of the diglycerides. The fatty acids which are released are believed to be bound to the serum proteins. Added [i-14C]stearic and Ii-14Cloleic acid are not incorporated into the resynthesized triglycerides. Pancreatin has very little action on tripalmitin when this is added to serum but rapidly hydrolyzes added triolein. When synthetic D- and L-dioleins are added to fresh serum these are slowly degraded to monoglycerides and other products. When both the dioleins and Pancreatin are added to serum, the dio]eins are more rapidly metabolized to other products. In this case, however, the dioleins stimulate the hydrolysis of serum lecithin to lysolecithin. Added I-monopalmitin also stimulates the Pancreatin hydrolysis of lecithin to lysolecithin during which time the monoglyceride is converted in part to diglycerides. Added triglycerides do not stimulate the Pancreatin hydrolysis of lecithin to lysolecithin. The data reported in this paper demonstrate the active synthetic ability of Pancreatin and indicate that enzymic transesterification reactions between serum phosphatides and glycerides m a y cccur.
INTRODUCTION The chromatographic method described previously 1 was used to study the time course of action of pancreatic lipase and venom phospholipase A on human serum (and plasma) lipoproteins. The effect of added triglycerides, diglycerides, monoglycerides, and free f a t t y acids was also investigated. The data not only demonstrate the hydrolytic activity of pancreatic lipase and phospholipase A on serum lipids but also show that pancreatic lipase synthesizes triglycerides from diglycerides. In addition transesterification between phospholipids and glycerides is indicated. Biochim. Biophy~. Acta, 46 (1961) 468-478
SERUM LIPID TRANSFORMATIONS
469
METHODS
Fresh human sera (8 : oo A. M. fasting samples) were obtained from the Blood Laboratory. The sera were prepared by clotting and centrifuging whole blood. In some experiments the sera were used immediately whereas in others the sera were frozen and used at appropriate times (up to several weeks). Fresh human plasma or frozen human plasma was also used in other experiments. The plasma was obtained from Versene-treated whole blood. Rattlesnake venom (Crotalus adarnanteus) was purchased from Ross Allen Reptile Farms, Silver Springs, Fla. (U.S.A.). U.S.P. Pancreatin (pancreatic lipase) was obtained from Fisher Scientific Co. (Cat. No. P-I3, lot No. 532165). 20 mg of venom or Pancreatin were dissolved in 2 ml of 0. 9 % saline. The snake venom dissolved completely but the Pancreatin did not. The latter was filtered through a coarse sintered glass filter before use. These enzyme solutions were used on the same day of preparation, usually within I h. They can be stored in the cold for several days and still retain activity. The D-afi-diolein and L-a,fi-diolein were gift samples from Dr. BAER, Univ. of Toronto. The [i-14C]stearic and [i-14Cloleic acid were obtained from Nuclear-Chicago. I-Monopalmitin (Lot L-34 , recrystallized) was purchased from Armour and Co., Chicago, Ill. Tripalmitin was obtained from Eastman Kodak Co. Triolein (Squibb) was a gift sample from Dr. C. WATERHOUSE.The monopalmitin, diolein, tripalmitin and triolein were suspended in saline and the serum or plasma then added and mixed thoroughly. The [I-14C]oleic acid was used as the albumin complex (dissolved in a 25 % solution of human serum albumin). The [i-14C]stearic acid was used alone, (suspended in saline), as the albumin complex and as the urea adduct. All reactions were carried out at room temperature (approx. 21-25 °) unless otherwise specified. Paper chromatography was carried out as described previously 1. In most experiments 20 t,1 of serum or plasma were spotted on the silicic acid impregnated paper and chromatography was carried out in wide mouth Mason jars. We have now found that monoglycerides (I-monopalmitin) remain at the origin in solvent B (see ref. i) and hence cannot be separated from the phospholipid and proteins. However, by changing the solvent ratio of heptane-diisobutyl ketone from (96:6) to (96:24) the monoglycerides move ahead of the phosphatides and proteins and are well separated from the diglycerides and triglycerides. The diagrams given in Figs. 1-8 represent actual tracings of the original chromatograms. All the lipids (except the fatty acids bound to protein) moved as discrete spots and are well separated.
RESULTS The direct chromatography of the serum or plasma lipids made possible the time study of action of venom phospholipase A and pancreatic lipase on the lipoproteinbound lipids. The effect of added lipids to serum (with and without added Pancreatin) was also studied. At the outset it is important to state that the results which were obtained were dependent on the concentration of added enzyme and on whether the serum or plasma was fresh or stored (stored at - - I O °) and whether the serum was obtained by clotting whole blood or by clotting Versene treated plasma with CaClz.
Biochim. Biophys. Acta, 46 (I96I) 468-478
470
G. V. MARINETTI
Action of venom phospholipase A Snake venom effected a rapid hydrolysis (within 5 rain) of lipoprotein-bound lecithin to yield lysolecithin (Fig. I). The venom also hydrolyzed the serum phosphatidylethanolamine to its corresponding lyso-compound. The lysolecithin which was formed contained mainly saturated fatty acids whereas the liberated fatty acids were predominantly unsaturated. The venom action was observed on both fresh and stored human or rat sera but not on Versene-containing plasma. Addition of CaC12 to the Versene-containing plasma yielded a serum in which tile venom phospholipase A activity was completely restored. Therefore the activity of venom phospholipase A is dependent of Ca ions, an observation noted previously 2. The tatty acids which are released are bound to protein (or some other plasma component) since in nearly all cases they are either not observed or are barely detectable on chromatograms which are run on the heptane--ketone solvent and since the serum remains clear during the hydrolysis. Addition of an equivalent amount of free stearic acid to the same volume of serum yields a cloudy solution. Moreover we have observed that when free fatty acids either alone or when suspended in serum are chromatographed in heptane-
Fig. i. C h r o m a t o g r a m s and s u p e r i m p o s e d a u t o r a d i o g r a m s showing the effect of v e n o m p h o s pholipase A on r a t s e r u m lipids. A white r a t was given 50o #C of E3ZP~orthophosphate s u b c u taneously. After i8 h the blood was collected and treated with a m m o n i u m oxalate to p r e v e n t coagulation. The p l a s m a was obtained by centrifugation. To each ml of p l a s m a were added o. 15 ml of o.i M CaC12. The s e r u m was obtained b y centrifugation. To 0.3 ml of s e r u m were added 500 # g of snake v e n o m in 50 /~1 of saline. A control t u b e was prepared containing o.3 ml of s e r u m a n d 5 °/~1 of saline. The t u b e s were incubated at r o o m t e m p e r a t u r e . At the times indicated in min. 20/~1 were r e m o v e d for p a p e r c h r o m a t o g r a p h i c analysis. C h r o m a t o g r a p h y 1 was carried o u t in diisobutyl k e t o n e - a c e t i c a c i d - w a t e r (40:2o:3). I t can be seen t h a t the lecithin is nearly completely hydrolyzed to lysoleeithin within 5 rain. If the incubation is prolonged to 24-48 h the lysolecithin is slowly degraded. By use of the p e r m a n g a n a t e test it was d e m o n s t r a t e d t h a t the lecithins contained a high c o n t e n t of u n s a t u r a t e d f a t t y acids b u t the lysolecithins contained p r e d o m i n a n t l y s a t u r a t e d f a t t y acids. The u n s a t u r a t e d f a t t y acids liberated b y the v e n o m m o v e d near the solvent front. The p e r m a n g a n a t e test and the n i n h y d r i n test also revealed t h a t the v e n o m hydrolyzed the s e r u m p h o s p h a t i d y l e t h a n o l a m i n e to yield l y s o p h o s p h a t i d y l e t h a n o l a m i n e and free f a t t y acids. The l y s o p h o s p h a t i d y l e t h a n o l a m i n e ran with the sphingomyelin spot or slightly ahead of it.
Biochim. Biophys. Acta, 46 (196I ! 468-478
471
SERUM LIPID TRANSFORMATIONS
diisobutyl ketone (96 : 6) they migrate as discrete spots (RF 0.43) but when these fatty acids are first complexed with albumin and then chromatographed, nearly all the fatty acid remains at the origin with the albumin. The venom had no demonstrable action on the other serum lipids (sphingomyelin, triglycerides, cholesterol esters) in a time interval of 24 h. Hence its action seems specific for serum lecithin and phosphatidylethanolamine. After 24-28 h the lysolecithin was slowly degraded indicating a much weaker lysolecithinase B activity. The venom phospholipase A was able to effect nearly a complete hydrolysis of all the lecithin contained in o.5 ml of serum when as little as 14 ~g of venom were used. The phospholipase A was completely inactivated b y the addition of IOO /xmoles of citric acid, and oxalic acid per o.5 ml of serum. These inhibitors like Versene are believed to act by binding calcium.
Action of pancreatin Pancreatin catalyzed several lipid transformations. The first effect was a very rapid hydrolysis (within 5 min) of lipoprotein-bound triglycerides to diglycerides and f a t t y acids (Fig. 2). Throughout this reaction time the serum (or plasma) remains clear*. Of special interest is our observation that the diglycerides are within 1-3 h
Q
@@®@@
@®@ @®@@
©
@®0@ © @ @@®®@ "/'W_/ '~4,' CON.
IO
4
']-:
','-~:
:3:'
','~
3
@®@®@@0
@
20 50 MINUTES
80
120
5
2
STD.
Fig. 2. C h r o m a t o g r a m s showing the time course of action of Pancreati-n on h u m a n serum lipids. To o. 5 ml of fresh h u m a n s e r u m were added 5oo # g of P a n c r e a t i n in 5 °/~1 of saline. At the time intervals s h o w n 15-/,1 aliquots were s p o t t e d on silicic acid i m p r e g n a t e d paper. C h r o m a t o g r a p h y was carried out as described previously 1. The solvent s y s t e m was h e p t a n e diisobutyl ketone (96:6). CON, control s e r u m at zero time. STD, s t a n d a r d m i x t u r e of dip a l m i t i n (spot I), stearic acid (spot 3) and trip a l m i t i n (spot 4). o, m i x t u r e of p l a s m a proteins and phosphatides. Monoglyceride also m a y occur at the origin. 2, free cholesterol; 4, triglycerides; 5, cholesterol esters. The decrease in triglycerides and c o n c o m i t a n t increase in diglycerides at the I o - 2 o min intervals and the s u b s e q u e n t decrease in diglycerides and res y n t h e s i s of triglycerides is a p p a r e n t . Free f a t t y acids were not detected on the c h r o m a t o g r a m s .
vdJY vd2Y CON.
2
IO
30 70 MINUTES
145
200
Fig. 3. C h r o m a t o g r a m s showing the time course of action of Pancreatin on h u m a n p l a s m a lipids. To 0. 5 ml of h u m a n p l a s m a (stored, Versene-treated p l a s m a was used; i volume of o.o 3 M Versene in saline/io v o l u m e s of blood) were added 5oo /~g of P a n c r e a t i n in 5 ° /21 of saline. The control t u b e (labeled CON.) contained o.5 ml of p l a s m a and 5o /~1 of saline. The p l a s m a samples were incubated at r o o m t e m p e r a t u r e and at the t i m e s indicated 15/* 1 were r e m o v e d for p a p e r c h r o m a t o g r a p h i c analysis. C h r o m a t o g r a p h y was carried o u t as given in Fig. 2. The identification of the spots is as follows: o, proteins, p h o s p h a t i d e s and m o n o glycerides; I, diglycerides; 2, free cholesterol; 4, froe f a t t y acids; 4, triglycerides ; 5, cholesterol esters.
* The diglycerides a n d / o r f a t t y acids released b y enzyme hydrolysis are p r e s u m e d to be b o u n d to p r o t e i n since an equivalent a m o u n t of diolein and stearic acid w h e n added to the same v o l u m e of s e r u m (or plasma) yields a cloudy suspension.
]3iochim. Biophys. Acla, 46 (196I) 468-478
472
G. V. MAR1NETTI
resynthesized to triglycerides. The synthetic activity of pancreatic lipase has been observed by others 3,4. The action of Pancreatin was observed on fresh and stored serum and on fresh and stored plasma (Figs. 2-4). Versene at a level of i to 20/zg did not inhibit the action of Pancreatin whereas it inhibited venom phospholipase A completely. Within a period of 6 h no alterations were observed on the other endogenous serum or plasma lipids when the amount of Pancreatin employed was ioo-500/~g/o.5 ml serum (or plasma). However, when the Pancreatin concentration was increased to I-IO mg the presence of a lecithinase A was demonstrable. Indeed, when higher levels of Pancreatin were used (20 mg of Pancreatin/o.5 ml serum) the lecithin was nearly completely hydrclyzed to lysolecithin within 4 h. Hence Pancreatin has lecithinase A activity ~-s but this is very low in comparison to the lipase activity associated with trigtyceride hydrolysis. As was the case with venom phospholipase A, the lysolecithins formed by both enzymes are predominantly saturated derivatives and the liberated fatty acids are highly unsaturated. In all instances except when serum was obtained by clotting frozen Versenetreated plasma with excess calcium chloride, the free fatty acids released by Pancreatin action were either not detected or just barely observed on chromatograms and hence are presumed to be bound to protein*. However, in the case where the serum which was used contained excess calcium ions the free fatty acids released by Pancreatin hydrolysis of the triglycerides were detected on chromatograms (Compare Figs. 2 and 3 with Fig. 4). Moreover, as the triglycerides were resynthesized there was a concomitant proportionate disappearance of diglycerides and the fatty acids (Fig. 4)These data suggest that the binding of fatty acids to the serum proteins is influenced by calcium ions. It is noteworthy however that when [I-14Cloleic acid or [I-1~C!stearic acid was added to fresh or stored serum and Pancreatin was then added, no incorporation of these labeled fatty acids occurred into the triglycerides under con-
@ ®®@
®@@
=
O
K/J
v.Z~
v.ZP v..~
v.z~
CON.
2
I0 30 MINUTES
70
v.z~ v . ~ 145
200
Fig. 4. C h r o m a t o g r a m s s h o w i n g the time course of action of P a n c r e a t i n on h u m a n s e r u m lipids. Stored, Versene-treated h u m a n p l a s m a (same as used in Fig. 3) was t r e a t e d with CaCI~ (o.15 ml of 0.2 M CaC12 were a d d e d / m l of the p l a s m a and centrifuged). To 0. 5 ml of s e r u m were added 500/zg of P a n c r e a t i n in 5o/~1 of saline. The control t u b e (labeled CON.) contained 0. 5 ml of s e r u m and 5° / , 1 of saline. The t u b e s were incubated at r o o m t e m p e r a t u r e and at the t i m e s indicated I5-/~1 aliquots were r e m o v e d for p a p e r c h r o m a t o g r a p h i c analysis. The identification of the spots is the same as t h a t given in Fig. 3. I n this e x p e r i m e n t the free f a t t y acids (spot 3) were observed as m a j o r components. * See footnote p. 471.
Biochim. Biophys..4eta, 46 (i96I) 468-478
SERUM LIPID TRANSFORMATIONS
473
ditions when the endogenous triglycerides were being hydrolyzed and resynthesized (Figs. 5 and 6). Hence the disappearance of the fatty acids seen in Fig. 4 is believed to be due to a coincidental reaction whereby the added fatty acids are being bound to the serum albumin or other serum proteins and hence remain at the origin on the chromatograms.
Fig. 5. C h r o m a t o g r a m s showing the effect of Pancreatin on s e r u m lipids in the presence of [I-14C]stearic acid. The s y s t e m s are as follows: To a series of 4 t u b e s (A-D) were added 0. 3 mg of [I-14C lstearic acid (i.5 /~C), io m g of urea and 0.3 ml of saline. The t u b e s were heated at 8o ° for 5 m i n a n d s h a k e n on a t u b e buzzer. After cooling, o.5 ml of s e r u m were added and the t u b e s s h a k e n again. To each t u b e A - D were added 500/~g of Pancreatin in 5 °/~1 of saline. The control t u b e (labeled Con.) contained 0. 5 ml of serum, IO m g urea and o.35 ml of saline. The s t a n d a r d [I-14C]stearic acid (io/~g) was also r u n (labeled STD.). This was applied to the p a p e r in chloroform. The s y s t e m s were i n c u b a t e d for 3 h at r o o m t e m p e r a t u r e . At various times 2o-/~1 aliquots were r e m o v e d for p a p e r c h r o m a t o g r a p h i c analysis. This showed t h a t the triglycerides were hydrolyzed and resynthesized. The a u t o r a d i o g r a m s h o w e v e r revealed no incorporation of radioactivity into the resynthesized triglycerides at the end of 3 h. I t is n o t e w o r t h y t h a t in s y s t e m s A - D m o s t of the f a t t y acid remains b o u n d a t the origin. Studies w i t h oleic acid (see Fig. 6) s h o w t h a t this is due to binding of the f a t t y acid to s e r u m proteins. A u t o r a d i o g r a m s were p r e p a r e d on X - r a y film and required 3 days exposure.
The activity of the Pancreatin with regard to the transformation of triglycerides and diglycerides is completely abolished by heating the saline solution of Pancreatin at 8o ° for 5 min. This heat treatment on tile other hand had no effect on the activity of venom phospholipase A. Attempts to heat the serum or plasma to determine possible enzyme activity in these fluids were unsuccessful because of the formation of gels which were difficult to manipulate. In order to further elucidate the mechanism whereby the diglycerides are resynthesized to triglycerides, various substrates were added to the system. These included D-~,~-diolein, L-~,/3-diolein, I-monopalmitin, and triolein*. These were added in amounts ranging from o.25 mg to I.O mg/o.5 ml of serum or plasma. The * These lipids were checked for p u r i t y b y p a p e r c h r o m a t o g r a p h y . All except t h e n- and Ldioleins migrated as single c o m p o n e n t s . B o t h the D- and L-diolein m i g r a t e d as 2 c o m p o n e n t s (see Fig. 7). The slower m o v i n g c o m p o n e n t occurred in higher a m o u n t and is believed to be the a,flisomer. The faster m o v i n g c o m p o n e n t is t h e n t a k e n to be the a,ce'-isomer. These samples were over i year old and hence isomerization d u r i n g storage a p p e a r s likely.
Biochim. Biophys. Acta, 46 (1961) 468-478
474
~. v. MARINETTI
results which were obtained were dependent on whether the serum or plasma was fresh or stored (Figs. 7 and 8). Addition of monopalmitin and Pancreatin to flesh serum effected a marked increase in breakdown of lecithin to lysolecithin. This transformation required I 2 24 h. In the same system added tripalmitin had no such effect. Control systems with
Fig. 6. C h r o m a t o g r a m s shouting the effect of Pancreatin on s e r u m lipids in tile presence of [I -llC]oleic acid. To a series of t u b e s (A-F) were added o. 5 ml of stored h u m a n s e r u m and o.i ml of [I-14C]oleic a c i d - a l b u m i n complex (I /~C of [i -14C]oleic acid/o.i ml of 2 5 % h u m a n s e r u m albumin). The following additions were m a d e : A, 5oo t~g of Pancreatin in 5 ° /~1 of saline; B, o. 5 mg of i m o n o p a l m i t i n + 5oo ktg P a n c r e a t i n in 5° /~1 of saline; C, 0. 5 mg of D-~,fl-diolein + 5oo /~g of P a n c r e a t i n in 5 ° ktl of saline; D, 5 ° #1 of saline, E, o. 5 mg of I - m o n o p a l m i t i n + 5 ° #1 of saline; F, o. 5 m g of n-~,fl-diolein plus 5 °/~1 of saline. The control tube (CON.) contained o. 5 ml of serum plus o.15 ml of saline. The s t a n d a r d (STD.) contained 2o/~1 of Ei-14C]oleic a c i d - a l b u m i n complex. The t u b e s were incubated at r o o m t e m p e r a t u r e for 3 h. Aliquots (2o kd) were r e m o v e d at o.5-h intervals a n d analyzed b y p a p e r c h r o m a t o g r a p h y in order to establish the fact t h a t the triglycerides were hydrolyzed and resynthesized. The c h r o m a t o g r a m s and superimposed a u t o r a d i o g r a m s depict the 3-h time interval. No i n c o r p o r a t i o n occurred in the triglycerides. The binding of the oleic acid to s e r u m a l b u m i n is also evident. This is in c o n t r a s t to the El J4C]stearic acid which moved as a distinct spot w h e n applied to the p a p e r in chloroform (see Fig. 5) r a t h e r t h a n in a l b u m i n solution. The 3 areas of radioactivity in A--F m a y be due to three different binding sites for the f a t t y acids on albumin w h e n it is in a m e d i u m containing serum.
or without Pancreatin but without added monopalmitin did not exhibit these changes. The added monopalmitin was in part converted to 3 different products which migrated on paper in the area characteristic for diglycerides. It was not ascertained whether any monopalmitin was converted to triglyceride. The 3 products may represent diglycerides which differ with respect to either the type or position of the two fatty acids. The added tripalmitin was not acted upon in serum without added Pancreatin and was only very slowly hydrolyzed when Pancreatin was supplied. Hence Pancreatin acts much more rapidly on endogenous triglycerides bound to lipoproteins. It is possible that Pancreatin acts preferentially on unsaturated triglycerides. This was supported by our finding that added triolein was hydrolyzed and resynthesized nearly as rapidly as the endogenous triglycerides. On the other hand Pancreatin effected only a very slow hydrolysis of triolein when suspended in saline or when emulsified in Biochi~n. Biophys. ~4c/a, 46 (I96t) t()8 47 ~
475
SERUM LIPID TRANSFORMATIONS
saline with Tween 2o. By way of comparison it m a y be noted that lipoprotein lipase has been shown to act more rapidly on triolein than on saturated triglycerides ~. Fresh serum differed from stored serum in that only in fresh serum were added D- and L-~,fi-diolein acted upon without added Pancreatin (Fig. 7). However, with both fresh and stored sera the added dioleins were acted upon when Pancreatin was also supplied. I t is noteworthy that both the D and L-s, fi-dioleins, although being pure synthetic compounds, showed 2 components when chromatographed* (see Fig. 7)- It is of further interest that these diglyceride components were not acted upon in the same manner (see below).
©00
:.::
..)
0
c~
::)
()
C!
',~:
(:..J r.-7-~ c / ~
A
:.
0000
'k/_/S
~,4./s
B
C
r/-h ,,R-x ,,..4..// w...U D
E
,<-A //-~ ,
STD
Fig. 7. C h r o m a t o g r a m s showing the effect of h u m a n s e r u m (with and w i t h o u t added P a n creatin) on s y n t h e t i c dioleins. To a series of six t u b e s were added o. 4 ml of fresh pooled h u m a n serum. The following additions were t h e n m a d e : A, 500 /~g of D-~,j~-diolein plus 500 /~g of P a n c r e a t i n in ioo ,ul of saline; B, 5oo/~g of L-,¢,/5-diolein plus 500 /~g of P a n c r e a t i n in ioo F,1 of saline; C, 5o0 /~g of D-~,j~-diolein plus IOO ~u] of saline; D, 500/~g of L-~,~-diolein plus IOO /~l of saline; E, 5o0 # g of Pancreatin in IOO gl of saline; F, IOO gl of saline (control). The dioleins were first suspended in the s e r u m by shaking on a t u b e buzzer before the enzyme was added. The t u b e s were incubated at r o o m t e m p e r a t u r e for 6 h a n d at 5 ° for 18 h. Aliquots (20 FI) were r e m o v e d for p a p e r c h r o m a t o graphic analysis at several time intervals. This figure shows the results obtained at the end of the incubation period. The identification of the s p o t s is the s a m e as t h a t given in Fig. 3. I n this e x p e r i m e n t the f a t t y acids (spot 3) released in the s y s t e m s A and B trailed into and below the cholesterol s p o t (spot 2). I n addition the dioleins (spot I) m i g r a t e d as 2 components. STD., 20 # g of s t a n d a r d n-o¢,jS-diolein which w a s applied to the p a p e r in chloroform*. L-~, jS-diolein gave the s a m e p a t t e r n as the Disomer. C h r o m a t o g r a p h i c analysis of the phosp h a t i d e s in these s y s t e m s is given in Fig. 8.
A
B
C
O
E
/~/~ F
STD.
Fig. 8. C h r o m a t o g r a m s showing the effect of synthetic dioleins on the Pancreatin-catalyzed hydrolysis of s e r u m lecithin. The s y s t e m s A - F are the same as those given in Fig. 7. At the end of the incubation period 2o /~1 were rem o v e d for p a p e r c h r o m a t o g r a p h i c analysis. C h r o m a t o g r a p h y was carried o u t in diisobutylk e t o n e - a c e t i c - a c i d - w a t e r (4° : 20 : 3). The identification of the spots is as follows: o, proteins; I, lysolecithin plus inosito] p h o s p h a t i d e (in A a n d B this s p o t is p r e d o m i n a n t l y lysolecithin as evidenced b y the staining test with R h o d amine 6G in which lysolecithin stains yelloworange and inositol p h o s p h a t i d e stains bluepurple) 2, sphingomyelin; 3, lecithin; 4, phosp h a t i d y l e t h a n o l a m i n e ; 5, m i x t u r e of the nonp h o s p h a t i d e s ; STD., 20 t*g of D-~,fl-diolein. The m a r k e d decrease in lecithin and c o n c o m i t a n t increase in lysolecithin in A and B is evident. The small c o m p o n e n t below spot i is unidentified. The lysolecithins which were formed were p r e d o m i n a n t l y s a t u r a t e d derivatives w h e r e a s the lecithins contained a high c o n t e n t of uns a t u r a t e d f a t t y acids. The liberated f a t t y acids in s y s t e m s A and B were highly u n s a t u r a t e d and m o v e d near the solvent f r o n t with the other n o n - p h o s p h a t i d e s .
* See footnote p. 473-
Biochim. Biophys. Acta, 46 (1961) 468-478
476
G.V. MARINETTI
In fresh serum without added Pancreatin the D-and L-dioleins slowly disappeared (6 h at 22-25 ° and 18 h at 5 °) (Fig. 7). Very little or no free fatty acids were observed on the chromatograms. The triglycerides appeared to increase but not enough to account for the simultaneous decrease in the dioleins. The other plasma lipids (lecithins, sphingomyelin, lysolecithin, cholesterol and cholesterol esters) did not undergo any detectable changes. A small amount of monoglycerides was formed. When the same fresh serum has Pancreatin added the D- and L-diolein are acted upon more rapidly (Fig. 7). Free fatty acids are released (observed on chromatograms within 6 h) and there is a concomitant hydrolysis of endogenous triglycerides. The endogenous triglycerides require 4 h for complete hydrolysis whereas in a similar "control" system without added diolein the triglycerides are completely hydrolyzed within 5 min. Either the added diolein inhibits the Pancreatin hydrolysis of endogenous triglyceride or the added diolein is being converted to triglyceride at the same time that the endogenous triglycerides are being hydrolyzed. The latter situation would make it appear that the endogenous triglycerides were being more slowly hydrolyzed than in the "control". It is of further interest however, that at the same time these changes occur, the lecithin is hydrolyzed to lysolecithin (Fig. 8). These changes are observed within 6 h incubation but are more pronounced by 24 tl. It is important to note that with the amount of Pancreatin used in this experiment (5oo/~g /o.5 ml serum) no lecithin is broken down to lysolecithin in the absence of added diglycerides. Hence, as was the case with I-monopalmitin, either the mono- or diglycerides stimulate pancreatic lecithinase A or transesterification is occurring between the phospholipids and glycerides such that f a t t y acids from lecithin are being utilized for the conversion of monoglycerides to diglycerides (or triglycerides) and diglycerides to triglycerides. When this latter system containing added diolein and Pancreatin is incubated for 18 h at 5 ° the triglycerides which were initially hydrolyzed are resynthesized. At no time is there any appreciable amount of monoglycerides formed. No detectable changes are observed in the other lipid fractions (cholesterol, cholesterol esters, and sphingomyelin) during this time. In the system which contains fresh serum and added diolein but without Pancreatin the slower moving diolein component is more rapidly metabolized. Contrariwise, in the system containing fresh serum, added diolein and added Pancreatin, the faster moving diolein component is more rapidly metabolized (Fig. 7). The diglyceride formed b y Pancreatin hydrolysis of endogenous serum triglyceride moves identically as the slower moving diolein. Since pancreatic lipase has been shown to be specific for the a-linked fatty acid 1° this supports our belief that the slower moving diolein is the a,~-isomer. The faster moving diolein would therefore be the a,~'-isomer. Appropriate controls were run at all times in which serum alone (plus saline) was incubated for the same time intervals. At no time were any significant changes observed in the serum lipids of these controls during incubation up to 24 h. Inhibition studies showed that sodium fluoride, iodoacetate and Versene at a level of io to 2o/,g /o.5 ml of serum did not inhibit pancreatic lipase but did effect a marked inhibition at a level of 2oo-25o ~g/o.5 ml serum. DISCUSSION
The experiments reported in the paper are mainly qualitative in nature but clearly show that Pancreatin contains an active lipase which rapidly hydrolyzes serum lipoBiochim. Biophys. Acla, 46 (196t) 468-478
SERUM LIPID TRANSFORMATIONS
477
protein-bound triglycerides to diglycerides but the diglycerides are resynthesized to triglycerides. The diglycerides are believed to remain bound to protein since the serum remains clear during the reaction. The d a t a furthermore indicate t h a t transesterification between the various serum lipids m a y occur. These reactions are as follows: Triglycerides ~ Diglyceride + Fatty acid
(I)
Lecithin ---~ Lysolecithin + Fatty acid
(2)
Lecithin + Diglyceride---~ Triglyceride + Lysolecithin
(3)
Lecithin + Monoglyceride--+ Diglyceride + Lysolecithin
(4)
Reaction (I) is rapid and reversible and catalyzed b y pancreatic lipase. Reaction (2) when catalyzed b y venom phospholipase A is very rapid but reaction (2) when catalyzed b y pancreatic phospholipase A is m u c h slower and requires a m u c h higher concentration of Pancreatin. However the d a t a indicate t h a t reaction (2) when catalyzed b y Pancreatin is stimulated b y addition of monoglycerides and diglycerides. This stimulation m a y occur as shown in reactions (3) and (4). These postulated transesterification reactions (3) and (4) are supported b y the finding t h a t the conversion of lecithin to lysolecithin is markedly stimulated b y the addition of monopalmitin with a concomitant increase in diglycerides. Although diolein (Fig. 7) stimulated the Pancreatin hydrolysis of serum lecithin to lysolecithin it was not ascertained whether the diolein had been converted to triglycerides. During the early period of these transformations very little or no free f a t t y acids can be detected on chromatograms. Hence the f a t t y acids are believed to be " b o u n d " to protein or to some other serum components. This "binding" of the liberated f a t t y acids m a y or m a y not be the same as FFA*. W h e n the serum reaction mixture is put on the chromatographic paper and the applied spots are then treated with methanol, no free f a t t y acids are liberated b y this procedure. This was done in order to rule out the possibility t h a t the chromatographic solvent heptane-diisobutylketone (96:6) was not polar enough to rupture the f a t t y acid-protein bonds. Yet it is of interest t h a t heptane-diisobutyl ketone (96:6) can free most of the serum lipoprotein-bound triglycerides, diglycerides, cholesterol, and cholesterol esters**. Only in the serum obtained by clotting Versene treated plasma with excess calcium ions were the f a t t y acids liberated b y Pancreatin hydrolysis of serum triglycerides observed on the chromatograms in appreciable amount. In this case the liberated f a t t y acids m a y exist as Ca salts and these cannot be converted to a " b o u n d " form as rapidly as the free acids. When Ii-14Clstearic acid and [i-14Cloleic acid were added to serum these were not incorporated into the triglycerides under conditions when triglycerides were being hydrolyzed and resynthesized b y Pancreatin (Figs. 5 and 6). It m a y be possible t h a t other means can be found to bring about this incorporation. However, a very slight incorporation of 14C was observed in both cases into the cholesterol ester fraction. The use of appropriate labeled triglycerides, diglycerides, monoglycerides and * FFA signifies the albumin bound fatty acids which are believed to play a role in lipid transport. Some of the neutral lipids remain at the origin. Chromatograms can first be run in chloroformmethanol (i :i) for 2- 3 cm, dried for IO min and then run in the heptane-ketone system. This liberates all the neutral lipids and is important for quantitative work. * *
Biochim. Biophys. Acts, 46 (1961) 468-478
478
G . V . MARINETTI
lecithin may elucidate the postulated transesterification reactions given above. Such transesterification reactions would be in harmony with the other well established transfer reactions (transamination, transglucosidation, transamidation, transmethylation) which have been shown to occur in living ceils and if confirmed would demonstrate new metabolic pathways for interconversion of glycerides and phosphatides. ACKNOWLEDGEMENT
This work was supported in part by funds from a grant H 2063 from the National Heart Institute, National Institute of Health, U.S. Public Health Service. REFERENCES 1 G. V. MARINETTI AND E. STOTZ,Biochim. Biophys. Acta, 37 (196o) 571. H. WHITTCOFF, The Phosphatides, Reinhold Publ. Corp. N. Y. 1951, p. lO5. 3 H. POTTEVlN, Compt. rend., 136 (19o3) 767. 4 C. ARTOM AND L. REALE, Bull. soc. chim. biol., 18 (1936) 959. 5 S. BELFANTI, Fisiole reed. (Rome), 12 (1933) 821. 6 A. CONTARDI AND A. ERCOLI, Biochem. Z., 261 (1933) 275. 7 V. GRONCI-II, Sperimentale, 90 (1936) 233. 8 D. J. HANAHAN, J. Biol. Chem., 195 (1952) 199. 9 B. SnoRE, O. M. COLVlN AND V. G. SHORE, Biochim. Biophys. Acta, 36 (1959) 563 • 10 F. H. MATTSON AND L. W. BECK, J, Biol. Chem., 214 (1955) 115.
Biochim. Biophys. Acta, 46 (1961) 468-478
BIOCHIMICA ET BIOPHYSICA ACTA
I N V I T R O L I P I D TRANSFORMATIONS IN SERUM G. V. MARINETTI Department o/Biochemistry, University o/Rochester School o] Medicine and Dentistry, Rochester, N. Y. (U.S.A.)
(Received July 23rd, 196o)
SUMMARY Snake venom phospholipase A effects a rapid hydrolysis of serum lecithin to yield lysolecithin which has predominantly saturated fatty acids. The fatty acids which are released are highly unsaturated. The venom also hydrolyzes serum phosphatidylethanolamine to its lyso-compound. The activity of the venom is abolished byVersene, citrate, and oxalate and reactivated by calcium ions. Pancreatin effects a rapid hydrolysis of serum triglycerides to yield diglycerides. The triglycerides are resynthesized within 1-3 h with a concomitant disappearance of the diglycerides. The fatty acids which are released are believed to be bound to the serum proteins. Added [i-14C]stearic and Ii-14Cloleic acid are not incorporated into the resynthesized triglycerides. Pancreatin has very little action on tripalmitin when this is added to serum but rapidly hydrolyzes added triolein. When synthetic D- and L-dioleins are added to fresh serum these are slowly degraded to monoglycerides and other products. When both the dioleins and Pancreatin are added to serum, the dio]eins are more rapidly metabolized to other products. In this case, however, the dioleins stimulate the hydrolysis of serum lecithin to lysolecithin. Added I-monopalmitin also stimulates the Pancreatin hydrolysis of lecithin to lysolecithin during which time the monoglyceride is converted in part to diglycerides. Added triglycerides do not stimulate the Pancreatin hydrolysis of lecithin to lysolecithin. The data reported in this paper demonstrate the active synthetic ability of Pancreatin and indicate that enzymic transesterification reactions between serum phosphatides and glycerides m a y cccur.
INTRODUCTION The chromatographic method described previously 1 was used to study the time course of action of pancreatic lipase and venom phospholipase A on human serum (and plasma) lipoproteins. The effect of added triglycerides, diglycerides, monoglycerides, and free f a t t y acids was also investigated. The data not only demonstrate the hydrolytic activity of pancreatic lipase and phospholipase A on serum lipids but also show that pancreatic lipase synthesizes triglycerides from diglycerides. In addition transesterification between phospholipids and glycerides is indicated. Biochim. Biophy~. Acta, 46 (1961) 468-478
SERUM LIPID TRANSFORMATIONS
469
METHODS
Fresh human sera (8 : oo A. M. fasting samples) were obtained from the Blood Laboratory. The sera were prepared by clotting and centrifuging whole blood. In some experiments the sera were used immediately whereas in others the sera were frozen and used at appropriate times (up to several weeks). Fresh human plasma or frozen human plasma was also used in other experiments. The plasma was obtained from Versene-treated whole blood. Rattlesnake venom (Crotalus adarnanteus) was purchased from Ross Allen Reptile Farms, Silver Springs, Fla. (U.S.A.). U.S.P. Pancreatin (pancreatic lipase) was obtained from Fisher Scientific Co. (Cat. No. P-I3, lot No. 532165). 20 mg of venom or Pancreatin were dissolved in 2 ml of 0. 9 % saline. The snake venom dissolved completely but the Pancreatin did not. The latter was filtered through a coarse sintered glass filter before use. These enzyme solutions were used on the same day of preparation, usually within I h. They can be stored in the cold for several days and still retain activity. The D-afi-diolein and L-a,fi-diolein were gift samples from Dr. BAER, Univ. of Toronto. The [i-14C]stearic and [i-14Cloleic acid were obtained from Nuclear-Chicago. I-Monopalmitin (Lot L-34 , recrystallized) was purchased from Armour and Co., Chicago, Ill. Tripalmitin was obtained from Eastman Kodak Co. Triolein (Squibb) was a gift sample from Dr. C. WATERHOUSE.The monopalmitin, diolein, tripalmitin and triolein were suspended in saline and the serum or plasma then added and mixed thoroughly. The [I-14C]oleic acid was used as the albumin complex (dissolved in a 25 % solution of human serum albumin). The [i-14C]stearic acid was used alone, (suspended in saline), as the albumin complex and as the urea adduct. All reactions were carried out at room temperature (approx. 21-25 °) unless otherwise specified. Paper chromatography was carried out as described previously 1. In most experiments 20 t,1 of serum or plasma were spotted on the silicic acid impregnated paper and chromatography was carried out in wide mouth Mason jars. We have now found that monoglycerides (I-monopalmitin) remain at the origin in solvent B (see ref. i) and hence cannot be separated from the phospholipid and proteins. However, by changing the solvent ratio of heptane-diisobutyl ketone from (96:6) to (96:24) the monoglycerides move ahead of the phosphatides and proteins and are well separated from the diglycerides and triglycerides. The diagrams given in Figs. 1-8 represent actual tracings of the original chromatograms. All the lipids (except the fatty acids bound to protein) moved as discrete spots and are well separated.
RESULTS The direct chromatography of the serum or plasma lipids made possible the time study of action of venom phospholipase A and pancreatic lipase on the lipoproteinbound lipids. The effect of added lipids to serum (with and without added Pancreatin) was also studied. At the outset it is important to state that the results which were obtained were dependent on the concentration of added enzyme and on whether the serum or plasma was fresh or stored (stored at - - I O °) and whether the serum was obtained by clotting whole blood or by clotting Versene treated plasma with CaClz.
Biochim. Biophys. Acta, 46 (I96I) 468-478
470
G. V. MARINETTI
Action of venom phospholipase A Snake venom effected a rapid hydrolysis (within 5 rain) of lipoprotein-bound lecithin to yield lysolecithin (Fig. I). The venom also hydrolyzed the serum phosphatidylethanolamine to its corresponding lyso-compound. The lysolecithin which was formed contained mainly saturated fatty acids whereas the liberated fatty acids were predominantly unsaturated. The venom action was observed on both fresh and stored human or rat sera but not on Versene-containing plasma. Addition of CaC12 to the Versene-containing plasma yielded a serum in which tile venom phospholipase A activity was completely restored. Therefore the activity of venom phospholipase A is dependent of Ca ions, an observation noted previously 2. The tatty acids which are released are bound to protein (or some other plasma component) since in nearly all cases they are either not observed or are barely detectable on chromatograms which are run on the heptane--ketone solvent and since the serum remains clear during the hydrolysis. Addition of an equivalent amount of free stearic acid to the same volume of serum yields a cloudy solution. Moreover we have observed that when free fatty acids either alone or when suspended in serum are chromatographed in heptane-
Fig. i. C h r o m a t o g r a m s and s u p e r i m p o s e d a u t o r a d i o g r a m s showing the effect of v e n o m p h o s pholipase A on r a t s e r u m lipids. A white r a t was given 50o #C of E3ZP~orthophosphate s u b c u taneously. After i8 h the blood was collected and treated with a m m o n i u m oxalate to p r e v e n t coagulation. The p l a s m a was obtained by centrifugation. To each ml of p l a s m a were added o. 15 ml of o.i M CaC12. The s e r u m was obtained b y centrifugation. To 0.3 ml of s e r u m were added 500 # g of snake v e n o m in 50 /~1 of saline. A control t u b e was prepared containing o.3 ml of s e r u m a n d 5 °/~1 of saline. The t u b e s were incubated at r o o m t e m p e r a t u r e . At the times indicated in min. 20/~1 were r e m o v e d for p a p e r c h r o m a t o g r a p h i c analysis. C h r o m a t o g r a p h y 1 was carried o u t in diisobutyl k e t o n e - a c e t i c a c i d - w a t e r (40:2o:3). I t can be seen t h a t the lecithin is nearly completely hydrolyzed to lysoleeithin within 5 rain. If the incubation is prolonged to 24-48 h the lysolecithin is slowly degraded. By use of the p e r m a n g a n a t e test it was d e m o n s t r a t e d t h a t the lecithins contained a high c o n t e n t of u n s a t u r a t e d f a t t y acids b u t the lysolecithins contained p r e d o m i n a n t l y s a t u r a t e d f a t t y acids. The u n s a t u r a t e d f a t t y acids liberated b y the v e n o m m o v e d near the solvent front. The p e r m a n g a n a t e test and the n i n h y d r i n test also revealed t h a t the v e n o m hydrolyzed the s e r u m p h o s p h a t i d y l e t h a n o l a m i n e to yield l y s o p h o s p h a t i d y l e t h a n o l a m i n e and free f a t t y acids. The l y s o p h o s p h a t i d y l e t h a n o l a m i n e ran with the sphingomyelin spot or slightly ahead of it.
Biochim. Biophys. Acta, 46 (196I ! 468-478
471
SERUM LIPID TRANSFORMATIONS
diisobutyl ketone (96 : 6) they migrate as discrete spots (RF 0.43) but when these fatty acids are first complexed with albumin and then chromatographed, nearly all the fatty acid remains at the origin with the albumin. The venom had no demonstrable action on the other serum lipids (sphingomyelin, triglycerides, cholesterol esters) in a time interval of 24 h. Hence its action seems specific for serum lecithin and phosphatidylethanolamine. After 24-28 h the lysolecithin was slowly degraded indicating a much weaker lysolecithinase B activity. The venom phospholipase A was able to effect nearly a complete hydrolysis of all the lecithin contained in o.5 ml of serum when as little as 14 ~g of venom were used. The phospholipase A was completely inactivated b y the addition of IOO /xmoles of citric acid, and oxalic acid per o.5 ml of serum. These inhibitors like Versene are believed to act by binding calcium.
Action of pancreatin Pancreatin catalyzed several lipid transformations. The first effect was a very rapid hydrolysis (within 5 min) of lipoprotein-bound triglycerides to diglycerides and f a t t y acids (Fig. 2). Throughout this reaction time the serum (or plasma) remains clear*. Of special interest is our observation that the diglycerides are within 1-3 h
Q
@@®@@
@®@ @®@@
©
@®0@ © @ @@®®@ "/'W_/ '~4,' CON.
IO
4
']-:
','-~:
:3:'
','~
3
@®@®@@0
@
20 50 MINUTES
80
120
5
2
STD.
Fig. 2. C h r o m a t o g r a m s showing the time course of action of Pancreati-n on h u m a n serum lipids. To o. 5 ml of fresh h u m a n s e r u m were added 5oo # g of P a n c r e a t i n in 5 °/~1 of saline. At the time intervals s h o w n 15-/,1 aliquots were s p o t t e d on silicic acid i m p r e g n a t e d paper. C h r o m a t o g r a p h y was carried out as described previously 1. The solvent s y s t e m was h e p t a n e diisobutyl ketone (96:6). CON, control s e r u m at zero time. STD, s t a n d a r d m i x t u r e of dip a l m i t i n (spot I), stearic acid (spot 3) and trip a l m i t i n (spot 4). o, m i x t u r e of p l a s m a proteins and phosphatides. Monoglyceride also m a y occur at the origin. 2, free cholesterol; 4, triglycerides; 5, cholesterol esters. The decrease in triglycerides and c o n c o m i t a n t increase in diglycerides at the I o - 2 o min intervals and the s u b s e q u e n t decrease in diglycerides and res y n t h e s i s of triglycerides is a p p a r e n t . Free f a t t y acids were not detected on the c h r o m a t o g r a m s .
vdJY vd2Y CON.
2
IO
30 70 MINUTES
145
200
Fig. 3. C h r o m a t o g r a m s showing the time course of action of Pancreatin on h u m a n p l a s m a lipids. To 0. 5 ml of h u m a n p l a s m a (stored, Versene-treated p l a s m a was used; i volume of o.o 3 M Versene in saline/io v o l u m e s of blood) were added 5oo /~g of P a n c r e a t i n in 5 ° /21 of saline. The control t u b e (labeled CON.) contained o.5 ml of p l a s m a and 5o /~1 of saline. The p l a s m a samples were incubated at r o o m t e m p e r a t u r e and at the t i m e s indicated 15/* 1 were r e m o v e d for p a p e r c h r o m a t o g r a p h i c analysis. C h r o m a t o g r a p h y was carried o u t as given in Fig. 2. The identification of the spots is as follows: o, proteins, p h o s p h a t i d e s and m o n o glycerides; I, diglycerides; 2, free cholesterol; 4, froe f a t t y acids; 4, triglycerides ; 5, cholesterol esters.
* The diglycerides a n d / o r f a t t y acids released b y enzyme hydrolysis are p r e s u m e d to be b o u n d to p r o t e i n since an equivalent a m o u n t of diolein and stearic acid w h e n added to the same v o l u m e of s e r u m (or plasma) yields a cloudy suspension.
]3iochim. Biophys. Acla, 46 (196I) 468-478
472
G. V. MAR1NETTI
resynthesized to triglycerides. The synthetic activity of pancreatic lipase has been observed by others 3,4. The action of Pancreatin was observed on fresh and stored serum and on fresh and stored plasma (Figs. 2-4). Versene at a level of i to 20/zg did not inhibit the action of Pancreatin whereas it inhibited venom phospholipase A completely. Within a period of 6 h no alterations were observed on the other endogenous serum or plasma lipids when the amount of Pancreatin employed was ioo-500/~g/o.5 ml serum (or plasma). However, when the Pancreatin concentration was increased to I-IO mg the presence of a lecithinase A was demonstrable. Indeed, when higher levels of Pancreatin were used (20 mg of Pancreatin/o.5 ml serum) the lecithin was nearly completely hydrclyzed to lysolecithin within 4 h. Hence Pancreatin has lecithinase A activity ~-s but this is very low in comparison to the lipase activity associated with trigtyceride hydrolysis. As was the case with venom phospholipase A, the lysolecithins formed by both enzymes are predominantly saturated derivatives and the liberated fatty acids are highly unsaturated. In all instances except when serum was obtained by clotting frozen Versenetreated plasma with excess calcium chloride, the free fatty acids released by Pancreatin action were either not detected or just barely observed on chromatograms and hence are presumed to be bound to protein*. However, in the case where the serum which was used contained excess calcium ions the free fatty acids released by Pancreatin hydrolysis of the triglycerides were detected on chromatograms (Compare Figs. 2 and 3 with Fig. 4). Moreover, as the triglycerides were resynthesized there was a concomitant proportionate disappearance of diglycerides and the fatty acids (Fig. 4)These data suggest that the binding of fatty acids to the serum proteins is influenced by calcium ions. It is noteworthy however that when [I-14Cloleic acid or [I-1~C!stearic acid was added to fresh or stored serum and Pancreatin was then added, no incorporation of these labeled fatty acids occurred into the triglycerides under con-
@ ®®@
®@@
=
O
K/J
v.Z~
v.ZP v..~
v.z~
CON.
2
I0 30 MINUTES
70
v.z~ v . ~ 145
200
Fig. 4. C h r o m a t o g r a m s s h o w i n g the time course of action of P a n c r e a t i n on h u m a n s e r u m lipids. Stored, Versene-treated h u m a n p l a s m a (same as used in Fig. 3) was t r e a t e d with CaCI~ (o.15 ml of 0.2 M CaC12 were a d d e d / m l of the p l a s m a and centrifuged). To 0. 5 ml of s e r u m were added 500/zg of P a n c r e a t i n in 5o/~1 of saline. The control t u b e (labeled CON.) contained 0. 5 ml of s e r u m and 5° / , 1 of saline. The t u b e s were incubated at r o o m t e m p e r a t u r e and at the t i m e s indicated I5-/~1 aliquots were r e m o v e d for p a p e r c h r o m a t o g r a p h i c analysis. The identification of the spots is the same as t h a t given in Fig. 3. I n this e x p e r i m e n t the free f a t t y acids (spot 3) were observed as m a j o r components. * See footnote p. 471.
Biochim. Biophys..4eta, 46 (i96I) 468-478
SERUM LIPID TRANSFORMATIONS
473
ditions when the endogenous triglycerides were being hydrolyzed and resynthesized (Figs. 5 and 6). Hence the disappearance of the fatty acids seen in Fig. 4 is believed to be due to a coincidental reaction whereby the added fatty acids are being bound to the serum albumin or other serum proteins and hence remain at the origin on the chromatograms.
Fig. 5. C h r o m a t o g r a m s showing the effect of Pancreatin on s e r u m lipids in the presence of [I-14C]stearic acid. The s y s t e m s are as follows: To a series of 4 t u b e s (A-D) were added 0. 3 mg of [I-14C lstearic acid (i.5 /~C), io m g of urea and 0.3 ml of saline. The t u b e s were heated at 8o ° for 5 m i n a n d s h a k e n on a t u b e buzzer. After cooling, o.5 ml of s e r u m were added and the t u b e s s h a k e n again. To each t u b e A - D were added 500/~g of Pancreatin in 5 °/~1 of saline. The control t u b e (labeled Con.) contained 0. 5 ml of serum, IO m g urea and o.35 ml of saline. The s t a n d a r d [I-14C]stearic acid (io/~g) was also r u n (labeled STD.). This was applied to the p a p e r in chloroform. The s y s t e m s were i n c u b a t e d for 3 h at r o o m t e m p e r a t u r e . At various times 2o-/~1 aliquots were r e m o v e d for p a p e r c h r o m a t o g r a p h i c analysis. This showed t h a t the triglycerides were hydrolyzed and resynthesized. The a u t o r a d i o g r a m s h o w e v e r revealed no incorporation of radioactivity into the resynthesized triglycerides at the end of 3 h. I t is n o t e w o r t h y t h a t in s y s t e m s A - D m o s t of the f a t t y acid remains b o u n d a t the origin. Studies w i t h oleic acid (see Fig. 6) s h o w t h a t this is due to binding of the f a t t y acid to s e r u m proteins. A u t o r a d i o g r a m s were p r e p a r e d on X - r a y film and required 3 days exposure.
The activity of the Pancreatin with regard to the transformation of triglycerides and diglycerides is completely abolished by heating the saline solution of Pancreatin at 8o ° for 5 min. This heat treatment on tile other hand had no effect on the activity of venom phospholipase A. Attempts to heat the serum or plasma to determine possible enzyme activity in these fluids were unsuccessful because of the formation of gels which were difficult to manipulate. In order to further elucidate the mechanism whereby the diglycerides are resynthesized to triglycerides, various substrates were added to the system. These included D-~,~-diolein, L-~,/3-diolein, I-monopalmitin, and triolein*. These were added in amounts ranging from o.25 mg to I.O mg/o.5 ml of serum or plasma. The * These lipids were checked for p u r i t y b y p a p e r c h r o m a t o g r a p h y . All except t h e n- and Ldioleins migrated as single c o m p o n e n t s . B o t h the D- and L-diolein m i g r a t e d as 2 c o m p o n e n t s (see Fig. 7). The slower m o v i n g c o m p o n e n t occurred in higher a m o u n t and is believed to be the a,flisomer. The faster m o v i n g c o m p o n e n t is t h e n t a k e n to be the a,ce'-isomer. These samples were over i year old and hence isomerization d u r i n g storage a p p e a r s likely.
Biochim. Biophys. Acta, 46 (1961) 468-478
474
~. v. MARINETTI
results which were obtained were dependent on whether the serum or plasma was fresh or stored (Figs. 7 and 8). Addition of monopalmitin and Pancreatin to flesh serum effected a marked increase in breakdown of lecithin to lysolecithin. This transformation required I 2 24 h. In the same system added tripalmitin had no such effect. Control systems with
Fig. 6. C h r o m a t o g r a m s shouting the effect of Pancreatin on s e r u m lipids in tile presence of [I -llC]oleic acid. To a series of t u b e s (A-F) were added o. 5 ml of stored h u m a n s e r u m and o.i ml of [I-14C]oleic a c i d - a l b u m i n complex (I /~C of [i -14C]oleic acid/o.i ml of 2 5 % h u m a n s e r u m albumin). The following additions were m a d e : A, 5oo t~g of Pancreatin in 5 ° /~1 of saline; B, o. 5 mg of i m o n o p a l m i t i n + 5oo ktg P a n c r e a t i n in 5° /~1 of saline; C, 0. 5 mg of D-~,fl-diolein + 5oo /~g of P a n c r e a t i n in 5 ° ktl of saline; D, 5 ° #1 of saline, E, o. 5 mg of I - m o n o p a l m i t i n + 5 ° #1 of saline; F, o. 5 m g of n-~,fl-diolein plus 5 °/~1 of saline. The control tube (CON.) contained o. 5 ml of serum plus o.15 ml of saline. The s t a n d a r d (STD.) contained 2o/~1 of Ei-14C]oleic a c i d - a l b u m i n complex. The t u b e s were incubated at r o o m t e m p e r a t u r e for 3 h. Aliquots (2o kd) were r e m o v e d at o.5-h intervals a n d analyzed b y p a p e r c h r o m a t o g r a p h y in order to establish the fact t h a t the triglycerides were hydrolyzed and resynthesized. The c h r o m a t o g r a m s and superimposed a u t o r a d i o g r a m s depict the 3-h time interval. No i n c o r p o r a t i o n occurred in the triglycerides. The binding of the oleic acid to s e r u m a l b u m i n is also evident. This is in c o n t r a s t to the El J4C]stearic acid which moved as a distinct spot w h e n applied to the p a p e r in chloroform (see Fig. 5) r a t h e r t h a n in a l b u m i n solution. The 3 areas of radioactivity in A--F m a y be due to three different binding sites for the f a t t y acids on albumin w h e n it is in a m e d i u m containing serum.
or without Pancreatin but without added monopalmitin did not exhibit these changes. The added monopalmitin was in part converted to 3 different products which migrated on paper in the area characteristic for diglycerides. It was not ascertained whether any monopalmitin was converted to triglyceride. The 3 products may represent diglycerides which differ with respect to either the type or position of the two fatty acids. The added tripalmitin was not acted upon in serum without added Pancreatin and was only very slowly hydrolyzed when Pancreatin was supplied. Hence Pancreatin acts much more rapidly on endogenous triglycerides bound to lipoproteins. It is possible that Pancreatin acts preferentially on unsaturated triglycerides. This was supported by our finding that added triolein was hydrolyzed and resynthesized nearly as rapidly as the endogenous triglycerides. On the other hand Pancreatin effected only a very slow hydrolysis of triolein when suspended in saline or when emulsified in Biochi~n. Biophys. ~4c/a, 46 (I96t) t()8 47 ~
475
SERUM LIPID TRANSFORMATIONS
saline with Tween 2o. By way of comparison it m a y be noted that lipoprotein lipase has been shown to act more rapidly on triolein than on saturated triglycerides ~. Fresh serum differed from stored serum in that only in fresh serum were added D- and L-~,fi-diolein acted upon without added Pancreatin (Fig. 7). However, with both fresh and stored sera the added dioleins were acted upon when Pancreatin was also supplied. I t is noteworthy that both the D and L-s, fi-dioleins, although being pure synthetic compounds, showed 2 components when chromatographed* (see Fig. 7)- It is of further interest that these diglyceride components were not acted upon in the same manner (see below).
©00
:.::
..)
0
c~
::)
()
C!
',~:
(:..J r.-7-~ c / ~
A
:.
0000
'k/_/S
~,4./s
B
C
r/-h ,,R-x ,,..4..// w...U D
E
,<-A //-~ ,
STD
Fig. 7. C h r o m a t o g r a m s showing the effect of h u m a n s e r u m (with and w i t h o u t added P a n creatin) on s y n t h e t i c dioleins. To a series of six t u b e s were added o. 4 ml of fresh pooled h u m a n serum. The following additions were t h e n m a d e : A, 500 /~g of D-~,j~-diolein plus 500 /~g of P a n c r e a t i n in ioo ,ul of saline; B, 5oo/~g of L-,¢,/5-diolein plus 500 /~g of P a n c r e a t i n in ioo F,1 of saline; C, 5o0 /~g of D-~,j~-diolein plus IOO ~u] of saline; D, 500/~g of L-~,~-diolein plus IOO /~l of saline; E, 5o0 # g of Pancreatin in IOO gl of saline; F, IOO gl of saline (control). The dioleins were first suspended in the s e r u m by shaking on a t u b e buzzer before the enzyme was added. The t u b e s were incubated at r o o m t e m p e r a t u r e for 6 h a n d at 5 ° for 18 h. Aliquots (20 FI) were r e m o v e d for p a p e r c h r o m a t o graphic analysis at several time intervals. This figure shows the results obtained at the end of the incubation period. The identification of the s p o t s is the s a m e as t h a t given in Fig. 3. I n this e x p e r i m e n t the f a t t y acids (spot 3) released in the s y s t e m s A and B trailed into and below the cholesterol s p o t (spot 2). I n addition the dioleins (spot I) m i g r a t e d as 2 components. STD., 20 # g of s t a n d a r d n-o¢,jS-diolein which w a s applied to the p a p e r in chloroform*. L-~, jS-diolein gave the s a m e p a t t e r n as the Disomer. C h r o m a t o g r a p h i c analysis of the phosp h a t i d e s in these s y s t e m s is given in Fig. 8.
A
B
C
O
E
/~/~ F
STD.
Fig. 8. C h r o m a t o g r a m s showing the effect of synthetic dioleins on the Pancreatin-catalyzed hydrolysis of s e r u m lecithin. The s y s t e m s A - F are the same as those given in Fig. 7. At the end of the incubation period 2o /~1 were rem o v e d for p a p e r c h r o m a t o g r a p h i c analysis. C h r o m a t o g r a p h y was carried o u t in diisobutylk e t o n e - a c e t i c - a c i d - w a t e r (4° : 20 : 3). The identification of the spots is as follows: o, proteins; I, lysolecithin plus inosito] p h o s p h a t i d e (in A a n d B this s p o t is p r e d o m i n a n t l y lysolecithin as evidenced b y the staining test with R h o d amine 6G in which lysolecithin stains yelloworange and inositol p h o s p h a t i d e stains bluepurple) 2, sphingomyelin; 3, lecithin; 4, phosp h a t i d y l e t h a n o l a m i n e ; 5, m i x t u r e of the nonp h o s p h a t i d e s ; STD., 20 t*g of D-~,fl-diolein. The m a r k e d decrease in lecithin and c o n c o m i t a n t increase in lysolecithin in A and B is evident. The small c o m p o n e n t below spot i is unidentified. The lysolecithins which were formed were p r e d o m i n a n t l y s a t u r a t e d derivatives w h e r e a s the lecithins contained a high c o n t e n t of uns a t u r a t e d f a t t y acids. The liberated f a t t y acids in s y s t e m s A and B were highly u n s a t u r a t e d and m o v e d near the solvent f r o n t with the other n o n - p h o s p h a t i d e s .
* See footnote p. 473-
Biochim. Biophys. Acta, 46 (1961) 468-478
476
G.V. MARINETTI
In fresh serum without added Pancreatin the D-and L-dioleins slowly disappeared (6 h at 22-25 ° and 18 h at 5 °) (Fig. 7). Very little or no free fatty acids were observed on the chromatograms. The triglycerides appeared to increase but not enough to account for the simultaneous decrease in the dioleins. The other plasma lipids (lecithins, sphingomyelin, lysolecithin, cholesterol and cholesterol esters) did not undergo any detectable changes. A small amount of monoglycerides was formed. When the same fresh serum has Pancreatin added the D- and L-diolein are acted upon more rapidly (Fig. 7). Free fatty acids are released (observed on chromatograms within 6 h) and there is a concomitant hydrolysis of endogenous triglycerides. The endogenous triglycerides require 4 h for complete hydrolysis whereas in a similar "control" system without added diolein the triglycerides are completely hydrolyzed within 5 min. Either the added diolein inhibits the Pancreatin hydrolysis of endogenous triglyceride or the added diolein is being converted to triglyceride at the same time that the endogenous triglycerides are being hydrolyzed. The latter situation would make it appear that the endogenous triglycerides were being more slowly hydrolyzed than in the "control". It is of further interest however, that at the same time these changes occur, the lecithin is hydrolyzed to lysolecithin (Fig. 8). These changes are observed within 6 h incubation but are more pronounced by 24 tl. It is important to note that with the amount of Pancreatin used in this experiment (5oo/~g /o.5 ml serum) no lecithin is broken down to lysolecithin in the absence of added diglycerides. Hence, as was the case with I-monopalmitin, either the mono- or diglycerides stimulate pancreatic lecithinase A or transesterification is occurring between the phospholipids and glycerides such that f a t t y acids from lecithin are being utilized for the conversion of monoglycerides to diglycerides (or triglycerides) and diglycerides to triglycerides. When this latter system containing added diolein and Pancreatin is incubated for 18 h at 5 ° the triglycerides which were initially hydrolyzed are resynthesized. At no time is there any appreciable amount of monoglycerides formed. No detectable changes are observed in the other lipid fractions (cholesterol, cholesterol esters, and sphingomyelin) during this time. In the system which contains fresh serum and added diolein but without Pancreatin the slower moving diolein component is more rapidly metabolized. Contrariwise, in the system containing fresh serum, added diolein and added Pancreatin, the faster moving diolein component is more rapidly metabolized (Fig. 7). The diglyceride formed b y Pancreatin hydrolysis of endogenous serum triglyceride moves identically as the slower moving diolein. Since pancreatic lipase has been shown to be specific for the a-linked fatty acid 1° this supports our belief that the slower moving diolein is the a,~-isomer. The faster moving diolein would therefore be the a,~'-isomer. Appropriate controls were run at all times in which serum alone (plus saline) was incubated for the same time intervals. At no time were any significant changes observed in the serum lipids of these controls during incubation up to 24 h. Inhibition studies showed that sodium fluoride, iodoacetate and Versene at a level of io to 2o/,g /o.5 ml of serum did not inhibit pancreatic lipase but did effect a marked inhibition at a level of 2oo-25o ~g/o.5 ml serum. DISCUSSION
The experiments reported in the paper are mainly qualitative in nature but clearly show that Pancreatin contains an active lipase which rapidly hydrolyzes serum lipoBiochim. Biophys. Acla, 46 (196t) 468-478
SERUM LIPID TRANSFORMATIONS
477
protein-bound triglycerides to diglycerides but the diglycerides are resynthesized to triglycerides. The diglycerides are believed to remain bound to protein since the serum remains clear during the reaction. The d a t a furthermore indicate t h a t transesterification between the various serum lipids m a y occur. These reactions are as follows: Triglycerides ~ Diglyceride + Fatty acid
(I)
Lecithin ---~ Lysolecithin + Fatty acid
(2)
Lecithin + Diglyceride---~ Triglyceride + Lysolecithin
(3)
Lecithin + Monoglyceride--+ Diglyceride + Lysolecithin
(4)
Reaction (I) is rapid and reversible and catalyzed b y pancreatic lipase. Reaction (2) when catalyzed b y venom phospholipase A is very rapid but reaction (2) when catalyzed b y pancreatic phospholipase A is m u c h slower and requires a m u c h higher concentration of Pancreatin. However the d a t a indicate t h a t reaction (2) when catalyzed b y Pancreatin is stimulated b y addition of monoglycerides and diglycerides. This stimulation m a y occur as shown in reactions (3) and (4). These postulated transesterification reactions (3) and (4) are supported b y the finding t h a t the conversion of lecithin to lysolecithin is markedly stimulated b y the addition of monopalmitin with a concomitant increase in diglycerides. Although diolein (Fig. 7) stimulated the Pancreatin hydrolysis of serum lecithin to lysolecithin it was not ascertained whether the diolein had been converted to triglycerides. During the early period of these transformations very little or no free f a t t y acids can be detected on chromatograms. Hence the f a t t y acids are believed to be " b o u n d " to protein or to some other serum components. This "binding" of the liberated f a t t y acids m a y or m a y not be the same as FFA*. W h e n the serum reaction mixture is put on the chromatographic paper and the applied spots are then treated with methanol, no free f a t t y acids are liberated b y this procedure. This was done in order to rule out the possibility t h a t the chromatographic solvent heptane-diisobutylketone (96:6) was not polar enough to rupture the f a t t y acid-protein bonds. Yet it is of interest t h a t heptane-diisobutyl ketone (96:6) can free most of the serum lipoprotein-bound triglycerides, diglycerides, cholesterol, and cholesterol esters**. Only in the serum obtained by clotting Versene treated plasma with excess calcium ions were the f a t t y acids liberated b y Pancreatin hydrolysis of serum triglycerides observed on the chromatograms in appreciable amount. In this case the liberated f a t t y acids m a y exist as Ca salts and these cannot be converted to a " b o u n d " form as rapidly as the free acids. When Ii-14Clstearic acid and [i-14Cloleic acid were added to serum these were not incorporated into the triglycerides under conditions when triglycerides were being hydrolyzed and resynthesized b y Pancreatin (Figs. 5 and 6). It m a y be possible t h a t other means can be found to bring about this incorporation. However, a very slight incorporation of 14C was observed in both cases into the cholesterol ester fraction. The use of appropriate labeled triglycerides, diglycerides, monoglycerides and * FFA signifies the albumin bound fatty acids which are believed to play a role in lipid transport. Some of the neutral lipids remain at the origin. Chromatograms can first be run in chloroformmethanol (i :i) for 2- 3 cm, dried for IO min and then run in the heptane-ketone system. This liberates all the neutral lipids and is important for quantitative work. * *
Biochim. Biophys. Acts, 46 (1961) 468-478
478
G . V . MARINETTI
lecithin may elucidate the postulated transesterification reactions given above. Such transesterification reactions would be in harmony with the other well established transfer reactions (transamination, transglucosidation, transamidation, transmethylation) which have been shown to occur in living ceils and if confirmed would demonstrate new metabolic pathways for interconversion of glycerides and phosphatides. ACKNOWLEDGEMENT
This work was supported in part by funds from a grant H 2063 from the National Heart Institute, National Institute of Health, U.S. Public Health Service. REFERENCES 1 G. V. MARINETTI AND E. STOTZ,Biochim. Biophys. Acta, 37 (196o) 571. H. WHITTCOFF, The Phosphatides, Reinhold Publ. Corp. N. Y. 1951, p. lO5. 3 H. POTTEVlN, Compt. rend., 136 (19o3) 767. 4 C. ARTOM AND L. REALE, Bull. soc. chim. biol., 18 (1936) 959. 5 S. BELFANTI, Fisiole reed. (Rome), 12 (1933) 821. 6 A. CONTARDI AND A. ERCOLI, Biochem. Z., 261 (1933) 275. 7 V. GRONCI-II, Sperimentale, 90 (1936) 233. 8 D. J. HANAHAN, J. Biol. Chem., 195 (1952) 199. 9 B. SnoRE, O. M. COLVlN AND V. G. SHORE, Biochim. Biophys. Acta, 36 (1959) 563 • 10 F. H. MATTSON AND L. W. BECK, J, Biol. Chem., 214 (1955) 115.
Biochim. Biophys. Acta, 46 (1961) 468-478