- Email: [email protected]
The effect of intravenous papain on the plasma lipids and fatty acids of adult rabbits
533
JOURNAL OF ATHEROSCLEROSIS RESEARCH
THE EFFECT OF INTRAVENOUS PAPAIN* ON THE PLASMA LIPIDS AND FATTY ACIDS OF ADULT RABBITS
T. T. TSALTAS D~partment of
AND
C. E. LUTTON**
Pathology, Jefferson Medical College, Philadelphia, Pa. (U. S. A.j
(Received November 19th, 1964)
INTRODUCTION Intravenous injection of aqueous papain solution has been reported to produce lipemia in adult rabbits 1 •2 . TSALTAS3 described a metaplasia of aortic connective tissue to cartilage and bone following the intravenous administration of papain in rabbits. Since the changes in plasma lipid concentrations have been implicated in the etiology of atherosclerosis, we thought it of interest to investigate in detail the lipemia produced by the intravenous injection of papain in rabbits. MATERIALS AND METHODS A total of 167 animals of both sexes were utilized in this study. Sixty-two animals were used as controls and 105 animals received papain injections. They all were adult albino New Zealand rabbits weighing between 3.6 and 4.8 kg. The animals were maintained on Purina Rabbit Chow for at least seven days prior to experimentation. No food or water restrictions were imposed during the course of the study. Papain was injected in the form of a 2 % aqueous solution prepared from a highly purified powdered enzyrne*. The same papain powder was used throughout these experiments, kept at 4°C in a vacuum dessicator. Its activity was 1400 proteolytic Units/mg of powder or 200 Winthrop Milk Clotting Units/g of powder. Other commercially available papains were tested and it was found that crude, purified, and crystalline papain produced the same results on the blood lipids and the cartilage matrix of the animals. It was decided to use the purified papain prepared by the Sterling-Winthr'op Laboratories, because it gave more consistent results than the various commercially available papain preparations, and because the purified papain was much easier to handle than the relatively unstable crystalline papain. The very
Supported by USPHS Research Grant H-05647. * Papain powder was kindly donated by the Sterling-Winthrop Research Lab., Rensselear, New York. ** Post Doctoral Fellow, USPHS, NIH.
J. Atheroscler. Res., 5 (1965) 533-539
534
T. T. TSALTAS, C. E. LUTTON
high cost of the crystalline papain was also an additional factor. The injectable solution was prepared by placing 1 g of the purified powdered enzyme in 50 ml of 0.15 M solution of sodium chloride. The mixture was agitated for 30 min on a magnetic stirrer, transferred in presterilized Boerner centrifugal filters and centrifuged for 20 min at 800 X g. This produced a sterile, clear, straw-colored solution that was used for injections within 6 h after preparation. The animals were injected with 15 mg of papain/kg body weight. The control animals received an equal volume of 0.15 M saline. Blood for plasma lipid analysis was obtained from the central ear artery in a vacutainer apparatus containing 0.2 ml of 5 % sodium disequestrin. Blood for chromatographic determinations was obtained also in disequestrin solution by exsanguinating the animals by cardiac puncture. The plasma was immediately separated from the formed elements of the blood by centrifugation at 4°C for 20 min at 800 x g. All chemicals used were of reagent grade and all solvents were redistilled prior to use. Plasma lipoprotein lipase (LPL) activity was determined immediately following plasma separation by the method of KORN4. Total fatty acid (TFA) analyses were carried out on chloroform-methanol (2 : 1) extracts of plasma as described by ALBRINK5. Plasma unesterified fatty acid (UFA) concentrations were determined by the method of DOLE6. Chromatographic fractionation and subsequent analyses of the plasma lipids required the precipitation of the plasma proteins and lipids as described by SOMOGYI? Lipids were extracted from the precipitate with alcohol-ether (3 : 1), and appropriate aliquots were prepared for cholestero1 8 (C), phospholipid9 (PL), and total lipid (TL) determinations. Total lipids were determined gravimetrically. Glyceride concentrations were calculated by difference from the other fractions. Plasma for fatty acid analysis by chromatography was extracted directly with BLooR's solution, taken to dryness under vacuum, and purified according to the method described by FOLCHIO. The PL fraction was separated from the other lipid fractions on silicic acid columns following elution scheme B as described by HIRSCH AND AHRENSll. The solvents containing the other lipid fractions were pooled, taken to dryness, and reapplied to florisil columns to be eluted as described by CARROLL12 into 6 fractions; cholesterol esters (CE), triglycerides (TG) , free cholesterol (FC), diglycerides (DG), monoglycerides (MG), and unesterified fatty acids (UFA). The fatty acid composition of each lipid fraction was determined by gas-liquid chromatography (GLC) following methyl interesterification as suggested by FARQUHAR13. Analyses were carried out on a F-M model-609 gas-liquid chromatograph equipped with 5 foot by 1.25 mm aluminum columns. Ethylene glycol adipate and apiezon "L" were employed as the stationary phases. The columns were maintained at 197°C with a helium inlet pressure of 20 pounds/squ. in. The GLC was equipped with a flame ionization detector. Calculations of the areas under the curves were done by triangulation, and the results reported are as per cent total free fatty acid present.
J.
Atheroscler. Res., 5 (1965) 533-539
~
TABLE I
"'1 "'1
VALUES OF PLASMA LIPID CHANGES (MG/100 ML) PRODUCED BY SERIAL BLEEDINGS (30 ANIMALS REPORTED)
Fraction
Control
2 Hours
8 Hours
24 Hours
48 Hours
~
(") ...,
72 Hours
96 Hours
0
"'1 >-<
Tota11ipids 288.0 Glycerides 144.0 Phospholipids 75.0 Total cholesterol 40.0 Free cholesterol 8.8 Cholesterol esters 51.0 Unesterified fatty acids 3.8 Lipoprotein lipase 3.9
*t<
± 57 ± 44 ± 19 ± 9 ± 0.7 ± 13 ± 1.2 ± 0.8
279.0 133.0 95.0 47.0 11.2 60.0 3.8 3.6
± 51 ± 76 ± 39 ± 12 ± 1.1 ± 16 ± 1.5 ± 1.2
271.0 184.0 82.0 47.0 9.0 62.0 4.2 8.1
± 62 ± 75 ± 34 ± 10 ± 1.3 ± 13 ± 1.0 ± 2.7
288.0 128.0 103.0 48.0 13.0 58.0 3.4
-
± 82 ± 34 ± 36 ± 11 ± 1.1 ± 12 ± 1.7 -
300.0 124.0 95.0 50.0 10.1 66.0 3.7
-
± 82 ± 31 ± 17 ± 9 ± 0.8 ± 14 ± 1.0 -
413.0 191.0 116.0 59.0 16.7 70.0 3.6 3.3
± 120* ± 59* ± 24* ± 15 ± 0.8 ± 16 ± 0.8 ± 1.2
436.0 226.0 126.0 58.0 9.9 70.0 4.2 4.1
± 90* ± 64* ± 25* ± 15 ± 1.0 ± 15 ± 0.8 ± 2.0
~
'1:1
> '1:1 > >-<
z
0
Z
'1:1 t'"'
> (fl
~
> t'"' >-< '1:1
0.05.
>-<
tJ
(fl
':-<
TABLE II
> Z
VALUES OF SERIAL PLASMA LIPID CHANGES (MG/100 ML) FOLLOWING INTRAVENOUS PAPAIN (47 ANIMALS REPORTED)
"'1
Fraction
tJ
Control
2 Hours
8 HoU1's
24 Hours
48 Hours
> ..., ..., 72 Hours
96 Hours
;:,.
;;:.
'"cl Co
~
:"
~
'"
!'>
<:II
..... ttl
'"
..::!!
Tota11ipids 271.0 Glycerides 109,0 Phospholipids 104.0 Total cholesterol 35.0 Free cholesterol 10.0 Cholesterol esters 45.0 Unesterified fatty acids 3.4 Lipoprotein lipase 3.0
I
<:II
V>
± 54 ± 44 ± 35 ± 17 ± 3 ± 22 ± 1.3 ± 2.3
295.0 134.0 97.0 32.0 11.0 48.0 5.3 21.0
± 57 ± 49 ± 26 ± 15 ± 3 ± 22 ± 2.7 ± 9.6
278.0 134.0 89.0 39.0 11.0 38.0 5.6 8.1
± 51 ± 60 ± 29 ± 14 ± 3 ± 17 ± 2.8 ± 2.6
348.0 191.0 95.0 45.0 13.0 43.0 5.8 3.4
± 61 ± 61 * ± 22 ± 14 ± 4 ± 21 ± 3.1 ± 1.6
429.0 248.0 114.0 47.0 17.0 46.0 4.3 2.2
± 77* ± 66** ± 30 ± 14 ± 4 ± 17 ± 1.6 ± 0.8
547.0 321.0 148.0 47.0 15.0 59.0 4.4 1.6
± 82** ± 57** ± 30* ± 14 ± 3 ± 25 ± 1.9 ± 1.0
486.0 271.0 132.0 58.0 22.0 56.0 4.7 3.6
± 79* ± 73* ± 28* ± 18 ± 4 ± 19 ± 1.6 ± 2.0
>-<
tJ
(fl
0
"'1
> tJ c: ~ i:li > td td
>-< ..., (fl
<:II
V> V>
><
>
(")
* t ** t
< <
0.05. 0.02.
CJ1
<:0:1
CJ1
536
T. T. TSALTAS, C. E. LUTTON
RESULTS
All animals receiving papain demonstrated a slight dyspnea immediately following the injection. They remained quiet in their cages for 30-45 min after the injection demonstrating a moderate degree of hyperventilation. They returned to their normal activity and breathing within 12-24 h after papain. Animals were autopsied at 24 h intervals after papain to examine any gross and microscopic changes as the result of papain injection. The most consistent findings were from the lungs. They consisted of moderate pulmonary edema and small areas of hemorrhage at the periphery of the lungs. These changes occurred with higher frequency at 72 and 96 h post papain. All animals recovered spontaneously. No attempt was made to evaluate the foci of myocardial necrosis produced by papain since they have been reported previously in detail by other authors 14 . Before any data could be obtained on the effect of papain on the plasma lipids, it was thought necessary to establish the effect that repeated bleeding produces on the plasma lipids of normal rabbits. Table I shows the results obtained by such serial bleedings on the plasma lipid fractions. Changes begin to occur at the 48 hour period and become statistically significant by 72 hours. The most notable change occurs in the TL, G, and PL fractions, whereas no changes are noted in the UFA fraction or LPL activity. The change in glyceride is a cumulative effect of the TG, DG, and MG fractions. Table II demonstrates the changes produced in the plasma lipid fractions by serial bleedings, following the intravenous administration of papain. In this case, changes are noted as early as 24 hours after the injection of papain. A significant increase in the TL, G, and PL fraction concentrations was again noted. The increase in the glyceride concentration was significantly higher than that produced by bleeding alone. The increases produced in the PL, TC, FC, and CE were similar to those found when the animals were bled, but not receiving papain. Lipoprotein lipase activity increased significantly at the 2 hour post papain bleeding interval and remained increased at the 8 hour interval, but returned to control levels by 24 hours. No other increase was found at following bleeding intervals. Unesterified fatty acid concentration, although not significantly elevated, demonstrated a tendency toward increased concentrations at the 2, 8, and 24 hour bleeding interval with return toward control values at 48, 72, and 96 hours. The major changes in the plasma lipid concentrations occurred at 72 hours post papain. It was at this time interval that changes were noted in the plasma lipid levels of the control animals following serial bleedings. In order to study the effect of papain without interferences from a bleeding lipemia, rabbits were injected with papain and bled only at the 72 hour interval. Table III shows the effect of papain on plasma lipids at 72 hours without interference from the daily bleedings. In this case, the only rise observed to be statistically significant was the glycer]. Atheroscler. Res., 5 (1965) 533-539
EFFECT OF LV. PAPAIN ON PLASMA LIPIDS AND FATTY ACIDS OF ADULT RABBITS
537
ide fraction concentration. The other lipid fractions did not vary significantly from the control levels. Twenty-one fatty acids were found present in rabbit plasma, most of which
TABLE III VALUES OF PLASMA LIPIDS (MG/lOO ML) FOLLOWING INTRAVENOUS PAPAIN AND A SINGLE BLEEDING
(40
ANIMALS REPORTED).
Fraction
Control
72 Hours
Difference
Glycerides Phospholipids Total cholesterol Free cholesterol Cholesterol esters Unesterified fatty acids
124.0 95.0 35.0 10.5 47.0
± ± ± ± ±
41 28 14 3.1 20
174 102.0 32.4 10.6 36.2
± ± ± ± ±
44 31 13 2.7 16
50.0** 7.0 -2.6 0.1 -10.8
3.8
±
2.1
2.9
±
2.0
0.3
** t< 0.02.
were present in trace amounts. These consisted of 8:0, 10:0, 12:0, 13:0, 14:0, 14:1, 15:0, iso 15:0, 16:0, 16:1, 17:0, iso 17:0, 18:0, 18:1, 18:2, 18:3, 20:0, 20:1, 20:2, 20:3, 20:4. Since palmitic, palmholeic, stearic, oleic, and linoleic acids composed more than 90 % of the plasma total fatty acids, only the actual concentration of these fatty acids will be presented in the following tables. Table IV shows the comparison of the total lipid fatty acids of control animals with that of animals receiving papain. It can be seen that bleeding did not alter significantly the fatty acid concentration of the total lipids. Similarly, intravenous papain did not produce any statistically significant alterations in the fatty acid composition of the total lipids as compared to the control animals. Although the fatty acid composition of the total lipid extract was not effected to any significant extent by the injected papain, it was thought necessary to invest~gate if any possible alteration had occurred in the fatty acid composition of the individual plasma lipid fractions. The reason for this was that changes might have occurred in these fractions that counterbalanced each other when the total lipid extract was analysed for fatty acids. The results of the detailed fatty acid analysis by means of GLC showed that no statistically significant alterations in the fatty acid composition of the plasma lipid fractions could be demonstrated between control and 72 hour post papain samples. DISCUSSION
The findings reported here substantiate the earlier report by
J. Atheroscler. Res.,
BOGGS AND
5 (1965) 533-539
538
T. T. TSALTAS, C. E. LUTTON
MORRIS15 that daily bleedings produce lipemia in rabbits. The detailed fractionation and analysis of the plasma lipids of this lipemia as well as the fatty acid composition of the lipid fractions were undertaken and are presented in this report. This became necessary because if we were to evaluate in detail the lipemia produced by the injection of papain in rabbits, it was essential to have a firm basis of control. The data presented here indicate that the injection of papain in rabbits produces a significant elevation of the plasma lipids that reaches its maximal effect 72-96 hours after the injection. They also indicate that while repeated bleeding contributes to the magnitude of this lipemia, the injection of papain alone is capable of inducing statistically significant elevation of the plasma lipids. TABLE IV MAJOR FATTY ACIDS PRESENT IN PLASMA LIPIDS FOLLOWING PAPAIN INJECTION
(50
Fatty acid
Sample
Control
2 Hours
1(; : 0
Control Papain Control Papain Control Papain Control Papain
25.3 ± 1.4
23.8 22.5 11.2 12.0 22.6 24.8 33.2 32.0
18 : 0 18: 1 18 : 2
12.2 ± 1.6 21.5 ± 2.7 31.3±2.1
± ± ± ± ± ± ± ±
1.2 1.3 1.4 1.6 2.1 3.4 2.9 3.1
ANIMALS REPORTED)
8 Hours
24 Hours
48 Hours
72 Hours
25.1 24.4 11.7 14.3 22.9 25.3 31.2 28.5
25.6 25.2 12.0 12.5 24.2 24.9 30.1 30.6
23.8 23.8 9.4 11.6 24.0 24.9 33.1 32.7
25.7 23.8 10.1 12.4 22.8 24.9 32.1 32.0
± ± ± ± ± ± ± ±
1.6 1.2 0.9 2.0 2.4 3.0 1.8 2.2
± ± ± ± ± ± ± ±
1.4 1.6 1.5 1.2 2.1 2.6 2.2 2.5
± ± ± ± ± ± ± ±
0.9 1.4 1.0 1.5 1.9 3.0 2.4 1.8
± ± ± ± ± ± ± ±
All values shown are in per cent of total fatty acids present in the extract.
It is of interest to note that the change in plasma lipid concentration involves primarily the glyceride fraction, with the other lipids participating to a minor extent in the lipemia at levels that were found statistically not significant. The changes in the fatty acid composition of all lipid fractions in animals receiving papain were minimal and statistically not significant. This was also true for the fatty acids of the lipids present in the lipemia produced after repeated bleedings. As for the possible explanation of the papain-induced hyperlipemia, no concrete explanation can be offered at the present time. It is quite possible that the entire phenomenon is the result of a stress reaction to the injection of papain. This could be supported by the pulmonary edema and hemorrhage found at autopsy in animals receiving papain, and of the time coincidence of the maximal plasma glyceride elevation and the histologic finding mentioned above. Since this manuscript was submitted for publication, it has come to our attention a brief communication by HIRSCH et al. 2o on studies of a hyperlipemia produced in rabbits by the injection of pituitary gland extracts. This hyperlipemia is very similar to the one produced by papain and reported here. It would seem possible that the papain lipemia may be the result of a pituitary gland stimulation,
J.
Atheroscler. Res., 5 (1965)
533~539
1.6 0.8 2.1 1.7 2.0 2.2 2.5 2.0
EFFECT OF LV. PAPAIN ON PLASMA LIPIDS AND FATTY ACIDS OF ADULT RABBITS
539
since we have observed evidence of stress in our experimental animals following the injection of papain. This would come to a general agreement with the available evidence that the pituitary gland hormones playa major role in the "stress reaction" of experimental animals, and that rabbits receiving steroids react with plasma glyceride elevation 16 - 19 . SUMMARY
Papain administered intravenously in adult rabbits produces a rIse in the plasma glyceride concentration without significantly altering the concentrations of the other lipid fractions. The injection of papain does not alter the fatty acid composition of the plasma total lipids or the individual lipid fractions. This lipemia is accompanied by moderate pulmonary edema and other stress phenomena. This stress reaction of the injected papain could be considered as the major cause of the lipemia. REFERENCES 1 J. H. BRYANT,
1. G.
LEDER AND DEWITT STETTEN, JR.,
Arch. Biochem. Biophys., 76 (1958)
122-130.
Federation Proc., 20 (1961). Nature, 196 (1962) 1006-1007. E. D. KORN, Methods Biochem. Anal., 7 (1957). M. J. ALB RINK, ] . Lipid Res., 1 (1959) 53. V. P. DOLE, ] . Clin. Invest., 35 (1956) 150. M. SOMOGYI, J. Biol. Chem., 86 (1930) 658. W. M. SPERRY AND M. WEBB, J. Biol. Chem., 187 (1950) 97. C. P. STEWART AND E. B. HENDRY, Biochem. j., 29 (1935) 1683. J. FOLCH, M. LEES AND G. H. SLOANE-STANLEY, J. Biol. Chern., 22 (1915) 133. J. HIRSCH AND E. H. AHRENS, J. Biol. Chem., 233 (1958) 311. K. K. CARROLL, J. Lipid Res., 2 (1961) 135. I. W. FARQUHAR, W. INSULL, JR., P. ROSEN, W. STOFFEL AND E. H. AHRENS, JR., Nutr. Abstr. Rev., 17 (1959) Supp!. 1. A. KELLNER AND T. ROBERTSON, J. Exptl. Med., 99 (1954) 387. T. R. BOGGS AND R. S. MORRIS, j. Exptl. Med., 11 (1902) 553. D. RUDIMAN AND F. SEIDMAN, Circulation, 18 (1958) 486. K. E. SUSSMAN, E. SHAFER AND D. STEINBERG, Circulation, 18 (1958) 486. A. R. RICH, J. H. COCHRAN AND D. C. MCBOON, Bull. johns Hopkins Hosp., 88 (1951) 101. D. ADLERSBERG, S. R. DRACHM AN AND H. E. SCHAFER, Circulation,4 (1951) 475. R. L. HIRSCH et al., Circulation, 30 (1964) Supp!. 3.
T. TSALTAS, 3 T. TSALTAS, 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
J.!Atheroscler. Res., 5 (1965) 533-539
JOURNAL OF ATHEROSCLEROSIS RESEARCH
THE EFFECT OF INTRAVENOUS PAPAIN* ON THE PLASMA LIPIDS AND FATTY ACIDS OF ADULT RABBITS
T. T. TSALTAS D~partment of
AND
C. E. LUTTON**
Pathology, Jefferson Medical College, Philadelphia, Pa. (U. S. A.j
(Received November 19th, 1964)
INTRODUCTION Intravenous injection of aqueous papain solution has been reported to produce lipemia in adult rabbits 1 •2 . TSALTAS3 described a metaplasia of aortic connective tissue to cartilage and bone following the intravenous administration of papain in rabbits. Since the changes in plasma lipid concentrations have been implicated in the etiology of atherosclerosis, we thought it of interest to investigate in detail the lipemia produced by the intravenous injection of papain in rabbits. MATERIALS AND METHODS A total of 167 animals of both sexes were utilized in this study. Sixty-two animals were used as controls and 105 animals received papain injections. They all were adult albino New Zealand rabbits weighing between 3.6 and 4.8 kg. The animals were maintained on Purina Rabbit Chow for at least seven days prior to experimentation. No food or water restrictions were imposed during the course of the study. Papain was injected in the form of a 2 % aqueous solution prepared from a highly purified powdered enzyrne*. The same papain powder was used throughout these experiments, kept at 4°C in a vacuum dessicator. Its activity was 1400 proteolytic Units/mg of powder or 200 Winthrop Milk Clotting Units/g of powder. Other commercially available papains were tested and it was found that crude, purified, and crystalline papain produced the same results on the blood lipids and the cartilage matrix of the animals. It was decided to use the purified papain prepared by the Sterling-Winthr'op Laboratories, because it gave more consistent results than the various commercially available papain preparations, and because the purified papain was much easier to handle than the relatively unstable crystalline papain. The very
Supported by USPHS Research Grant H-05647. * Papain powder was kindly donated by the Sterling-Winthrop Research Lab., Rensselear, New York. ** Post Doctoral Fellow, USPHS, NIH.
J. Atheroscler. Res., 5 (1965) 533-539
534
T. T. TSALTAS, C. E. LUTTON
high cost of the crystalline papain was also an additional factor. The injectable solution was prepared by placing 1 g of the purified powdered enzyme in 50 ml of 0.15 M solution of sodium chloride. The mixture was agitated for 30 min on a magnetic stirrer, transferred in presterilized Boerner centrifugal filters and centrifuged for 20 min at 800 X g. This produced a sterile, clear, straw-colored solution that was used for injections within 6 h after preparation. The animals were injected with 15 mg of papain/kg body weight. The control animals received an equal volume of 0.15 M saline. Blood for plasma lipid analysis was obtained from the central ear artery in a vacutainer apparatus containing 0.2 ml of 5 % sodium disequestrin. Blood for chromatographic determinations was obtained also in disequestrin solution by exsanguinating the animals by cardiac puncture. The plasma was immediately separated from the formed elements of the blood by centrifugation at 4°C for 20 min at 800 x g. All chemicals used were of reagent grade and all solvents were redistilled prior to use. Plasma lipoprotein lipase (LPL) activity was determined immediately following plasma separation by the method of KORN4. Total fatty acid (TFA) analyses were carried out on chloroform-methanol (2 : 1) extracts of plasma as described by ALBRINK5. Plasma unesterified fatty acid (UFA) concentrations were determined by the method of DOLE6. Chromatographic fractionation and subsequent analyses of the plasma lipids required the precipitation of the plasma proteins and lipids as described by SOMOGYI? Lipids were extracted from the precipitate with alcohol-ether (3 : 1), and appropriate aliquots were prepared for cholestero1 8 (C), phospholipid9 (PL), and total lipid (TL) determinations. Total lipids were determined gravimetrically. Glyceride concentrations were calculated by difference from the other fractions. Plasma for fatty acid analysis by chromatography was extracted directly with BLooR's solution, taken to dryness under vacuum, and purified according to the method described by FOLCHIO. The PL fraction was separated from the other lipid fractions on silicic acid columns following elution scheme B as described by HIRSCH AND AHRENSll. The solvents containing the other lipid fractions were pooled, taken to dryness, and reapplied to florisil columns to be eluted as described by CARROLL12 into 6 fractions; cholesterol esters (CE), triglycerides (TG) , free cholesterol (FC), diglycerides (DG), monoglycerides (MG), and unesterified fatty acids (UFA). The fatty acid composition of each lipid fraction was determined by gas-liquid chromatography (GLC) following methyl interesterification as suggested by FARQUHAR13. Analyses were carried out on a F-M model-609 gas-liquid chromatograph equipped with 5 foot by 1.25 mm aluminum columns. Ethylene glycol adipate and apiezon "L" were employed as the stationary phases. The columns were maintained at 197°C with a helium inlet pressure of 20 pounds/squ. in. The GLC was equipped with a flame ionization detector. Calculations of the areas under the curves were done by triangulation, and the results reported are as per cent total free fatty acid present.
J.
Atheroscler. Res., 5 (1965) 533-539
~
TABLE I
"'1 "'1
VALUES OF PLASMA LIPID CHANGES (MG/100 ML) PRODUCED BY SERIAL BLEEDINGS (30 ANIMALS REPORTED)
Fraction
Control
2 Hours
8 Hours
24 Hours
48 Hours
~
(") ...,
72 Hours
96 Hours
0
"'1 >-<
Tota11ipids 288.0 Glycerides 144.0 Phospholipids 75.0 Total cholesterol 40.0 Free cholesterol 8.8 Cholesterol esters 51.0 Unesterified fatty acids 3.8 Lipoprotein lipase 3.9
*t<
± 57 ± 44 ± 19 ± 9 ± 0.7 ± 13 ± 1.2 ± 0.8
279.0 133.0 95.0 47.0 11.2 60.0 3.8 3.6
± 51 ± 76 ± 39 ± 12 ± 1.1 ± 16 ± 1.5 ± 1.2
271.0 184.0 82.0 47.0 9.0 62.0 4.2 8.1
± 62 ± 75 ± 34 ± 10 ± 1.3 ± 13 ± 1.0 ± 2.7
288.0 128.0 103.0 48.0 13.0 58.0 3.4
-
± 82 ± 34 ± 36 ± 11 ± 1.1 ± 12 ± 1.7 -
300.0 124.0 95.0 50.0 10.1 66.0 3.7
-
± 82 ± 31 ± 17 ± 9 ± 0.8 ± 14 ± 1.0 -
413.0 191.0 116.0 59.0 16.7 70.0 3.6 3.3
± 120* ± 59* ± 24* ± 15 ± 0.8 ± 16 ± 0.8 ± 1.2
436.0 226.0 126.0 58.0 9.9 70.0 4.2 4.1
± 90* ± 64* ± 25* ± 15 ± 1.0 ± 15 ± 0.8 ± 2.0
~
'1:1
> '1:1 > >-<
z
0
Z
'1:1 t'"'
> (fl
~
> t'"' >-< '1:1
0.05.
>-<
tJ
(fl
':-<
TABLE II
> Z
VALUES OF SERIAL PLASMA LIPID CHANGES (MG/100 ML) FOLLOWING INTRAVENOUS PAPAIN (47 ANIMALS REPORTED)
"'1
Fraction
tJ
Control
2 Hours
8 HoU1's
24 Hours
48 Hours
> ..., ..., 72 Hours
96 Hours
;:,.
;;:.
'"cl Co
~
:"
~
'"
!'>
<:II
..... ttl
'"
..::!!
Tota11ipids 271.0 Glycerides 109,0 Phospholipids 104.0 Total cholesterol 35.0 Free cholesterol 10.0 Cholesterol esters 45.0 Unesterified fatty acids 3.4 Lipoprotein lipase 3.0
I
<:II
V>
± 54 ± 44 ± 35 ± 17 ± 3 ± 22 ± 1.3 ± 2.3
295.0 134.0 97.0 32.0 11.0 48.0 5.3 21.0
± 57 ± 49 ± 26 ± 15 ± 3 ± 22 ± 2.7 ± 9.6
278.0 134.0 89.0 39.0 11.0 38.0 5.6 8.1
± 51 ± 60 ± 29 ± 14 ± 3 ± 17 ± 2.8 ± 2.6
348.0 191.0 95.0 45.0 13.0 43.0 5.8 3.4
± 61 ± 61 * ± 22 ± 14 ± 4 ± 21 ± 3.1 ± 1.6
429.0 248.0 114.0 47.0 17.0 46.0 4.3 2.2
± 77* ± 66** ± 30 ± 14 ± 4 ± 17 ± 1.6 ± 0.8
547.0 321.0 148.0 47.0 15.0 59.0 4.4 1.6
± 82** ± 57** ± 30* ± 14 ± 3 ± 25 ± 1.9 ± 1.0
486.0 271.0 132.0 58.0 22.0 56.0 4.7 3.6
± 79* ± 73* ± 28* ± 18 ± 4 ± 19 ± 1.6 ± 2.0
>-<
tJ
(fl
0
"'1
> tJ c: ~ i:li > td td
>-< ..., (fl
<:II
V> V>
><
>
(")
* t ** t
< <
0.05. 0.02.
CJ1
<:0:1
CJ1
536
T. T. TSALTAS, C. E. LUTTON
RESULTS
All animals receiving papain demonstrated a slight dyspnea immediately following the injection. They remained quiet in their cages for 30-45 min after the injection demonstrating a moderate degree of hyperventilation. They returned to their normal activity and breathing within 12-24 h after papain. Animals were autopsied at 24 h intervals after papain to examine any gross and microscopic changes as the result of papain injection. The most consistent findings were from the lungs. They consisted of moderate pulmonary edema and small areas of hemorrhage at the periphery of the lungs. These changes occurred with higher frequency at 72 and 96 h post papain. All animals recovered spontaneously. No attempt was made to evaluate the foci of myocardial necrosis produced by papain since they have been reported previously in detail by other authors 14 . Before any data could be obtained on the effect of papain on the plasma lipids, it was thought necessary to establish the effect that repeated bleeding produces on the plasma lipids of normal rabbits. Table I shows the results obtained by such serial bleedings on the plasma lipid fractions. Changes begin to occur at the 48 hour period and become statistically significant by 72 hours. The most notable change occurs in the TL, G, and PL fractions, whereas no changes are noted in the UFA fraction or LPL activity. The change in glyceride is a cumulative effect of the TG, DG, and MG fractions. Table II demonstrates the changes produced in the plasma lipid fractions by serial bleedings, following the intravenous administration of papain. In this case, changes are noted as early as 24 hours after the injection of papain. A significant increase in the TL, G, and PL fraction concentrations was again noted. The increase in the glyceride concentration was significantly higher than that produced by bleeding alone. The increases produced in the PL, TC, FC, and CE were similar to those found when the animals were bled, but not receiving papain. Lipoprotein lipase activity increased significantly at the 2 hour post papain bleeding interval and remained increased at the 8 hour interval, but returned to control levels by 24 hours. No other increase was found at following bleeding intervals. Unesterified fatty acid concentration, although not significantly elevated, demonstrated a tendency toward increased concentrations at the 2, 8, and 24 hour bleeding interval with return toward control values at 48, 72, and 96 hours. The major changes in the plasma lipid concentrations occurred at 72 hours post papain. It was at this time interval that changes were noted in the plasma lipid levels of the control animals following serial bleedings. In order to study the effect of papain without interferences from a bleeding lipemia, rabbits were injected with papain and bled only at the 72 hour interval. Table III shows the effect of papain on plasma lipids at 72 hours without interference from the daily bleedings. In this case, the only rise observed to be statistically significant was the glycer]. Atheroscler. Res., 5 (1965) 533-539
EFFECT OF LV. PAPAIN ON PLASMA LIPIDS AND FATTY ACIDS OF ADULT RABBITS
537
ide fraction concentration. The other lipid fractions did not vary significantly from the control levels. Twenty-one fatty acids were found present in rabbit plasma, most of which
TABLE III VALUES OF PLASMA LIPIDS (MG/lOO ML) FOLLOWING INTRAVENOUS PAPAIN AND A SINGLE BLEEDING
(40
ANIMALS REPORTED).
Fraction
Control
72 Hours
Difference
Glycerides Phospholipids Total cholesterol Free cholesterol Cholesterol esters Unesterified fatty acids
124.0 95.0 35.0 10.5 47.0
± ± ± ± ±
41 28 14 3.1 20
174 102.0 32.4 10.6 36.2
± ± ± ± ±
44 31 13 2.7 16
50.0** 7.0 -2.6 0.1 -10.8
3.8
±
2.1
2.9
±
2.0
0.3
** t< 0.02.
were present in trace amounts. These consisted of 8:0, 10:0, 12:0, 13:0, 14:0, 14:1, 15:0, iso 15:0, 16:0, 16:1, 17:0, iso 17:0, 18:0, 18:1, 18:2, 18:3, 20:0, 20:1, 20:2, 20:3, 20:4. Since palmitic, palmholeic, stearic, oleic, and linoleic acids composed more than 90 % of the plasma total fatty acids, only the actual concentration of these fatty acids will be presented in the following tables. Table IV shows the comparison of the total lipid fatty acids of control animals with that of animals receiving papain. It can be seen that bleeding did not alter significantly the fatty acid concentration of the total lipids. Similarly, intravenous papain did not produce any statistically significant alterations in the fatty acid composition of the total lipids as compared to the control animals. Although the fatty acid composition of the total lipid extract was not effected to any significant extent by the injected papain, it was thought necessary to invest~gate if any possible alteration had occurred in the fatty acid composition of the individual plasma lipid fractions. The reason for this was that changes might have occurred in these fractions that counterbalanced each other when the total lipid extract was analysed for fatty acids. The results of the detailed fatty acid analysis by means of GLC showed that no statistically significant alterations in the fatty acid composition of the plasma lipid fractions could be demonstrated between control and 72 hour post papain samples. DISCUSSION
The findings reported here substantiate the earlier report by
J. Atheroscler. Res.,
BOGGS AND
5 (1965) 533-539
538
T. T. TSALTAS, C. E. LUTTON
MORRIS15 that daily bleedings produce lipemia in rabbits. The detailed fractionation and analysis of the plasma lipids of this lipemia as well as the fatty acid composition of the lipid fractions were undertaken and are presented in this report. This became necessary because if we were to evaluate in detail the lipemia produced by the injection of papain in rabbits, it was essential to have a firm basis of control. The data presented here indicate that the injection of papain in rabbits produces a significant elevation of the plasma lipids that reaches its maximal effect 72-96 hours after the injection. They also indicate that while repeated bleeding contributes to the magnitude of this lipemia, the injection of papain alone is capable of inducing statistically significant elevation of the plasma lipids. TABLE IV MAJOR FATTY ACIDS PRESENT IN PLASMA LIPIDS FOLLOWING PAPAIN INJECTION
(50
Fatty acid
Sample
Control
2 Hours
1(; : 0
Control Papain Control Papain Control Papain Control Papain
25.3 ± 1.4
23.8 22.5 11.2 12.0 22.6 24.8 33.2 32.0
18 : 0 18: 1 18 : 2
12.2 ± 1.6 21.5 ± 2.7 31.3±2.1
± ± ± ± ± ± ± ±
1.2 1.3 1.4 1.6 2.1 3.4 2.9 3.1
ANIMALS REPORTED)
8 Hours
24 Hours
48 Hours
72 Hours
25.1 24.4 11.7 14.3 22.9 25.3 31.2 28.5
25.6 25.2 12.0 12.5 24.2 24.9 30.1 30.6
23.8 23.8 9.4 11.6 24.0 24.9 33.1 32.7
25.7 23.8 10.1 12.4 22.8 24.9 32.1 32.0
± ± ± ± ± ± ± ±
1.6 1.2 0.9 2.0 2.4 3.0 1.8 2.2
± ± ± ± ± ± ± ±
1.4 1.6 1.5 1.2 2.1 2.6 2.2 2.5
± ± ± ± ± ± ± ±
0.9 1.4 1.0 1.5 1.9 3.0 2.4 1.8
± ± ± ± ± ± ± ±
All values shown are in per cent of total fatty acids present in the extract.
It is of interest to note that the change in plasma lipid concentration involves primarily the glyceride fraction, with the other lipids participating to a minor extent in the lipemia at levels that were found statistically not significant. The changes in the fatty acid composition of all lipid fractions in animals receiving papain were minimal and statistically not significant. This was also true for the fatty acids of the lipids present in the lipemia produced after repeated bleedings. As for the possible explanation of the papain-induced hyperlipemia, no concrete explanation can be offered at the present time. It is quite possible that the entire phenomenon is the result of a stress reaction to the injection of papain. This could be supported by the pulmonary edema and hemorrhage found at autopsy in animals receiving papain, and of the time coincidence of the maximal plasma glyceride elevation and the histologic finding mentioned above. Since this manuscript was submitted for publication, it has come to our attention a brief communication by HIRSCH et al. 2o on studies of a hyperlipemia produced in rabbits by the injection of pituitary gland extracts. This hyperlipemia is very similar to the one produced by papain and reported here. It would seem possible that the papain lipemia may be the result of a pituitary gland stimulation,
J.
Atheroscler. Res., 5 (1965)
533~539
1.6 0.8 2.1 1.7 2.0 2.2 2.5 2.0
EFFECT OF LV. PAPAIN ON PLASMA LIPIDS AND FATTY ACIDS OF ADULT RABBITS
539
since we have observed evidence of stress in our experimental animals following the injection of papain. This would come to a general agreement with the available evidence that the pituitary gland hormones playa major role in the "stress reaction" of experimental animals, and that rabbits receiving steroids react with plasma glyceride elevation 16 - 19 . SUMMARY
Papain administered intravenously in adult rabbits produces a rIse in the plasma glyceride concentration without significantly altering the concentrations of the other lipid fractions. The injection of papain does not alter the fatty acid composition of the plasma total lipids or the individual lipid fractions. This lipemia is accompanied by moderate pulmonary edema and other stress phenomena. This stress reaction of the injected papain could be considered as the major cause of the lipemia. REFERENCES 1 J. H. BRYANT,
1. G.
LEDER AND DEWITT STETTEN, JR.,
Arch. Biochem. Biophys., 76 (1958)
122-130.
Federation Proc., 20 (1961). Nature, 196 (1962) 1006-1007. E. D. KORN, Methods Biochem. Anal., 7 (1957). M. J. ALB RINK, ] . Lipid Res., 1 (1959) 53. V. P. DOLE, ] . Clin. Invest., 35 (1956) 150. M. SOMOGYI, J. Biol. Chem., 86 (1930) 658. W. M. SPERRY AND M. WEBB, J. Biol. Chem., 187 (1950) 97. C. P. STEWART AND E. B. HENDRY, Biochem. j., 29 (1935) 1683. J. FOLCH, M. LEES AND G. H. SLOANE-STANLEY, J. Biol. Chern., 22 (1915) 133. J. HIRSCH AND E. H. AHRENS, J. Biol. Chem., 233 (1958) 311. K. K. CARROLL, J. Lipid Res., 2 (1961) 135. I. W. FARQUHAR, W. INSULL, JR., P. ROSEN, W. STOFFEL AND E. H. AHRENS, JR., Nutr. Abstr. Rev., 17 (1959) Supp!. 1. A. KELLNER AND T. ROBERTSON, J. Exptl. Med., 99 (1954) 387. T. R. BOGGS AND R. S. MORRIS, j. Exptl. Med., 11 (1902) 553. D. RUDIMAN AND F. SEIDMAN, Circulation, 18 (1958) 486. K. E. SUSSMAN, E. SHAFER AND D. STEINBERG, Circulation, 18 (1958) 486. A. R. RICH, J. H. COCHRAN AND D. C. MCBOON, Bull. johns Hopkins Hosp., 88 (1951) 101. D. ADLERSBERG, S. R. DRACHM AN AND H. E. SCHAFER, Circulation,4 (1951) 475. R. L. HIRSCH et al., Circulation, 30 (1964) Supp!. 3.
T. TSALTAS, 3 T. TSALTAS, 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
J.!Atheroscler. Res., 5 (1965) 533-539