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Lipid peroxides and the diet-induced Shwartzman reaction
Lipid peroxides and the diet-induced Shwartzman reaction HOWARD
B.
GOLDSTEIN,
DONALD
G.
MCKAY,
New
York,
New
M.D. M.D.
York
T H E M E c H A N I s M of induction of the generalized Shwartzman reaction in pregnant rats by dietary means has recently rebeceived study. I-3 The many similarities tween this syndrome and human eclampsia have been noted.4 The diet used is low in its tocopherol content” and contains oxidized lipids. It has been shown that feeding polyunsaturated fatty acids increases the need for tocopherol in the rat.6-s Addition of alpha tocopherol (vitamin E), a potent antioxidant, to the diet will prevent the occurrence of the generalized Shwartzman reaction. The specific role of vitamin E in metabolism remains obscure but it has been suggested to be an inhibitor of lipid peroxidation.!’ These considerations have prompted the present study of tissue lipid peroxidation during the diet-induced generalized Shwartzman reaction. In addition, the erythrocytes of pregnant rats eating the toxemic diet were tested for susceptibility to lipid peroxidation during in vitro stress with dialuric acid or with H,O,. Materials
and
pregnancy they were fed a diet containing equal parts of fractions VI and VII derived from molecular distillation of oxidized cod liver oil.3 The animals were sacrificed on the twenty-first day of gestation. Control animals were fed a Rockland pellet diet. Measurements of lipid peroxides in the distillate fractions of oxidized cod liver oil and in tissue were made by determination of the malonaldehyde content with the P-thiobarbituric acid (TBA) reaction”’ as modified by Blackard, Ball, and Engel.‘l One hundred milligram samples of each of the eight molecular distillate fractions of oxidized cod liver oil were weighed out under nitrogen atmosphere, and the TBA reaction performed. For tissue studies, organs to be tested were removed immediately at the time of death and were frozen with liquid nitrogen or dry ice. Three hundred milligram samples were then macerated in a tissue homogenizer containing 10 per cent trichloroacetic acid prior to TBA testing by the method cited.ll The color developed was measured with a Beckman-DU spectrophotometer at 532 rnp. For the erythrocyte studies blood was collected by cardiac puncture in saline buffered at pH 7.4 and containing 1 per cent heparin. The cells were washed twice with 20 volumes of buffered saline prior to resuspending at a hematocrit of 10 per cent. The dialuric acid test was performed by the method of Rose and Gyorgy.” In addition, the hydrogen peroxide diffusion system of Cohen and Hochstein13 was utilized for testing the sensitivity of the cells. Cells were incubated, in this system, at 37’ C. with
methods
Female albino rats of the Columbia-Sherman strain weighing between 180 and 230 grams were used. During the period of their
From the Department the College of Physicians of Columbia Uniuersity.
of
Pathology of and Surgeons
Aided by Grants 5 TI GM-865-02 and H&0.5666-04 from the Diuision of General Medical Sciences and the Heart Institute of the National Institutes of Health, United States Public Health Service. 843
844
Goldstein
and
McKay
MALONALDEHYDE VALUES OF DISTILLATE FRACTIONS OF OXIDIZED COD LIVER OIL 2-Thiobarbituric acid reaction DerfOrmed on IOOmg of sample
FRACTION
MA VALUE [O.D 532my 0.175 0.230
0.400 0.435 0.680 I.400 2.000 I ,300 Untreated Cod Liver Oil
0.015
Fig. I. Lipid peroxide content of distillate fractions of oxidized cod li\fer oil compared with unoxidized cod liver oil.
shaking. The degree of lysis was evaluated on measured aliquots of the red cell suspensions which were converted to cyanomethemoglobin with K,Fe (CN),, and KCN prior to reading in a spectrophotometer at 540 mp.’ i Results Malonaldehyde content of diet distillate fractions. The highest lipid peroxide content was observed in fractions VI, VII, and VIII while unoxidized cod liver oil was relatively unreactive (Fig. 1). Tissue studies. Lipid peroxides existed in all tissues tested but were significantly elevated above control levels only in the kidney (p < .Ol), liver (p < .02). and the placenta (p < .Ol) (Fig. 2). The spleens and uteri of pregnant and nonpregnant animals of both control and experimental diets were found to be quite silnilar. Erythrocyte stress studies. In the dialuric acid test, significantly ,yrcat~r lysis of red hlood cells was ohscrvrd in thr toxemic series (Fiq. 3). The mean degrt,es of lysis were 65 per cent for the toxemic versus 2-1 per cent for the controls. The hydrogen
peroxide diffusion test provided a clearer, separation between the toxemic diet group and the controls (Fig. 3). The mean degrees of lysis were 47 per cent versus 7 per cent after 2 hours of diffusion and 97 per cent versus 12 per cent after 3 hours. Lipid prrosidc
content
hi,yh in the as is shown gamma of s!rstem fully mals on the
was
similarly
found
to
bc
experimental series crythrocytcs in Fig. 4. The addition of :i alpha tocopherol in the in vitro protected the cells from the anitosemic diet.
Comment The conclusion drawn from these studies is that a state of relative vitamin E deficiency exists at the preterm period in the pregnant rats fed fractions of osidized cod liver oil. There appears to be no doubt that the feeding of polyunsaturated fats stresses the vitamin E status of animals. Although the chemical nature and mode of action of toxic lipid peroxides is not yet certain, lipid peroxidation has been shown to be toxic for several enzyme systems’“, ‘I’ and has been proposed as a likely basis for the damage done h) vitamin E deficiency. Structures such as mitochondria and microsomes which are rich in lipoprotein may be especially susceptible to lipid peroxidation. Kecent studies show that
ubichronlenol,”
an
antioxidant
sub-
stance, is able to maintain pregnancy in the tocopherol deficient rat. It is conceivable ttlat the lack of the antioxidant properties of vitamin E may he felt acutely under the metabolic stresses of pregnancy and be the source of cellular damage leading to eclampsia. Analysis of the lipid peroxide content of the
oxidized
cod
that
the
highest
isted
in
precisely
o&y
found’”
of frtal
liver
oil
fractions
Inalonaldehyde those to
resorptions,
\.ield
lipid the
revealed content
fractions highest
spontaneous
Wpre\i-
incidence deaths.
and
cortical lesions. Si,qnificant clta\,ations in lipid peroxides wrre found in kidney. li\.cr, and placenta of the expc~rimental aniulals. The reasons for s&=ctivr~ organ elcvarrnal
tions
The
arc
not
clear.
beha\G)r
of the crythrocytes
of thcl
Lipid peroxides
and Shwartzman
reaction
845
MALONALDEHYDE VALUES OF RAT TISSUES (2-Thioborbituric acid reaction)
1000
FRACTION
900
I m PELLET
XI-YII
pregnont nonpregnont DIET
B3 pregnant El nonpregnant
800 700 600 500 400 300 200 I 00 0 SPLEEN
KIDNEY
LIVER
UTERUS
PLACENTA
Fig. 2. The spleen, kidney, liver, and placenta of all pregnant animals on the oxidized cod liver oil diet showed increases in their reactivity to thiobarbituric acid. This indicates an increased malonaldehyde content which may be due to an increase in lipid peroxides.
H202
DIFFUSION
DIALURIC
ACID TEST
100 0
0
80
0
l
l
60
= FroctionXI-m Diet
a= Control 0 0
03 iii 3 40 I5 ii w 20 / 0.
n j0
0 0 0 r
L
8 I
2
3
HOURS Fig. 3. A comparison of the degrees of red cell lysis occurring in normal toxemic rat cells by the HzO: diffusion test and by the dialuric acid Each point indicates an individual animal at 21 days’ gestation.
and test.
846
Goldstein
and
MALONALDEHYDE FOLLOWING (21 0.D.532mA 0.7 0.6
day
= Fraction o= Control l
McKay
VALUES OF RBC: Hz02 DIFFUSION gestation
rots)
XI-m
Diet
0.5
-
0.4
-
0.3
-
0.2
-
0,I
-
$
f 8 ’
toxemic rats under the stresses of dialuric acid are what one would expect from a vitamin E deficient rat.“. ‘!I Horwitt”” has sug-
2. 3. 4. 5. 6.
7. a. 9. 10. 11. 12.
REFERENCES McKay, D. G., and Goldenberg. 1’. E.: Obst. & Gynec. 21: 651. 1963. McKay, D. G., and Wong, T. C.: J. Exper. Med. 115: 1117, 1962. Kaunitz, H., Malins, D. C.. and McKay, D. G.: J. Exper. Med. 115: 1127, 1962. McKay, D. G.: Obst. & Gynec. 20: 1, 1962. Stamler, F. W.: Am. J. Path. 35: 1207, 1959. Horwitt, M. K.. Harvey, C. C.. Century. B.. and Witting, L. A.: J. Am. Dietet. A. 38: 231. 1961. Moore, T., and Sharman, 1. M.: Brit. J. Nutrition 15: 297, 1961. Century, B., and Horwitt, M. K;.: J. Nutrition 72: 357, 1960. Horwitt. M. K.: Borden‘s Rev. Nutrition Res. 22: 1, 1961. Bernheim, F., Bernheim, M. L. C.. and Wilbur, K. M.: J. Biol. Chem. 174: 257, 1948. Blackard. W. G., Ball, M. F., and Engel. F. L.: J. Clin. Invest. 41: 1288, 1962. Rose. C:. S.. and Gyorgy, P.: Blood 5: 1062, 1950.
that
Summary
0
Fig. 4. Lipid peroxide content of red cells following 2 and 3 hours of H,O, diffusion. Each point indicates an individual animal at 21 days of gestation.
1.
the degree of hemolpsis is directly related to the ratio of tocophcrol to peroxidizable lipid. Analysis of the lipid components of the erythrocytes of the toxemic rats has not been made at this time. ,g:ested
Fractions of oxidized cod liver oil previously found to produce experimental eclampsia have been shown to have high concentrations of lipid peroxides in the more potent fractions. Pregnant rats fed fractions VI and VII derived from molecular distillations of oxidized cod liver oil had increased amounts of lipid peroxides in their kidneys, livers, and placentas. The erythrocytes of these animals Iysed under the stresses of hydroqn peroside diffusion and dialuric acid in a manner similar to that of known vitamin E deficient animals. It is concluded that a state of relativ,e \.itamin E deficiency exists in these toxemic rats and that the deficiency of the antioxidant properties of v.itamin E may be the basis of cellular damage leading to the eclamptic state.
13. 14. 15.
16. 17.
la. 19. 20.
Cohen, G., ,rnd Hochstein, P.: Science 134: 1756, 1961. Cohen. G.. and Hochstein, P.: Hiochem. 2: 1420. 1963. Caputto. R.. M&a);, P. 8.. and Carpenter. M. P. Am. J. Clin. Nutrition 9: 61. 1961. (Part II.) Schwarz, K.: Am. J. Clin. Nutrition. 9: 71, 1961. (Part II.) Johnson. B. C., Crider, Q., Schunk, C. I-I., Linn, B. O., Wong. E. L., and Folkars. Ii.: Biochem. Biophys. Res. Communications 5: 309. 1961. Kaunitz, H.. Gauglitz, E., Jr., and McKay, D. G.: Metabolism 12: 3’71. 1963. Goldstein. H. B., and Cohen, G.: In preparetion. Horwitt. M. K.: Am. J. (Xn. Nutrition 8: -1-51. 1960.
630 Wed 168th h’erc, I-ark, Nuw
Street Fork
10032
B.
GOLDSTEIN,
DONALD
G.
MCKAY,
New
York,
New
M.D. M.D.
York
T H E M E c H A N I s M of induction of the generalized Shwartzman reaction in pregnant rats by dietary means has recently rebeceived study. I-3 The many similarities tween this syndrome and human eclampsia have been noted.4 The diet used is low in its tocopherol content” and contains oxidized lipids. It has been shown that feeding polyunsaturated fatty acids increases the need for tocopherol in the rat.6-s Addition of alpha tocopherol (vitamin E), a potent antioxidant, to the diet will prevent the occurrence of the generalized Shwartzman reaction. The specific role of vitamin E in metabolism remains obscure but it has been suggested to be an inhibitor of lipid peroxidation.!’ These considerations have prompted the present study of tissue lipid peroxidation during the diet-induced generalized Shwartzman reaction. In addition, the erythrocytes of pregnant rats eating the toxemic diet were tested for susceptibility to lipid peroxidation during in vitro stress with dialuric acid or with H,O,. Materials
and
pregnancy they were fed a diet containing equal parts of fractions VI and VII derived from molecular distillation of oxidized cod liver oil.3 The animals were sacrificed on the twenty-first day of gestation. Control animals were fed a Rockland pellet diet. Measurements of lipid peroxides in the distillate fractions of oxidized cod liver oil and in tissue were made by determination of the malonaldehyde content with the P-thiobarbituric acid (TBA) reaction”’ as modified by Blackard, Ball, and Engel.‘l One hundred milligram samples of each of the eight molecular distillate fractions of oxidized cod liver oil were weighed out under nitrogen atmosphere, and the TBA reaction performed. For tissue studies, organs to be tested were removed immediately at the time of death and were frozen with liquid nitrogen or dry ice. Three hundred milligram samples were then macerated in a tissue homogenizer containing 10 per cent trichloroacetic acid prior to TBA testing by the method cited.ll The color developed was measured with a Beckman-DU spectrophotometer at 532 rnp. For the erythrocyte studies blood was collected by cardiac puncture in saline buffered at pH 7.4 and containing 1 per cent heparin. The cells were washed twice with 20 volumes of buffered saline prior to resuspending at a hematocrit of 10 per cent. The dialuric acid test was performed by the method of Rose and Gyorgy.” In addition, the hydrogen peroxide diffusion system of Cohen and Hochstein13 was utilized for testing the sensitivity of the cells. Cells were incubated, in this system, at 37’ C. with
methods
Female albino rats of the Columbia-Sherman strain weighing between 180 and 230 grams were used. During the period of their
From the Department the College of Physicians of Columbia Uniuersity.
of
Pathology of and Surgeons
Aided by Grants 5 TI GM-865-02 and H&0.5666-04 from the Diuision of General Medical Sciences and the Heart Institute of the National Institutes of Health, United States Public Health Service. 843
844
Goldstein
and
McKay
MALONALDEHYDE VALUES OF DISTILLATE FRACTIONS OF OXIDIZED COD LIVER OIL 2-Thiobarbituric acid reaction DerfOrmed on IOOmg of sample
FRACTION
MA VALUE [O.D 532my 0.175 0.230
0.400 0.435 0.680 I.400 2.000 I ,300 Untreated Cod Liver Oil
0.015
Fig. I. Lipid peroxide content of distillate fractions of oxidized cod li\fer oil compared with unoxidized cod liver oil.
shaking. The degree of lysis was evaluated on measured aliquots of the red cell suspensions which were converted to cyanomethemoglobin with K,Fe (CN),, and KCN prior to reading in a spectrophotometer at 540 mp.’ i Results Malonaldehyde content of diet distillate fractions. The highest lipid peroxide content was observed in fractions VI, VII, and VIII while unoxidized cod liver oil was relatively unreactive (Fig. 1). Tissue studies. Lipid peroxides existed in all tissues tested but were significantly elevated above control levels only in the kidney (p < .Ol), liver (p < .02). and the placenta (p < .Ol) (Fig. 2). The spleens and uteri of pregnant and nonpregnant animals of both control and experimental diets were found to be quite silnilar. Erythrocyte stress studies. In the dialuric acid test, significantly ,yrcat~r lysis of red hlood cells was ohscrvrd in thr toxemic series (Fiq. 3). The mean degrt,es of lysis were 65 per cent for the toxemic versus 2-1 per cent for the controls. The hydrogen
peroxide diffusion test provided a clearer, separation between the toxemic diet group and the controls (Fig. 3). The mean degrees of lysis were 47 per cent versus 7 per cent after 2 hours of diffusion and 97 per cent versus 12 per cent after 3 hours. Lipid prrosidc
content
hi,yh in the as is shown gamma of s!rstem fully mals on the
was
similarly
found
to
bc
experimental series crythrocytcs in Fig. 4. The addition of :i alpha tocopherol in the in vitro protected the cells from the anitosemic diet.
Comment The conclusion drawn from these studies is that a state of relative vitamin E deficiency exists at the preterm period in the pregnant rats fed fractions of osidized cod liver oil. There appears to be no doubt that the feeding of polyunsaturated fats stresses the vitamin E status of animals. Although the chemical nature and mode of action of toxic lipid peroxides is not yet certain, lipid peroxidation has been shown to be toxic for several enzyme systems’“, ‘I’ and has been proposed as a likely basis for the damage done h) vitamin E deficiency. Structures such as mitochondria and microsomes which are rich in lipoprotein may be especially susceptible to lipid peroxidation. Kecent studies show that
ubichronlenol,”
an
antioxidant
sub-
stance, is able to maintain pregnancy in the tocopherol deficient rat. It is conceivable ttlat the lack of the antioxidant properties of vitamin E may he felt acutely under the metabolic stresses of pregnancy and be the source of cellular damage leading to eclampsia. Analysis of the lipid peroxide content of the
oxidized
cod
that
the
highest
isted
in
precisely
o&y
found’”
of frtal
liver
oil
fractions
Inalonaldehyde those to
resorptions,
\.ield
lipid the
revealed content
fractions highest
spontaneous
Wpre\i-
incidence deaths.
and
cortical lesions. Si,qnificant clta\,ations in lipid peroxides wrre found in kidney. li\.cr, and placenta of the expc~rimental aniulals. The reasons for s&=ctivr~ organ elcvarrnal
tions
The
arc
not
clear.
beha\G)r
of the crythrocytes
of thcl
Lipid peroxides
and Shwartzman
reaction
845
MALONALDEHYDE VALUES OF RAT TISSUES (2-Thioborbituric acid reaction)
1000
FRACTION
900
I m PELLET
XI-YII
pregnont nonpregnont DIET
B3 pregnant El nonpregnant
800 700 600 500 400 300 200 I 00 0 SPLEEN
KIDNEY
LIVER
UTERUS
PLACENTA
Fig. 2. The spleen, kidney, liver, and placenta of all pregnant animals on the oxidized cod liver oil diet showed increases in their reactivity to thiobarbituric acid. This indicates an increased malonaldehyde content which may be due to an increase in lipid peroxides.
H202
DIFFUSION
DIALURIC
ACID TEST
100 0
0
80
0
l
l
60
= FroctionXI-m Diet
a= Control 0 0
03 iii 3 40 I5 ii w 20 / 0.
n j0
0 0 0 r
L
8 I
2
3
HOURS Fig. 3. A comparison of the degrees of red cell lysis occurring in normal toxemic rat cells by the HzO: diffusion test and by the dialuric acid Each point indicates an individual animal at 21 days’ gestation.
and test.
846
Goldstein
and
MALONALDEHYDE FOLLOWING (21 0.D.532mA 0.7 0.6
day
= Fraction o= Control l
McKay
VALUES OF RBC: Hz02 DIFFUSION gestation
rots)
XI-m
Diet
0.5
-
0.4
-
0.3
-
0.2
-
0,I
-
$
f 8 ’
toxemic rats under the stresses of dialuric acid are what one would expect from a vitamin E deficient rat.“. ‘!I Horwitt”” has sug-
2. 3. 4. 5. 6.
7. a. 9. 10. 11. 12.
REFERENCES McKay, D. G., and Goldenberg. 1’. E.: Obst. & Gynec. 21: 651. 1963. McKay, D. G., and Wong, T. C.: J. Exper. Med. 115: 1117, 1962. Kaunitz, H., Malins, D. C.. and McKay, D. G.: J. Exper. Med. 115: 1127, 1962. McKay, D. G.: Obst. & Gynec. 20: 1, 1962. Stamler, F. W.: Am. J. Path. 35: 1207, 1959. Horwitt, M. K.. Harvey, C. C.. Century. B.. and Witting, L. A.: J. Am. Dietet. A. 38: 231. 1961. Moore, T., and Sharman, 1. M.: Brit. J. Nutrition 15: 297, 1961. Century, B., and Horwitt, M. K;.: J. Nutrition 72: 357, 1960. Horwitt. M. K.: Borden‘s Rev. Nutrition Res. 22: 1, 1961. Bernheim, F., Bernheim, M. L. C.. and Wilbur, K. M.: J. Biol. Chem. 174: 257, 1948. Blackard. W. G., Ball, M. F., and Engel. F. L.: J. Clin. Invest. 41: 1288, 1962. Rose. C:. S.. and Gyorgy, P.: Blood 5: 1062, 1950.
that
Summary
0
Fig. 4. Lipid peroxide content of red cells following 2 and 3 hours of H,O, diffusion. Each point indicates an individual animal at 21 days of gestation.
1.
the degree of hemolpsis is directly related to the ratio of tocophcrol to peroxidizable lipid. Analysis of the lipid components of the erythrocytes of the toxemic rats has not been made at this time. ,g:ested
Fractions of oxidized cod liver oil previously found to produce experimental eclampsia have been shown to have high concentrations of lipid peroxides in the more potent fractions. Pregnant rats fed fractions VI and VII derived from molecular distillations of oxidized cod liver oil had increased amounts of lipid peroxides in their kidneys, livers, and placentas. The erythrocytes of these animals Iysed under the stresses of hydroqn peroside diffusion and dialuric acid in a manner similar to that of known vitamin E deficient animals. It is concluded that a state of relativ,e \.itamin E deficiency exists in these toxemic rats and that the deficiency of the antioxidant properties of v.itamin E may be the basis of cellular damage leading to the eclamptic state.
13. 14. 15.
16. 17.
la. 19. 20.
Cohen, G., ,rnd Hochstein, P.: Science 134: 1756, 1961. Cohen. G.. and Hochstein, P.: Hiochem. 2: 1420. 1963. Caputto. R.. M&a);, P. 8.. and Carpenter. M. P. Am. J. Clin. Nutrition 9: 61. 1961. (Part II.) Schwarz, K.: Am. J. Clin. Nutrition. 9: 71, 1961. (Part II.) Johnson. B. C., Crider, Q., Schunk, C. I-I., Linn, B. O., Wong. E. L., and Folkars. Ii.: Biochem. Biophys. Res. Communications 5: 309. 1961. Kaunitz, H.. Gauglitz, E., Jr., and McKay, D. G.: Metabolism 12: 3’71. 1963. Goldstein. H. B., and Cohen, G.: In preparetion. Horwitt. M. K.: Am. J. (Xn. Nutrition 8: -1-51. 1960.
630 Wed 168th h’erc, I-ark, Nuw
Street Fork
10032