443
Special Communication
Free Radical Pathology
JACK D. BUTTERFIELD, JR., B.A. AND C. PATRICK MCGRAW, PH.D.
Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017
OVER the past several years free radical pathology,
especially as it pertains to experimentally induced
cerebral lesions, has been the subject of renewed interest in several investigative laboratories. Its possible
role in the development of ischemic cerebral lesions,
i.e., stroke, is suggested.
For most chemicals, the electron orbitals are filled
with paired electrons, which spin in opposite directions and cancel each other's magnetic fields. For a
free radical, however, the outer orbital has a lone, unpaired electron, which spins unopposed. In most cases,
this unpaired electron creates an extremely unstable
electronic distribution and the free radical is quite
reactive.111 The pattern of its chemical reactions is
referred to as "anti-Markovnikov" as opposed to the
usual pattern of organic reactions known as "Markovnikov."12 Many compounds can be converted into a
free radical state by x-ray or ultraviolet irradiation,
or by chemical agents known as initiators. The most
significant aspect of radical chemistry is a marked
alteration in the size and shape of the molecules, leading to altered function of the chemicals involved.
The physical-chemical properties of molecular oxygen (O2) are central to the theory of free radical
pathology. First, the molecular structure of O2 is such
that it can be written as having a covalent double
bond, O = O, or as being a diradical, O-O. It is this
diradical characteristic that provides 0 2 with the
potential to undergo initiation and addition reactions.
Reactions such as those depicted in figure 1 are typical
of the free radical reactions that occur in the classic
rancidification of fats. The presence of iron complexes
or copper complexes can catalyze further radical reactions, as shown in figure 2.
Molecular oxygen is 7 to 8 times more soluble in
nonpolar media than in polar media, a characteristic
that significantly affects the structure of the plasma
membrane. Quite simplified, the plasma membrane is
a bimolecular leaflet of phospholipids, with hydrophobic, hydrophilic, and amphipathic substances interdigitated into and interacting with the leaflet by
hydrophobic and ionic forces. As shown in figure 3,
From the Department of Neurology and Section on
Neurosurgery, Department of Surgery, Bowman Gray School of
Medicine of Wake Forest University, Winston-Salem, NC 27103.
Supported in part by a Student Clerkship in Cerebrovascular
Disease from the American Heart Association and, in part, by
NINDS Grant NS06655-11.
Reprints: C. Patrick McGraw, Ph.D., Department of Neurology,
Bowman Gray School of Medicine, Winston-Salem, NC 27103.
the plasma membrane lipid bilayer has a hydrophobic
midzone area. This is the nonpolar medium in which
O2 is so soluble. It is also the location of the polyunsaturated fatty acids with their allylic carbonhydrogen bonds which are so susceptible to free
radical attack (fig. 2). Thus, the normal membrane
has the highest concentration of O2 (with its diradical
potential) in the hydrophobic midzone area, where it
has the potential for doing the most damage to the
membrane's polyunsaturated fatty acids, i.e., the
membrane is poised for disruption.
The question that logically follows this line of
reasoning is, "What prevents the normal plasma
membrane from being destroyed by lipid peroxidation?" Demopoulos et al.2 have suggested that
cholesterol has a protective antioxidant function in
normal cell membranes. Cholesterol is an
amphipathic substance that enters the hydrophobic
midzone area perpendicular to the surface of the
membrane. The fatty acid "tails" on the
phospholipids (fig. 3) are free to wave and, via hydrophobic forces, wrap themselves tightly around the
nonpolar end of the cholesterol radical and "fit" or intercalate the cholesterol into the membrane. Since the
steroid is firmly bound to the fatty acid tails, it serves
as a physical barrier between the free radical and the
allylic bonds of the fatty acids. Mitamura et al.13 offer
evidence in support of this theory, showing that the
cholesterol content of membranes is decreased in coldinduced cerebral edema. They attributed this finding
to chemical alteration of the steroid by the free
radicals formed in the damaged tissue. Thus it appears
that one of cholesterol's many roles may be to protect
normal plasma membranes from free radical attacks.
With this background, one can speculate on the role
of free radical pathology in acute cerebral ischemia.
The cellular damage caused by cerebral ischemia
might include destruction of the integrity of the
capillary endothelium, which, in turn, would allow extravasation of red blood cells and fluid into the
ischemic area. As the neurons, glial cells, and extravasated red blood cells die, iron and copper complexes
(heme and cytochromes) would be released into the
tissue spaces. Since oxygen is soluble in the
hydrophobic midzone area of the membrane, and
since it acts as a diradical, it could initiate free radical
reactions that would contribute to cholesterol depletion. In the absence of sufficient cholesterol and in the
presence of iron and copper complexes, catalysis of
free radical reactions would proceed unchecked, disrupting membrane integrity. And anything that dis-
444
(l)
STROKE
H H H H
H H H H
i
i
i
i
i
-c-c=c-cI
I
H
H
O-O
i
i
H
i
•
»--c-c=c-c-
(2)
I
i
-c-c=c-c-
(D
Fe
H
.
pertly droxy
radical
I
I
I
i
(2)
I
(3)
-c-c=c-c-
i
i
i
I
-c-c=c-c-
I
I
I
O-O- H
free radical
i
i
i
-c-c=c-cI
I
0
H
i
OH H H
H
.
Fe
2+
H H H H
I
*• -c-c=c-c-
hydroperoxide
i
H H H H
i
hydroxyperoxide
O-OH
H HH
1978
H H H H
i
o-o-
i
-c-c=c-c- I
H
I
i
O-OH H
I
I
i
3+
H
H H H H
i
H H H H
i
O-O
alkyl radical
i
VOL 9, No 5, SEPTEMBER-OCTOBER
H H H H
i
I
I
i
1
I
I
I
O-O- H
H
i
H H H H
i
i
2H
-C-C=C-C-
-c-c=c-c-
O-O-
I
i
i
i
H
H
i
i
i
2 -C-C=C-Ci
OH
i
H
Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017
alkyl peroxide
alkyl perhydroxy
radical
2. Sequences like (I) produce more free radicals
to further propagate the reactions. Sequences like (2) cause
the breakdown of lipid peroxides already formed and cause
further membrane disruption.
FIGURE
H H H H
I
(4)
i
i
i
-C-C=C-C-
-C-C=C-C
O-O- H
0 HH H
1
I
-c-c=c-c-
alkyl peroxide
FIGURE 1. Two possible courses for the progression of free
radical initiation and addition reactions. Note that only
allylic hydrogen bonds are depicted as being attacked. This
is because the allylic hydrogen bond is relatively weak due to
the electron delocalization within the double bond, and is,
therefore, most susceptible to attack. This particular weak
bond is the reason polyunsaturated fatty acids in the membrane are most susceptible to derangement from free radical
attack.
rupts the normal integrity of the plasma membrane
(such as lipid peroxidation) must, as a consequence,
also disrupt that membrane's functions, e.g.,
permeability, transport, barrier capacity, bioenergetics. The ultimate result would be cellular damage,
necrosis, cerebral edema, and all the complications
thereof.
References
1. Ransohoff J: The effect of steroids on edema in man, pp
211-217. In Reulen HJ and SchQrmann K (eds) Steroids and
Brain Edema. New York, Springer-Verlag, 1972
2. Demopoulos HB, Jorgensen E, Mitamura J et al: Control of
free radical damage to membrane lipids by barbiturates. Paper
No 32 presented at Annual Meeting American Association
Neurological Surgeons, Toronto, Canada, 1977
3. Michenfelder JD, Milde J H , Sundt TM Jr: Cerebral protection by barbiturate anesthesia. Use after middle cerebral artery
INTRAEXTRACELLULAR a S - s = D CELLULAR
WATER
c C ~ - 3 0 WATER
HYDROPHOBIC
MIOZONE
AREA
PROTEIN
PHOSPHOLIPID
FATTY ACID TAILS
FIGURE 3. The lipid bilayer. Substances like protein and
cholesterol are found in, and as part of, the plasma membrane. Hydrophobic associations keep the nonpolar
molecules close together when they are surrounded by
aqueous media. Since cell membranes have aqueous media
on both sides of their surfaces, the polar end groups are attracted outward and the hydrophobic end groups are
directed inward, toward each other and away from the
water.
occlusion in Java monkeys. Arch Neurol 33: 345-350, 1976
4. Ortega BD, Demopoulos HB, Ransohoh : Effect of antioxidants on experimental cold-induced cerebral edema, pp
167-175. In Reulen HJ and Schflrmann K (eds) Steroids and
Brain Edema, New York, Springer-Verlag, 1972
5. Pappius HM: Effects of steroids on cold injury edema, pp
57-63. In Reulen HJ and Schflrmann K (eds): Steroids and
Brain Edema, New York, Springer-Verlag, 1972
6. Aaes-Jorgensen E: Antioxidation of fatty compounds in living
tissue, biological antioxidants. In Lundberg WO (ed): Auto-
FREE RADICAL PATHOLOGY/Butterfield.
oxidation and Antioxidants, Vol II, New York, Interscience
Publishers, 1962
7. Demopoulos HB: Control of free radicals in biologic systems.
Fed Proc 32: 1903-1908, 1973
8. Demopoulos HB: The basis of free radical pathology. Fed Proc
32: 1859-1861, 1973
9. Demopoulos HB, Milvey P, Kakari S et al: Molecular aspects
of membrane structure in cerebral edema, pp 29-39. In Reulen
HJ and Schflrmann K (eds): Steroids and Brain Edema, New
York, Springer-Verlag, 1972
McGraw
445
10. Tappel AL: Free-radical lipid peroxidation damage and its inhibition by vitamin E and selenium. Fed Proc 24: 73-78, 1965
11. Tappel AL: Lipid peroxidation damage to cell components. Fed
Proc 32: 1870-1874, 1973
12. Morrison RT, Boyde RN: Organic Chemistry, 2nd ed, pp
185-188. Boston, Allyn and Bacon Inc, 1966
13. Mitamura J, Iappolo A, Seligmon ML et al: Loss of cerebral
cholesterol in CNS injury and modulation by corticosteroids.
Paper No 25 presented at the Annual Meeting American
Association Neurological Surgeons, Toronto, Canada, 1977
Free Radicals in Cerebral Ischemia
EUGENE S. FLAMM, M.D.,
HARRY B. DEMOPOULOS, M.D.,
MYRON L. SELIGMAN, PH.D.,
RICHARD G. POSER, AND JOSEPH RANSOHOFF,
M.D.
Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017
SUMMARY The possibility that cerebral ischemia may initiate a series of pathological free radical reactions within the
membrane components of the CNS was investigated in the cat. The normally occurring electron transport radicals require
adequate molecular oxygen for orderly transport of electrons and protons. A decrease in tissue oxygen removes the controls
over the electron transport radicals, and allows them to initiate pathologic radical reactions among cell membranes such as
mitochondria. Pathologic radical reactions result in multiple products, each of which may be present in too small a concentration to permit their detection at early time periods. It is possible to follow the time course, however, by the decrease of a
major antioxidant as it is consumed by the pathologic radical reactions. For this reason, ascorbic acid was measured in
ischemic and control brain following middle cerebral artery occlusion. There was a progressive decrease in the amount of
detectable ascorbic acid ranging from 25% at 1 hour to 65% at 24 hours after occlusion. The reduction of this normally occurring antioxidant and free radical scavenger may indicate consumption of ascorbic acid in an attempt to quench pathologic
free radical reactions occurring within the components of cytomembranes.
RECENT STUDIES of the effects of decreased oxygen supply to the central nervous system (CNS) have
examined the ischemic or hypoxic brain for histologic,
histochemical and ultrastructural changes.16 Extensive studies have focused attention on alterations of
the bioenergetic pathways in response to ischemia or
anoxia.79 Only recently, however, has emphasis been
given to the changes produced at the molecular level
prior to the onset of frank subcellular and cellular
pathology.10- n Since neuronal integrity and function
depend on competent cellular membranes, an early
response to an insult such as ischemia might well be
reflected by molecular changes in membrane components.12
The concept that CNS cytomembranes can be disturbed by membrane lipid free radical reactions has
been discussed in prior work from this laboratory.1316
This work has also suggested that the similarly complex and variegated events observed in cerebral edema
and spinal cord trauma may be tied together by an
hypothesis that centers on molecular pathologic per-
From the Departments of Neurosurgery (Drs. Flamm, Seligman
and Ransohoff) and Pathology (Drs. Demopoulos, Seligman and
Mr. Poser), New York University Medical Center, Milbank
Research Laboratories, 340 East 24th St., New York, NY 10010.
Presented, in part, at the Annual Meeting of the American
Association of Neurological Surgeons, St. Louis, MO, April 21-25,
1974.
turbations of the many membrane systems that comprise a cell.14-17"20
Free radicals are compounds that have a lone electron in an outer orbital and are therefore highly reactive. They occur normally in the mitochondrial electron transport chain of all cells and are kept under
control by virtue of tight physical and chemical coupling of the intermediates. Oxygen, at the terminus of
the electron transport chain, serves as the natural terminator of these free radical reactions by reduction to
water. Hunter et al. have demonstrated in vitro that
mitochondrial swelling is accompanied by the production of hydrogen peroxide, as well as malonaldehyde,
a product of free radical initiated lipid peroxidation.4
The question that arises is how lowered oxygen levels
might permit free radical intermediates in the normal
electron transport chain to react with membrane lipids
and start a series of pathologic free radical reactions
within the mitochondrial membranes.
The hypothesis that electron transport free radicals
may increase in concentration with diminished oxygen
tension, and thereby reach the levels needed to initiate
lipid free radical reactions in mitochondrial membranes, was tested in a model employing middle
cerebral artery occlusion in the cat. The ischemic
tissue was studied at different time intervals for the
content of a normally occurring tissue antioxidant,
ascorbic acid. We interpret the consumption of
available ascorbic acid as an indication that radical
mechanisms are operative.13
Free radical pathology.
J D Butterfield, Jr and C P McGraw
Stroke. 1978;9:443-445
doi: 10.1161/01.STR.9.5.443
Downloaded from http://stroke.ahajournals.org/ by guest on June 17, 2017
Stroke is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1978 American Heart Association, Inc. All rights reserved.
Print ISSN: 0039-2499. Online ISSN: 1524-4628
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://stroke.ahajournals.org/content/9/5/443.citation
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in
Stroke can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office.
Once the online version of the published article for which permission is being requested is located, click Request
Permissions in the middle column of the Web page under Services. Further information about this process is
available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Stroke is online at:
http://stroke.ahajournals.org//subscriptions/