331
Biochimica et Biophysics Acta, 388 (1975) 331-338 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BBA 56617
THE EFFECT OF ESSENTIAL FATTY ACID DEFICIENCY FATTY ACID UPTAKE BY THE BRAIN
UPON
DOUGLAS S. LYLES, L.L. SULYA and H.B. WHITE, JR Department of Biochemistry, Miss. 39216 (U.S.A.)
University of Mississippi Medical Center, Jackson,
(Received December 18th, 1974)
Summary Young adult rats, either control or essential fatty acid deficient, were administered either [ 3 H] oleic acid or [’ H] arachidonic acid by stomach tube. In addition, a group of control rats was given [ 3 H] palmitic acid. The rats were killed at various times thereafter, and the radioactivity of the lipids of brain and plasma was examined. In confirmation of previous work, the blood lipid label was found to rise rapidly and then fall, whereas the activity of brain lipids increased slowly and did not show a decline through the 24-h period studied. Analysis of the brain uptake data according to first-order kinetics confirmed the impressions gained from visual inspection of the data. The initial rate of uptake of arachidonic acid was about 4.5 times that of oleic acid in control animals and in deficient animals. Essential fatty acid deficiency, however, did not induce an altered rate of uptake for either oleic acid or arachidonic acid. The rate of uptake of palmitic acid by control rats was not significantly different from that of oleic acid. Even though the initial rates of incorporation of oleic and arachidonic acids were not changed during essential fatty acid deficiency, the final levels of radioactivity obtained in brain lipids were higher in deficient rats with both fatty acids. The plateau value obtained with oleic acid was 1.5 times higher in deficient animals, while the plateau value for arachidonic acid was 1.7 times higher. An experiment in which deficient animals were allowed access to a control diet for 12 or 24 h prior to the labeling experiment suggested that the higher levels of radioactivity found in brain lipids of deficient animals was not due to an isotope dilution effect. Such animals still displayed. the labeling pattern of deficient animals with arachidonic acid, while the results with oleic acid varied somewhat. Our results suggest that essential fatty acid deficiency does not alter the ability of the brain to take up the fatty acids studied. However, the fatty acids, especially arachidonic, are retained in the brain to a greater extent in the deficient animals.
332
Introduction Labeled fatty acids, like many other metabolites, are taken up by the brain of adult rats at a much slower rate than the brain of younger rats in which the nervous system is still developing [l] . Such observations led to the postulate that the so-called “blood-brain barrier” applied to fatty acids as well as other molecules. Presumably the brain acquired its complement of fatty acids early in development and conserved them throughout life by recycling the degradation products of brain lipids [2]. This view has been challenged by those who maintain that the decrease in the uptake of metabolites with age represents the decreased metabolic activity accompanying the end of development rather than some restriction imposed by the blood-brain barrier [3]. Recently, experiments performed by Mead and coworkers [4-71 have been reviewed [8]. In these experiments, the direct uptake of several fatty acids by the adult rat brain was demonstrated. That such an uptake occurs is consistent with the fact that in essential fatty acid deficient rats, the fatty acid composition of the brain phospholipids returns to normal after the rats are restored to a normal diet [9,10]. Our experiments were designed to test whether essential fatty acid deficiency alters the ability of the brain to take up fatty acids, or alternatively, whether the normal rate of uptake is sufficient to reconstitute the brain fatty acid composition as essential fatty acid deficient rats recover from the deficiency. Methods Young adult Sprague-Dawley rats approx. 45 days old were used. The essential fatty acid-deficient regimen was initiated about 5 days before birth. The control diet contained 24% casein, 4.5% crude fiber, 63.5% sucrose, 4% vegetable fat, and 4% salt mixture. The essential fatty acid-deficient diet was fat free and contained 21.1% casein, 16.4% Alphacel cellulose, 58.4% sucrose, and 4% salt mixture. Each diet was supplemented with a complete complement of the necessary vitamins. Isotope administration To administer the isotopically labeled fatty acids, rats were lightly anesthetized with ether. Each isotope was administered by stomach tube in 0.5 ml of a clear solution containing 50 mg of glucose and approx. 15 PCi of labeled fatty acid prepared using the non-toxic emulsifier Pluronic F-68 (kindly supplied by Wyandotte Chemicals Corporation of Wyandotte, Michigan). After various time periods, the animals (which weighed about 150 g each) were anesthetized with 0.2 ml of Nembutal given intraperitoneally and opened from lower abdomen to the base of the neck. A 2.0-ml blood sample was taken by heart puncture, and then 0.1 ml of a sodium heparin solution (1000 U.S.P. units/ml) was injected into the heart. The animals were perfused with isotonic saline for 5 min at a pressure of about 65 cm water. The needle was placed in the left ventricle and the inferior vena cava was cut to allow drainage. Perfusion was judged complete by “tanning” of the liver and clearing of the perfusate. The whole brain was removed and frozen until analyses could be performed.
333
The radioactive fatty acids used were [9,10-’ H2 ] palmitic acid (393 Ci/mol), [9,10-3 H2 ] oleic acid (2.29 Ci/mmol), and [5,6,8,9,11,12,14,15’ H, ] arachidonic acid (18.2 Ci/mmol), all obtained from New England Nuclear. 3 H-labeled fatty acids were used in preference to a ’ ‘C label in order to circumvent the isotope recyclization problem. In the oxidative degradation of fatty acids to acetyl-CoA, a large portion of the 3 H label would be lost and would ultimately exchange with and be diluted by body water. That such degradation and reincorporation of label does not occur to a significant extent is shown by thin-layer chromatographic analysis of the label distribution in brain lipids. 24 h after the administration of [ 3 H] palmitic acid, the sterol fraction of a brain lipid aliquot contained only 34 cpm of a total 1150 cpm. There was a significant direct conversion of [’ H] palmitic acid to other fatty acids, as one-fourth of the counts in the fatty acid fraction was associated with monoenoic fatty acids. Lipid analysis Total lipid was extracted from the tissues with chloroform/methanol (2 : 1, v/v) and washed once by the method of Folch et al. [ll] . Aliquots of the lipid extract were taken and the solvent was evaporated for gravimetric determination of total lipid weight or for determination of radioactivity by liquid scintillation counting in 10 ml of 0.5% 2,5-bis-(2-(5-t-butylbenzoxazolyl))-thiophene in toluene. All counts were converted to dpm values by comparison with an external standard. Calculations The calculation of radioactivity per mg of lipid has been normalized for the activity of the dose administered and for the dilution due to the weight of the rat. This quantity, which we refer to as relative specific activity (A), has the units (dpm/mg lipid)/(dpm administered/g weight of rat). Thus comparisons between the various fatty acids can be made. For purposes of statistical comparison, the brain uptake data were analysed according to first-order kinetics. The assumptions made in this analysis are (1) that in each instance there is a flux (J) of fatty acid into (Ji,) and out of (Jout) the brain; therefore, the kinetics of labeling are determined by both Jin and J out9. and (2) that this system approximates first-order kinetics, thus A, =Aeq(l
-ePkL)
(1)
where A, = relative specific activity at any time t, A,, = the plateau relative specific activity (obtained before the level of radioactivity in the brain begins to fall), and k = the rate constant for the approach to equilibrium, which can be shown to be proportional to J,, t if assumption (1) above is true. In these examples in which A,, was known (i.e. the palmitate and oleate experiments), k was calculated from the equation
Wk,
-A,)
by plotting
=lnA,, log (A,,
-kt - A,) versus t, the slope being -k/2.303.
(2) In those experi-
334
ments in which a plateau was not reached by 24 h (the arachidonate experiments), the plateau value A,, was obtained by a computer fit of the data and the Jz determined from the semi-log plot as before. In this analysis the initial slope of the At vs t plot is proportional to Jirl, since at the earliest time points, loss of label due to Jo, t is insignificant, since brain lipids are not labeled at t = 0. The initial slope (Ji, ,n ) can be derived from Eqn 1 above as
Jin,A = (dAIldt),=,
= A,,
.k
(3)
Standard error estimates can be obtained from the least-squares fit of the semi-log plots for h and A,, from the slope and intercept, respectively [12]. Thus a t value can be calculated for differences in Ji,, or rather (dAt/dt), = 0 which is proportional to Ji,, and these differences can be tested for significance. Likewise, differences in k can be tested for significance from error estimates obtained in the least squares fit. Results Fig. 1 illustrates the incorporation of the radioactive fatty acids into plasma and brain lipids of control and essential fatty acid-deficient rats. Large differences were seen in comparing the plasma activity levels of different fatty acids. Comparing the plasma activity levels of control and deficient animals, gross differences are noted in the levels of the oleic acid label with the deficient being higher at all time points. No such difference is seen with the arachidonic acid label, the control and deficient being comparable throughout. The incorporation of labeled fatty acids into brain lipids reveals significant effects of fatty acid structure, just as did incorporation into plasma lipids. At early time points, there is little difference between control and deficient animals in the uptake of either oleic or arachidonic acid. However, at later times, the level of activity is higher in deficient animals with both fatty acids. These general impressions on the nature of brain uptake can be quantitated and tested for significance by analysing the data according to first-order kinetics. Semi-log plots of the brain data in Fig. 1 are shown in Fig. 2. The fact that in each instance the semi-log plots showed very little deviation from linearity supports the validity of assumption (2) above. Table I shows the relevant kinetic parameters derived from Fig. 2. Error estimates derived from the least squares fit were used to test for significance by the t-test. The higher influx of arachidonic was significant (P < 0.05) but the influx of oleic was not significantly different from that of palmitic acid. In animals fed the fat-free diet, influx rates of arachidonic and oleic acids were not significantly different from corresponding values from control animals, but were, of course, significantly different from each other. Control and deficient rats did differ in some respects. The rate constant for the approach to equilibrium, k, is significantly higher in control animals with both oleic and arachidonic acids. Given the virtual equivalence of influx rates from control and deficient animals, this necessarily means that the asymptotic value A,, will be higher in deficient animals, as is observed with both fatty acids.
335
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-.
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3
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9
TIME
12
15
18
21
24
(hours)
Fig. 1. Labeled fatty acid uptake into plasma and brain lipids of control and deficient brain. c --3, control rats fed [3H]oleate; n -m. deficient rats fed [ 3HI &ate: rats fed L3Hl arachidonate; l - - - - - -0, deficient rats fed L3Hl arachidonate: il. . . . . C3Hl palmitate. Each point is the average of five rats.
rats. A, plasma; B. co- - - - - -3, control .^,, control rats fed
To test whether the higher final relative specific activity in brain lipids of deficient animals was due to an isotope dilution effect, i.e. due to lower fatty acid levels in blood resulting in higher specific activities being presented to the brain, the following experiment was performed. A group of deficient animals was placed on a control diet for 12 or 24 h prior to the administration of a 24-h label plus. Such a change in diet should drastically alter the blood levels of arachidonic acid especially, since it is a member of the essential w6 family and thus occurs in very low levels in the blood of deficient animals. The results of this experiment are presented in Table II. The labeling of plasma and brain lipids by [“HI palmitic acid was not significantly altered by essential fatty acid deficiency or by deficiency followed
-4.2’
I 3
I 6
1 9
, 12
I 18
I 15
I 21
1 24
t ( hour 1 Fig.
2.
First-order
deficient [3H]
rats
fed
arachidonate:
TABLE
[3Hl
of
brain
oleate:
Au’,
fatty
acid
i-----i‘, control
rats
rats fed
[ 3Hl
PARAMETERS
FOR
THE
UPTAKE
OF
control
X,
fed
[ 3Hl
palmitate.
rats
arachidonate;
Each
point
fed
[xHl
l-m,
oleate:
0 ~0,
deficient
is the average
of five
BY
THE
BRAIN
X 103)
Jin,A
0.122
+ 0.003
0.388
! 0.008
0.0475
i 0.0017
0.217
?- 0.021
0.283
f 0.020
0.0614
i 0.081
Deficient
0.115
i 0.010
0.432
+ 0.014
0.0497
i 0.050
Control
0.108
i 0.0034
2.65
+ 0.11
0.287
+ 0.015
Deficient
0.0608
? 0.0024
4.48
f 0.14
0.273
? 0.013
DEFICIENCY
AND
k + S.D.
Palmitic
Control
Oleic
Control
TABLE
ACIDS
A eq
acid supplied
Arachidonic
FATTY
(h-l)
Diet
rats fed
rats.
f
S.D.
(A
S.D.
i
(A
X 103/h)
II
INFLUENCE FATTY
Fatty
X-
uptake.
control
I
KINETIC
Fatty
plot
OF
ACID
acid
Pabnitic
ESSENTIAL
ENTRY
supplied
INTO
FATTY
Plasma
Diet
TO
A CONTROL
lipid
A (X
103)
Brain
25
0.40
Deficient
28
0.37
28
0.39
+ 24 h control
Control Deficient
Arachidonic
RETURN
Control Deficient
Oleic
ACID
DIET
BRAIN
9.9
0.31
25.7
0.45
Deficient
+ 12 h control
20.1
0.18
Deficient
+ 24 h control
6.6
0.38
Control
181
2.3
Deficient
195
3.3
Deficient
+ 12 h control
156
3.5
Deficient
+ 24 h control
173
3.0
lipid
A (X
103)
ON
by 24 h on a control diet. The labeling pattern with [.’ H] oleic acid was altered substantially by a return from a fat-free diet, to a control diet. Such fluctuations in labeling may be a result of the rapidly changing metabolic state of these animals. The most important result of this experiment occurred in the arachidonic experiment, the one in which the label dilution would be the greatest upon return to a control diet. The brain lipid relative specific activities of deficient animals placed on a control diet for 12 or 24 h do not differ significantly from those of deficient animals, and, as in deficient, animals, are significantly higher t,han controls after a 24-h labeling period. Discussion In our investigations the labeled fatty acid was presented to the brain in its most nearly normal physiologic form, i.e. by iI~testina1 adsorption. The importance of the route of administration of metabolites to the brain has been stressed [ 13,141. Despite the added complexity of the intestinal route, we find it to be the preferred method of comparing normal and essential fatty aciddeficient brain uptake for the following reasons: (1) There is no difference in the rate of intestinal absorption of lipids of essentiat fatty acid-deficient rats compared to normal rats when the lower body weights of deficient rats is taken into account [15]. (2) The uptake and distribution of labeled fatty acid in complex lipids of brain is similar whether the label is administered orally or bound to albumin and injected intravenously [1,8], This is consistent with the idea that the preferred form for fatty acid uptake by the brain is free fatty acid bound to albumin [ 16,171. The analysis of brain fatty acid uptake by first-order kinetics is an oversimplification. The quantity we have identified as influx is actually a composite of rates of the various processes which limit the entry of fatty acids into the brain, such as crossing the cerebrospinal fluid, uptake by brain cells, and incorporation into complex lipids. It is apparent from visual inspection of Fig. 1 that brain relative specific activities at early time points are very similar in deficient and control animals. Use of the simplifying assumption that the brain uptake approaches first-order kinetics allows this impression to be quantitated and tested for significance. The points at which the two groups differ are in the later time periods, as the brain activity levels off. Deficient animals show higher activities for both oleic and ~achidonic acids. Three hypotheses which would account for the higher final activities of deficient animals are as follows: (1) The flux of fatty acid out of the brain is reduced in deficient rats. Thus higher brain lipid specific activities must be reached in order for the isotope leaving the brain to balance that coming in. (2) The specific activity of the fatty acid presented to the brain is higher in deficient animals due to a lower blood level of fatty acid, hence less isotope dilution. The results of Table II argue against this possibility. (3) The fall in the level of isotope in the plasma, rather than the flux out of the brain is responsible for the plateau in brain uptake, and the plasma level falls slower in deficient animals. Such an argument might account for the differences observed in the brain activities with oleic acid, since the plasma
338
activity levels are so much higher in deficient animals, but with arachidonic acid, the plasma levels are quite comparable throughout the experiment. That the plasma activity level is not the limiting factor in causing the plateau in brain uptake is suggested by the fact that plasma activities are orders of magnitude higher than brain activities even after 24 h, when brain levels are highest and plasma levels are lowest. At isotopic equilibrium, the label distribution between the two tissues should reflect the relative content of the fatty acid in each, which certainly is not the case here. The most likely expIanation for the higher levels of label in the deficient rats appears to be that deficient rats are incorporating fatty acids into stable complex lipids to a greater extent, so that the outflow of fatty acids is reduced. Thus our studies indicate that essential fatty acid deficiency does not alter the blood-brain barrier, that is, whatever mechanism is rate-limiting for fatty acid uptake into brain remains unchanged in deficiency. However, higher levels of labeling are obtained, especially with arachidonic acid, a member of the essential w6 fatty acid family, consistent with the tendency of the brain fatty acid composition to return to normal upon restoration to a normal diet. Acknowledgement The assistance of Mrs Anne C. Turner acknowledged. Financial support was provided
and Philip Balaski is gratefully by U.S.P.H.S. Grant NB 08892.
References 1 2 3 4 5 6 7 8 Q 10 11 12 13 14 15 16 17
Carroll, K.K. (1962) Can. J. Biochem. Physiol. 40. 1229-1238 Sun, G.Y. and Horrocks, L.A. (1969) J. Neurochem. 16. 181-189 Dobbing, J. (1968) Prog. Brain Res. 29. 417-427 Dhopeshwarkar. G.H., Maier, R. and Mead, J.F. (1969) Biochim. Binphss. Acta 187, 6-12 Dhopeshwarkar, G.H. and Mead. J.F. (1969) Biochim. Biophys. Acta 187, 461-467 Dhopeshwarkar. G.H. and Mead, J.F. (lQ70) Biochim. Biophys. Acta 210, 250-256 Dhopeshwarkar, G.H., Subramanian, C. and Mead. J.F. (1971) Biochim. Biophys. Acta 231, 8-14 Dhopeshwarkar. G.H. and Mead, J.F. (1972) Adv. Lipid Rcs. 11, 109-142 White, .Jr, H.B., Galii, C. and Paoletti, R. (1971) J. Neurochem. 18, 869-882 Galli, C.. White, Jr, H.B. and Paoletti. R. (1971) Lipids 6, 378-387 Folch, J., Lees. M. and Sloane-Stanley. G.H. (1957) J. Biol. Chem. 226, 497-509 Mather, K. (1947) Statistical Analysis in Biology, pp. 113-119. Interscience, New York L&n, E. and Scicli, G. (1969) Brain Res. 13, l--12 Levin, E:. and Kleeman, CR. (1970) Proc. Sot. Esp. Biol. Med. 135, 685-689 Barnes, R.H., Miller, E.S. and Burr, G.O. (1941) J. Bioi. Chem. 140, 773-778 Spitzer, J.J. and Wolf, E.N. (1971) Am. J. Physiol. 221, 1426-1430 Dhopeshwarkar, G.H., Subramanian, C., McConnell, D.H. and Mend. J.F. (1972) Biochim. Biophys. hcta 255, 572--579
Biochimica et Biophysics Acta, 388 (1975) 331-338 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BBA 56617
THE EFFECT OF ESSENTIAL FATTY ACID DEFICIENCY FATTY ACID UPTAKE BY THE BRAIN
UPON
DOUGLAS S. LYLES, L.L. SULYA and H.B. WHITE, JR Department of Biochemistry, Miss. 39216 (U.S.A.)
University of Mississippi Medical Center, Jackson,
(Received December 18th, 1974)
Summary Young adult rats, either control or essential fatty acid deficient, were administered either [ 3 H] oleic acid or [’ H] arachidonic acid by stomach tube. In addition, a group of control rats was given [ 3 H] palmitic acid. The rats were killed at various times thereafter, and the radioactivity of the lipids of brain and plasma was examined. In confirmation of previous work, the blood lipid label was found to rise rapidly and then fall, whereas the activity of brain lipids increased slowly and did not show a decline through the 24-h period studied. Analysis of the brain uptake data according to first-order kinetics confirmed the impressions gained from visual inspection of the data. The initial rate of uptake of arachidonic acid was about 4.5 times that of oleic acid in control animals and in deficient animals. Essential fatty acid deficiency, however, did not induce an altered rate of uptake for either oleic acid or arachidonic acid. The rate of uptake of palmitic acid by control rats was not significantly different from that of oleic acid. Even though the initial rates of incorporation of oleic and arachidonic acids were not changed during essential fatty acid deficiency, the final levels of radioactivity obtained in brain lipids were higher in deficient rats with both fatty acids. The plateau value obtained with oleic acid was 1.5 times higher in deficient animals, while the plateau value for arachidonic acid was 1.7 times higher. An experiment in which deficient animals were allowed access to a control diet for 12 or 24 h prior to the labeling experiment suggested that the higher levels of radioactivity found in brain lipids of deficient animals was not due to an isotope dilution effect. Such animals still displayed. the labeling pattern of deficient animals with arachidonic acid, while the results with oleic acid varied somewhat. Our results suggest that essential fatty acid deficiency does not alter the ability of the brain to take up the fatty acids studied. However, the fatty acids, especially arachidonic, are retained in the brain to a greater extent in the deficient animals.
332
Introduction Labeled fatty acids, like many other metabolites, are taken up by the brain of adult rats at a much slower rate than the brain of younger rats in which the nervous system is still developing [l] . Such observations led to the postulate that the so-called “blood-brain barrier” applied to fatty acids as well as other molecules. Presumably the brain acquired its complement of fatty acids early in development and conserved them throughout life by recycling the degradation products of brain lipids [2]. This view has been challenged by those who maintain that the decrease in the uptake of metabolites with age represents the decreased metabolic activity accompanying the end of development rather than some restriction imposed by the blood-brain barrier [3]. Recently, experiments performed by Mead and coworkers [4-71 have been reviewed [8]. In these experiments, the direct uptake of several fatty acids by the adult rat brain was demonstrated. That such an uptake occurs is consistent with the fact that in essential fatty acid deficient rats, the fatty acid composition of the brain phospholipids returns to normal after the rats are restored to a normal diet [9,10]. Our experiments were designed to test whether essential fatty acid deficiency alters the ability of the brain to take up fatty acids, or alternatively, whether the normal rate of uptake is sufficient to reconstitute the brain fatty acid composition as essential fatty acid deficient rats recover from the deficiency. Methods Young adult Sprague-Dawley rats approx. 45 days old were used. The essential fatty acid-deficient regimen was initiated about 5 days before birth. The control diet contained 24% casein, 4.5% crude fiber, 63.5% sucrose, 4% vegetable fat, and 4% salt mixture. The essential fatty acid-deficient diet was fat free and contained 21.1% casein, 16.4% Alphacel cellulose, 58.4% sucrose, and 4% salt mixture. Each diet was supplemented with a complete complement of the necessary vitamins. Isotope administration To administer the isotopically labeled fatty acids, rats were lightly anesthetized with ether. Each isotope was administered by stomach tube in 0.5 ml of a clear solution containing 50 mg of glucose and approx. 15 PCi of labeled fatty acid prepared using the non-toxic emulsifier Pluronic F-68 (kindly supplied by Wyandotte Chemicals Corporation of Wyandotte, Michigan). After various time periods, the animals (which weighed about 150 g each) were anesthetized with 0.2 ml of Nembutal given intraperitoneally and opened from lower abdomen to the base of the neck. A 2.0-ml blood sample was taken by heart puncture, and then 0.1 ml of a sodium heparin solution (1000 U.S.P. units/ml) was injected into the heart. The animals were perfused with isotonic saline for 5 min at a pressure of about 65 cm water. The needle was placed in the left ventricle and the inferior vena cava was cut to allow drainage. Perfusion was judged complete by “tanning” of the liver and clearing of the perfusate. The whole brain was removed and frozen until analyses could be performed.
333
The radioactive fatty acids used were [9,10-’ H2 ] palmitic acid (393 Ci/mol), [9,10-3 H2 ] oleic acid (2.29 Ci/mmol), and [5,6,8,9,11,12,14,15’ H, ] arachidonic acid (18.2 Ci/mmol), all obtained from New England Nuclear. 3 H-labeled fatty acids were used in preference to a ’ ‘C label in order to circumvent the isotope recyclization problem. In the oxidative degradation of fatty acids to acetyl-CoA, a large portion of the 3 H label would be lost and would ultimately exchange with and be diluted by body water. That such degradation and reincorporation of label does not occur to a significant extent is shown by thin-layer chromatographic analysis of the label distribution in brain lipids. 24 h after the administration of [ 3 H] palmitic acid, the sterol fraction of a brain lipid aliquot contained only 34 cpm of a total 1150 cpm. There was a significant direct conversion of [’ H] palmitic acid to other fatty acids, as one-fourth of the counts in the fatty acid fraction was associated with monoenoic fatty acids. Lipid analysis Total lipid was extracted from the tissues with chloroform/methanol (2 : 1, v/v) and washed once by the method of Folch et al. [ll] . Aliquots of the lipid extract were taken and the solvent was evaporated for gravimetric determination of total lipid weight or for determination of radioactivity by liquid scintillation counting in 10 ml of 0.5% 2,5-bis-(2-(5-t-butylbenzoxazolyl))-thiophene in toluene. All counts were converted to dpm values by comparison with an external standard. Calculations The calculation of radioactivity per mg of lipid has been normalized for the activity of the dose administered and for the dilution due to the weight of the rat. This quantity, which we refer to as relative specific activity (A), has the units (dpm/mg lipid)/(dpm administered/g weight of rat). Thus comparisons between the various fatty acids can be made. For purposes of statistical comparison, the brain uptake data were analysed according to first-order kinetics. The assumptions made in this analysis are (1) that in each instance there is a flux (J) of fatty acid into (Ji,) and out of (Jout) the brain; therefore, the kinetics of labeling are determined by both Jin and J out9. and (2) that this system approximates first-order kinetics, thus A, =Aeq(l
-ePkL)
(1)
where A, = relative specific activity at any time t, A,, = the plateau relative specific activity (obtained before the level of radioactivity in the brain begins to fall), and k = the rate constant for the approach to equilibrium, which can be shown to be proportional to J,, t if assumption (1) above is true. In these examples in which A,, was known (i.e. the palmitate and oleate experiments), k was calculated from the equation
Wk,
-A,)
by plotting
=lnA,, log (A,,
-kt - A,) versus t, the slope being -k/2.303.
(2) In those experi-
334
ments in which a plateau was not reached by 24 h (the arachidonate experiments), the plateau value A,, was obtained by a computer fit of the data and the Jz determined from the semi-log plot as before. In this analysis the initial slope of the At vs t plot is proportional to Jirl, since at the earliest time points, loss of label due to Jo, t is insignificant, since brain lipids are not labeled at t = 0. The initial slope (Ji, ,n ) can be derived from Eqn 1 above as
Jin,A = (dAIldt),=,
= A,,
.k
(3)
Standard error estimates can be obtained from the least-squares fit of the semi-log plots for h and A,, from the slope and intercept, respectively [12]. Thus a t value can be calculated for differences in Ji,, or rather (dAt/dt), = 0 which is proportional to Ji,, and these differences can be tested for significance. Likewise, differences in k can be tested for significance from error estimates obtained in the least squares fit. Results Fig. 1 illustrates the incorporation of the radioactive fatty acids into plasma and brain lipids of control and essential fatty acid-deficient rats. Large differences were seen in comparing the plasma activity levels of different fatty acids. Comparing the plasma activity levels of control and deficient animals, gross differences are noted in the levels of the oleic acid label with the deficient being higher at all time points. No such difference is seen with the arachidonic acid label, the control and deficient being comparable throughout. The incorporation of labeled fatty acids into brain lipids reveals significant effects of fatty acid structure, just as did incorporation into plasma lipids. At early time points, there is little difference between control and deficient animals in the uptake of either oleic or arachidonic acid. However, at later times, the level of activity is higher in deficient animals with both fatty acids. These general impressions on the nature of brain uptake can be quantitated and tested for significance by analysing the data according to first-order kinetics. Semi-log plots of the brain data in Fig. 1 are shown in Fig. 2. The fact that in each instance the semi-log plots showed very little deviation from linearity supports the validity of assumption (2) above. Table I shows the relevant kinetic parameters derived from Fig. 2. Error estimates derived from the least squares fit were used to test for significance by the t-test. The higher influx of arachidonic was significant (P < 0.05) but the influx of oleic was not significantly different from that of palmitic acid. In animals fed the fat-free diet, influx rates of arachidonic and oleic acids were not significantly different from corresponding values from control animals, but were, of course, significantly different from each other. Control and deficient rats did differ in some respects. The rate constant for the approach to equilibrium, k, is significantly higher in control animals with both oleic and arachidonic acids. Given the virtual equivalence of influx rates from control and deficient animals, this necessarily means that the asymptotic value A,, will be higher in deficient animals, as is observed with both fatty acids.
335
A
4.0-
B
3.53.05 -
2.5-
4
2.G
I’
,’
/’
,’
I’
__-,,’ ,I p
1.5-
.’
I’
.’
__--
_/-
-.
__---o
_/-/Yr
,/P
3
6
9
TIME
12
15
18
21
24
(hours)
Fig. 1. Labeled fatty acid uptake into plasma and brain lipids of control and deficient brain. c --3, control rats fed [3H]oleate; n -m. deficient rats fed [ 3HI &ate: rats fed L3Hl arachidonate; l - - - - - -0, deficient rats fed L3Hl arachidonate: il. . . . . C3Hl palmitate. Each point is the average of five rats.
rats. A, plasma; B. co- - - - - -3, control .^,, control rats fed
To test whether the higher final relative specific activity in brain lipids of deficient animals was due to an isotope dilution effect, i.e. due to lower fatty acid levels in blood resulting in higher specific activities being presented to the brain, the following experiment was performed. A group of deficient animals was placed on a control diet for 12 or 24 h prior to the administration of a 24-h label plus. Such a change in diet should drastically alter the blood levels of arachidonic acid especially, since it is a member of the essential w6 family and thus occurs in very low levels in the blood of deficient animals. The results of this experiment are presented in Table II. The labeling of plasma and brain lipids by [“HI palmitic acid was not significantly altered by essential fatty acid deficiency or by deficiency followed
-4.2’
I 3
I 6
1 9
, 12
I 18
I 15
I 21
1 24
t ( hour 1 Fig.
2.
First-order
deficient [3H]
rats
fed
arachidonate:
TABLE
[3Hl
of
brain
oleate:
Au’,
fatty
acid
i-----i‘, control
rats
rats fed
[ 3Hl
PARAMETERS
FOR
THE
UPTAKE
OF
control
X,
fed
[ 3Hl
palmitate.
rats
arachidonate;
Each
point
fed
[xHl
l-m,
oleate:
0 ~0,
deficient
is the average
of five
BY
THE
BRAIN
X 103)
Jin,A
0.122
+ 0.003
0.388
! 0.008
0.0475
i 0.0017
0.217
?- 0.021
0.283
f 0.020
0.0614
i 0.081
Deficient
0.115
i 0.010
0.432
+ 0.014
0.0497
i 0.050
Control
0.108
i 0.0034
2.65
+ 0.11
0.287
+ 0.015
Deficient
0.0608
? 0.0024
4.48
f 0.14
0.273
? 0.013
DEFICIENCY
AND
k + S.D.
Palmitic
Control
Oleic
Control
TABLE
ACIDS
A eq
acid supplied
Arachidonic
FATTY
(h-l)
Diet
rats fed
rats.
f
S.D.
(A
S.D.
i
(A
X 103/h)
II
INFLUENCE FATTY
Fatty
X-
uptake.
control
I
KINETIC
Fatty
plot
OF
ACID
acid
Pabnitic
ESSENTIAL
ENTRY
supplied
INTO
FATTY
Plasma
Diet
TO
A CONTROL
lipid
A (X
103)
Brain
25
0.40
Deficient
28
0.37
28
0.39
+ 24 h control
Control Deficient
Arachidonic
RETURN
Control Deficient
Oleic
ACID
DIET
BRAIN
9.9
0.31
25.7
0.45
Deficient
+ 12 h control
20.1
0.18
Deficient
+ 24 h control
6.6
0.38
Control
181
2.3
Deficient
195
3.3
Deficient
+ 12 h control
156
3.5
Deficient
+ 24 h control
173
3.0
lipid
A (X
103)
ON
by 24 h on a control diet. The labeling pattern with [.’ H] oleic acid was altered substantially by a return from a fat-free diet, to a control diet. Such fluctuations in labeling may be a result of the rapidly changing metabolic state of these animals. The most important result of this experiment occurred in the arachidonic experiment, the one in which the label dilution would be the greatest upon return to a control diet. The brain lipid relative specific activities of deficient animals placed on a control diet for 12 or 24 h do not differ significantly from those of deficient animals, and, as in deficient, animals, are significantly higher t,han controls after a 24-h labeling period. Discussion In our investigations the labeled fatty acid was presented to the brain in its most nearly normal physiologic form, i.e. by iI~testina1 adsorption. The importance of the route of administration of metabolites to the brain has been stressed [ 13,141. Despite the added complexity of the intestinal route, we find it to be the preferred method of comparing normal and essential fatty aciddeficient brain uptake for the following reasons: (1) There is no difference in the rate of intestinal absorption of lipids of essentiat fatty acid-deficient rats compared to normal rats when the lower body weights of deficient rats is taken into account [15]. (2) The uptake and distribution of labeled fatty acid in complex lipids of brain is similar whether the label is administered orally or bound to albumin and injected intravenously [1,8], This is consistent with the idea that the preferred form for fatty acid uptake by the brain is free fatty acid bound to albumin [ 16,171. The analysis of brain fatty acid uptake by first-order kinetics is an oversimplification. The quantity we have identified as influx is actually a composite of rates of the various processes which limit the entry of fatty acids into the brain, such as crossing the cerebrospinal fluid, uptake by brain cells, and incorporation into complex lipids. It is apparent from visual inspection of Fig. 1 that brain relative specific activities at early time points are very similar in deficient and control animals. Use of the simplifying assumption that the brain uptake approaches first-order kinetics allows this impression to be quantitated and tested for significance. The points at which the two groups differ are in the later time periods, as the brain activity levels off. Deficient animals show higher activities for both oleic and ~achidonic acids. Three hypotheses which would account for the higher final activities of deficient animals are as follows: (1) The flux of fatty acid out of the brain is reduced in deficient rats. Thus higher brain lipid specific activities must be reached in order for the isotope leaving the brain to balance that coming in. (2) The specific activity of the fatty acid presented to the brain is higher in deficient animals due to a lower blood level of fatty acid, hence less isotope dilution. The results of Table II argue against this possibility. (3) The fall in the level of isotope in the plasma, rather than the flux out of the brain is responsible for the plateau in brain uptake, and the plasma level falls slower in deficient animals. Such an argument might account for the differences observed in the brain activities with oleic acid, since the plasma
338
activity levels are so much higher in deficient animals, but with arachidonic acid, the plasma levels are quite comparable throughout the experiment. That the plasma activity level is not the limiting factor in causing the plateau in brain uptake is suggested by the fact that plasma activities are orders of magnitude higher than brain activities even after 24 h, when brain levels are highest and plasma levels are lowest. At isotopic equilibrium, the label distribution between the two tissues should reflect the relative content of the fatty acid in each, which certainly is not the case here. The most likely expIanation for the higher levels of label in the deficient rats appears to be that deficient rats are incorporating fatty acids into stable complex lipids to a greater extent, so that the outflow of fatty acids is reduced. Thus our studies indicate that essential fatty acid deficiency does not alter the blood-brain barrier, that is, whatever mechanism is rate-limiting for fatty acid uptake into brain remains unchanged in deficiency. However, higher levels of labeling are obtained, especially with arachidonic acid, a member of the essential w6 fatty acid family, consistent with the tendency of the brain fatty acid composition to return to normal upon restoration to a normal diet. Acknowledgement The assistance of Mrs Anne C. Turner acknowledged. Financial support was provided
and Philip Balaski is gratefully by U.S.P.H.S. Grant NB 08892.
References 1 2 3 4 5 6 7 8 Q 10 11 12 13 14 15 16 17
Carroll, K.K. (1962) Can. J. Biochem. Physiol. 40. 1229-1238 Sun, G.Y. and Horrocks, L.A. (1969) J. Neurochem. 16. 181-189 Dobbing, J. (1968) Prog. Brain Res. 29. 417-427 Dhopeshwarkar. G.H., Maier, R. and Mead, J.F. (1969) Biochim. Binphss. Acta 187, 6-12 Dhopeshwarkar, G.H. and Mead. J.F. (1969) Biochim. Biophys. Acta 187, 461-467 Dhopeshwarkar. G.H. and Mead, J.F. (lQ70) Biochim. Biophys. Acta 210, 250-256 Dhopeshwarkar, G.H., Subramanian, C. and Mead. J.F. (1971) Biochim. Biophys. Acta 231, 8-14 Dhopeshwarkar. G.H. and Mead, J.F. (1972) Adv. Lipid Rcs. 11, 109-142 White, .Jr, H.B., Galii, C. and Paoletti, R. (1971) J. Neurochem. 18, 869-882 Galli, C.. White, Jr, H.B. and Paoletti. R. (1971) Lipids 6, 378-387 Folch, J., Lees. M. and Sloane-Stanley. G.H. (1957) J. Biol. Chem. 226, 497-509 Mather, K. (1947) Statistical Analysis in Biology, pp. 113-119. Interscience, New York L&n, E. and Scicli, G. (1969) Brain Res. 13, l--12 Levin, E:. and Kleeman, CR. (1970) Proc. Sot. Esp. Biol. Med. 135, 685-689 Barnes, R.H., Miller, E.S. and Burr, G.O. (1941) J. Bioi. Chem. 140, 773-778 Spitzer, J.J. and Wolf, E.N. (1971) Am. J. Physiol. 221, 1426-1430 Dhopeshwarkar, G.H., Subramanian, C., McConnell, D.H. and Mend. J.F. (1972) Biochim. Biophys. hcta 255, 572--579
