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Thyroxine degradation
ARCHIVES
OF
BIOCHEMISTRY
AND
126, 880-891 (1968)
BIOPHYSICS
Thyroxine Antioxidant
Function
and
Microsomal
Degradation Nonenzymatic lipid
JAMES
Degradation
during
Peroxidation’ WYNN
Department of Medicine, University of Arkansas Medical Center, Little Rock, Arkansas Received
February
29, 1968; accepted
June 19, 1968
These studies were undertaken with the aim of demonstrating an interaction of the phenoxyl free radical of thyroxine with a component of liver microsomes. The probability of such an interaction had been suggested by the prior study of the degradation of thyroxine by rat liver microsomes. The microsomal degradation reactions of thyroxine are initiated by ferrous iron. Rapid oxidation in microsomes is also initiated by ferrous iron. Such rapid oxidation is terminated by thyroxine and similar compounds. The material rapidly oxidized in microsomes in the presence of ferrous iron is a lecithin. Thyroxine and related compounds function as highly specific antioxidants exceeding the effectiveness of vitamin E, aecorbate, cysteine, glutat,hione or epinephrine. Study of the features of the antioxidant reaction suggests that thyroxine terminates the oxidations by reaction with the propagating species of a chain oxidation. During the course of such chain termination in peroxidizing lecithin, thyroxine is itself nonenzymatically degraded to yield the same products as those formed during microsomal degradation initiated by ferrous iron. The study supports the hypothesis that thyroxine participates in a system of free radical reactions, and as a consequence of these reactions it is degraded.
The phenoxyl radical of thyroxine may be the reactive species in the microsomal reactions leading to thyroxine degradation (1). In this paper, investigations are presented which demonstrate an antioxidant function of thyroxine during oxidation of microsomal lecithin. During the antioxidant reaction, thyroxine is nonenzymatically degraded to yield products which are identical to those previously found to occur during the microsomal degradation of thyroxine (2, 3). Thyroxine participates in these reactions by a mechanism compatible with its function as a free radical chain terminator supporting the hypothesis that the formation of a thyroxine radical precedes its degradation. 1 This work was supported by Grant AM 1240701 from the National Institutes of Health to the University of Arkansas.
EXPERIMENTAL
PROCEDURE
Materials
L-Thyroxine, L-thyroxine (3’, 5’ -1311), (1-9 -14C) and (lo-15 -14C), thyroxine analogues and related phenols were all obtained from sources or prepared by methods previously described (14). Standard fatty acids, phospholipids, lysolecithin, cholesterol, cerebrosides, sulfatides and sphingomyelin were purchased from Applied Science Laboratories. nn-ar-Glycerophosphate disodium salt and a-glycerophosphate dehydrogenase were purchased from Sigma Chemical Company. Cumene hydroperoxide, t-butyl hydroperoxide and 8-p-menthyl hydroperoxide were purchased from Matheson, Coleman and Bell. Aluminum oxide, neutral, activity grade 1 was purchased from M. Woelm, Eschwege, Germany.
THYROXINE
DEGRADATION
881
acid:ether. Neither was eluted after beginning the chloroform elution. Microsomes were prepared from rat livers Assay for long chain aldehydes derived as previously described (4). from plasmalogens was assessed by the pFractionation of lipid, protein and water sol- nitrophenylhydrazine method of Rapport uble components of microsomes. Two ml of and Alonzo (5). microsome suspension was packed at 8OOOg Determination of cu-punsaturated linkage in (after freezing, microsomes pack easily under phospholipids was measured by the method these conditions), and the sediment was re- of Gottfried and Rapport (6). suspended in 100 ml of chloroform: methDetermination of acyl ester groups in phosanol 50 : 50 (v/v). After stirring for 1 hr, in- pholipids was measured by the method of soluble residue was removed by filtration, Rapport and Alonzo with the following moddried under nitrogen and resuspended in 0.25 ification (7). The alkaline hydroxylamine sucrose (fraction No. 1). The chloroform: was added prior to ether and allowed to react methanol filtrate was dried under nitrogen for 5 minutes with lipid previously dried and the residue was mixed with 15-20 ml of under nitrogen in the reaction tube. ethanol. The portion of the material insoluTitration of unsaturated carbon bonds with ble in ethanol was removed by filtration and ICI was carried out by a modification of the dissolved in 5 ml of chloroform:methanol procedure of Wijs (8). Results are expressed (fraction No. 2). The clear, ethanol filtrate in terms of moles of ICl taken up per mole (fraction No. 3) was dried under nitrogen, of lipid phosphorus added. and a suspension of the residue was prepared Lipid phosphorus was determined by the in 5 ml of hexane: ethyl ether : acetic acid 85 : method of Chen, Toribara, and Warner (9). 15 : 1 (v/v). A column of dry aluminum oxide Lipid nitrogen was determined by the as supplied, 0.9 cm in diameter and 20 cm tall micro Kjeldahl method of Lang (10). was prepared, washed with 200 ml of hexane: Anaerobic, alkaline hydrolysis of phosphoethyl ether: acetic acid and onto this was lipid was carried out as follows : lo-30 pmoles placed the hexane: ethyl ether: acetic acid of phospholipid phosphorus was dried in a suspension of ethanol soluble, lipid sub- test tube under nitrogen. One to five millistances. The column was washed with 150 liters of 1.0 N NaOH previously gassed with ml of hexane:ethyl ether:acetic acid, the nitrogen was added and the tube was sealed effluent was collected in one batch, dried anaerobically. After incubating at 100” for under a stream of nitrogen, and the residue 3 hr and at 55” for 24 hr, material prepared was dissolved in 5 ml of absolute ethanol for thin-layer chromatography was solu(fraction No. 3a). The column was then bilized by the addition of an equal volume washed with 150 ml of chloroform collected of absolute ethanol and stored under nitroin a batch which was dried and the residue gen until used. Hydrolyzate prepared for was dissolved in 5 ml of ethanol (fraction No. the cY-glycerophosphate assay was passed 3b). The chloroform wash was followed by 150 through a column of Dowex 50 ‘H’ form mlof chloroform: methanol 50: 50, theeffluent sufficient to remove 100 mEq of cation. of whichwas fractionated into 3-ml aliquots Effluent and washes were dried, extracted (fraction No. 3~). These were individually three times with hexane and reconstituted dried under nitrogen, and each was dissolved in a l-ml volume of water. A standard soluin 5 ml of ethanol. After having been mixed tion of m-a-glycerophosphate was subjected with appropriate solvent each fraction was ex- to this same procedure with no loss of the starting material. amined as outlined below for contained Estimation of L-a-glycerophosphate in material susceptible to rapid rates of oxidation. Standard FMN and FAD were added anaerobic, alkaline hydrolysate was carried out by the procedure of Bublitz and Kento the lipid preparation in order to ascertain whether they might be co-eluted with the nedy (11). A control procedure was set up to lipid. Small amounts of these flavins were determine whether the hydrolysis of lecithin in hot NaOH would lead to complete eluted during the wash with hexane:acetic Methods
882
JAMES
racemization of the freed cu-glycerophosphate. n-a-Glycerophosphate was prepared by allowing L-a-glycerophosphate dehydrogenase to react to completion on a DL substrate. The n-cu-glycerophosphate remaining was then processed as the lecithin was in hot 1 N NaOH. Following recovery of the aglycerophosphate, half of that which was originally D isomer could now be accounted for as the L-a-glycerophosphate as measured by the enzyme assay. Methylation of fatty acids for gas chromatography was carried out according to the method of Morrison and Smith using boron trifluoride and methyl alcohol as the methylating agents (12). Gas chromatography of fatty acid methyl esters derived from phospholipid was carried out on Research Specialties Model 600 apparatus using an Argon ionization chamber with strontium 90 as the source. The procedure was carried out using benzene as the solvent at 180”. The stationary phase was ethylene glycol succinate and the support was Anachrome A-B 60-80 mesh. The column was 6 feet long and 0.25 inches in diameter. The gas was argon at 15 lb/square inch. Thin-layer chromatography was carried out only in silica gel G. Lecithin separation was best demonstrated in chloroform: methanol: water 65: 25:4. Choline in the hydrolyzate was demonstrated in a two-dimensional solvent system prepared as follows: the first solvent was 70 % acetic acid developed at 4’; after drying, the second solvent was 90 % acetic acid at room temperature. Fatty acids, lecithin and choline were located by iodine staining. Perchloric acid-ammonium molybdate in HCl was used to demonstrate aglycerophosphate (13). Paper chromatography of thyroxine and its labeled derivatives was carried out using methods previously described (2). Synthesis of 1,4-hydroquinone dibenzoate and recrystallization methods using this compound are previously described (3). Electrophoresis of presumed iodide and 3,5diiodotyrosine derived from labeled thyroxine was carried out in 0.2 M ammonium carbonate solution at room temperature and 500 V. Thiobarbituric acid reaction for the detec-
WYNN
tion of malonyl dialdehyde was carried out by a modification of the method described by Dohle, Elden, and Holman (14). Xanthine oxidase reaction: milk xanthine oxidase was kindly supplied by Dr. K. V. Rajagopalen of the Department of Biochemistry, Duke University. The reaction was carried out in Tris buffer at pH 7.8 made 1 X 10v4 with versene. Oxygen consumption was usually measured polarographically, but in certain instances the Warburg apparatus was used. The oxygen electrode was a teflon membrane covered platinum electrode with internal KC1 bridge to a saturated KC1 calomel reference electrode. Measurements were made in temperature-controlled, narrow-necked 2- or lo-ml vessels at 23’. Microsomes were added to make the final concentration such that 1 ml contained the microsomes derived from 20 mg of original liver. Lecithin oxidation was examined at several concentrations but the concentration at which most of the studies were carried out was 4 X 1O-5 M. Lecithin was added in 0.2 ml ethanol. Controls contained the same quantity of ethanol. Buffer was either 5 X low2 M citrate at pH 4.7 or 1 X 10m2M phosphate at pH 7.0. Ferrous iron was added in aqueous solution as ferrous ammonium sulfate using various quantities as indicated in the legends of the figures. Thyroxine and analogues were added in 0.01 ml of ethanol. The Warburg apparatus was used in experiments in which attempts were made to oxidize large amounts of lecithin. The side arm contained 4 X 10m6moles of Fez+. The central compartment contained the same molar amount of lecithin. The volume was made up to a final total of 2.0 ml with 2.5 X lo+! M citrate buffer at pH 4.7. At time zero the iron solution was tipped into the lecithin mixture. Incubations were carried out for 15 min at 37”. Assay of radioactive material: CarbonI was measured in a liquid scintillation spectrometer and by a GM tube strip scanner. Iodinem was measured in a gamma spectrometer at the 364 KEV peak and by a GM tube strip scanner.
THYROXINE RESULTS
AND
DISCUSSION
The Relationship of Microsomal Oxidation and Thyroxine Degradation Although oxygen is necessary in the Fez+stimulated reactions leading to thyroxine degradation (4), it is not established that microsomal oxidation and thyroxine degradationare related reactions. In Fig. 1 isshown a graph of the simultaneous plot of Fez+stimulated thyroxine degradation and oxygen uptake as a function of time. Thyroxine degradation was studied as in previous studies (4). The parallel course of these time curves suggests but does not establish a relationship. Isolation of the Rapidly Oxidized Substance in Microsomes In an attempt to simplify the system studied, the microsomal material which is rapidly oxidized in the presence of Fez+ was isolated. I
301
2 -0.4 3\ P -0.3 ::& I5 b -0.2 0 x -0.1 *
t =
0 10
50
100
150
200
Time-Seconds
FIG. 1. Oxygen uptake by 0.2 ml microsomes and the simultaneous degradation of thyroxine is shown. The left-hand ordinate indicates the amount of oxygen uptake; the right-hand ordinate indicates the pmoles of thyroxine degraded as measured by the amount of inorganic iodide produced (4). The reaction was carried out in 0.05 M citrate buffer at pH 4.7 containing 1 X 10-C M thyroxine and was initiated by 2 X 10-d M Fez+. The curve of oxygen uptake has been corrected by subtracting a blank value for the oxidation of Fez+ alone. The degradation reaction shows an initial lag but then parallels the course of the oxidation. In Figs. 5 and 6, studies are shown in which thyroxine added to microsomes and microsomal lecithin stopped the oxidation. If the amount of thyroxine is small and the amount of Fee+ large, oxidation is favored. If the amount of thyroxine is large and the amount of Fez+ is small, oxidation can be virtually entirely stopped.
DEGRADATION
883
Fortuitously, the procedure carried out developed into a very reproducible method for isolating a relatively pure lecithin fraction. The lipid-free protein containing fraction No. 1 contained no materials susceptible to such oxidation, and the ethanol soluble lipid fraction No. 3 contained all such reactive material. Fractionation of the ethanol soluble lipids on alumina gel allowed the separation of a discrete fraction among the lipids which was susceptible to rapidly induced Fe2+ oxidation. This fraction was eluted from alumina immediately behind the front of the chloroform: methanol 50: 50 eluant or in fraction No. 3c. Fractions No. 3a and No. 3b contained no such rapidly oxidized material. Chromatography showed the pertinent material to be composed of lipid inseparable from standard lecithin. The ninhydrin reaction was negative. Two minor components seen after prolonged iodine staining were also present. These latter components did not have mobility characteristics associated with any of the standard lipids used for identification. They did not contain stainable phosphate. Further characterization of this lipid material was then undertaken. In Table I is shown a resume of the pracedures carried out to identify this lipid material. The attempts to demonstrate long chain aldehyde and a-0 unsaturated bonds were negative. The phosphorus :nitrogen ratio was 1 .O: 1.05. The phosphorus : acyl ester ratio was 1.0 : 1.94. The phosphorus: ICl molar ratio was 1.0:2.5. The theoretical molecular weight based on the assumption of one phosphorus atom per molecule after drying and weighing a sample was 764. In Fig. 2 is shown a drawing of a chromatogram of choline and cu-glycerophosphate tentatively identified by thin-layer chromatography in silica gel following alkaline hydrolysis of the lecithin-like fraction. The content of L-a-glycerophosphate in an alkaline hydrolyzate was measured enzymatically. Thirty micromoles of organic phosphorus was subjected to alkaline hydrolysis. Twenty-four micromoles of organic phosphorus was recovered after passage through Dowex 50. Sixteen micromoIes of m-a-glycerophosphate was accounted for enzymatically representing a 67 % recovery
884
JAMES
WYNN
TABLE
I
PROCEDURES CARRIED OUT ATTEMPTING TO IDENTIFY THE PHOSPHOLIPID DERIVED FROM M~c~oso~~s SUXZEPTIBLE TO RAPID OXIDATION Result
Procedure Silica gel G-chromatography Special Stains (a) Ninhydrin (b) Malachite green (lysolecithin) (c) Phosphomolybdic acid (choline) p-nitrophenylhydrazine (G-B unsaturated aldehyde) Iodine uptake (a-p unsaturated aldehyde) Phosphorus:Nitrogen rat.io Phosphorous:acyl ester ratio Phosphorous:ICl uptake ratio Dried weight of sample containing 3.1 mg Phosphorous Alkaline hydrolyzate (a) Products demonstrated chromatographically (b) Recovery of organic Phosphorous as L-W glycerophosphate Gas chromatography of fatty acid methyl esters
70%
L-a -GI
Acetic
Inseparable
from standard
lecithin
Negative Negative Positive Negative Negative 1:1.05 1:1.94 1:2.50 76.4 mg Choline, 67% See Table
L-cu-glycerophosphate
II
Acid at 4O
hots Q Origin
FIG. 2. Silica gel chromatography of the alkaline hydrolysate of microsomal lecithin is shown. Spots identified as choline and L-cu-glycerophosphate were verified by co-chromatography of known standards on the plates with unknown. The standard and unknown spots were inseparable.
of the expected compound. The remaining 33% was not identified. In Fig. 3 is shown a graph of the gas chromatography of the fatty acid methyl esters prepared from the lecithin-like material. The fatty acid esters isolated by this procedure
FIG. 3. Gas chromatography of the fatty acid methyl esters derived from microsomal lecithin is shown. The larger peaks were tentatively identified by co-chromatography of standard materials. The smaller peaks A, B, C, D, and E are unidentified. The time scale is distorted between 18 and 20 and 45 and 120 min during which no peaks were eluted.
were inseparable from palmitic, steric, oleic, linoleic, and arachidonic esters. Several small peaks of unidentified materials were isolated as well. On the basis of the cochromatography of known quantities of standard materials and the calculated area developed for these known quantities, the recovery of each tentatively identified fatty acid ester was
THYROXINE TABLE MOLAR RECOVERY ESTERS DTJRING
On the basis of the extent of ferric the original sample lipid phosphorous ester.
Palmitate Stearate Oleate Linoleate Arachidonate
II
OF FATTY ACID METHYL Gas CHROMATOGRAPHY
the phosphorous content and per-chlorate reacting acyl ester contained 12.8 X lo+ moles of and 24.8 X lo-” moles of acyl
1.26 1.24 0.49 0.79 1.45
4.66 4.16 1.66 2.69 4.46
26.4 23.6 9.4 15.3 25.3
calculated. In Table II these absolute recoveries and the percentage of the total fatty acid methyl ester recovery are shown with comparisons on a molar basis. The yield of recovered fatty acid methyl ester was 70% of the theoret’ical yield based on the quantity of ester calculated to be present by the ferric perchlorate reaction. Assuming that the 30 % lost during methylation was evenly distributed among the component fatty acids, the average number of double bonds per molecule of lecithin containing one saturated and one unsaturated fatty acid is 2.7, and this agrees closely with the phosphorus : ICl ratio of 1:2.5. Two hundred and ninety mg of lecithin were recovered from 1.72 g of dry weight of microsomes or 17% of the dry weight of microsomes was lecithin. This compares to a recovery of 15-21% obtained by other workers comparing dry weight of lecithin to dry weight of microsomes (15). Following these studies, it was concluded that the lipid material isolated as the rapidly oxidizable fraction was predominantly lecithin without measurable plasmalogen or lysolecithin contaminants. There was minor contamination by other lipid substances shown by thin-layer and gas chromatography, but these were not identified. The systems contained no FMN or FAD for reasons noted in the METHODS section. On the basis of the phosphorus to nitrogen to ester ratio, the absence of a ninhydrin reaction, the chromatography studies and
DEGRADATION
885
the theoretical molecular weight, it is felt that the material isolated is a lecithin which is 95 % pure, has an average fatty acid chain length of 18 carbons in which there are an average of 2.5 unsaturated bonds per molecule of lecithin. There are no large amounts of ususual fatty acid or other constitutents. The concentrations of lecithin used in the oxidation studies were small and the presence of iron precluded the direct measurement of small amounts of hydroperoxide product (16). Measurements of malonyl dialdehyde present before and following oxidation of lecithin were attempted with negative results. Unable to detect either hydroperoxides or malonyl dialdehyde, an attempt was then made to make a less specific observation which would confirm the fact that the lecithin was being oxidized. Two solutions of lecithin were prepared. Into one the usual addition of Fez+ was made. Into the second, oxygen was bubbled at 40” for 1 hr. Chromatograms were prepared of unaltered, oxygen-treat’ed and Fe+treated lecithin solution. The Fez+-treated and the 02-treated samples showed a relative decrease in the density of the lecithin spots and the appearance of a new, more polar group of phosphate containing spots not present in the original lecithin sample. This supports the view that it is the lecithin itself which is oxidized by the addition of Fez+. In Fig. 4, strips 1, 2, and 3 are reproductions of chromatograms illustrating the changes mentioned. Antioxidant E$ect of Thyroxine During Iron Stimulated Oxidation The influence of thyroxine on the course of microsomal oxidation was next examined. In Fig. 5 is shown a graph of the rapid oxidation of liver microsomes following the addition of small amounts of ferrous iron and the inhibition of such oxidation by 5 X 1O-6 M thyroxine. The difference between the rate of oxygen consumed by iron alone and iron plus microsomes is indicative of the rate of oxygen uptake by microsomes. If the microsomal lecithin isolated is the agent which is rapidly oxidized in the microsome, then thyroxine may inhibit iron stimulated oxidation in the lecithin preparation as well,
JAMES
WYNN
Products Formed from Thyroxine During the Oxidation-Antioxidant Reaction
.0nl
1
(3)
(4)
i---i
1 Origin
(5)
FIG. 4. A diagram is shown of the silica gel chromatography of lecithin treated several ways. In strip No. 1 lecithin was simply added to .05 M citrate buffer, pH 4.7, and gassed immediately with Nz. In strip No. 2 a similar sample was treated with Fez+ and incubated aerobically for 15 min. In strip No. 3 a similar sample was treated with O2 for 1 hr at 40”. In strip No. 4, Fe*+ was added as in strip No. 2 but in addition a final concent,ration of 1 x 1O-6 M thyroxine was added before the iron. Strip No. 5 is a standard containing several phospholipids. The solvent system and the demonstrations of phosphate containing spots are described in the methods section. The dark-hatched areas indicate dense stain. The light-hatched areas indicate a light stain. In strips No. 2 and No. 3 phosphate-containing material was identified near the origin of the chromatograms. The spots are labeled A and B. In strip No. 4, thyroxine prevented the appearance of spot A.
This is shown in Fig. 6. If repeated additions of Fez+ are made to the lecithin system containing thyroxine, the antioxidant effect is dissipated and the lecithin may then be oxidized suggesting that the thyroxine is altered during the antioxidant reaction. If it is lecithin oxidation which thyroxine prevents, the antioxidant effect of thyroxine should prevent the chromatographic appearance of the more polar oxidation products of lecithin seen following the addition of Fez+. This is shown in strip 4 of Fig. 4.
The degradation products of thyroxine in the Fez+-stimulated microsomal system are inorganic iodide, 3,5-diiodotyrosine and an acid hydrolyzable product of 1,4-hydroquinone (2, 3). Studies were undertaken to demonstrate whether the oxidation-antioxidant reaction involving oxygen, lecithin, and thyroxine might yield similar products. In separate studies products derived from thyroxine labeled with 13’1 in the ,8 phenyl ring, with 14carbon in the (Y phenyl ring and with 14carbon in the ,f3ring were determined. Chromatography in three solvents was carried out with comparable results in each system, but only the collidine-NH,-HZ0 chromatograms are shown in Fig. 7. The P phenyl ring iodine was converted to inorganic iodide; the 0 phenyl ring itself yielded a new product dissociated from the a! phenyl ring and containing no iodine; and the cr phenyl ring-alanine side chain yielded material which is inseparable from 3,5-diiodotyrosine. Inorganic iodide and 3,5-diiodotyrosine were further identified by their electrophoretic characteristics. The acid hydro-
/ 100 I
68I FIG. 5. Percentage oxygen change effected by microsomes in the presence of Fez+ with and without thyroxine is shown. The oxygen uptake of Fez+ without microsomes is shown as a blank value. Microsomes without Fez+ took up no oxygen. Thyroxine added to the Fez+ blank reaction did not retard oxygen uptake. Reactions were carried out in a final volume of 10.0 ml of 0.025 M citrate at pH 4.7. Each addition of Fe2+ indicated by an arrow contained 5 X lo-’ moles of Fez+. Microsomal volumes were 0.2 ml of the preparation described in the methods section. Thyroxine concentration was 5 x 10-G M.
THYROXINE
92: ‘Z 0 842 :: & 76L 6 <0 6860 _
DEGRADATION
887
w--w-. II-~= --I -100
No lecithin, No T4 4X 10v5M lecithin, No T4 4X10e5M lecilhin+lX10‘7M I 0
I 100
T4
I 300 Time-Seconds
I 500
I 700
c 90
FIG. 6. A graph is shown of the effect of increments of Fez+ added to solution containing 4 X 10-6 M microsomal lecithin and 1 X 10-TM thyroxine. Each increment of Fez+ was 2 X 1CP moles and is indicated by an arrow. The reaction volume was 10 ml in .025 M citrate pH 4.7. The effect of such increments of Fez+ on microsomal oxidation without thyroxine is shown for comparison.
FIG. 7. Graphs are shown of the strip counting of chromatograms developed in collidine:water: NH8 prepared following the reaction of variously labeled thyroxines with peroxidizing lecithin. The underlined captions indicate the location of standard markers on the chromatogram. Diiodotyrosine is abbreviated as DIT; monoiodotyrosine ae MIT; and thyroxine as T4. It is apparent that fl phenyl ring iodine is converted to inorganic iodide, the 01phenyl ring yields a product inseparable from diiodotyrosine, and the @phenyl ring yields a new product demonstrated to be a 1,4-hydroquinone derivative in other studies.
lyzed 14carbon product of the 0 phenyl ring was inseparable chromatographically from 1,4-hydroquinone. Prior to such hydrolysis, the labeled compound had solubility characteristics similar to those of hydroquinone dibenzoate. The unhydrolyzed, labeled product was treated with an excess of benzoyl chloride, mixed in dioxane solution with carrier hydroquinone dibenzoate and reprecipitated nine times from solution by the addition of water. Aliquots of each re-solution were studied for radioactive content and optical density at 234 rnp as a measure of hydroquinone dibenzoate recovery. These studies are shown in Table III. Specific activity was constant after the second precipitation. Thus, during Fez+ stimulated oxidation in both liver microsomes and derived lecithin, thyroxine serves as an antioxidant, is degraded during the reaction and yields products which are identical in both situations. Isolation of the Reaction Leading to Thyroxine Degradation The precise reaction in which thyroxine may participate is not, indicated by these studies. The value of having related the degradation of thyroxine to an oxidizing lecithin preparation is plain. The degradation system can be reduced to four primary
888
JAMES TABLE
REPRECIPITATION OF p HYDROQUINONE
III PHENYL PRODUCT DIAEKZOATE
AS
The p phenyl ring product formed during the reaction of thyroxine with peroxidizing lecithin was treated with an excess of benzoyl chloride. The derived labeled material was dissolved in dioxane with carrier hydroquinone dibenzoate and precipitated from solution with water. Nine precipitations and resolutions were carried out. An aliquot of the dioxane solution prepared followingeachprecipitation was studied. The 14C content and optical density at 234 rnp were measured. At 234 rnp hydroquinone dibenzoate has an absorption maximum the extinction coefficient of which is 5700. The CPM recovered relative to calculated mg recovery of hydroquinone dibenzoate is shown. Precipitation 1
2 3 4 5 6 7 8 9
Total Mg/sample
Yellow Yellow 4.72 4.69 3.81 3.52 2.91 1.86 1.19
CPM/mg hydroquinone dibenzoate
color interference color interference 2770 2850 2700 2610 2680 2850 2600
reactants (lecithin, Fez+, oxygen and thyroxine), the possible intermediate products, and the end products. None of the primary reactants alone will degrade thyroxine. The end products of Fez+-stimulated oxidation of lecithin will not degrade thyroxine nor will simple organic hydroperoxides effect this degradation. It seems probable then that thyroxine effects its antioxidant reaction and is itself degraded by some intermediate reactant in the oxidizing mixture. Mechanism of the Antioxidant
Reaction
In order to develop an understanding of the possible mechanism by which thyroxine exerts its antioxidant effect and is itself degraded, some surmise must be made in regard to the mechanism of oxidation of the lecithin. Attempts have been made to initiate rapid oxidation of lecithin in oxygen saturated water by elevating the temperature to 40”, by the addition of Fe3+, Cu2+, Cul+, Mn2+, Co2+, Co3+, Sn2+, Sn4+, hydrogen peroxide,
WYNN
various organic hydroperoxides, by the reaction of benzoyl peroxide and light and by carrying the reaction out in the presence of the xanthine oxidase reaction which generates per hydroxyl radical (17). None of these attempts have been successful. Thus rapid oxidation seems rather specifically related to Fez+. Since the system contained no FMN or FAD, the reactions described by Reinwein are excluded (18). The possibility that Fez+ may function catalytically forming an easily oxidized ligand or chelate with lecithin cannot be excluded from the data discussed above. Since the Fez+ is rapidly oxidized to Fe3f (which has no effect on lecithin oxidation), such a catalytic effect would disappear rapidly after each addition of Fez+ as it may in Fig. 6. A more probable explanation, however, is that the Fez+ initiates a chain peroxidation in lecithin. The study of the influence of various concentrations of thyroxine on the rate of oxidation of lecithin clarifies this problem to some extent. In Fig. 8 a group of studies are shown indicating the rate of lecithin oxidation at various thyroxine concentrations and several initiating concentrations of Fe2+. In Table IV are shown the tabulation of the intercept values and the standard deviations of the intercepts of these curves on the AO,/ dt axis. The thyroxine concentrations are plotted as reciprocals so t’hat oxidation rate at infinite thyroxine concentration may be estimated. At infinit’e thyroxine concentration the oxidation rate is directly related to the Fe2+ concentration. This demonstration is not compatible with the notion that Fe2+ catalyzes the oxidation of lecithin. If thyroxine were to function as an antioxidant because it dissociated an easily oxidized complex of Fe2+ and lecithin, then at infinite thyroxine concentration there should be virtually no such complex. On the other hand, these results are compatible with a free radical initiated lecithin chain oxidation. Thyroxine in such a scheme may function as a terminator of the propagation species. Although at infinite concentration thyroxine may effectively remove the propagation species, it is unable to prevent the initiating reaction, the initial electron abstraction which involves lecithin, Fez+ and oxygen.
THYROXINE EXPERIMENT
I
EXPERIMENT
2
EXPERIMENT
4
EXPERIMENT
5
EXPERIMENT
3
EXPERIMENT
6
1
1
"1
61
DEGRADATION
1 .
A
‘A
I x 10-e T4
MOLARITY
FIG. 8. A composite of graphs is shown of the oxidation rate in a solution of 4 X 10-b M lecithin initiated by three different concentrations of Fe*+ in the presence of increasing concentrations of thyroxine. The three concentrations of Fez+ used were: A = 2.0 X 10-S M; B= 1.5 X 10-5 M; and C = 1.0 X 10-S M. Final volume of reactions was 10 ml in 0.05 M citrate buffer at pH 4.7. Thyroxine concentrations are plotted as reciprocals so that an extrapolation to infinte thyroxine concentration can be assessed. The blank values of Fez+ oxidation alone have been subtracted from the values comprising the points on these curves. Six experiments were run on different days. Each point was assessed by two measurements as shown. Small differences in daily slope of these curves and absolute rates under any given set of conditions are due to day to day variations which could not be controlled. They probably relate to the exact physical form of the aqueous suspension of lecithin at the time that each set of experiments were carried out. Despite such daily variations, at infinite thyroxine concentration, a small but definite oxygen uptake persists, and on each day this oxygen uptake at infinite thyroxine concentration is approximat.ely proportionate to the amount of Fe”+ used to initiate the oxidation. Intercepts were estimated by the method of least squares. In Table V is shown a tabulation of the intercept values and the standard deviation of the intercepts of these curves on the AO,/dt axis.
The rate of formation of the initiating free radical is directly related to the Fez+ concentration; this radical reacts immediately with oxygen to form the propagating species, and the reaction is then terminated by reduction of the radical by thyroxine. Limited to kinetic studiesof oxygen uptake it is not possible to prove that these are chain oxidations or that thyroxine functions as an antioxidant by virtue of its termination of the propagating free radical species. Yet
the inferential data seems consistent with this as the best hypothesis at this time. The chemical mechanism by which thyroxine is then further degraded after having functioned as an antioxidant is obscure. Biologic Significance of These Reactions In Table V are shown ratio of percent inhibition concentration of several culated as follows: percent
the values of the of oxidation to the antioxidants calinhibition relative
JAMES WYNN
890
TABLE IV The AOz/dt intercept values of the curves shown in Fig. 8 and the calculated standard deviations of these intercepts are shown in this table. AOOt/dt &oms 02 uptake/liter/min
Experiment 1 1.0 X lo-&~ Fez+’ 1 5 X UY6~ Fe*+ 210 X ~O+M Fez+ Experiment 2 1 .O X lO+nn Fez+ 1.5 X lo-% Fez+ 2.0 X ~O+M Fez+ Experiment 3 1 .O X 10-6~ Fez+ 1.5 X 10M601 Fez+ 2.0 X 10-6~ Fez+ Experiment 4 1 .O X ~O+‘M Fez+’ 1.5 X 10-s~ Fez+ 2.0 X 10-6~ Fez+ Experiment 5 1 .O X KVM Fez+ 1.5 X 10d5~ Fez+ 2.0 X 10V~ Fez+ Experiment 6 1 .O X 10-6~ Fez+ 1.5 X 10-6~ Fez+ 2.0 X 10-5~ Fez+
Standard deviation
1.0 1.46 2.03
.17 .17
1.60 2.54 3.05
.14 .13 .26
.97 1.46 2.03
.ll .ll .13
1.69 2.34 3.08
.12 .13 .19
1.36 2.09 2.77
.22 .13 .14
1.15 1.65 2.06
.ll .19 .16
.lO
to uninhibited oxidation rate/concentration of antioxidant. The compounds selected for this portion of study are either structurally related to thyroxine in some way or are recognized biologic reducing agents. The more effective an antioxidant, the higher the ratio will be. It is evident that those compounds which predictably may form more stable free radical structures, are also those compounds which serve as the better antioxidants. By this rough assessment, thyroxine is at least 7300 times more effective than ascorbate, vitamin E, epinephrine and cysteine in this system. 0-methyl-N-acetyl thyroxine which may not form a phenoxyl radical has no measured antioxidant effect. Phenols with electron attracting para subsitutents forming unstable phenoxyl radicals and reducing agents which are primarily water or lipid soluble are relatively ineffective. The most effective
antioxidant examined, 2,6-diiodo 4-methoxyphenol, is a stable compound with solubility in both oil and water. Three areas of biologic importance are apparent in assigning this type of reactivity to thyroxine. This formulation describes a reactivity dependent on the peculiar structure of thyroxine. In previous papers the features of the substituents in the phenolic ring which impart a stabilized free radical capacity have been discussed (1). Secondly, thyroxine actually has a highly specialized antioxidant function in these in. vitro reactions. It is superior to several common biologic reducing agents with far more favorable oxidation potentials. Finally, if free radical reactions of thyroxine are biologically functional reactions, then such functional reactions may result in its degradation. This is consistent with in viva observations that the rate of thyroxine degradation and the apparent clinical effect of thyroxine seem closely related (19). There is a small body of other information which suggests an antioxidant function of thyroxine. The ablity of thyroxine to serve TABLE
V
COMPARISON OF RELATIVE ANTIOXIDANT EFFECT SEVERAL THYRONINE DERIVATIVES, OF PHENOLIC ANALOGUES AND A GROUP OF STRUCTURALLY DISSIMILAR REDUCING AGENTS
Percentage inhibition is calculated on the basis of the observed, uninhibited -dOz/dt. Lecithin concentration was 4 X lo-6 M. Fez+ initiator was 2 x lo- M. Antioxidant
2,6-Diiodo 4-methoxyphenol L-Thyroxine ~-3,3’,5’-Triiodothyronine 2-t-butyl4-Methoxyphenol L3,5 ,3’-Triiodothyronine O-Methyl N-acetyl thyroxine L-3,5-Diiodotyrosine 2,6-Diiodo 4-nitrophenol 2,4-Dinitrophenol L-Cysteine Ascorbic acid nn-Epinephrine Vitamin E
Percent COIlCelltration of inhibition antioxidant of. upin(y;;:;’ hlblted -dOl/dt
1.0 1.0 1.0
1.0 1.0
1.0 1.0 1.0 1.0 l.cklOO 1.0-100 1.0-100 1.0-100
80 73 50 27 26
THYROXINE
DEGRADATION
as an uncoupling agent has been related to its capacity to form a stable free radical or an oxidized species (20). It has been shown to serve as an antioxidant during metalcatalyzed peroxidation of mitochondria (21). It has the critical structure requisite of a phenolic antioxidant, and a stable free radical signal has been demonstrated (22). Because peroxidative reactions may have been shown to occur in vivo, and because such reactions would seem detrimental, the biologic utility of a highly specific lipid antioxidant is plain (23). It is suggested that a biologic function of thyroxine may be to serve to limit propagation of chain oxidation in lipid structures and that in serving this function it may be degraded. REFERENCES 1. WYNN, J., AND GIBBS, R., J. Biol. Chem. 238, 3490, (1963). 2. WYNN, J., AND GIBBS, R., J. Biol. Chem. 237, 3499, (1962). 3. WYNN, J., AND GIBBS, R., J. Biol. Chem. 239, 527, (1964). 4. WYNN, J., GIBBS, R., AND ROYSTER, B., J. Biol. Chem. 237, 1892, (1962). 5. RAPPORT, M. M., AND ALONZO, N., J. Biol. Chem. 217, 199, (1955). 6. GOTTFRIED, E. L., AND RAPPORT, M. M., J. Biol. Chem. 237, 329, (1962). 7. RAPPORT, M. M., AND ALONZO, N., J. Biol. Chem. 217, 193, (1955).
891
8. WIJS, J. J. A., 2. Anger. Chem. Heft 13, 290, (1898). 9. CHEN, P. S., TORIBARA, T. Y., AND WARNER, H., Analytical Chem. 28, 1756, (1956). 10. LANG, C. A., Analytical Chem. 30, 1962, (1958). 11. BUBLITZ, C., AND KENNEDY, E. P., J. Biol. Chem. 211, 951, (1954). 12. MORRISON, W. R., AND SMITH, L. M., J. of Lipid Research 6,600, (1964). 13. HANES, C. S., AND ISHERWOOD, F. A., Nature 164, 1107, (1949). 14. DAHLE, L. K., ELDEN, G. H., AND HOLMAN, R. T., Arch. Biochem. Biophys. 88, 253, (1962). 15. GETZ, G. S., BARTLEY, W., STIRPE, F., NOTTON, B. M., AND RENSHAW, A., Biochem. J. 83, 181, (1962). 16. FORDHAM, J. W., AND WILLIAMS, H. L., J. Am. Chem. Sot. 72, 4465, (1950). 17. MACLEOD, R. M., FRIDOVICH, I., AND HANDLER, P., J. Biol. Chem. 236, 1847, (1961). 18. REINWEIN, D., AND RALL, J. E., J. BioZ. Chem. 241, 1636, (1966). 19. GALTON, V. A., AND INGB-&R, S. H., Endocrinology 70. 622, (1962). 20. WYNN, J., AND FORE, W., J. BioZ. Chem. 240, 1766, (1965). 21. CASH, W. P., GARDY, M., CARLSON, H. E., AND EKONG, E. A., J. Biol. Chem. 241, 1745, (1966). 22. BERG, D. C., Proc. NatZ. Acad. Sciences 63, 829, (1965). and 23. AAES-JORGENSEN, E., in “Autoxidation Antioxidants” (W. 0. Lundberg, ed.), p. 1045. Wiley (Interscience), New York (1961).
OF
BIOCHEMISTRY
AND
126, 880-891 (1968)
BIOPHYSICS
Thyroxine Antioxidant
Function
and
Microsomal
Degradation Nonenzymatic lipid
JAMES
Degradation
during
Peroxidation’ WYNN
Department of Medicine, University of Arkansas Medical Center, Little Rock, Arkansas Received
February
29, 1968; accepted
June 19, 1968
These studies were undertaken with the aim of demonstrating an interaction of the phenoxyl free radical of thyroxine with a component of liver microsomes. The probability of such an interaction had been suggested by the prior study of the degradation of thyroxine by rat liver microsomes. The microsomal degradation reactions of thyroxine are initiated by ferrous iron. Rapid oxidation in microsomes is also initiated by ferrous iron. Such rapid oxidation is terminated by thyroxine and similar compounds. The material rapidly oxidized in microsomes in the presence of ferrous iron is a lecithin. Thyroxine and related compounds function as highly specific antioxidants exceeding the effectiveness of vitamin E, aecorbate, cysteine, glutat,hione or epinephrine. Study of the features of the antioxidant reaction suggests that thyroxine terminates the oxidations by reaction with the propagating species of a chain oxidation. During the course of such chain termination in peroxidizing lecithin, thyroxine is itself nonenzymatically degraded to yield the same products as those formed during microsomal degradation initiated by ferrous iron. The study supports the hypothesis that thyroxine participates in a system of free radical reactions, and as a consequence of these reactions it is degraded.
The phenoxyl radical of thyroxine may be the reactive species in the microsomal reactions leading to thyroxine degradation (1). In this paper, investigations are presented which demonstrate an antioxidant function of thyroxine during oxidation of microsomal lecithin. During the antioxidant reaction, thyroxine is nonenzymatically degraded to yield products which are identical to those previously found to occur during the microsomal degradation of thyroxine (2, 3). Thyroxine participates in these reactions by a mechanism compatible with its function as a free radical chain terminator supporting the hypothesis that the formation of a thyroxine radical precedes its degradation. 1 This work was supported by Grant AM 1240701 from the National Institutes of Health to the University of Arkansas.
EXPERIMENTAL
PROCEDURE
Materials
L-Thyroxine, L-thyroxine (3’, 5’ -1311), (1-9 -14C) and (lo-15 -14C), thyroxine analogues and related phenols were all obtained from sources or prepared by methods previously described (14). Standard fatty acids, phospholipids, lysolecithin, cholesterol, cerebrosides, sulfatides and sphingomyelin were purchased from Applied Science Laboratories. nn-ar-Glycerophosphate disodium salt and a-glycerophosphate dehydrogenase were purchased from Sigma Chemical Company. Cumene hydroperoxide, t-butyl hydroperoxide and 8-p-menthyl hydroperoxide were purchased from Matheson, Coleman and Bell. Aluminum oxide, neutral, activity grade 1 was purchased from M. Woelm, Eschwege, Germany.
THYROXINE
DEGRADATION
881
acid:ether. Neither was eluted after beginning the chloroform elution. Microsomes were prepared from rat livers Assay for long chain aldehydes derived as previously described (4). from plasmalogens was assessed by the pFractionation of lipid, protein and water sol- nitrophenylhydrazine method of Rapport uble components of microsomes. Two ml of and Alonzo (5). microsome suspension was packed at 8OOOg Determination of cu-punsaturated linkage in (after freezing, microsomes pack easily under phospholipids was measured by the method these conditions), and the sediment was re- of Gottfried and Rapport (6). suspended in 100 ml of chloroform: methDetermination of acyl ester groups in phosanol 50 : 50 (v/v). After stirring for 1 hr, in- pholipids was measured by the method of soluble residue was removed by filtration, Rapport and Alonzo with the following moddried under nitrogen and resuspended in 0.25 ification (7). The alkaline hydroxylamine sucrose (fraction No. 1). The chloroform: was added prior to ether and allowed to react methanol filtrate was dried under nitrogen for 5 minutes with lipid previously dried and the residue was mixed with 15-20 ml of under nitrogen in the reaction tube. ethanol. The portion of the material insoluTitration of unsaturated carbon bonds with ble in ethanol was removed by filtration and ICI was carried out by a modification of the dissolved in 5 ml of chloroform:methanol procedure of Wijs (8). Results are expressed (fraction No. 2). The clear, ethanol filtrate in terms of moles of ICl taken up per mole (fraction No. 3) was dried under nitrogen, of lipid phosphorus added. and a suspension of the residue was prepared Lipid phosphorus was determined by the in 5 ml of hexane: ethyl ether : acetic acid 85 : method of Chen, Toribara, and Warner (9). 15 : 1 (v/v). A column of dry aluminum oxide Lipid nitrogen was determined by the as supplied, 0.9 cm in diameter and 20 cm tall micro Kjeldahl method of Lang (10). was prepared, washed with 200 ml of hexane: Anaerobic, alkaline hydrolysis of phosphoethyl ether: acetic acid and onto this was lipid was carried out as follows : lo-30 pmoles placed the hexane: ethyl ether: acetic acid of phospholipid phosphorus was dried in a suspension of ethanol soluble, lipid sub- test tube under nitrogen. One to five millistances. The column was washed with 150 liters of 1.0 N NaOH previously gassed with ml of hexane:ethyl ether:acetic acid, the nitrogen was added and the tube was sealed effluent was collected in one batch, dried anaerobically. After incubating at 100” for under a stream of nitrogen, and the residue 3 hr and at 55” for 24 hr, material prepared was dissolved in 5 ml of absolute ethanol for thin-layer chromatography was solu(fraction No. 3a). The column was then bilized by the addition of an equal volume washed with 150 ml of chloroform collected of absolute ethanol and stored under nitroin a batch which was dried and the residue gen until used. Hydrolyzate prepared for was dissolved in 5 ml of ethanol (fraction No. the cY-glycerophosphate assay was passed 3b). The chloroform wash was followed by 150 through a column of Dowex 50 ‘H’ form mlof chloroform: methanol 50: 50, theeffluent sufficient to remove 100 mEq of cation. of whichwas fractionated into 3-ml aliquots Effluent and washes were dried, extracted (fraction No. 3~). These were individually three times with hexane and reconstituted dried under nitrogen, and each was dissolved in a l-ml volume of water. A standard soluin 5 ml of ethanol. After having been mixed tion of m-a-glycerophosphate was subjected with appropriate solvent each fraction was ex- to this same procedure with no loss of the starting material. amined as outlined below for contained Estimation of L-a-glycerophosphate in material susceptible to rapid rates of oxidation. Standard FMN and FAD were added anaerobic, alkaline hydrolysate was carried out by the procedure of Bublitz and Kento the lipid preparation in order to ascertain whether they might be co-eluted with the nedy (11). A control procedure was set up to lipid. Small amounts of these flavins were determine whether the hydrolysis of lecithin in hot NaOH would lead to complete eluted during the wash with hexane:acetic Methods
882
JAMES
racemization of the freed cu-glycerophosphate. n-a-Glycerophosphate was prepared by allowing L-a-glycerophosphate dehydrogenase to react to completion on a DL substrate. The n-cu-glycerophosphate remaining was then processed as the lecithin was in hot 1 N NaOH. Following recovery of the aglycerophosphate, half of that which was originally D isomer could now be accounted for as the L-a-glycerophosphate as measured by the enzyme assay. Methylation of fatty acids for gas chromatography was carried out according to the method of Morrison and Smith using boron trifluoride and methyl alcohol as the methylating agents (12). Gas chromatography of fatty acid methyl esters derived from phospholipid was carried out on Research Specialties Model 600 apparatus using an Argon ionization chamber with strontium 90 as the source. The procedure was carried out using benzene as the solvent at 180”. The stationary phase was ethylene glycol succinate and the support was Anachrome A-B 60-80 mesh. The column was 6 feet long and 0.25 inches in diameter. The gas was argon at 15 lb/square inch. Thin-layer chromatography was carried out only in silica gel G. Lecithin separation was best demonstrated in chloroform: methanol: water 65: 25:4. Choline in the hydrolyzate was demonstrated in a two-dimensional solvent system prepared as follows: the first solvent was 70 % acetic acid developed at 4’; after drying, the second solvent was 90 % acetic acid at room temperature. Fatty acids, lecithin and choline were located by iodine staining. Perchloric acid-ammonium molybdate in HCl was used to demonstrate aglycerophosphate (13). Paper chromatography of thyroxine and its labeled derivatives was carried out using methods previously described (2). Synthesis of 1,4-hydroquinone dibenzoate and recrystallization methods using this compound are previously described (3). Electrophoresis of presumed iodide and 3,5diiodotyrosine derived from labeled thyroxine was carried out in 0.2 M ammonium carbonate solution at room temperature and 500 V. Thiobarbituric acid reaction for the detec-
WYNN
tion of malonyl dialdehyde was carried out by a modification of the method described by Dohle, Elden, and Holman (14). Xanthine oxidase reaction: milk xanthine oxidase was kindly supplied by Dr. K. V. Rajagopalen of the Department of Biochemistry, Duke University. The reaction was carried out in Tris buffer at pH 7.8 made 1 X 10v4 with versene. Oxygen consumption was usually measured polarographically, but in certain instances the Warburg apparatus was used. The oxygen electrode was a teflon membrane covered platinum electrode with internal KC1 bridge to a saturated KC1 calomel reference electrode. Measurements were made in temperature-controlled, narrow-necked 2- or lo-ml vessels at 23’. Microsomes were added to make the final concentration such that 1 ml contained the microsomes derived from 20 mg of original liver. Lecithin oxidation was examined at several concentrations but the concentration at which most of the studies were carried out was 4 X 1O-5 M. Lecithin was added in 0.2 ml ethanol. Controls contained the same quantity of ethanol. Buffer was either 5 X low2 M citrate at pH 4.7 or 1 X 10m2M phosphate at pH 7.0. Ferrous iron was added in aqueous solution as ferrous ammonium sulfate using various quantities as indicated in the legends of the figures. Thyroxine and analogues were added in 0.01 ml of ethanol. The Warburg apparatus was used in experiments in which attempts were made to oxidize large amounts of lecithin. The side arm contained 4 X 10m6moles of Fez+. The central compartment contained the same molar amount of lecithin. The volume was made up to a final total of 2.0 ml with 2.5 X lo+! M citrate buffer at pH 4.7. At time zero the iron solution was tipped into the lecithin mixture. Incubations were carried out for 15 min at 37”. Assay of radioactive material: CarbonI was measured in a liquid scintillation spectrometer and by a GM tube strip scanner. Iodinem was measured in a gamma spectrometer at the 364 KEV peak and by a GM tube strip scanner.
THYROXINE RESULTS
AND
DISCUSSION
The Relationship of Microsomal Oxidation and Thyroxine Degradation Although oxygen is necessary in the Fez+stimulated reactions leading to thyroxine degradation (4), it is not established that microsomal oxidation and thyroxine degradationare related reactions. In Fig. 1 isshown a graph of the simultaneous plot of Fez+stimulated thyroxine degradation and oxygen uptake as a function of time. Thyroxine degradation was studied as in previous studies (4). The parallel course of these time curves suggests but does not establish a relationship. Isolation of the Rapidly Oxidized Substance in Microsomes In an attempt to simplify the system studied, the microsomal material which is rapidly oxidized in the presence of Fez+ was isolated. I
301
2 -0.4 3\ P -0.3 ::& I5 b -0.2 0 x -0.1 *
t =
0 10
50
100
150
200
Time-Seconds
FIG. 1. Oxygen uptake by 0.2 ml microsomes and the simultaneous degradation of thyroxine is shown. The left-hand ordinate indicates the amount of oxygen uptake; the right-hand ordinate indicates the pmoles of thyroxine degraded as measured by the amount of inorganic iodide produced (4). The reaction was carried out in 0.05 M citrate buffer at pH 4.7 containing 1 X 10-C M thyroxine and was initiated by 2 X 10-d M Fez+. The curve of oxygen uptake has been corrected by subtracting a blank value for the oxidation of Fez+ alone. The degradation reaction shows an initial lag but then parallels the course of the oxidation. In Figs. 5 and 6, studies are shown in which thyroxine added to microsomes and microsomal lecithin stopped the oxidation. If the amount of thyroxine is small and the amount of Fee+ large, oxidation is favored. If the amount of thyroxine is large and the amount of Fez+ is small, oxidation can be virtually entirely stopped.
DEGRADATION
883
Fortuitously, the procedure carried out developed into a very reproducible method for isolating a relatively pure lecithin fraction. The lipid-free protein containing fraction No. 1 contained no materials susceptible to such oxidation, and the ethanol soluble lipid fraction No. 3 contained all such reactive material. Fractionation of the ethanol soluble lipids on alumina gel allowed the separation of a discrete fraction among the lipids which was susceptible to rapidly induced Fe2+ oxidation. This fraction was eluted from alumina immediately behind the front of the chloroform: methanol 50: 50 eluant or in fraction No. 3c. Fractions No. 3a and No. 3b contained no such rapidly oxidized material. Chromatography showed the pertinent material to be composed of lipid inseparable from standard lecithin. The ninhydrin reaction was negative. Two minor components seen after prolonged iodine staining were also present. These latter components did not have mobility characteristics associated with any of the standard lipids used for identification. They did not contain stainable phosphate. Further characterization of this lipid material was then undertaken. In Table I is shown a resume of the pracedures carried out to identify this lipid material. The attempts to demonstrate long chain aldehyde and a-0 unsaturated bonds were negative. The phosphorus :nitrogen ratio was 1 .O: 1.05. The phosphorus : acyl ester ratio was 1.0 : 1.94. The phosphorus: ICl molar ratio was 1.0:2.5. The theoretical molecular weight based on the assumption of one phosphorus atom per molecule after drying and weighing a sample was 764. In Fig. 2 is shown a drawing of a chromatogram of choline and cu-glycerophosphate tentatively identified by thin-layer chromatography in silica gel following alkaline hydrolysis of the lecithin-like fraction. The content of L-a-glycerophosphate in an alkaline hydrolyzate was measured enzymatically. Thirty micromoles of organic phosphorus was subjected to alkaline hydrolysis. Twenty-four micromoles of organic phosphorus was recovered after passage through Dowex 50. Sixteen micromoIes of m-a-glycerophosphate was accounted for enzymatically representing a 67 % recovery
884
JAMES
WYNN
TABLE
I
PROCEDURES CARRIED OUT ATTEMPTING TO IDENTIFY THE PHOSPHOLIPID DERIVED FROM M~c~oso~~s SUXZEPTIBLE TO RAPID OXIDATION Result
Procedure Silica gel G-chromatography Special Stains (a) Ninhydrin (b) Malachite green (lysolecithin) (c) Phosphomolybdic acid (choline) p-nitrophenylhydrazine (G-B unsaturated aldehyde) Iodine uptake (a-p unsaturated aldehyde) Phosphorus:Nitrogen rat.io Phosphorous:acyl ester ratio Phosphorous:ICl uptake ratio Dried weight of sample containing 3.1 mg Phosphorous Alkaline hydrolyzate (a) Products demonstrated chromatographically (b) Recovery of organic Phosphorous as L-W glycerophosphate Gas chromatography of fatty acid methyl esters
70%
L-a -GI
Acetic
Inseparable
from standard
lecithin
Negative Negative Positive Negative Negative 1:1.05 1:1.94 1:2.50 76.4 mg Choline, 67% See Table
L-cu-glycerophosphate
II
Acid at 4O
hots Q Origin
FIG. 2. Silica gel chromatography of the alkaline hydrolysate of microsomal lecithin is shown. Spots identified as choline and L-cu-glycerophosphate were verified by co-chromatography of known standards on the plates with unknown. The standard and unknown spots were inseparable.
of the expected compound. The remaining 33% was not identified. In Fig. 3 is shown a graph of the gas chromatography of the fatty acid methyl esters prepared from the lecithin-like material. The fatty acid esters isolated by this procedure
FIG. 3. Gas chromatography of the fatty acid methyl esters derived from microsomal lecithin is shown. The larger peaks were tentatively identified by co-chromatography of standard materials. The smaller peaks A, B, C, D, and E are unidentified. The time scale is distorted between 18 and 20 and 45 and 120 min during which no peaks were eluted.
were inseparable from palmitic, steric, oleic, linoleic, and arachidonic esters. Several small peaks of unidentified materials were isolated as well. On the basis of the cochromatography of known quantities of standard materials and the calculated area developed for these known quantities, the recovery of each tentatively identified fatty acid ester was
THYROXINE TABLE MOLAR RECOVERY ESTERS DTJRING
On the basis of the extent of ferric the original sample lipid phosphorous ester.
Palmitate Stearate Oleate Linoleate Arachidonate
II
OF FATTY ACID METHYL Gas CHROMATOGRAPHY
the phosphorous content and per-chlorate reacting acyl ester contained 12.8 X lo+ moles of and 24.8 X lo-” moles of acyl
1.26 1.24 0.49 0.79 1.45
4.66 4.16 1.66 2.69 4.46
26.4 23.6 9.4 15.3 25.3
calculated. In Table II these absolute recoveries and the percentage of the total fatty acid methyl ester recovery are shown with comparisons on a molar basis. The yield of recovered fatty acid methyl ester was 70% of the theoret’ical yield based on the quantity of ester calculated to be present by the ferric perchlorate reaction. Assuming that the 30 % lost during methylation was evenly distributed among the component fatty acids, the average number of double bonds per molecule of lecithin containing one saturated and one unsaturated fatty acid is 2.7, and this agrees closely with the phosphorus : ICl ratio of 1:2.5. Two hundred and ninety mg of lecithin were recovered from 1.72 g of dry weight of microsomes or 17% of the dry weight of microsomes was lecithin. This compares to a recovery of 15-21% obtained by other workers comparing dry weight of lecithin to dry weight of microsomes (15). Following these studies, it was concluded that the lipid material isolated as the rapidly oxidizable fraction was predominantly lecithin without measurable plasmalogen or lysolecithin contaminants. There was minor contamination by other lipid substances shown by thin-layer and gas chromatography, but these were not identified. The systems contained no FMN or FAD for reasons noted in the METHODS section. On the basis of the phosphorus to nitrogen to ester ratio, the absence of a ninhydrin reaction, the chromatography studies and
DEGRADATION
885
the theoretical molecular weight, it is felt that the material isolated is a lecithin which is 95 % pure, has an average fatty acid chain length of 18 carbons in which there are an average of 2.5 unsaturated bonds per molecule of lecithin. There are no large amounts of ususual fatty acid or other constitutents. The concentrations of lecithin used in the oxidation studies were small and the presence of iron precluded the direct measurement of small amounts of hydroperoxide product (16). Measurements of malonyl dialdehyde present before and following oxidation of lecithin were attempted with negative results. Unable to detect either hydroperoxides or malonyl dialdehyde, an attempt was then made to make a less specific observation which would confirm the fact that the lecithin was being oxidized. Two solutions of lecithin were prepared. Into one the usual addition of Fez+ was made. Into the second, oxygen was bubbled at 40” for 1 hr. Chromatograms were prepared of unaltered, oxygen-treat’ed and Fe+treated lecithin solution. The Fez+-treated and the 02-treated samples showed a relative decrease in the density of the lecithin spots and the appearance of a new, more polar group of phosphate containing spots not present in the original lecithin sample. This supports the view that it is the lecithin itself which is oxidized by the addition of Fez+. In Fig. 4, strips 1, 2, and 3 are reproductions of chromatograms illustrating the changes mentioned. Antioxidant E$ect of Thyroxine During Iron Stimulated Oxidation The influence of thyroxine on the course of microsomal oxidation was next examined. In Fig. 5 is shown a graph of the rapid oxidation of liver microsomes following the addition of small amounts of ferrous iron and the inhibition of such oxidation by 5 X 1O-6 M thyroxine. The difference between the rate of oxygen consumed by iron alone and iron plus microsomes is indicative of the rate of oxygen uptake by microsomes. If the microsomal lecithin isolated is the agent which is rapidly oxidized in the microsome, then thyroxine may inhibit iron stimulated oxidation in the lecithin preparation as well,
JAMES
WYNN
Products Formed from Thyroxine During the Oxidation-Antioxidant Reaction
.0nl
1
(3)
(4)
i---i
1 Origin
(5)
FIG. 4. A diagram is shown of the silica gel chromatography of lecithin treated several ways. In strip No. 1 lecithin was simply added to .05 M citrate buffer, pH 4.7, and gassed immediately with Nz. In strip No. 2 a similar sample was treated with Fez+ and incubated aerobically for 15 min. In strip No. 3 a similar sample was treated with O2 for 1 hr at 40”. In strip No. 4, Fe*+ was added as in strip No. 2 but in addition a final concent,ration of 1 x 1O-6 M thyroxine was added before the iron. Strip No. 5 is a standard containing several phospholipids. The solvent system and the demonstrations of phosphate containing spots are described in the methods section. The dark-hatched areas indicate dense stain. The light-hatched areas indicate a light stain. In strips No. 2 and No. 3 phosphate-containing material was identified near the origin of the chromatograms. The spots are labeled A and B. In strip No. 4, thyroxine prevented the appearance of spot A.
This is shown in Fig. 6. If repeated additions of Fez+ are made to the lecithin system containing thyroxine, the antioxidant effect is dissipated and the lecithin may then be oxidized suggesting that the thyroxine is altered during the antioxidant reaction. If it is lecithin oxidation which thyroxine prevents, the antioxidant effect of thyroxine should prevent the chromatographic appearance of the more polar oxidation products of lecithin seen following the addition of Fez+. This is shown in strip 4 of Fig. 4.
The degradation products of thyroxine in the Fez+-stimulated microsomal system are inorganic iodide, 3,5-diiodotyrosine and an acid hydrolyzable product of 1,4-hydroquinone (2, 3). Studies were undertaken to demonstrate whether the oxidation-antioxidant reaction involving oxygen, lecithin, and thyroxine might yield similar products. In separate studies products derived from thyroxine labeled with 13’1 in the ,8 phenyl ring, with 14carbon in the (Y phenyl ring and with 14carbon in the ,f3ring were determined. Chromatography in three solvents was carried out with comparable results in each system, but only the collidine-NH,-HZ0 chromatograms are shown in Fig. 7. The P phenyl ring iodine was converted to inorganic iodide; the 0 phenyl ring itself yielded a new product dissociated from the a! phenyl ring and containing no iodine; and the cr phenyl ring-alanine side chain yielded material which is inseparable from 3,5-diiodotyrosine. Inorganic iodide and 3,5-diiodotyrosine were further identified by their electrophoretic characteristics. The acid hydro-
/ 100 I
68I FIG. 5. Percentage oxygen change effected by microsomes in the presence of Fez+ with and without thyroxine is shown. The oxygen uptake of Fez+ without microsomes is shown as a blank value. Microsomes without Fez+ took up no oxygen. Thyroxine added to the Fez+ blank reaction did not retard oxygen uptake. Reactions were carried out in a final volume of 10.0 ml of 0.025 M citrate at pH 4.7. Each addition of Fe2+ indicated by an arrow contained 5 X lo-’ moles of Fez+. Microsomal volumes were 0.2 ml of the preparation described in the methods section. Thyroxine concentration was 5 x 10-G M.
THYROXINE
92: ‘Z 0 842 :: & 76L 6 <0 6860 _
DEGRADATION
887
w--w-. II-~= --I -100
No lecithin, No T4 4X 10v5M lecithin, No T4 4X10e5M lecilhin+lX10‘7M I 0
I 100
T4
I 300 Time-Seconds
I 500
I 700
c 90
FIG. 6. A graph is shown of the effect of increments of Fez+ added to solution containing 4 X 10-6 M microsomal lecithin and 1 X 10-TM thyroxine. Each increment of Fez+ was 2 X 1CP moles and is indicated by an arrow. The reaction volume was 10 ml in .025 M citrate pH 4.7. The effect of such increments of Fez+ on microsomal oxidation without thyroxine is shown for comparison.
FIG. 7. Graphs are shown of the strip counting of chromatograms developed in collidine:water: NH8 prepared following the reaction of variously labeled thyroxines with peroxidizing lecithin. The underlined captions indicate the location of standard markers on the chromatogram. Diiodotyrosine is abbreviated as DIT; monoiodotyrosine ae MIT; and thyroxine as T4. It is apparent that fl phenyl ring iodine is converted to inorganic iodide, the 01phenyl ring yields a product inseparable from diiodotyrosine, and the @phenyl ring yields a new product demonstrated to be a 1,4-hydroquinone derivative in other studies.
lyzed 14carbon product of the 0 phenyl ring was inseparable chromatographically from 1,4-hydroquinone. Prior to such hydrolysis, the labeled compound had solubility characteristics similar to those of hydroquinone dibenzoate. The unhydrolyzed, labeled product was treated with an excess of benzoyl chloride, mixed in dioxane solution with carrier hydroquinone dibenzoate and reprecipitated nine times from solution by the addition of water. Aliquots of each re-solution were studied for radioactive content and optical density at 234 rnp as a measure of hydroquinone dibenzoate recovery. These studies are shown in Table III. Specific activity was constant after the second precipitation. Thus, during Fez+ stimulated oxidation in both liver microsomes and derived lecithin, thyroxine serves as an antioxidant, is degraded during the reaction and yields products which are identical in both situations. Isolation of the Reaction Leading to Thyroxine Degradation The precise reaction in which thyroxine may participate is not, indicated by these studies. The value of having related the degradation of thyroxine to an oxidizing lecithin preparation is plain. The degradation system can be reduced to four primary
888
JAMES TABLE
REPRECIPITATION OF p HYDROQUINONE
III PHENYL PRODUCT DIAEKZOATE
AS
The p phenyl ring product formed during the reaction of thyroxine with peroxidizing lecithin was treated with an excess of benzoyl chloride. The derived labeled material was dissolved in dioxane with carrier hydroquinone dibenzoate and precipitated from solution with water. Nine precipitations and resolutions were carried out. An aliquot of the dioxane solution prepared followingeachprecipitation was studied. The 14C content and optical density at 234 rnp were measured. At 234 rnp hydroquinone dibenzoate has an absorption maximum the extinction coefficient of which is 5700. The CPM recovered relative to calculated mg recovery of hydroquinone dibenzoate is shown. Precipitation 1
2 3 4 5 6 7 8 9
Total Mg/sample
Yellow Yellow 4.72 4.69 3.81 3.52 2.91 1.86 1.19
CPM/mg hydroquinone dibenzoate
color interference color interference 2770 2850 2700 2610 2680 2850 2600
reactants (lecithin, Fez+, oxygen and thyroxine), the possible intermediate products, and the end products. None of the primary reactants alone will degrade thyroxine. The end products of Fez+-stimulated oxidation of lecithin will not degrade thyroxine nor will simple organic hydroperoxides effect this degradation. It seems probable then that thyroxine effects its antioxidant reaction and is itself degraded by some intermediate reactant in the oxidizing mixture. Mechanism of the Antioxidant
Reaction
In order to develop an understanding of the possible mechanism by which thyroxine exerts its antioxidant effect and is itself degraded, some surmise must be made in regard to the mechanism of oxidation of the lecithin. Attempts have been made to initiate rapid oxidation of lecithin in oxygen saturated water by elevating the temperature to 40”, by the addition of Fe3+, Cu2+, Cul+, Mn2+, Co2+, Co3+, Sn2+, Sn4+, hydrogen peroxide,
WYNN
various organic hydroperoxides, by the reaction of benzoyl peroxide and light and by carrying the reaction out in the presence of the xanthine oxidase reaction which generates per hydroxyl radical (17). None of these attempts have been successful. Thus rapid oxidation seems rather specifically related to Fez+. Since the system contained no FMN or FAD, the reactions described by Reinwein are excluded (18). The possibility that Fez+ may function catalytically forming an easily oxidized ligand or chelate with lecithin cannot be excluded from the data discussed above. Since the Fez+ is rapidly oxidized to Fe3f (which has no effect on lecithin oxidation), such a catalytic effect would disappear rapidly after each addition of Fez+ as it may in Fig. 6. A more probable explanation, however, is that the Fez+ initiates a chain peroxidation in lecithin. The study of the influence of various concentrations of thyroxine on the rate of oxidation of lecithin clarifies this problem to some extent. In Fig. 8 a group of studies are shown indicating the rate of lecithin oxidation at various thyroxine concentrations and several initiating concentrations of Fe2+. In Table IV are shown the tabulation of the intercept values and the standard deviations of the intercepts of these curves on the AO,/ dt axis. The thyroxine concentrations are plotted as reciprocals so t’hat oxidation rate at infinite thyroxine concentration may be estimated. At infinit’e thyroxine concentration the oxidation rate is directly related to the Fe2+ concentration. This demonstration is not compatible with the notion that Fe2+ catalyzes the oxidation of lecithin. If thyroxine were to function as an antioxidant because it dissociated an easily oxidized complex of Fe2+ and lecithin, then at infinite thyroxine concentration there should be virtually no such complex. On the other hand, these results are compatible with a free radical initiated lecithin chain oxidation. Thyroxine in such a scheme may function as a terminator of the propagation species. Although at infinite concentration thyroxine may effectively remove the propagation species, it is unable to prevent the initiating reaction, the initial electron abstraction which involves lecithin, Fez+ and oxygen.
THYROXINE EXPERIMENT
I
EXPERIMENT
2
EXPERIMENT
4
EXPERIMENT
5
EXPERIMENT
3
EXPERIMENT
6
1
1
"1
61
DEGRADATION
1 .
A
‘A
I x 10-e T4
MOLARITY
FIG. 8. A composite of graphs is shown of the oxidation rate in a solution of 4 X 10-b M lecithin initiated by three different concentrations of Fe*+ in the presence of increasing concentrations of thyroxine. The three concentrations of Fez+ used were: A = 2.0 X 10-S M; B= 1.5 X 10-5 M; and C = 1.0 X 10-S M. Final volume of reactions was 10 ml in 0.05 M citrate buffer at pH 4.7. Thyroxine concentrations are plotted as reciprocals so that an extrapolation to infinte thyroxine concentration can be assessed. The blank values of Fez+ oxidation alone have been subtracted from the values comprising the points on these curves. Six experiments were run on different days. Each point was assessed by two measurements as shown. Small differences in daily slope of these curves and absolute rates under any given set of conditions are due to day to day variations which could not be controlled. They probably relate to the exact physical form of the aqueous suspension of lecithin at the time that each set of experiments were carried out. Despite such daily variations, at infinite thyroxine concentration, a small but definite oxygen uptake persists, and on each day this oxygen uptake at infinite thyroxine concentration is approximat.ely proportionate to the amount of Fe”+ used to initiate the oxidation. Intercepts were estimated by the method of least squares. In Table V is shown a tabulation of the intercept values and the standard deviation of the intercepts of these curves on the AO,/dt axis.
The rate of formation of the initiating free radical is directly related to the Fez+ concentration; this radical reacts immediately with oxygen to form the propagating species, and the reaction is then terminated by reduction of the radical by thyroxine. Limited to kinetic studiesof oxygen uptake it is not possible to prove that these are chain oxidations or that thyroxine functions as an antioxidant by virtue of its termination of the propagating free radical species. Yet
the inferential data seems consistent with this as the best hypothesis at this time. The chemical mechanism by which thyroxine is then further degraded after having functioned as an antioxidant is obscure. Biologic Significance of These Reactions In Table V are shown ratio of percent inhibition concentration of several culated as follows: percent
the values of the of oxidation to the antioxidants calinhibition relative
JAMES WYNN
890
TABLE IV The AOz/dt intercept values of the curves shown in Fig. 8 and the calculated standard deviations of these intercepts are shown in this table. AOOt/dt &oms 02 uptake/liter/min
Experiment 1 1.0 X lo-&~ Fez+’ 1 5 X UY6~ Fe*+ 210 X ~O+M Fez+ Experiment 2 1 .O X lO+nn Fez+ 1.5 X lo-% Fez+ 2.0 X ~O+M Fez+ Experiment 3 1 .O X 10-6~ Fez+ 1.5 X 10M601 Fez+ 2.0 X 10-6~ Fez+ Experiment 4 1 .O X ~O+‘M Fez+’ 1.5 X 10-s~ Fez+ 2.0 X 10-6~ Fez+ Experiment 5 1 .O X KVM Fez+ 1.5 X 10d5~ Fez+ 2.0 X 10V~ Fez+ Experiment 6 1 .O X 10-6~ Fez+ 1.5 X 10-6~ Fez+ 2.0 X 10-5~ Fez+
Standard deviation
1.0 1.46 2.03
.17 .17
1.60 2.54 3.05
.14 .13 .26
.97 1.46 2.03
.ll .ll .13
1.69 2.34 3.08
.12 .13 .19
1.36 2.09 2.77
.22 .13 .14
1.15 1.65 2.06
.ll .19 .16
.lO
to uninhibited oxidation rate/concentration of antioxidant. The compounds selected for this portion of study are either structurally related to thyroxine in some way or are recognized biologic reducing agents. The more effective an antioxidant, the higher the ratio will be. It is evident that those compounds which predictably may form more stable free radical structures, are also those compounds which serve as the better antioxidants. By this rough assessment, thyroxine is at least 7300 times more effective than ascorbate, vitamin E, epinephrine and cysteine in this system. 0-methyl-N-acetyl thyroxine which may not form a phenoxyl radical has no measured antioxidant effect. Phenols with electron attracting para subsitutents forming unstable phenoxyl radicals and reducing agents which are primarily water or lipid soluble are relatively ineffective. The most effective
antioxidant examined, 2,6-diiodo 4-methoxyphenol, is a stable compound with solubility in both oil and water. Three areas of biologic importance are apparent in assigning this type of reactivity to thyroxine. This formulation describes a reactivity dependent on the peculiar structure of thyroxine. In previous papers the features of the substituents in the phenolic ring which impart a stabilized free radical capacity have been discussed (1). Secondly, thyroxine actually has a highly specialized antioxidant function in these in. vitro reactions. It is superior to several common biologic reducing agents with far more favorable oxidation potentials. Finally, if free radical reactions of thyroxine are biologically functional reactions, then such functional reactions may result in its degradation. This is consistent with in viva observations that the rate of thyroxine degradation and the apparent clinical effect of thyroxine seem closely related (19). There is a small body of other information which suggests an antioxidant function of thyroxine. The ablity of thyroxine to serve TABLE
V
COMPARISON OF RELATIVE ANTIOXIDANT EFFECT SEVERAL THYRONINE DERIVATIVES, OF PHENOLIC ANALOGUES AND A GROUP OF STRUCTURALLY DISSIMILAR REDUCING AGENTS
Percentage inhibition is calculated on the basis of the observed, uninhibited -dOz/dt. Lecithin concentration was 4 X lo-6 M. Fez+ initiator was 2 x lo- M. Antioxidant
2,6-Diiodo 4-methoxyphenol L-Thyroxine ~-3,3’,5’-Triiodothyronine 2-t-butyl4-Methoxyphenol L3,5 ,3’-Triiodothyronine O-Methyl N-acetyl thyroxine L-3,5-Diiodotyrosine 2,6-Diiodo 4-nitrophenol 2,4-Dinitrophenol L-Cysteine Ascorbic acid nn-Epinephrine Vitamin E
Percent COIlCelltration of inhibition antioxidant of. upin(y;;:;’ hlblted -dOl/dt
1.0 1.0 1.0
1.0 1.0
1.0 1.0 1.0 1.0 l.cklOO 1.0-100 1.0-100 1.0-100
80 73 50 27 26
THYROXINE
DEGRADATION
as an uncoupling agent has been related to its capacity to form a stable free radical or an oxidized species (20). It has been shown to serve as an antioxidant during metalcatalyzed peroxidation of mitochondria (21). It has the critical structure requisite of a phenolic antioxidant, and a stable free radical signal has been demonstrated (22). Because peroxidative reactions may have been shown to occur in vivo, and because such reactions would seem detrimental, the biologic utility of a highly specific lipid antioxidant is plain (23). It is suggested that a biologic function of thyroxine may be to serve to limit propagation of chain oxidation in lipid structures and that in serving this function it may be degraded. REFERENCES 1. WYNN, J., AND GIBBS, R., J. Biol. Chem. 238, 3490, (1963). 2. WYNN, J., AND GIBBS, R., J. Biol. Chem. 237, 3499, (1962). 3. WYNN, J., AND GIBBS, R., J. Biol. Chem. 239, 527, (1964). 4. WYNN, J., GIBBS, R., AND ROYSTER, B., J. Biol. Chem. 237, 1892, (1962). 5. RAPPORT, M. M., AND ALONZO, N., J. Biol. Chem. 217, 199, (1955). 6. GOTTFRIED, E. L., AND RAPPORT, M. M., J. Biol. Chem. 237, 329, (1962). 7. RAPPORT, M. M., AND ALONZO, N., J. Biol. Chem. 217, 193, (1955).
891
8. WIJS, J. J. A., 2. Anger. Chem. Heft 13, 290, (1898). 9. CHEN, P. S., TORIBARA, T. Y., AND WARNER, H., Analytical Chem. 28, 1756, (1956). 10. LANG, C. A., Analytical Chem. 30, 1962, (1958). 11. BUBLITZ, C., AND KENNEDY, E. P., J. Biol. Chem. 211, 951, (1954). 12. MORRISON, W. R., AND SMITH, L. M., J. of Lipid Research 6,600, (1964). 13. HANES, C. S., AND ISHERWOOD, F. A., Nature 164, 1107, (1949). 14. DAHLE, L. K., ELDEN, G. H., AND HOLMAN, R. T., Arch. Biochem. Biophys. 88, 253, (1962). 15. GETZ, G. S., BARTLEY, W., STIRPE, F., NOTTON, B. M., AND RENSHAW, A., Biochem. J. 83, 181, (1962). 16. FORDHAM, J. W., AND WILLIAMS, H. L., J. Am. Chem. Sot. 72, 4465, (1950). 17. MACLEOD, R. M., FRIDOVICH, I., AND HANDLER, P., J. Biol. Chem. 236, 1847, (1961). 18. REINWEIN, D., AND RALL, J. E., J. BioZ. Chem. 241, 1636, (1966). 19. GALTON, V. A., AND INGB-&R, S. H., Endocrinology 70. 622, (1962). 20. WYNN, J., AND FORE, W., J. BioZ. Chem. 240, 1766, (1965). 21. CASH, W. P., GARDY, M., CARLSON, H. E., AND EKONG, E. A., J. Biol. Chem. 241, 1745, (1966). 22. BERG, D. C., Proc. NatZ. Acad. Sciences 63, 829, (1965). and 23. AAES-JORGENSEN, E., in “Autoxidation Antioxidants” (W. 0. Lundberg, ed.), p. 1045. Wiley (Interscience), New York (1961).