Reduced triphosphopyridine nucleotide oxidase-catalyzed alterations of membrane phospholipids. VI. Structural changes in mitochondria associated with inactivation of electron transport activity.

Abstract These studies are part of an effort to determine how the activities of certain membrane-bound oxidoreductase systems affect the structure and function of the membranes in which they are situated. This report shows that the mitochondrial oxidation of TPNH in the presence of physiological quantities of Fe3+ results in reproducible polyunsaturated fatty acid degradation which is accompanied by a marked loss in mitochondrial capacity to oxidize Krebs cycle intermediates and to perform oxidative phosphorylation. The enzyme responsible for the changes appears to be the mitochondrial non-energy-dependent transhydrogenase. The phenomenon may account for some of the turnover of lipids in mitochondrial membranes.

TPNH oxidase activity in normal rat liver microsomes has been shown to cause significant, reproducible changes in the composition of the endogenous phospholipids of microsomal membranes (1). The alterations involve the loss of major portions of portions of the polyunsaturated fatty acids situated at the p position of the phospholipids.
Considerable oxygen is taken up during this reaction.
Small amounts of malondialdehyde are formed, and the amount formed has been shown to be an index of the extent of the reaction (2). The system which promotes * This work was supported by Grants AM-06978 and AM-08397 from the National Institutes of Health. United States Public Health Service. Some of the data reported herein were taken from a thesis submitted by H. E. May to the University of Oklahoma School of Medicine in nartial fulfillment of the reauirements for the Ph.D. degree. The preceding paper in this series is Reference 1.
$ Present address, Department of Chemistry, Oral Roberts University, Tulsa, Oklahoma. these lipid alterations is probably an integral part of the microsomal electron transport complex which catalyzes the oxidative metabolism of drugs and other organic substances foreign to mammalian cells (3). The studies described in the present report were designed (a) to establish the lipid alterations are the result of enzymic activity, (5) to investigate the conditions required for maximum activity, (c) to observe the effect of various inhibitors, and (d) to determine the stoichiometry of the system insofar as possible.

Matetials
AnimalsAdult male albino rats originally derived from the Holtzman-Sprague-Dawley strain were used. The animals were fed a commercial laboratory ration obtained from Rockland Laboratories, Teckland, Inc., Monmouth, Illinois.
Reagents and Xolvents-All chemicals were reagent grade and were used as obtained except where specified. TPNf, TPNH, sodium n-glucose&P, yeast, glucose-6-P dehydrogenase (Type VI; yeast) and HMBr were obtained from Sigma Chemical Company, St. Louis, Missouri.
ADP was a product of P-L Biochemicals, Milwaukee, Wisconsin. Tris, chloroform, methanol, other organic solvents, and inorganic salts were purchased from Fisher Scientific. EDTA and 2thiobarbituric acid were obtained from Eastman.
Boron trifluoride-methanol reagent and some fatty acid methyl ester standards were products of Applied Sciences Laboratories, State College, Pennsylvania. Some methyl ester standards were purchased from the Hormel Institute, Austin, Minnesota.
The "chromatoquality" reagent, n-hexane, was obtained from Matheson Coleman and Bell (Divisionof the Matheson Company, East Rutherford, New Jersey).

Methods
Microsomes were prepared as described in the accompanying paper (1).
Issue of May 10, 1968 H. E. May and P. B. McCay Enzyme Incubation SystemsAssay conditions are given in each table or figure.
All quantities of addition to the assay system are given as the final concentration values in the incubation system. Microsomes present in the reaction systems contained about 1 mg of protein per ml of final incubation volume, except in the case of the microsome concentration study (Fig. 5,below).
Photometric Assay-MDA formation was estimated by the thiobarbituric acid reaction (4) with l-ml reaction systems incubated in a Dubnoff apparatus as described previously (1). TPNH oxidation was also determined with l-ml reaction systems incubated in the same way. After removal of protein with 3 volumes of ethanol, the absorbance change at 340 rnk was measured in a Beckman DU-2 spectrophotometer.
Fatty Acid Analyses and Oxygen Uptalce-Systems (5 ml) were incubated in a differential respirometer for 15 min, or until thermal equilibrium was established.
The reaction was initiated by tipping the substrate (TPNH or a mixture of TPNH, glucose+P, and glucose-6-P dehydrogenase) into the reaction chamber from the vessel side arm. Oxygen uptake was recorded until the indicated incubation time had elapsed. The vessels were promptly removed from the respirometer and the reaction was stopped by the addition of 25 ml of methanol.
After the small quantity of silicone grease had been carefully removed from the ground glass fitting of the vessel, the contents of the flasks were transferred to 250-ml separatory funnels.
The incubation vessels, which contained some residue, were then filled with chloroform-methanol (2:1, v/v). After the residual material had been extracted for 15 min, the solvent was transferred to the appropriate separatory funnel. The chloroform and methanol were then added to the separatory funnels, so that the final solvent composition was chloroform-methanol (2: 1) and the ratio of the solvent volume to the original incubation volume was 20: 1. This extraction procedure is essentially the same as that of Folch,Lees,and Sloane Stanley (5). At this point exactly 300 pg of highly purified erucic acid (22:l)'J were added to each system as an internal standard.
After the extraction systems were shaken thoroughly to ensure complete mixing of the internal standard and complete extraction of lipid from the microsomal membranes, 20 ml of 0.5% (w/v) aqueous NaCl solution were added to each separatory funnel.
The mixture was shaken and, after phase separation, the lower (chloroform) layer was isolated and evaporated under reduced pressure.
The lipid residue from each system was then taken up in 2 ml of chloroform.
Aliquots were removed for various chemical analyses and for the preparation of fatty acid methyl esters for gasliquid chromatography.
The methyl esters were prepared by the boron trifluoride method (6).
Fatty Acid Composition-The fatty acid compositions of the total lipids extracted from the incubation systems were determined by gas-liquid chromatography of the methyl esters derived from the lipid.
A description of the instrument conditions and the polyester columns used is given in Table II of the preceding paper (1). Methyl esters were identified by comparison of their relative retention times with those of known standard esters chromatographed under the same conditions. The identity of esters was confirmed by comparison of the retention times of hydrogenated sample esters with those of standard saturated esters as described in the preceding paper (1). Quantitative determination of individual fatty acids was accomplished by the addition of a standardized amount (300 pg) of erucic acid to sample lipids at the time of extraction.
This acid was not present in detectable amounts in the microsomal lipid. The detector response to this acid was determined to be linear over the sample size range used in the gas-liquid chromatographic analyses.

RESULTS
Enzymic Nature of MDA Production-The chromogen produced in this enzyme system yields a colored derivative when allowed to react with 2-thiobarbituric acid. The chromogen has been shown to be MDA (2). The enzymic nature of MDA production is shown in Figs. 1 to 3. Both MDA production by microsomal TPNH oxidase ( Fig. 1A) and the oxidation of TPNH itself (Fig. 1B) were sensitive to mild heat treatment. Almost complete loss of these activities occurred when microsomes were heated for 30 set at 65". It was also observed that continuous oxidation of TPNH was required for MDA formation ( Fig. 2). Curve 1 shows that when a limiting amount of TPNH was added to the incubation system, MDA production stopped after all TPNH was oxidized.
After the initial quantity of TPNH was fully oxidized (in about 10 min), supplementation with additional TPNH (Curve z?) or a TPNH-generating enzyme system (Curve S) led to a continuation of MDA formation. Fig. 3 shows that both the formation of MDA and the O2 uptake associated with this system were prevented by the addition of HMB.
Addition of HMB 5 min after the enzyme activity had been initiated caused an immediate inhibition of MDA production and O2 uptake.
Correlation among MDA Fomnation, O2 Uptake, and Polyun-saturate&FA Loss-It was reported in the preceding paper (1)  uptake, and the amount of polyunsaturated-FA lost during TPNH oxidase activity is shown in Fig. 4.4. On a molar basis, the final amount of O2 uptake was about 4 times polyunsaturated-FA loss, and the polyunsaturated-FA loss was 5 to 7 times greater than the MDA formation. Thus, on a molar basis, MDA is a minor product. However, when the data were expressed as as percentage of total O2 uptake, total MDA formation, and total polyunsaturated-FA loss (Fig. 4B), the three curves were similar.
All of them were hyperbolic in shape. Thus, MDA production, O2 uptake, and polyunsaturated-FA loss were qualitatively related but the quantitative relationship was complex and changed during the time course of the enzyme reaction.
E uptake; 0, total polyunsaturated fatty acid lost; a, malondialdehyde formation. Incubation system and conditions were the same as in Fig. 3. Polyunsaturated fatty acid losses and 0, uptake were determined on the same 5-ml system; malondialdehyde was determined in l-ml systems. The temperature was 37". Uptake- Fig.  5 shows the effect of the concentration of microsomes on MDA formation and 02 uptake.
The data indicated that the rates of MDA formation (B) and 02 uptake (D) were proportional to the concentration of microsomes present. It will be noted that both MDA production and O2 uptake reached a maximum value (B and 0) in spite of the fact that TPNH levels were maintained.
These maximum values were also proportional to the quantity of microsomes present. From the evidence we have obtained there is reason to believe that certain membrane lipids were acting as substrate in this reaction and that the depletion of these lipids accounted for the limiting values obtained.
Effect of TPNH Concentration on MDA Production and Polyunsaturated-FA LossesTheeffect of TPNH concentration on the rate of MDA production is shown in Fig. 6. The double reciprocal Lineweaver-Burk plot (7) was linear, and the K, for TPNH from this plot was 1 PM.
The kinetic plot for fatty acid loss could not be done since it was not feasible to obtain initial rates from the disappearance of the fatty acids. Table I shows the effect of the TPNH level, maintained with a TPNH-generating system, on the fatty acid composition of the total microsomal lipid.
The data indicated that the amount of polyunsaturated-FA lost from the microsomal membrane after a 60-min incubation was about the same for all levels of TPNH tested, provided that a TPNH-generating system was present. EJect of Temperature of Incubation cm MDA Forma&m- Fig.  7 shows that even at 0" the rate of MDA formation was 30 to 50% of the rate at 37". Furthermore, the rates at 37" and 21~ were were very similar.
A similar maximum amount of MDA was formed at all three temperatures, and this was not due to a limiting amount of TPNH since the latter was maintained at 300 PM by a TPNH-generating system. Effect of Variation of Incubation Parameters on Loss of iVicrosomal Membrane Polyunsaturated-FA-Temperature: Experiment 1 in Table II shows that even at 0" large losses of polyunsaturated-FA occurred in the systems containing TPNH.
The decreases of 18 : 2, 20 : 4, and 22 : 6 in this experiment were approximately 30, 70, and 90%, respectively, and the decreases were about the same for both the 0" and the 37" incubations (compare  formation. The incubation system was the same as in Fig. 3. Theincubation temperatures were 0,O"; A, 21"; l ,37". Experiments 1 and 2). These data represented the maximum amounts of polyunsaturated-FA loss under the conditions given (see Fig. 4), and implied nothing with regard to rates of polyunsaturated-FA loss. Effect of ADP-Fe+++: Experiment 2 shows that there were only minor quantitative differences in polyunsaturated-FA lost when the lipid compositions of systems incubated in the absence and in the presence of ADP-Fe+++ were compared.
Thus, ADP-Fe+++ was also not required for the    Table I. Values represent the quantity of each fatty acid in the total 5-ml incubation volume.
TPNH, 6 mM glucose-B-P, and 0.5 Kornberg unit of purified glucose-6-P dehydrogenase per ml of incubation mixture. c Microsomes were heated at 65" for 2 min. 5 Addition of a TPNH-generating system composed of 0.3 mM d 02 uptake was determined in a differential respirometer.
associated MDA production and O2 uptake (data not shown). ADP-Fe+* was routinely added, however, since it had an effect on rates and for the sake of uniformity of experimental conditions. Heat inactivation: The effect of heating microsomes at 65" for 1 mm is shown in Experiment 3. When the lipid composition of control systems containing heated microsomes was compared with that of systems containing untreated microsomes, it was apparent that the heat treatment had little if any effect on the polyunsaturated-FA content. Furthermore, the lipid from heated microsomes incubated in the presence of TPNH and a TPNH-generating system showed only small losses of polyunsaturated-FA when compared to its control and the fully active system. Thus, the activity responsible for the polyunsaturated-FA losses, as well as for MDA formation and O2 uptake, is heat sensitive.
Reduced O2 pressure: Experiment 4 shows that the losses of polyunsaturated-FA also occurred at 0.04 atm, although at a somewhat slower rate. Systems incubated in air exhibited a hyperbolic oxygen uptake curve which was reaching a plateau (see Fig. 50) by about 30 min. However, systems incubated at 0.04 atm of O2 pressure displayed a linear O2 uptake for at least 45 mm. Experiment 5 shows that a 60-min incubation of microsomes without TPNH (control system) had no effect on the fatty acid composition, since the latter was essen-tially identical with that of nonincubated microsomes.
Thus, the TPNH-dependent reaction utilizing polyunsaturated-FA from microsomal membrane lipid takes place under very mild temperature conditions, at low O2 pressures, and at very low TPNH concentrations (see Table I). All conditions and requirements for the reaction were at least compatible with physiological parameters whether the reaction takes place in viva or not, unless it is argued that microsomes in vitro are not in a physiological state.

Effects of Inhibitors on MDA Formation-HMB
is an inhibitor of microsomal electron transport (9-11) and also of lipid peroxidation (12). The effects of various kinds of inhibitors on MDA formation are shown in Table III Since inhibition by HMB is considered to be the result of a reaction with essential sulfhydryl groups, other sulfhydryl reagents were also tested.
IIMB not only prevented formation of MDA when added at zero time, but it also immediately stopped formation of MDA when added after the initiation of the MDA-forming reaction. However, other sulfhydryl reagents, such as N-ethylmaleimide, iodoacetate, and iodoacetamide, had little or no effect on MDA formation. Thus, the effect of HMB may or may not be an effect on sulfhy-dry1 groups.
Inorganic cations, such as Mn++ and Co*, are known to inhibit lipid peroxidation reactions (  Inhibitors of MDA formation in microsomal TPNH oxidafe system The control incubation system contained 0.1 ml of microsomes (approximately 1 mg of protein), 4 mM ADP, 0.012 mM FeC13, and 10 mM nicotinamide, all in 0.1 M Tris-HCl, pH 7.5. The experimental system contained all components of the control plus 0.3 mM TPNH, 6 mM glucose-6-P, and 0.5 Kornberg unit of glucose-6-P dehydrogenase.
Incubations were carried out in air at 37" in l-ml incubation volumes. All inhibitors were present at a level of 1 mM except a-tocopherol and sodium sulfite (50 mM 1 both of these cations prevented MDA formation, there did not appear to be an instantaneous inhibition of this process by these cations when the latter were added 5 min after the initiation of the MDA-forming reaction. Similarly, EDTA was able to prevent MDA formation if added prior to the addition of TPNH, but did not stop MDA production immediately if added 5 min after TPNH was added. a-Tocopherol was also able to prevent MDA formation by the TPNH oxidase system when it was homogenized with the liver during preparation of the microsomes (13). The effect of dietary or-tocopherol on this enzyme system is the subject of a forthcoming report.3 Sulfite is known to be oxidized aerobically by a free radical mechanism (12)) and has been used to detect free radical intermediates produced by certain enzyme reactions (14). Addition of small quantities of sulfite, which were insufficient to maintain the free radical chain reactions necessary for its oxidation, were without effect on MDA production.
However, higher concentrations of sulfite (12 W) were able to reduce MDA formation greatly and were also able to 3 H. E. May and P. B. McCay, manuscript in preparation, sustain the free radical reactions required for sulfite oxidation. These data implied that sulfhydryl groups, a metal, and a free radical are probably involved in some part of the enzyme system.
Effect of Inhibitors on Polyunsaturated-FA LossesTable IV shows the effect of Co* and HMB on the amount of polyunsaturated-FA lost from microsomal membrane lipids as a result of the action of TPNH oxidase. Experiment 1 shows that Co++ caused very little inhibition of the polyunsaturated-FA-utilizing activity at low concentration (0.01 mM). However, polyunsaturated-FL4 utilization was markedly inhibited when the system contained 1 mM Co*.
The residual O2 uptake observed with the higher level of Co* was presumably due to the oxidation of TPNH, which is independent of lipid utilization.
The effect of the addition of HMB to the enzyme system is shown in Experiment 2. The OX uptake was completely stopped and the polyunsaturated-FA losses were greatly reduced by HMB. Other experiments have shown that HMB induces some MDA formation in the control incubation system (see Table III Duplicate values are shown, and it can be seen that reproducibility was reasonably good. The content of 16:0, 18:0, and 18: I in the system remained constant throughout the time course of the reaction.
The value of 18 : 2 decreased somewhat during the 15-min period depicted, but greater losses are known to occur at 30 and 60 min. Both 20:4 and 22:6 were 50% utilized at the end of the 15-min incubation period.
Since the molar quantity of each fatty acid in the system could be determined, it was clear that the contents of saturated and monenoic fatty acids were not altered by this reaction.
The process appeared to be specific for the more highly unsaturated fatty acids, especially 2O:4 and 22:6.
Xtoichiometric XtudiesThe stoichiometric relationships among polyunsaturated-FA loss, TPNH oxidation, and O2 uptake are shown in Table V. All quantities are expressed on the basis of microsomal protein.
The loss of each fatty acid for the 60-mm time period is shown for five separate experiments.
The total polyunsaturated-FA lost was taken as the sum (in millimicromoles) of the losses of 18: 2, 20:4, and 22: 6. Losses of other polyunsaturated-FA, present in the microsome in trace amounts, were quantitatively insignificant. Comparison of the molar ratios tabulated in the last column of Table V divulges that the  stoichiometry was rather consistently fatty acids to TPNH to 02, 1:1:4.
The 1:l ratio between O2 uptake and polyunsaturated-FA loss shown in Table V, however, was a limiting value, and the time relationship between these two quantities is shown in Fig. 9. The O2 to fatty acid ratio changed throughout the time course of the reaction, and approached the limiting value of approximately 4. As indicated in Fig. 9, when the ratio was extrapolated to zero time, the value was approximately 1. Table VI shows that there was good agreement between the number of double bonds lost calculated from polyunsaturated-FA disappearance and the actual number lost, as indicated by bromination (15). This indicated that the product contained none of the unsaturation of the polyunsaturated-FA precursors, and was in general agreement with previous hydrogenation studies (16).   Since it was known that carbonyl compounds were among the ever, as already shown in these studies, other organic substances and inorganic cations apparently penetrated the inicrosome sufficiently to act as inhibitors of the system. Semicarbazide has been used in other enzyme systems to trap carbonyl compounds (17). DISCUSSION products formed in the TPNH oxidase-catalyzed lipid alteration excluded from reacting with the carbonyl compounds. How-reactions (I), it was thought that O2 might be utilized to further oxidize aldehydes to acids. With this possibility in mind, an experiment was done in which semicarbazide was added to the reaction system as a carbonyl "trap" to prevent a possible further oxidation. Accordingly, that portion of the 02 consumed which might be associated with the postulated oxidation should be prevented. Table VII shows that semicarbazide had no effect on the O2 to fatty acid stoichiometry.
It may be argued that semicarbazide, owing to solubility or steric factors, might be Three aspects of the phenomenon described above and in the preceding paper (1) seem particularly significant. First, essentially all of the fatty acids involved in the reaction are situated at the @ position of microsomal phospholipids.
Second, the conditions under which the membrane linids are ranidlv altered mav by guest on March 24, 2020 http://www.jbc.org/ Downloaded from exist in a number of TPNH-requiring enzymic assays in which the microsomal fraction of rat liver cell particulates is utilized as an enzyme.
The influence, therefore, of possible extensive and rapid changes in the microsomal lipid composition on such systems in unknown.
It would seem particularly pertinent to determine possible effects in enzymic systems containing microsomes and TPNH in which studies on fatty acid biosynthesis and interconversions are being carried out,. Third, the conditions which resulted in maximum activity of the lipid-altering system could apparently exist, in viva. There is a possibility, therefore, that a role for this phenomenon in the turnover of the phospholipid portion of microsomal membranes may exist. Omura,Siekevitz,and Palade (18) have shown that the turnover of phospholipids in the microsomal membranes is somewhat more rapid than that of the protein portion.
Our studies have shown that the microsomal TPNH oxidasecatalyzed lipid changes were inhibited in vitro when the animals had been given very high dietary levels of a-tocopherol, but elimination of these high levels of a-tocopherol from the diet for as little as 7 hours totally reversed the inhibition observed in vitro (16). Studies from this laboratory (19) have also shown that 20:4 has a faster turnover rate in the liver of &ocopheroldeficient animals relative to other fatty acids as compared to control animals.
TPNH oxidase-catalyzed alterations of membrane lipids could account for this observation.
In spite of the quantity of the data shown in this report and in the preceding paper (I), the mechanism of the enzymic reaction utilizing polyunsaturated fatty acids of phospholipids remains obscure.
It may be that all glycerophosphatides containing polyunsaturated-FA are substrates for this reaction, but the data in these studies provide sufficient evidence only to show that at least phosphatidylethanolamine and phosphatidylcholine are utilized.
The results indicate that the utilization of polyunsaturated-FA in the microsomal lipid is enzymic and occurs at, a rapid rate. From 15 to 20% of the total fatty acid content of the phospholipid was commonly consumed in the reaction, most of which occurred during the first 15 min of the incubation.
The lipid-altering reaction appears to be limited by the amount of polyunsaturated-FA present in the phospholipid, since 80 to 90 % of these acids were utilized when the reaction was allowed to go to fatty a&id (PUPA) losses. Incubation system and conditions were the same as in Fig. 3. Each point represents a 5-ml incubation system in which O2 uptake and f.atty acid analyses were done as described under "Methods." Polyunsaturated-FA losses were the sum of the losses of 18: 2,20:4, and 22:6. The ratio given in this graph was determined by dividing the number of micromoles of Oe uptake for a given system by the total micromoles of polyunsaturated-FA lost in that system. completion. The process was accompained by O2 consumption and the formation of a stoichiometrieally small amount of MDA.
That MDA is probably a minor (although consistent) product of this reaction can be supported by the fact that although over 400 mpmoles of fatty acid were utilized, only about, 45 mpmoles of MDA were produced (see Fig. 4).
The production of MDA followed typical enzyme kinetic patterns.
The double reciprocal Lineweaver-Burk plot was linear and gave an apparent K, of 1 X 10e6 M. This value is of the order one would expect for a pyridine nucleotide, and is in essential agreement with values obtained by others for various microsomal TPNH-dehydrogenating enzyme activities (20). It may be noteworthy that essentially the same quantity of fatty acid was altered when microsomes were incubated in the presence of quantities of TPNH approximating the K, value (e.g. 0.6 PM, Table I) as at higher levels of TPNH (e.g. 300 PM) provided that a TPNH-regenerating system was added to maintain the nucleotide in the reduced state.
The studies with sulfhydryl reagents, EDTA, metal ions, and free radical-trapping agents imply (a) that the enzyme system contains a site (or sites) susceptable to HMB which may or may not. involve a sulfhydryl group (or groups), (b) that a metal component is required, and (c) that a free radical component may be essential.
The time course study of polyunsaturated-FA utilization in this system indicates that loss of double bonds from microsomal TPNH Oxidase-catalyzed Alterations of Membrane Phospholipds. II Vol. 243,No. 9 lipid cannot be explained on the basis of saturation reactions, since the amounts of the saturated acids remained constant. Staudinger and Zubrzycki (21) have reported that microsomes reduce unsaturated fatty acids under similar conditions. The stoichiometric studies indicate that there is an initial 1: 1 molar relationship between 02 consumption and fatty acid loss from membrane lipids.
This ratio continues to increase to a limiting value of approximately 4: 1 as the reaction proceeds. The results indicate that an initial attack on lipid, requiring oxygen, occurs and that subsequent secondary reactions which consume 02 must follow almost immediately, thereby modifying the initial product further.
In order to gain some insight as to what a limiting stoichiometry of 4:1, O2 to fatty acid, might mean, calculations of the total number of double bonds present in the fatty acids utilized were made according to Table VI. For over 20 experiments in which the incubation period was long enough so that O2 uptake had ceased (i.e. the limiting condition), the ratio of O2 to the lipid double bonds contained in the polyunsaturated-FA that were utilized was always 1: 1. The agreement between these calculated double bond values and unsaturation values determined by bromination indicates that the products of the reaction contained none of the unsaturation of their polyunsaturated-FA precursors. It appears, therefore, that there is a stoichiometric relationship between O2 uptake and actual double bonds lost or between 02 uptake and some molecular feature of polyunsaturated-FA that is correlated with the number of double bonds in the molecule. At present it is not possible to say what portion of the 02 taken up is actually incorporated into the lipid. MDA is a minor product (on a molar basis, equivalent to approximately 12y0 of the fatty acid loss and only 3% of the O2 uptake).
It is also a minor product in lipid peroxidation in model systems (22). It may be pertinent to mention a similarity between this system and the biosynthesis of prostaglandins from polyunsaturated-FA.
The latter system requires a donor of reducing equivalents, consumes 02, utilizes polyunsaturated-FA, and produces stoichiometrically small quantities of MDA (23). It is fortunate that MDA formation and lipid alterations described in this work are closely correlated in this microsomal system, since the determination of MDA is simple and rapid, but we feel it is hazardous to design experiments and draw conclusions on the basis of an assay which measures as little as 3% of the products involved in a reaction. Lipid peroxidation in model systems is autocatalytic (24), whereas TPNH oxidase-catalyzed lipid alterations appear not to be. That the reaction is not autocatalytic after the first oxygen attack is indicated in Fig. 9, which shows that once the enzyme reaction is stopped at any point during the process, the particular ratio of O2 to fatty acid reached at the instant remains stable. All investigated methods of stopping enzymic oxidation of TPNH in this system also simultaneously stop the lipid alterations.
The tight "coupling" between active TPNH oxidation and lipid alteration