Proline isomer-specific antibodies reveal the early pathogenic tau conformation in Alzheimer's disease

cis-trans isomerization of proteins phosphorylated by proline-directed kinases is proposed to control numerous signaling molecules and is implicated in the pathogenesis of Alzheimer's and other diseases. However, there is no direct evidence for the existence of cis-trans protein isomers in?vivo or for their conformation-specific function or regulation. Here we develop peptide chemistries that allow the generation of cis- and trans-specific antibodies and use them to raise antibodies specific for isomers of phosphorylated tau. cis, but not trans, p-tau appears early?in the brains of humans with mild cognitive impairment, accumulates exclusively in degenerated neurons, and localizes to dystrophic neurites during Alzheimer's progression. Unlike trans p-tau, the cis isomer cannot promote microtubule assembly, is more resistant to dephosphorylation and degradation, and is more prone to aggregation. Pin1 converts cis to trans p-tau to prevent Alzheimer's tau pathology. Isomer-specific antibodies and vaccines may therefore have value for the early diagnosis and treatment of Alzheimer's disease.     

Enzymatic catalysis of prolyl isomerization in an unfolding protein.

Prolyl isomerases are able to accelerate slow steps in protein refolding that are limited in rate by cis/trans isomerizations of Xaa-Pro peptide bonds. We show here that prolyl isomerizations in the course of protein unfolding are also well catalyzed. To demonstrate catalysis we use cytoplasmic prolyl isomerase from Escherichia coli as the enzyme and reduced and carboxymethylated ribonuclease T1 as the substrate. This form of ribonuclease T1 without disulfide bonds is nativelike folded only in the presence of moderate concentrations of NaCl. Unfolding can be induced by reducing the NaCl concentration at ambient temperature and in the absence of denaturants. Under these conditions prolyl isomerase retains its activity and it catalyzes prolyl cis/trans isomerization in the unfolding protein. Under identical conditions within the NaCl-induced transition unfolding and refolding are catalyzed with equal efficiency. The stability of the protein and thus the final distribution of unfolded and folded molecules attained at equilibrium is unchanged in the presence of prolyl isomerase. These results demonstrate that prolyl isomerase functions in protein folding as an enzyme and catalyzes prolyl isomerization in either direction.     

Photoreactions of metarhodopsin III

Meta III is an inactive intermediate thermally formed following light activation of the visual pigment rhodopsin. It is produced from the Meta I/Meta II photoproduct equilibrium of rhodopsin by a thermal isomerization of the protonated Schiff base C=N bond of Meta I, and its chromophore configuration is therefore all-trans 15-syn. In contrast to the dark state of rhodopsin, which catalyzes exclusively the cis to trans isomerization of the C11=C12 bond of its 11-cis 15-anti chromophore, Meta III does not acquire this photoreaction specificity. Instead, it allows for light-dependent syn to anti isomerization of the C15=N bond of the protonated Schiff base, yielding Meta II, and for trans to cis isomerizations of C11=C12 and C9=C10 of the retinal polyene, as shown by FTIR spectroscopy. The 11-cis and 9-cis 15-syn isomers produced by the latter two reactions are not stable, decaying on the time scale of few seconds to dark state rhodopsin and isorhodopsin by thermal C15=N isomerization, as indicated by time-resolved FTIR methods. Flash photolysis of Meta III produces therefore Meta II, dark state rhodopsin, and isorhodopsin. Under continuous illumination, the latter two (or its unstable precursors) are converted as well to Meta II by presumably two different mechanisms.     

Interaction of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pili strain PAK with a cross-reactive antibody: conformation of the bound peptide

The C-terminal receptor binding region of Pseudomonas aeruginosa pilin protein strain PAK (residues 128-144) has been the target for the design of a vaccine effective against P. aeruginosa infections. We have recently cloned and expressed a (15)N-labeled PAK pilin peptide spanning residues 128-144 of the PAK pilin protein. The peptide exists as a major (trans) and minor (cis) species in solution, arising from isomerization around a central Ile(138)-Pro(139) peptide bond. The trans isomer adopts two well-defined turns in solution, a type I beta-turn spanning Asp(134)-Glu-Gln-Phe(137) and a type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142). The cis isomer adopts only one well-defined type II beta-turn spanning Pro(139)-Lys-Gly-Cys(142) but displays evidence of a less ordered turn spanning Asp(132)-Gln-Asp-Glu(135). These turns have been implicated in cross-reactive antibody recognition. (15)N-edited NMR spectroscopy was used to study the binding of the (15)N-labeled PAK pilin peptide to an Fab fragment of a cross-reactive monoclonal antibody, PAK-13, raised against the intact PAK pilus. The results of these studies are as follows: the trans and cis isomers bind with similar affinity to the Fab, despite their different topologies; both isomers maintain the conformational integrity of their beta-turns when bound; binding leads to the preferential stabilization of the first turn over the second turn in each isomer; and binding leads to the perturbation of resonances within regions of the trans and cis backbone that undergo microsecond to millisecond motions. These slow motions may play a role in induced fit binding of the first turn to Fab PAK-13, which would allow the same antibody combining site to accommodate either trans or cis topology. More importantly for vaccine design, these motions may also play a role in the development of a broad-spectrum vaccine capable of generating an antibody therapeutic effective against the multiple strains of P. aeruginosa.     

An electron spin resonance investigation and molecular orbital calculation of the anion radical intermediate in the enzymatic cis-trans isomerization of furylfuramide, a nitrofuran derivative of ethylene

The enzymatic cis-trans isomerization of nitrofuran derivatives has been proposed to occur via the formation of a radical anion intermediate. ESR investigations, in conjunction with intermediate neglect of differential overlap (INDO) molecular orbital calculations, support this concept by demonstrating the enzymatic generation of cis and trans radical anions of 3-(5-nitro-2-furyl)-2-(2-furyl) acrylamide. The INDO calculations further indicate that the rotational barrier between the cis and trans anion radicals of this compound is only 5--10 kcal/mol, whereas a 70 kcal/mol barrier exists for the parent geometric isomers. Hyperfine splitting constants for the cis-trans conformers have been assigned on the basis of INDO calculations. Surprisingly, only the nitrogen hyperfine splitting of the nitro group is distinguishably different in the two conformers, a result which is not inconsistent with the INDO calculations.     

Facile geometric isomerization of phenolic non-steroidal estrogens and antiestrogens: limitations to the interpretation of experiments characterizing the activity of individual isomers

In many estrogen responsive systems the isomers of tamoxifen are known to have different biological character-the trans isomer is generally an antagonist and the cis isomer an agonist. Attempts to similarly characterize the isomers of hydroxytamoxifen (which differ greatly in their affinity for the estrogen receptor) are shown to be complicated by their facile isomerization. This isomerization was studied in cultures of estrogen receptor positive MCF-7 human breast cancer cells and monitored by HPLC under reversed phase conditions. Hydroxytamoxifen isomers that are initially 99% pure, undergo a time and temperature dependent isomerization, so that after 2 days in tissue culture medium at 37 degrees C they have isomerized to the extent of 20%. This isomerization occurs in the cell-free medium alone and cannot be attributed to a metabolic conversion by the cells. The isomerization occurs much more slowly at 4 than at 37 degrees C and can be reduced considerably by various antioxidants (butylated hydroxytoluene, ascorbate, alpha-tocopherol, retinoic acid and retinal); however, at concentrations that block isomerization, these antioxidants are toxic to the cells. Although the medium contains both the cis and trans isomers of hydroxytamoxifen, the MCF-7 cells preferentially accumulate the trans isomer and the material associated with the nuclear estrogen receptor is, in all cases, mainly the higher affinity trans isomer. A similar preference of the estrogen receptor for the trans isomer is seen with diethylstilbestrol, resulting again in almost exclusive accumulation of the trans isomer in the receptor binding site. These experiments indicate the importance of verifying the isomer compositions of easily isomerizable non-steroidal estrogens and antiestrogens, such as diethylstilbestrol and hydroxytamoxifen, both in stock solutions and in experimental samples (especially those derived from receptor-associated material), so as to ascertain that the activity of the individual isomers is being correctly assigned.     

Photocyclization of trans-1-(1'-naphthyl)-2-(3-hydroxyphenyl)ethene: evidence for adiabatic 1trans*-->1cis* photoisomerization.

The photochemistry of trans- and cis-1-(1'-naphthyl)-2-(3-hydroxyphenyl)ethene in cyclohexane and acetonitrile was examined. In cyclohexane fluorescence is the main deactivation channel for the 1trans* isomer while photocyclization is the main reaction of the 1cis* isomer. The weighty formation of hydroxychrysene following one photon absorption by the trans isomer furnished evidence of an adiabatic 1trans*-->1cis* isomerization. The photoreactivity data in acetonitrile indicated the influence of solvent polarity on the shape of the excited state surface.     

Escherichia coli thioredoxin folds into two compact forms of different stability to urea denaturation

The urea-induced denaturation of Escherichia coli thioredoxin and thioredoxin variants has been examined by electrophoresis on urea gradient slab gels by the method of Creighton [Creighton, T. (1986) Methods Enzymol. 131, 156-172]. Thioredoxin has only two cysteine residues, and these form a redox-active disulfide at the active site. Oxidized thioredoxin-S2 and reduced thioredoxin-(SH)2 each show two folded isomers with a large difference in stability to urea denaturation. The difference in stability is greater for the isomers of oxidized than for the isomers of reduced thioredoxin. At 2 degrees C, the urea concentrations at the denaturation midpoint are approximately 8 and 4.3 M for the oxidized isomers and 4.8 and 3.7 M for the reduced isomers. The difference between the gel patterns of samples applied in native versus denaturing buffer, and at 2 and 25 degrees C, is characteristic for the involvement of a cis-proline-trans-proline isomerization. The data very strongly suggest that the two folded forms of different stabilities correspond to the cis and trans isomers of the highly conserved Pro 76 peptide bond, which is cis in the crystal structure of oxidized thioredoxin. Urea gel experiments with the mutant thioredoxin P76A, with alanine substituted for proline at position 76, corroborate this interpretation. The electrophoretic banding pattern diagnostic for an involvement of proline isomerization in urea denaturation is not observed for oxidized P76A. In broad estimates of delta G degree for the native-denatured transition, the difference in delta G degree (no urea) between the putative cis and trans isomers of the Ile 75-Pro 76 peptide bond is approximately 3 kcal/mol for oxidized thioredoxin and approximately 1.5 kcal/mol for reduced thioredoxin. Since cis oxidized thioredoxin is much more stable than trans, folded oxidized thioredoxin is essentially all cis. In folded reduced thioredoxin, cis and trans interconvert slowly, on the minute time scale at 2 and 25 degrees C. In the absence of urea, the folded reduced thioredoxin is less than a few percent trans. Three additional mutants with additions or substitutions at the active site also show electrophoresis banding patterns consistent with a difference in stability between cis and trans isomers.     

Interactions of platinum complexes with the essential and nonessential sulfhydryl groups of thymidylate synthetate.

Thymidylate synthetase (methylenetetrahydrofolate:deoxyuridylate C-methyltransferase) from Lactobacillus casei was progressively inactivated when incubated at 25 degrees C, pH 6.8, in the presence of trans-Pt(NH3)2Cl2. The inhibition appeared to be irreversible, and the rate ofa ctivity loss was dependent on the inhibitor concentration. The corresponding cis isomer was incapable of inhibiting the enzyme under the same conditions. The presence of 2-mercaptoethanol protected the enzyme from inhibition, but did not reactivate enzyme preparations which had been inhibited prior to the addition of the thiol. The interactions of cis- and trans-Pt(NH3)2Cl2 with the enzyme's sulfhydryl (-SH) groups were inferred from the results of spectrophotometric titrations of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) and p-hydroxymercuribenzoate. The results suggested that the cis isomer reacted with an average of 1.3 of the enzyme's 4-SH groups and that these were not essential for catalysis. The trans isomer reacted with a total of approximately 2.5 -SH groups, 1.2 of which are essential for catalysis. Neither the trans isomer nor a combination of both isomers was able to react with 1.2 of the 4 -SH groups. Further evidence that the Pt complexes are interacting with enzyme's -SH groups was obtained by reversibly blocking the -SH groups of thymidylate synthetase, and demonstrating the resistance of these preparations to inhibition by the trans Pt complex. Possible explanations for the preferential inhibition of thymidylate synthetase by only one of the two geometric isomers of Pt(NH3)2Cl2 are considered.     

Isomerization of stable isotopically labeled elaidic acid to cis and trans monoenes by ruminal microbes

A previous study showed that oleic acid was converted by mixed ruminal microbes to stearic acid and also converted to a multitude of trans octadecenoic acid isomers. This study traced the metabolism of one of these trans C18:1 isomers upon its incubation with mixed ruminal microbes. Unlabeled and labeled (18-[13C]trans-9 C18:1) elaidic acid were each added to four in vitro batch cultures with three cultures inoculated with mixed ruminal bacteria and one uninoculated culture. Samples were taken at 0, 12, 24, and 48 h and analyzed for 13C enrichment in component fatty acids by gas chromatography-mass spectrometry. At 0 h of incubation, enrichment was detected only in elaidic acid. By 48 h of incubation, 13C enrichment was 18% (P < 0.01) for stearic acid, 7% to 30% (P < 0.01) for all trans C18:1 isomers having double bonds between carbons six through 16, and 5% to 10% for cis-9 and cis-11 monoenes. After 48 h, 13C enrichment in the uninoculated cultures was only detected in the added elaidic acid. This study shows trans fatty acids exposed to active ruminal cultures are converted to stearic acid but also undergo enzymic isomerization yielding a multitude of positional and geometric isomers.     

NMR analysis of staphylococcal nuclease thermal quench refolding kinetics.

Thermally unfolded staphylococcal nuclease has been rapidly quenched to temperatures near 0 degree C and the refolding behavior examined using an NMR kinetic experiment. Unfolded protein, exhibiting random coil chemical shifts, persists following the quench and refolds in two distinct kinetic phases. A protein folding intermediate with a trans Lys 116-Pro 117 peptide bond is transiently overpopulated and relaxes to the predominantly cis native cis-trans equilibrium. The rate of trans-->cis isomerization in the native-like nuclease intermediate is approximately 100-fold faster than that observed in a Lys-Pro model peptide. The activation enthalpy of 20 kcal/mol observed for the nuclease Lys 116-Pro 117 peptide bond is comparable to that observed for other X-Pro isomerizations.     

Evidence that the substrate backbone conformation is critical to phosphorylation by p42 MAP kinase

The effect of prolyl bond isomers on the substrate recognition capabilities of various endoproteases may be investigated in a reaction where both cis/trans isomers co-exist. Here we address the question of whether enzyme reactions at the side chain of an amino acid preceding proline proceed through an isomer specific pathway. The proline-directed p42 mitogen-activated protein kinase (ERK2) was used to phosphorylate the serine side chain in Pro-Arg-Ser-Pro-Phe-4-nitroanilide under conditions where different amounts of cis prolyl isomer of the substrate were present. Initial phosphorylation rates were calculated ranging between zero at 100% cis isomer and around 60 pM/min at the equilibrium content of 83.5% trans isomer. In the presence of the peptidyl-prolyl cis/trans isomerase human hFKBP12 (500 nM), cis/trans isomerization proceeds rapidly, permitting the maximal phosphorylation rate to be observed in the dead time of the experiment. Results show that correct signature sequences are not sufficient to render potential substrates reactive to proline-directed enzymatic phosphorylations, but that the conformational state of the peptide bond following serine (threonine) is a critical determinant. Therefore, catalysis by peptidyl-prolyl cis/trans isomerases may add a new level of control to intracellular protein phosphorylations.     

Enzymic cis-trans isomerization of nitrofuran derivatives: isomerizing activity of xanthine oxidase, lipoyl dehydrogenase, DT-diaphorase and liver microsomes.

Xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) supplemented with an electron donor could catalyze the cis-trans isomerization of 3-(5-nitro-2-furyl)-2-(2-furyl)acrylamide, 3-(5-nitro-2-furyl)-2-phenylacrylamide and 3-(5-nitro-2-furyl)-2-(2-furyl)acrylonitrile. The direction of isomerization (cis leads to trans, cis in equilibrium trans or trans leads to cis) is dependent on the chemical structure of these nitrofuran derivatives. Lipoyl dehydrogenase (NADH:lipoamide oxidereductase, EC 1.6.4.3), DT-diaphorase (NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2) and liver microsomes could also catalyze the conversion of cis-3-(5-nitro-2-furyl)-2-(2-furyl)acrylamide to its trans isomer in the presence of an appropriate electron donor. Such isomerizing activity of these enzymes is much higher than their nitro-reducing activity. In addition, the cis-trans isomerization of some nitrofuran derivatives was demonstrated with the liver slices and the small intestines of rats. A new cis-trans isomerization mechanism which is based on transfer of a single electron by an enzyme system to a nitrofuran derivative to give the radical-anion was proposed. This postulated mechanism was supported by the preliminary experiments using pulse radiolysis technique.     

The isomers of the bis(1-thia-4,7-diazacyclononane)cobalt(III) ion: separation and characterization

The two geometric isomers cis and trans of the bis(1-thia-4,7-diazacyclononane)cobalt(III) ion have been separated and crystallized as the pure isomers by fractional crystallization, aided by cation- exchange chromatography on SP-Sephadex. The pure isomers are characterized by ¹³C NMR and UV-Vis spectroscopy. The isomers undergo a base- catalyzed isomerization to an equilibrium mixture cis/trans ca. 4/1. This isomerization is unusually facile, and occurs at a significant rate at pH down to about 5 at room temperature, so that chromatographic separations require acidic conditions. Separation by IE-HPLC has been demonstrated as a valuable tool to separate (3+)-charged complexes on an analytical scale, and can indicate the optimum conditions for preparative scale separations on SP- Sephadex columns. The isomerization process has been investigated as a function of temperature and pH using IE-HPLC, and ¹³C NMR and visible spectroscopy.     

13C nuclear magnetic resonance study of the cis-trans isomerism in X-Pro-Pro tripeptides.

13C nuclear magnetic resonance has been used to characterize quantitatively the cis-trans isomerism about both peptide bonds in the tripeptides Ser-Pro-Pro and Arg-Pro-Pro. Detailed pH titration data indicate that the configuration about both peptide bonds is closely linked to titration of the terminal carboxyl group and, to a lesser extent, to titration of the terminal amino group. The Pro2 C-3 resonance has been found particularly useful for interpretation due to its sensitivity to the isomerization about both peptide bonds. Analysis of the probabilities of the trans-trans, cic-cis, cis-trans, and trans-cis isomers in aqueous solution indicates a stability decrease in the order given. Similarities in the isomerization behavior of the two peptides indicate that side chain interactions involving the first residue have very little effect on the observed cis/trans ratios. The sensitivity of the cis/trans ratio to titration of the terminal amino group is most readily explained on the basis of an indirect effect on carbonyl-carbonyl repulsion.     

Energetic coupling between native-state prolyl isomerization and conformational protein folding

In folded proteins, prolyl peptide bonds are usually thought to be either trans or cis because only one of the isomers can be accommodated in the native folded protein. For the N-terminal domain of the gene-3 protein of the filamentous phage fd (N2 domain), Pro161 resides at the tip of a beta hairpin and was found to be cis in the crystal structure of this protein. Here we show that Pro161 exists in both the cis and the trans conformations in the folded form of the N2 domain. We investigated how conformational folding and prolyl isomerization are coupled in the unfolding and refolding of N2 domain. A combination of single-mixing and double-mixing unfolding and refolding experiments showed that, in unfolded N2 domain, 7% of the molecules contain a cis-Pro161 and 93% of the molecules contain a trans-Pro161. During refolding, the fraction of molecules with a cis-Pro161 increases to 85%. This implies that 10.3 kJ mol(-1) of the folding free energy was used to drive this 75-fold change in the Pro161 cis/trans equilibrium constant during folding. The stabilities of the forms with the cis and the trans isomers of Pro161 and their folding kinetics could be determined separately because their conformational folding is much faster than the prolyl isomerization reactions in the native and the unfolded proteins. The energetic coupling between conformational folding and Pro161 isomerization is already fully established in the transition state of folding, and the two isomeric forms are thus truly native forms. The folding kinetics are well described by a four-species box model, in which the N2 molecules with either isomer of Pro161 can fold to the native state and in which cis/trans isomerization occurs in both the unfolded and the folded proteins.     

Radiosensitizing effect of conjugated linoleic acid on tumor cells MCF-7 and MDA-MB-231

Apoptotic pathways in breast cancer cells are frequently altered, reducing the efficiency of radiotherapy. Conjugated linoleic acid (CLA), known to trigger apoptosis, was tested as radiosensitizer in breast cancer cells MCF-7 and MDA-MB-231. The CLA-mix, made up of the isomers CLA-9cis 11trans and CLA-10trans 12cis, was compared to three purified isomers, i.e., the CLA-9cis 11cis, CLA-9cis 11trans, and CLA-10trans 12cis. Using the apoptotic marker YO-PRO-1, the CLA-9cis 11cis at 50 micro mol/L turned out to be the best apoptotic inducer leading to a 10-fold increase in MCF-7 cells and a 2,5-fold increase in MDA-MB-231 cells, comparatively to the CLA-mix. Contrary to previous studies on colorectal and prostate cancer cells, CLA-10trans 12cis does not lead to an apoptotic response on breast cancer cell lines MCF-7 and MDA-MB-231. Our results also suggest that the main components of the CLA-mix (CLA-9cis 11trans and CLA-10trans 12cis) are not involved in the induction of apoptosis in the breast cancer cells studied. A dose of 5 Gy did not induce apoptosis in MCF-7 and MDA-MB-231 cells. The addition of CLA-9cis 11cis or CLA-mix has allowed us to observe a radiation-induced apoptosis, with the CLA-9cis 11cis being about 8-fold better than the CLA-mix. CLA-9cis 11cis turned out to be the best radiosensitizer, although the isomers CLA-9cis 11trans and CLA-10trans 12cis have also reduced the cell survival following irradiation, but using a mechanism not related to apoptosis. In conclusion, the radiosensitizing property of CLA-9cis 11cis supports its potential as an agent to improve radiotherapy against breast carcinoma.     

"Plasmalogen-type" cyclic acetals: formation and conformation of the 1,3-dioxanes and 1,3-dioxolanes from 1-0-cis-alk-1'-enyl-sn-glycerols

Acid-catalyzed cyclization of 1-O-cis-alk-1'-enyl-sn-glycerol produced four structurally and geometrically isomeric long-chain cyclic acetals of glycerol. The isomers were isolated by adsorption and gas-liquid chromatography and were identified as cis-2-alkyl-5-hydroxy-1,3-dioxane (Ia), trans-2-alkyl-5-hydroxy-1,3-dioxane (IIa), cis-2-alkyl-4-hydroxymethyl-1,3-dioxolane (IIIa), and trans-2-alkyl-4-hydroxymethyl-1,3-dioxolane (IVa). The structure of each isomer was established by chemical and spectroscopic methods. Cyclization with p-toluenesulfonic acid in boiling benzene led to a thermodynamically equilibrated mixture of isomers Ia-IVa in which the cis isomers predominated. Cyclization in acetic acid was found to be kinetically controlled, and formation of the trans isomers was relatively favored. Rearrangement of the cyclic acetal isomers did not occur in acetic acid; hence, optically active five-membered ring acetals were prepared.     

Effect of radical stress and ageing on the occurrence of trans fatty acids in rats fed a trans-free diet

In a previous paper, we demonstrated that tissue trans fatty acids can not only derive from the diet but also be endogenously formed. The central focus of this study was to prove that the in vivo isomerization occurs via a radical process. Two different models of radical insult were used: CCl(4) and AAPH injection to rats fed a diet completely free of trans isomers. Following this acute radical stress, a significant increase in unnatural trans fatty acid content of erythrocyte, kidney, and heart, but not liver, was observed. These results can be mainly explained by the high content, particularly in the liver, of antioxidant vitamins A and E that exhibit also an "anti-isomerizing" effect. Since during ageing cellular components are exposed to increasing radical insults, the observation of a significant trans fatty acid accumulation in 30-month-old rats could confirm that the in vivo formation of unnatural isomers is due to a radical process. Trans fatty acids can influence the physical characteristics of bilayer microdomains, affecting membrane properties and functions; thus, knowledge of biological radical species responsible for cis/trans isomerization and their possible sources can provide protective systems for preserving lipid geometry.     

Arachidonate geometrical isomers generated by thiyl radicals: the relationship with trans lipids detected in biological samples

The presence of trans fatty acids in mammalians is attributed to exogenous sources; nevertheless, trans isomers could be easily formed by free radical-catalyzed isomerization processes in vivo. The isomerization of methyl arachidonate (all-cis isomer) catalyzed by thiyl radical is proposed as a methodology applicable in biochemical laboratories, which produces mono- and di-trans isomers. Carbon-13 nuclear magnetic resonance spectroscopy shows that the carbon atom in position 15 is characteristic for each mono- and di-trans isomer. Antioxidants, such as alpha-tocopherol and all-trans-retinol acetate, inhibited the isomerization process. Trans phospholipids are formed in erythrocyte membranes by exposing blood to gamma-irradiation in the presence of thiols, which is in contradiction with the known role of these compounds as radioprotectors. Trans isomers are also analyzed in tissues harvested from breast cancer patients and compared to the adipose breast tissue taken a few centimeters from the edge of the tumor from the same patient. This work is generally aimed at contributing to the debate on trans fatty acids and stimulating a reconsideration of the current view on the exclusive presence of cis double bonds in cell membranes by studying radical processes that could affect or protect this natural configuration.