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Purpose: Progressive choroid and retinal pigment epithelial (RPE) degeneration causing vision loss is a unique characteristic of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), a fatty acid oxidation disorder caused by a common c.1528G>C pathogenic variant in HADHA, the α subunit of the mitochondrial trifunctional protein (TFP). We established and characterized an induced pluripotent stem cell (iPSC)-derived RPE cell model from cultured skin fibroblasts of patients with LCHADD and tested whether addition of wildtype (WT) HAHDA could rescue the phenotypes identified in LCHADD-RPE., Methods: We constructed an rAAV expression vector containing 3' 3xFLAG-tagged human HADHA cDNA under the transcriptional control of the cytomegalovirus (CMV) enhancer-chicken beta actin (CAG) promoter (CAG-HADHA-3XFLAG). LCHADD-RPE were cultured, matured, and transduced with either AAV-GFP (control) or AAV-HADHA-3XFLAG., Results: LCHADD-RPE express TFP subunits and accumulate 3-hydroxy-acylcarnitines, cannot oxidize palmitate, and release fewer ketones than WT-RPE. When LCHADD-RPE are exposed to docosahexaenoic acid (DHA), they have increased oxidative stress, lipid peroxidation, decreased viability, and are rescued by antioxidant agents potentially explaining the pathologic mechanism of RPE loss in LCHADD. Transduced LCHADD-RPE expressing a WT copy of TFPα incorporated TFPα-FLAG into the TFP complex in the mitochondria and accumulated significantly less 3-hydroxy-acylcarnitines, released more ketones in response to palmitate, and were more resistant to oxidative stress following DHA exposure than control., Conclusions: iPSC-derived LCHADD-RPE are susceptible to lipid peroxidation mediated cell death and are rescued by exogenous HADHA delivered with rAAV. These results are promising for AAV-HADHA gene addition therapy as a possible treatment for chorioretinopathy in patients with LCHADD.

A new approach to attenuating pathological inflammatory reactions by buffering the eicosanoid pathways with oxidation-resistant hexadeuterated arachidonic acid (D-ARA) is discussed. Enzymatic processing of ARA, released by phospholipase A2, by lipoxygenases, cyclooxygenases, and cytochromes yields a wide range of bioactive eicosanoids, including pro-inflammation, pro-angiogenesis and pro-thrombosis species that, when produced in excess, are an underlying cause of pathology. Conversely, some products of ARA oxidation possess pro-resolving properties. Non-enzymatic free radical oxidation of ARA generates another large group of products such as isoprostanes and their metabolites, associated with inflammation, ischemia-reperfusion stress, and atherosclerosis. A separate group comprises reactive carbonyl derivatives that irreversibly damage diverse biomolecules. Being resistant to both enzymatic and non-enzymatic oxidation pathways due to large kinetic isotope effects, D-ARA may play a role in mitigating inflammation-related disorders and conditions, including inflammaging.

Ferroptosis, an iron-dependent form of cell death driven by lipid peroxidation, provides a potential treatment avenue for drug-resistant cancers and may play a role in the pathology of some degenerative diseases. Identifying the subcellular membranes essential for ferroptosis and the sequence of their peroxidation will illuminate drug discovery strategies and ferroptosis-relevant disease mechanisms. In this study, we employed fluorescence and stimulated Raman scattering imaging to examine the structure-activity-distribution relationship of ferroptosis-modulating compounds. We found that, although lipid peroxidation in various subcellular membranes can induce ferroptosis, the endoplasmic reticulum (ER) membrane is a key site of lipid peroxidation. Our results suggest an ordered progression model of membrane peroxidation during ferroptosis that accumulates initially in the ER membrane and later in the plasma membrane. Thus, the design of ER-targeted inhibitors and inducers of ferroptosis may be used to optimally control the dynamics of lipid peroxidation in cells undergoing ferroptosis., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Ru-based catalysis results in highly unsaturated fatty acid (HUFA) ethyl esters (EE) deuterated to various extents. The products carry 2 H (D) mainly at their bis -allylic positions, where they are resistant to autoxidation compared to natural HUFA and are promising as neurological and retinal drugs. We characterized the extent of deuteration at each allylic position of docosa-4,7,10,13,16,19-hexaenoic acid deuterated to completion at bis -allylic and allylic positions (D-DHA) by two-dimensional (2D) and high-field (600 and 950 MHz) NMR. In separate experiments, the kinetics of docosahexaenoic acid (DHA) EE deuteration was evaluated using Paternò-Büchi (PB) reaction tandem mass spectrometry (MS/MS) analysis, enabling deuteration to be quantitatively characterized for isotopologues (D0-D14 DHA) at each internal allylic position. NMR analysis shows that the net deuteration of the isotopologue mixture is about 94% at the bis -allylic positions, and less than 1% remained as the protiated -CH 2 -. MS analysis shows that deuteration kinetics follow an increasing curve at bis -allylic positions with higher rate for internal bis -allylic positions. Percent D of bis -allylic positions increases linearly from D1 to D9 in which all internal bis -allylic positions (C9, C12, C15) deuterate uniformly and more rapidly than external bis -allylic positions (C6, C18). The mono -allylic positions near the methyl end (C21) show a steep increase of D only after the D10 isotopologue has been deuterated to >90%, while the mono -allylic position near the carboxyl position, C3, deuterates last and least. These data establish detailed methods for the characterization of Ru-catalyzed deuteration of HUFA as well as the phenomenological reaction kinetics as net product is formed.

Docosahexaenoic acid (DHA; 22:6n-3) rich photoreceptors function in a highly oxidizing microenvironment. Lipid peroxidation and inflammation contribute to initiation and progression of eye diseases including age-related macular degeneration (AMD). Deuteration of DHA at the bis-allylic positions (D-DHA) increases its resilience to oxidative damage in vitro. We studied the pharmacokinetics of dietary D-DHA as a therapy for replacing natural retinal DHA in vivo. Mice were fed 0.5% D-DHA for 77 days then switched to natural DHA (H-DHA) for 74 days. Tissue were harvested for analyses at various time points. D-DHA substitution levels were 75%-80% in the CNS and above 90% in all other tissues by day 77. D-DHA accretion was rapid in plasma and liver (t 1/2a ∼2.8 d), followed by heart and red blood cells (t 1/2a ∼8.5 d), then ocular tissues (choroid-RPE, neural retina, and optic nerve with t 1/2a of 10.1, 23.4, and 26.3 days, respectively), while CNS accretion was slowest (t 1/2a of 29.0-44.3 days). D-DHA elimination rates were comparable to, or slower than, accretion rates except for optic nerve. Retina had very long chain D-PUFA (D-VLC-PUFA) with 5 and 6 double bonds up to C36, as well as D-EPA and D-DPA derived metabolically from D-DHA. The neural retina and optic nerve reached the therapeutic target window (20%-50%) in 2-4 weeks. Biosynthesis of D-VLC-PUFA is consistent with normal metabolism. D-DHA crosses the blood-retina-barrier, enters visually active tissues, and is metabolized as its natural DHA parent where, as shown previously (Liu et al., 2022), it protects against lipid peroxidation., Competing Interests: Declaration of competing interest MSS and KS are employed by, and shareholders in, Retrotope. JTB is a shareholder in, and receives research support from, Retrotope. MPA receives research support from Retrotope. All other authors have no conflicts., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Polyunsaturated fatty acyl chains (PUFAs) concentrate in the brain and give rise to numerous oxidative chemical degradation products. It is widely assumed that these products are the result of free radical chain reactions, and reactions of this type have been demonstrated in preparations where a single PUFA substrate species predominates. However, it is unclear whether such reactions can occur in the biologically complex milieu of lipid membranes where PUFA substrates are a minority species, and where diverse free radical scavengers or other quenching mechanisms are present. It is of particular interest to know whether they occur in brain, where PUFAs are concentrated and where PUFA oxidation products have been implicated in the pathogenesis of neurodegenerative disorders. To ascertain whether free radical chain reactions can occur in a complex brain lipid mixture, mouse brain lipids were extracted, formed into vesicles, and treated with a fixed number of hydroxyl radicals under conditions wherein the concentrations and types of PUFA-containing phospholipids were varied. Specific phospholipid species in the mixture were assayed by tandem mass spectrometry to quantify the oxidative losses of endogenous PUFA-containing phospholipids. Results reveal crosstalk between the oxidative degradation of ω3 and ω6 PUFAs that can only be explained by the occurrence of free radical chain reactions. These results demonstrate that PUFAs in a complex brain lipid mixture can participate in free radical chain reactions wherein the extent of oxidative degradation is not limited by the number of reactive oxygen species available to initiate such reactions. These reactions may help explain otherwise puzzling in vivo interactions between ω3 and ω6 PUFAs in mouse brain., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Background: Long-chain polyunsaturated fatty acids (PUFAs) are important modulators of red blood cell (RBC) rheology. Dietary PUFAs are readily incorporated into the RBC membrane, improving RBC deformability, fluidity, and hydration. However, enriching the lipid membrane with PUFAs increases the potential for peroxidation in oxidative environments (e.g., refrigerated storage), resulting in membrane damage. Substitution of bis-allylic hydrogens with deuterium ions in PUFAs decreases hydrogen abstraction, thereby inhibiting peroxidation. If lipid peroxidation is a causal factor in the RBC storage lesion, incorporation of deuterated linoleic acid (DLA) into the RBC membrane should decrease lipid peroxidation, thereby improving RBC lifespan, deformability, filterability, and post-transfusion recovery (PTR) after cold storage. Study Design and Methods: Mice associated with good (C57BL/6J) and poor (FVB) RBC storage quality received diets containing 11,11-D2-LA Ethyl Ester (1.0 g/100 g diet; deuterated linoleic acid) or non-deuterated LA Ethyl Ester (control) for 8 weeks. Deformability, filterability, lipidomics, and lipid peroxidation markers were evaluated in fresh and stored RBCs. Results: DLA was incorporated into RBC membranes in both mouse strains. DLA diet decreased lipid peroxidation (malondialdehyde) by 25.4 and 31% percent in C57 mice and 12.9 and 79.9% in FVB mice before and after cold storage, respectively. In FVB, but not C57 mice, deformability filterability, and post-transfusion recovery were significantly improved. Discussion: In a mouse model of poor RBC storage, with elevated reactive oxygen species production, DLA attenuated lipid peroxidation and significantly improved RBC storage quality., Competing Interests: MS was employed by Retrotope Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kim, Johnson, Peltier, Spitalnik, Hod, Francis, Hudson, Stone, Gordy, Fu, Zimring, Amireault, Buehler, Wilson, D’Alessandro, Shchepinov and Thomas.)

Oxidative stress plays a central role in age-related macular degeneration (AMD). Iron, a potent generator of hydroxyl radicals through the Fenton reaction, has been implicated in AMD. One easily oxidized molecule is docosahexaenoic acid (DHA), the most abundant polyunsaturated fatty acid in photoreceptor membranes. Oxidation of DHA produces toxic oxidation products including carboxyethylpyrrole (CEP) adducts, which are increased in the retinas of AMD patients. In this study, we hypothesized that deuterium substitution on the bis-allylic sites of DHA in photoreceptor membranes could prevent iron-induced retinal degeneration by inhibiting oxidative stress and lipid peroxidation. Mice were fed with either DHA deuterated at the oxidation-prone positions (D-DHA) or control natural DHA and then given an intravitreal injection of iron or control saline. Orally administered D-DHA caused a dose-dependent increase in D-DHA levels in the neural retina and retinal pigment epithelium (RPE) as measured by mass spectrometry. At 1 week after iron injection, D-DHA provided nearly complete protection against iron-induced retinal autofluorescence and retinal degeneration, as determined by in vivo imaging, electroretinography, and histology. Iron injection resulted in carboxyethylpyrrole conjugate immunoreactivity in photoreceptors and RPE in mice fed with natural DHA but not D-DHA. Quantitative PCR results were consistent with iron-induced oxidative stress, inflammation, and retinal cell death in mice fed with natural DHA but not D-DHA. Taken together, our findings suggest that DHA oxidation is central to the pathogenesis of iron-induced retinal degeneration. They also provide preclinical evidence that dosing with D-DHA could be a viable therapeutic strategy for retinal diseases involving oxidative stress., (© 2022 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Lipid peroxidation (LPO) plays a key role in many age-related neurodegenerative conditions and other disorders. Light irradiation can initiate LPO through various mechanisms and is of importance in retinal and dermatological pathologies. The introduction of deuterated polyunsaturated fatty acids (D-PUFA) into membrane lipids is a promising approach for protection against LPO. Here, we report the protective effects of D-PUFA against the photodynamically induced LPO, using illumination in the presence of the photosensitizer trisulfonated aluminum phthalocyanine (AlPcS 3 ) in liposomes and giant unilamellar vesicles (GUV), as assessed in four experimental models: 1) sulforhodamine B leakage from liposomes, detected with fluorescence correlation spectroscopy (FCS); 2) formation of diene conjugates in liposomal membranes, measured by absorbance at 234 nm; 3) membrane leakage in GUV assessed by optical phase-contrast intensity observations; 4) UPLC-MS/MS method to detect oxidized linoleic acid (Lin)-derived metabolites. Specifically, in liposomes or GUV containing H-PUFA (dilinoleyl-sn-glycero-3-phosphatidylcholine), light irradiation led to an extensive oxidative damage to bilayers. By contrast, no damage was observed in lipid bilayers containing 20% or more D-PUFA (D2-Lin or D10-docosahexanenoic acid). Remarkably, addition of tocopherol increased the dye leakage from liposomes in H-PUFA bilayers compared to photoirradiation alone, signifying tocopherol's pro-oxidant properties. However, in the presence of D-PUFA the opposite effect was observed, whereby adding tocopherol increased the resistance to LPO. These findings suggest a method to augment the protective effects of D-PUFA, which are currently undergoing clinical trials in several neurological and retinal diseases that involve LPO., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Arachidonic acid (ARA) is a major component of lipid bilayers as well as the key substrate for the eicosanoid cascades. ARA is readily oxidized, and its non-enzymatic and enzymatic oxidation products induce inflammatory responses in nearly all tissues, including lung tissues. Deuteration at bis-allylic positions substantially decreases the overall rate of ARA oxidation when hydrogen abstraction is an initiating event. To compare the effects of dosing of arachidonic acid (H-ARA) and its bis-allylic hexadeuterated form (D-ARA) on lungs in conventionally healthy mice and in an acute lung injury model, mice were dosed with H-ARA or D-ARA for six weeks through dietary supplementation and then challenged with intranasal lipopolysaccharide (LPS) for subsequent analysis of bronchoalveolar lavage fluid and lung tissue. Dosing on D-ARA resulted in successful incorporation of D-ARA into various tissues. D-ARA significantly reduced LPS-induced adverse effects on alveolar septal thickness and the bronchoalveolar area. Oral deuterated ARA is taken up efficiently and protects against adverse LPS-induced pathology. This suggests novel therapeutic avenues for reducing lung damage during severe infections and other pathological conditions with inflammation in the pulmonary system and other inflammatory diseases.

Glutathione peroxidase 4 (GPX4), as the only enzyme in mammals capable of reducing esterified phospholipid hydroperoxides within a cellular context, protects cells from ferroptosis. We identified a homozygous point mutation in the GPX4 gene, resulting in an R152H coding mutation, in three patients with Sedaghatian-type spondylometaphyseal dysplasia. Using structure-based analyses and cell models, including patient fibroblasts, of this variant, we found that the missense variant destabilized a critical loop, which disrupted the active site and caused a substantial loss of enzymatic function. We also found that the R152H variant of GPX4 is less susceptible to degradation, revealing the degradation mechanism of the GPX4 protein. Proof-of-concept therapeutic treatments, which overcome the impaired R152H GPX4 activity, including selenium supplementation, selective antioxidants and a deuterated polyunsaturated fatty acid were identified. In addition to revealing a general approach to investigating rare genetic diseases, we demonstrate the biochemical foundations of therapeutic strategies targeting GPX4., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: Progressive supranuclear palsy (PSP) is a progressive movement disorder associated with lipid peroxidation and intracerebral accumulation of tau. RT001 is a deuterium reinforced isotopologue of linoleic acid that prevents lipid peroxidation (LPO) through the kinetic isotope effect., Methods: The effects of RT001 pre-treatment on various oxidative and bioenergetic parameters were evaluated in mesenchymal stem cells (MSC) derived from patients with PSP compared to controls. In parallel, 3 patients with PSP were treated with RT001 and followed clinically., Results: MSCs derived from PSP patients had a significantly higher rate of LPO (161.8 ± 8.2% of control; p < 0.001). A 72-h incubation with RT001 restored the PSP MSCs to normal levels. Mitochondrial reactive oxygen species (ROS) overproduction in PSP-MSCs significantly decreased the level of GSH compared to control MSCs (to 56% and 47% of control; p < 0.05). Incubation with RT001 significantly increased level of GSH in PSP MSCs. The level of mitochondrial DNA in the cells was significantly lower in PSP-MSCs (67.5%), compared to control MSCs. Changes in mitochondrial membrane potential, size, and shape were also observed. Three subjects with possible or probable PSP were treated with RT001 for a mean duration of 26 months. The slope of the PSPRS changed from the historical decline of 0.91 points/month to a mean decline of 0.16 points/month (+/-0.23 SEM). The UPDRS slope changed from an expected increase of 0.95 points/month to an average increase in score of 0.28 points/month (+/-0.41 SEM)., Conclusions: MSCs derived from patients with PSP have elevated basal levels of LPO, ROS, and mitochondrial dysfunction. These findings are reversed after incubation with RT001. In PSP patients, the progression of disease may be reduced by treatment with RT001.

No general method currently is available for the quantitative determination of deuterium (D) at C positions along a hydrocarbon chain. Bis -allylic deuterated highly unsaturated fatty acids (D-HUFA) are a novel class of drugs stabilized against H-abstraction-mediated oxidation by deuteration at the most labile positions. Ru-based catalytic deuteration overcomes the limited scale of bis -allylic D-HUFA production by total organic synthesis; however, it produces a complex mixture of bis -allylic D isotopologues and isotopomers, requiring detailed sequencing for characterization. We report here adaptation and application of the Paternó-Büchi (PB) reaction of 2-acetylpyridine to a series of D-HUFA with analysis by shotgun lipidomics to determine position-specific quantitative D abundances. Sodiated PB D-HUFA result in diagnostic ions of high abundance upon collision-induced dissociation (CID) activation, enabling sensitive differentiation and quantification of D fraction at each bis - and mono -allylic position for each isotopologue. Catalytically deuterated isotopologues D5-7 linolenic acid (D5-7 LnA), D6-8 arachidonic acid (D6-8 ARA), D7-9 eicosapentaenoic acid (D7-9 EPA), and D9-11 docosahexaenoic acid (D9-11 DHA) incorporate 80-98, 95-100, 81-100, and 83-100% D at their bis -allylic positions, respectively. D-HUFA isotopologues having D number greater than or equal to bis -allylic sites (e.g., D10-DHA or D11-DHA) deuterated >95% at bis -allylic positions, except for D-LnA. The mono- allylic position near the methyl end deuterates to a much greater extent than the mono- allylic position near the carboxyl end, and both positions deuterate only when bis -allylic D is near-saturated. This method enables rapid, accurate characterization of position and isotopomer-specific D composition and enables sequencing along the chain.

Lipid peroxidation is a key to a portfolio of neurodegenerative diseases and plays a central role in α-synuclein (α-syn) toxicity, mitochondrial dysfunction and neuronal death, all key processes in the pathogenesis of Parkinson's disease (PD). Polyunsaturated fatty acids (PUFAs) are important constituents of the synaptic and mitochondrial membranes and are often the first molecular targets attacked by reactive oxygen species (ROS). The rate-limiting step of the chain reaction of ROS-initiated PUFAs autoxidation involves hydrogen abstraction at bis-allylic sites, which can be slowed down if hydrogens are replaced with deuteriums. In this study, we show that targeted overexpression of human A53T α-syn using an AAV vector unilaterally in the rat substantia nigra reproduces some of pathological features seen in PD patients. Chronic dietary supplementation with deuterated PUFAs (D-PUFAs), specifically 0.8% D-linoleic and 0.3% H-linolenic, produced significant disease-modifying beneficial effects against α-syn-induced motor deficits, synaptic pathology, oxidative damage, mitochondrial dysfunction, disrupted trafficking along axons, inflammation and DA neuronal loss. These findings support the clinical evaluation of D-PUFAs as a neuroprotective therapy for PD.

RT001 is the di-deutero isotopologue of linoleic acid ethyl ester (D2-LA). Resistance to oxidative damage at the carbon-deuterium bond depends upon the concentration of D2-LA as a percentage of total LA. We report here on the plasma and red cell (RBC) pharmacokinetics (PK) of D2-LA, and its metabolite 13,13-D2-arachidonic acid (D2-AA), in patients with multiple neurodegenerative diseases (total of 59 participants). In Friedreich's ataxia patients, D2-LA was absorbed and transported similarly to dietary LA, peaking at about 6 h after oral dosing. Plasma D2-LA concentrations approached steady state after 28 days of dosing. After 6 months of daily dosing in subjects with other disorders, D2-LA and D2-AA levels were at or above the 20% of total (D2-LA/total LA, or D2-AA/total AA) therapeutic targets for most subjects. We conclude that chronic dosing of RT001 and associated dietary guidance can be maintained over many months to achieve target plasma and RBC levels, forming a basis for therapeutic dosing across a broad range of conditions. RT001 has been safe and well-tolerated in 59 different participants treated across 10 different neurodegenerative diseases in multiple clinical trials for up to 36 months with no significant drug related adverse events limiting use., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Protein aggregation and abnormal lipid homeostasis are both implicated in neurodegeneration through unknown mechanisms. Here we demonstrate that aggregate-membrane interaction is critical to induce a form of cell death called ferroptosis. Importantly, the aggregate-membrane interaction that drives ferroptosis depends both on the conformational structure of the aggregate, as well as the oxidation state of the lipid membrane. We generated human stem cell-derived models of synucleinopathy, characterized by the intracellular formation of α-synuclein aggregates that bind to membranes. In human iPSC-derived neurons with SNCA triplication, physiological concentrations of glutamate and dopamine induce abnormal calcium signaling owing to the incorporation of excess α-synuclein oligomers into membranes, leading to altered membrane conductance and abnormal calcium influx. α-synuclein oligomers further induce lipid peroxidation. Targeted inhibition of lipid peroxidation prevents the aggregate-membrane interaction, abolishes aberrant calcium fluxes, and restores physiological calcium signaling. Inhibition of lipid peroxidation, and reduction of iron-dependent accumulation of free radicals, further prevents oligomer-induced toxicity in human neurons. In summary, we report that peroxidation of polyunsaturated fatty acids underlies the incorporation of β-sheet-rich aggregates into the membranes, and that additionally induces neuronal death. This suggests a role for ferroptosis in Parkinson's disease, and highlights a new mechanism by which lipid peroxidation causes cell death.

Oxidative stress is a common feature of genetic and idiopathic neurological diseases that thus far have been intractable to drug therapy. Polyunsaturated fatty acids (PUFAs) form cellular, mitochondrial, retinal, and other membranes highly important in neuronal function. However, PUFAs are susceptible to the noxious lipid peroxidation (LPO) chain reaction, which is a common feature of various neurological and age-related pathologies, making this pathway an attractive target for therapeutic intervention. Regioselective deuteration that reinforces oxidation-prone, bis-allylic sites of PUFAs is a novel, nonantioxidant treatment modality that dramatically reduces LPO, potentially mitigating numerous diseases through preservation of membrane properties and amelioration of oxidative stress. Animal disease models and several ongoing human clinical trials highlight the potential of the deuterated-PUFA (D-PUFA) drug candidates currently in development., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Infantile neuroaxonal dystrophy (INAD) is a rare, autosomal recessive disease due to defects in PLA2G6 and is associated with lipid peroxidation. RT001 is a di-deuterated form of linoleic acid that protects lipids from oxidative damage., Methods: We evaluated the pharmacokinetics (PK), safety, and effectiveness of RT001 in two subjects with INAD (subject 1: 34 months; subject 2: 10 months). After screening and baseline evaluations, subjects received 1.8 g of RT001 BD. PK analysis and clinical evaluations were made periodically., Main Findings: Plasma levels of deuterated linoleic acid (D2-LA), deuterated arachidonic acid (D2-AA), D2-LA to total LA, and D2-AA to total AA ratios were measured. The targeted plasma D2-LA ratio (>20%) was achieved by month 1 and maintained throughout the study. RBC AA-ratios were 0.11 and 0.18 at 6 months for subjects 1 and 2; respectively. No treatment-related adverse events occurred. Limited slowing of disease progression and some return of lost developmental milestones were seen., Conclusions: Oral RT001 was administered safely in two subjects with INAD. Early findings suggest that the compound was well tolerated, metabolized and incorporated in the RBC membrane. A clinical trial is underway to assess efficacy., Competing Interests: D. A., J. D., and C. F. declare no conflict of interest. P. M., F. H., M. M., P. A., and M. S., are employed by and holds stock in Retrotope. R. M. is a founder and holds stock in Retrotope, Inc. Sarah Endemann is employed by Retrotope. All authors confirm that the content of the article has not been influenced by the sponsor., (© 2020 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Oxidative stress plays a crucial role in developing and accelerating retinal diseases including age-related macular degeneration (AMD). Docosahexaenoic acid (DHA, C22:6, n-3), the main lipid constituent of retinal epithelial cell membranes, is highly prone to radical and enzymatic oxidation leading to deleterious or beneficial metabolites for retinal tissue. To inhibit radical oxidation while preserving enzymatic metabolism, deuterium was incorporated at specific positions of DHA, resulting in D 2 -DHA when incorporated at position 6 and D 4 -DHA when incorporated at the 6,9 bis -allylic positions. Both derivatives were able to decrease DHAs' toxicity and free radical processes involved in lipid peroxidation, in ARPE-19 cells (Adult Retinal Pigment Epithelial cell line), under pro-oxidant conditions. Our positive results encouraged us to prepare lipophenolic-deuterated-DHA conjugates as possible drug candidates for AMD treatment. These novel derivatives proved efficient in limiting lipid peroxidation in ARPE-19 cells. Finally, we evaluated the underlying mechanisms and the enzymatic conversion of both deuterated DHA. While radical abstraction was affected at the deuterium incorporation sites, enzymatic conversion by the lipoxygenase 15s-LOX was not impacted. Our results suggest that site-specifically deuterated DHA could be used in the development of DHA conjugates for treatment of oxidative stress driven diseases, or as biological tools to study the roles, activities and mechanisms of DHA metabolites., Competing Interests: The authors declare no conflict of interest.

Autoxidation of polyunsaturated fatty acids (PUFAs) damages lipid membranes and generates numerous toxic by-products implicated in neurodegeneration, aging, and other pathologies. Abstraction of bis-allylic hydrogen atoms is the rate-limiting step of PUFA autoxidation, which is inhibited by replacing bis-allylic hydrogens with deuterium atoms (D-PUFAs). In cells, the presence of a relatively small fraction of D-PUFAs among natural PUFAs is sufficient to effectively inhibit lipid peroxidation (LPO). Here, we investigate the effect of various D-PUFAs on the stability of liposomes under oxidative stress conditions. The permeability of vesicle membranes to fluorescent dyes was measured as a proxy for bilayer integrity, and the formation of conjugated dienes was monitored as a proxy for LPO. Remarkably, both approaches reveal a similar threshold for the protective effect of D-PUFAs in liposomes. We show that protection rendered by D-PUFAs depends on the structure of the deuterated fatty acid. Our findings suggest that protection of PUFAs against autoxidation depends on the total level of deuterated bi-sallylic (CD 2 ) groups present in the lipid bilayer. However, the phospholipid containing 6,6,9,9,12,12,15,15,18,18-d 10 -docosahexaenoic acid exerts a stronger protective effect than should be expected from its deuteration level. These findings further support the application of D-PUFAs as preventive/therapeutic agents in numerous pathologies that involve LPO., (© 2019 Federation of European Biochemical Societies.)

Chemically reinforced essential fatty acids (FAs) promise to fight numerous age-related diseases including Alzheimer's, Friedreich's ataxia and other neurological conditions. The reinforcement is achieved by substituting the atoms of hydrogen at the bis-allylic methylene of these essential FAs with the isotope deuterium. This substitution leads to a significantly slower oxidation due to the kinetic isotope effect, inhibiting membrane damage. The approach has the advantage of preventing the harmful accumulation of reactive oxygen species (ROS) by inhibiting the propagation of lipid peroxidation while antioxidants potentially neutralize beneficial oxidative species. Here, we developed a model system to mimic the human dietary requirement of omega-3 in Caenorhabditis elegans to study the role of deuterated polyunsaturated fatty acids (D-PUFAs). Deuterated trilinolenin [D-TG(54:9)] was sufficient to prevent the accumulation of lipid peroxides and to reduce the accumulation or ROS. Moreover, D-TG(54:9) significantly extended the lifespan of worms under normal and oxidative stress conditions. These findings demonstrate that D-PUFAs can be used as a food supplement to decelerate the aging process, resulting in extended lifespan.

The synthesis of signal lipids, including eicosanoids, is not fully understood, although it is key to the modulation of various inflammatory states. Recently, isotopologues of essential polyunsaturated fatty acids (PUFAs) deuterated at bis-allylic positions (D-PUFAs) have been proposed as inhibitors of non-enzymatic lipid peroxidation (LPO) in various disease models. Arachidonic acid (AA, 20:4 n-6) is the main precursor to several classes of eicosanoids, which are produced by cyclooxygenases (COX) and lipoxygenases (LOX). In this study we analyzed the relative activity of human recombinant enzymes COX-2, 5-LOX, and 15-LOX-2 using a library of arachidonic acids variably deuterated at the bis-allylic (C7, C10, and C13) positions. Kinetic parameters (KM, V max ) and isotope effects calculated from kH/kD for seven deuterated arachidonic acid derivatives were obtained. Spectroscopic methods have shown that deuteration at the 13th position dramatically affects the kinetic parameters of COX-2 and 15-LOX-2. The activity of 5-LOX was evaluated by measuring hydroxyeicosatetraenoic acids (8-HETE and 5-HETE) using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Deuteration at the seventh and 10th positions affects the performance of the 5-LOX enzyme. A flowchart is proposed suggesting how to modulate the synthesis of selected eicosanoids using the library of deuterated isotopologues to potentially fine-tune various inflammation stages.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease which has no effective treatment and is characterized by psychiatric disorders, motor alterations, and dementia, with the cognitive deficits representing a devastating aspect of the disorder. Oxidative stress and elevated levels of lipid peroxidation (LPO) products are found in mouse models and patients with HD, suggesting that strategies to reduce LPO may be beneficial in HD. In contrast with traditional antioxidants, substituting hydrogen with deuterium at bis-allylic sites in polyunsaturated fatty acids (D-PUFA) decreases the rate-limiting initiation step of PUFA autoxidation, a strategy that has shown benefits in other neurodegenerative diseases. Here, we investigated the effect of D-PUFA treatment in a knock-in mouse model of HD (Q140) which presents motor deficits and neuropathology from a few months of age, and progressive cognitive decline. Q140 knock-in mice were fed a diet containing either D- or H-PUFAs for 5 months starting at 1 month of age. D-PUFA treatment significantly decreased F 2 -isoprostanes in the striatum by approximately 80% as compared to H-PUFA treatment and improved performance in novel object recognition tests, without significantly changing motor deficits or huntingtin aggregation. Therefore, D-PUFA administration represents a promising new strategy to broadly reduce rates of LPO, and may be useful in improving a subset of the core deficits in HD., (© 2018 Federation of European Biochemical Societies.)

Background: RT001 is a deuterated ethyl linoleate that inhibits lipid peroxidation and is hypothesized to reduce cellular damage and recover mitochondrial function in degenerative diseases such as Friedreich's ataxia., Objective: To evaluate the safety, pharmacokinetics, and preliminary efficacy of RT001 in Friedreich's ataxia patients., Design/methods: We conducted a phase I/II double-blind, comparator-controlled trial with 2 doses of RT001 in Friedreich's ataxia patients (9 subjects each cohort). Subjects were randomized 2:1 to receive either RT001 (1.8 or 9.0 g/day), or a matching dose of nondeuterated ethyl linoleate as comparator for 28 days. The primary endpoints were safety, tolerability, and pharmacokinetic analysis. Secondary endpoints included cardiopulmonary exercise testing and timed 25-foot walk., Results: Nineteen patients enrolled in the trial, and 18 completed all safety and efficacy measurements. RT001 was found to be safe and tolerable, with plasma levels approaching saturation by 28 days. One subject with a low body mass index experienced steatorrhea taking a high dose and discontinued the study. Deuterated arachidonic acid (a brain-penetrant metabolite of RT001) was found to be present in plasma on day 28. There was an improvement in peak workload in the drug group compared to placebo (0.16 watts/kg; P = 0.008), as well as an improvement trend in peak oxygen consumption (change of 0.16 L/min; P = 0.116), and in stride speed (P = 0.15)., Conclusions: RT001 was found to be safe and tolerable over 28 days, and improved peak workload. Further research into the effect of RT001 in Friedreich's ataxia is warranted. © 2018 International Parkinson and Movement Disorder Society., (© 2018 International Parkinson and Movement Disorder Society.)

Alzheimer's disease (AD) involves progressive deposition of amyloid β-peptide (Aβ), synapse loss, and neuronal death, which occur in brain regions critical for learning and memory. Considerable evidence suggests that lipid peroxidation contributes to synaptic dysfunction and neuronal degeneration, both upstream and downstream of Aβ pathology. Recent findings suggest that lipid peroxidation can be inhibited by replacement of polyunsaturated fatty acids (PUFA) with isotope-reinforced (deuterated) PUFA (D-PUFA), and that D-PUFA can protect neurons in experimental models of Parkinson's disease. Here, we determined whether dietary D-PUFA would ameliorate Aβ pathology and/or cognitive deficits in a mouse model of AD (amyloid precursor protein/presenilin 1 double mutant transgenic mice). The D-PUFA diet did not ameliorate spatial learning and memory deficits in the AD mice. Compared to mice fed an hydrogenated-PUFA control diet, those fed D-PUFA for 5 months exhibited high levels of incorporation of deuterium into arachidonic acid and docosahexaenoic acid, and reduced concentrations of lipid peroxidation products (F2 isoprostanes and neuroprostanes), in the brain tissues. Concentrations of Aβ40 and Aβ38 in the hippocampus were significantly lower, with a trend to reduced concentrations of Aβ42, in mice fed D-PUFA compared to those fed hydrogenated-PUFA. We conclude that a D-PUFA diet reduces the brain tissue concentrations of both arachidonic acid and docosahexaenoic acid oxidation products, as well as the concentration of Aβs., (Published by Elsevier Inc.)

Ferroptosis is a non-apoptotic form of regulated cell death caused by the failure of the glutathione-dependent lipid-peroxide-scavenging network. FINO 2 is an endoperoxide-containing 1,2-dioxolane that can initiate ferroptosis selectively in engineered cancer cells. We investigated the mechanism and structural features necessary for ferroptosis initiation by FINO 2 . We found that FINO 2 requires both an endoperoxide moiety and a nearby hydroxyl head group to initiate ferroptosis. In contrast to previously described ferroptosis inducers, FINO 2 does not inhibit system x c - or directly target the reducing enzyme GPX4, as do erastin and RSL3, respectively, nor does it deplete GPX4 protein, as does FIN56. Instead, FINO 2 both indirectly inhibits GPX4 enzymatic function and directly oxidizes iron, ultimately causing widespread lipid peroxidation. These findings suggest that endoperoxides such as FINO 2 can initiate a multipronged mechanism of ferroptosis.

Lipoxygenases (LOXs) have been implicated as central players in ferroptosis, a recently characterized cell death modality associated with the accumulation of lipid hydroperoxides: the products of LOX catalysis. To provide insight on their role, human embryonic kidney cells were transfected to overexpress each of the human isoforms associated with disease, 5-LOX, p12-LOX, and 15-LOX-1, which yielded stable cell lines that were demonstrably sensitized to ferroptosis. Interestingly, the cells could be rescued by less than half of a diverse collection of known LOX inhibitors. Furthermore, the cytoprotective compounds were similarly potent in each of the cell lines even though some were clearly isoform-selective LOX inhibitors. The cytoprotective compounds were subsequently demonstrated to be effective radical-trapping antioxidants, which protect lipids from autoxidation, the autocatalytic radical chain reaction that produces lipid hydroperoxides. From these data (and others reported herein), a picture emerges wherein LOX activity may contribute to the cellular pool of lipid hydroperoxides that initiate ferroptosis, but lipid autoxidation drives the cell death process., Competing Interests: The authors declare the following competing financial interest(s): M.S.S. owns shares in Retrotope, Inc.

Arachidonic acid (AA, 20:4) is an omega-6 polyunsaturated fatty acid (PUFA) and the main precursor to the class of lipid mediators known as eicosanoids. The enzymes that catalyze the oxygenation of AA begin by abstracting hydrogen from one of three bis-allylic carbons within 1,4-cis,cis-diene units. Substitution of deuterium for hydrogen has been shown to lead to massive kinetic isotope effects (KIE) for soybean lipoxygenase (sLOX) oxygenation of linoleic acid (LA, 18:2). Yet, experimental determination of the KIE during oxygenation of AA and LA by mammalian enzymes including cyclooxygenase (COX) and lipoxygenase (LOX) has revealed far lower values. All prior studies investigating the KIE of PUFA oxygenation have relied on in vitro systems using purified enzymes and were limited by availability of deuterated substrates. Here we demonstrate the use of macrophages as an ex vivo model system to study the physiological KIE (PKIE) during enzymatic AA oxygenation by living cells using a newly synthesized library of deuterated AA isotopologues. By extending lipidomic UPLC-MS/MS approaches to simultaneously quantify native and deuterated lipid products, we were able to demonstrate that the magnitude of the PKIE measured in macrophages for COX and LOX oxygenation of AA is similar to KIEs determined in previous reports using the AA isotopologue deuterated at carbon 13 (C13). However, for the first time we show that increasing the number of deuterated bis-allylic carbons to include both C10 and C13 leads to a massive increase in the PKIE for COX oxygenation of AA. We provide evidence that hydrogen(s) present at C10 of AA play a critical role in the catalysis of prostaglandin and thromboxane synthesis. Furthermore, we discovered that deuteration of C10 promotes the formation of the resolving lipid mediator lipoxin B4, likely by interfering with AA cyclization and shunting AA to the LOX pathway under physiological conditions.

Selective deuteration of drugs and biologically relevant molecules is becoming increasingly important in the pharmaceutical industry. Site-selective isotopic reinforcement of polyunsaturated fatty acids (PUFAs) at their bis-allylic sites has been identified as a unique approach in preventing oxidative damage in these molecules, which had been linked to neuronal and retinal diseases, atherosclerosis, and aging. Typical methods for preparation of site-selectively deuterated PUFAs require rather long, laborious, and expensive syntheses. In this report, we disclose a very efficient catalytic protocol for site-specific deuteration of PUFAs and analogous poly-alkenes under exceptional kinetic control. Deuterium oxide (D 2 O) has been identified not only as a deuterium source but also as a crucial component in the overall reaction mechanism responsible for averting the formation of thermodynamically favored side-products.

Oxidative damage resulting from increased lipid peroxidation (LPO) is considered an important factor in the development of late onset/age-related Alzheimer's disease (AD). Deuterium-reinforced polyunsaturated fatty acids (D-PUFAs) are more resistant to the reactive oxygen species-initiated chain reaction of LPO than regular hydrogenated (H-) PUFAs. We investigated the effect of D-PUFA treatment on LPO and cognitive performance in aldehyde dehydrogenase 2 (Aldh2) null mice, an established model of oxidative stress-related cognitive impairment that exhibits AD-like pathologies. Mice were fed a Western-type diet containing either D- or H-PUFAs for 18 weeks. D-PUFA treatment markedly decreased cortex and hippocampus F 2 -isoprostanes by approximately 55% and prostaglandin F 2α by 20-25% as compared to H-PUFA treatment. D-PUFAs consistently improved performance in cognitive/memory tests, essentially resetting performance of the D-PUFA-fed Aldh2 -/- mice to that of wild-type mice fed a typical laboratory diet. D-PUFAs therefore represent a promising new strategy to broadly reduce rates of LPO, and combat cognitive decline in AD., (© 2017 Federation of European Biochemical Societies.)

Background and Aims: Oxidative modification of lipoproteins is a crucial step in atherosclerosis development. Isotopic-reinforced polyunsaturated fatty acids (D-PUFAs) are more resistant to reactive oxygen species-initiated chain reaction of lipid peroxidation than regular hydrogenated (H-)PUFAs. We aimed at investigating the effect of D-PUFA treatment on lipid peroxidation, hypercholesterolemia and atherosclerosis development., Methods: Transgenic APOE*3-Leiden.CETP mice, a well-established model for human-like lipoprotein metabolism, were pre-treated with D-PUFAs or control H-PUFAs-containing diet (1.2%, w/w) for 4 weeks. Thereafter, mice were fed a Western-type diet (containing 0.15% cholesterol, w/w) for another 12 weeks, while continuing the D-/H-PUFA treatment., Results: D-PUFA treatment markedly decreased hepatic and plasma F 2 -isoprostanes (approx. -80%) and prostaglandin F 2 α (approx. -40%) as compared to H-PUFA treatment. Moreover, D-PUFAs reduced body weight gain during the study (-54%) by decreasing body fat mass gain (-87%) without altering lean mass. D-PUFAs consistently reduced plasma total cholesterol levels (approx. -25%), as reflected in reduced plasma non-HDL-cholesterol (-28%). Additional analyses of hepatic cholesterol metabolism indicated that D-PUFAs reduced the hepatic cholesterol content (-21%). Sterol markers of intestinal cholesterol absorption and cholesterol breakdown were decreased. Markers of cholesterol synthesis were increased. Finally, D-PUFAs reduced atherosclerotic lesion area formation throughout the aortic root of the heart (-26%)., Conclusions: D-PUFAs reduce body weight gain, improve cholesterol handling and reduce atherosclerosis development by reducing lipid peroxidation and plasma cholesterol levels. D-PUFAs, therefore, represent a promising new strategy to broadly reduce rates of lipid peroxidation, and combat hypercholesterolemia and cardiovascular diseases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Effects exerted by heavy isotope substitution in biopolymers on the functioning of whole organisms have not been investigated. We report on the decrease of permissive temperature of nematodes fed with bacteria containing 5,5-D2-lysine. We synthesized 5,5-dideuterolysine and, taking advantage of lysine being an essential amino acid, showed that C. elegans with modified lysine poorly develop from larvae into fertile adult hermaphrodites. This effect occurs only at high temperature within the permissible range for C. elegans (25 °C) and completely vanishes at 15 °C. The only known metabolic involvement of C5 in lysine is in post-translational modification through lysyl hydoxylases. Indeed, siRNA experiments showed that deficiency of let-268/plod lysyl-hydroxylase/glycosydase further amplifies the isotope effect making it apparent even at 20 °C, whereas control siRNAs as well as another lysyl-hydroxylase (psr-1/jmjdD) siRNA do not. We report for the first time that a site-specific deuteration may strongly affect the development of the whole animal organism especially under the conditions of deficiency of the corresponding enzyme. These findings provide the basis for our ongoing efforts to employ isotope effects for fine tuning of metabolic pathways to mitigate pathological processes.

The human DNA methyltransferase 3A (DNMT 3A) is responsible for de novo epigenetic regulation, which is essential for mammalian viability and implicated in diverse diseases. All DNA cytosine C5 methyltransferases follow a broadly conserved catalytic mechanism. We investigated whether C5 β-elimination contributes to the rate-limiting step in catalysis by DNMT3A and the bacterial M.HhaI by using deuterium substitutions of C5 and C6 hydrogens. This substitution caused a 1.59-1.83 fold change in the rate of catalysis, thus suggesting that β-elimination is partly rate-limiting for both enzymes. We used a multisite substrate to explore the consequences of slowing β-elimination during multiple cycles of catalysis. Processive catalysis was slower for both enzymes, and deuterium substitution resulted in DNMT 3A dissociating from its substrate. The decrease in DNA methylation rate by DNMT 3A provides the basis of our ongoing efforts to alter cellular DNA methylation levels without the toxicity of currently used methods., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Racemization in proteins and peptides at sites of L-asparaginyl and L-aspartyl residues contributes to their spontaneous degradation, especially in the biological aging process. Amino acid racemization involves deprotonation of the alpha carbon and replacement of the proton in the opposite stereoconfiguration; this reaction is much faster for aspartate/asparagine than for other amino acids because these residues form a succinimide ring in which resonance stabilizes the carbanion resulting from proton loss. To determine if the replacement of the hydrogen atom on the alpha carbon with a deuterium atom might decrease the rate of racemization and thus stabilize polypeptides, we synthesized a hexapeptide, VYPNGA, in which the three carbon-bound protons in the asparaginyl residue were replaced with deuterium atoms. Upon incubation of this peptide in pH 7.4 buffer at 37 °C, we found that the rate of deamidation via the succinimide intermediate was unchanged by the presence of the deuterium atoms. However, the accumulation of the D-aspartyl and D-isoaspartyl-forms resulting from racemization and hydrolysis of the succinimide was decreased more than five-fold in the deuterated peptide over a 20 day incubation at physiological temperature and pH. Additionally, we found that the succinimide intermediate arising from the degradation of the deuterated asparaginyl peptide was slightly less likely to open to the isoaspartyl configuration than was the protonated succinimide. These findings suggest that the kinetic isotope effect resulting from the presence of deuteriums in asparagine residues can limit the accumulation of at least some of the degradation products that arise as peptides and proteins age., Competing Interests: M. S. Shchepinov declares a competing financial interest as the Chief Scientific Officer of Retrotope, Inc.

Ferroptosis is form of regulated nonapoptotic cell death that is involved in diverse disease contexts. Small molecules that inhibit glutathione peroxidase 4 (GPX4), a phospholipid peroxidase, cause lethal accumulation of lipid peroxides and induce ferroptotic cell death. Although ferroptosis has been suggested to involve accumulation of reactive oxygen species (ROS) in lipid environments, the mediators and substrates of ROS generation and the pharmacological mechanism of GPX4 inhibition that generates ROS in lipid environments are unknown. We report here the mechanism of lipid peroxidation during ferroptosis, which involves phosphorylase kinase G2 (PHKG2) regulation of iron availability to lipoxygenase enzymes, which in turn drive ferroptosis through peroxidation of polyunsaturated fatty acids (PUFAs) at the bis-allylic position; indeed, pretreating cells with PUFAs containing the heavy hydrogen isotope deuterium at the site of peroxidation (D-PUFA) prevented PUFA oxidation and blocked ferroptosis. We further found that ferroptosis inducers inhibit GPX4 by covalently targeting the active site selenocysteine, leading to accumulation of PUFA hydroperoxides. In summary, we found that PUFA oxidation by lipoxygenases via a PHKG2-dependent iron pool is necessary for ferroptosis and that the covalent inhibition of the catalytic selenocysteine in Gpx4 prevents elimination of PUFA hydroperoxides; these findings suggest new strategies for controlling ferroptosis in diverse contexts., Competing Interests: The authors declare no conflict of interest.

Parkinson's disease is the second most common neurodegenerative disease and its pathogenesis is closely associated with oxidative stress. Deposition of aggregated α-synuclein (α-Syn) occurs in familial and sporadic forms of Parkinson's disease. Here, we studied the effect of oligomeric α-Syn on one of the major markers of oxidative stress, lipid peroxidation, in primary co-cultures of neurons and astrocytes. We found that oligomeric but not monomeric α-Syn significantly increases the rate of production of reactive oxygen species, subsequently inducing lipid peroxidation in both neurons and astrocytes. Pre-incubation of cells with isotope-reinforced polyunsaturated fatty acids (D-PUFAs) completely prevented the effect of oligomeric α-Syn on lipid peroxidation. Inhibition of lipid peroxidation with D-PUFAs further protected cells from cell death induced by oligomeric α-Syn. Thus, lipid peroxidation induced by misfolding of α-Syn may play an important role in the cellular mechanism of neuronal cell loss in Parkinson's disease. We have found that aggregated α-synuclein-induced production of reactive oxygen species (ROS) that subsequently stimulates lipid peroxidation and cell death in neurons and astrocytes. Specific inhibition of lipid peroxidation by incubation with reinforced polyunsaturated fatty acids (D-PUFAs) completely prevented the effect of α-synuclein on lipid peroxidation and cell death., (© 2015 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of The International Society for Neurochemistry.)

Polyunsaturated fatty acid (PUFA) peroxidation is initiated by hydrogen atom abstraction at bis-allylic sites and sets in motion a chain reaction that generates multiple toxic products associated with numerous disorders. Replacement of bis-allylic hydrogens of PUFAs with deuterium atoms (D-PUFAs), termed site-specific isotope reinforcement, inhibits PUFA peroxidation and confers cell protection against oxidative stress. We demonstrate that structurally diverse deuterated PUFAs similarly protect against oxidative stress-induced injury in both yeast and mammalian (myoblast H9C2) cells. Cell protection occurs specifically at the lipid peroxidation step, as the formation of isoprostanes, immediate products of lipid peroxidation, is drastically suppressed by D-PUFAs. Mitochondrial bioenergetics function is a likely downstream target of oxidative stress and a subject of protection by D-PUFAs. Pretreatment of cells with D-PUFAs is shown to prevent inhibition of maximal uncoupler-stimulated respiration as well as increased mitochondrial uncoupling, in response to oxidative stress induced by agents with diverse mechanisms of action, including t-butylhydroperoxide, ethacrynic acid, or ferrous iron. Analysis of structure-activity relationships of PUFAs harboring deuterium at distinct sites suggests that there may be a mechanism supplementary to the kinetic isotope effect of deuterium abstraction off the bis-allylic sites that accounts for the protection rendered by deuteration of PUFAs. Paradoxically, PUFAs with partially deuterated bis-allylic positions that retain vulnerable hydrogen atoms (e.g., monodeuterated 11-D1-Lin) protect in a manner similar to that of PUFAs with completely deuterated bis-allylic positions (e.g., 11,11-D2-Lin). Moreover, inclusion of just a fraction of deuterated PUFAs (20-50%) in the total pool of PUFAs preserves mitochondrial respiratory function and confers cell protection. The results indicate that the therapeutic potential of D-PUFAs may derive from the preservation of mitochondrial function., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Substitution of -CD2- at the reactive centers of linoleic and linolenic acids reduces the rate of abstraction of D by a tocopheryl radical by as much as 36-fold, compared to the abstraction of H from a corresponding -CH2- center. This H atom transfer reaction is the rate-determining step in the tocopherol-mediated peroxidation of lipids in human low-density lipoproteins, a process that has been linked to coronary artery disease. The unanticipated large kinetic isotope effects reported here for the tocopherol-mediated oxidation of linoleic and linolenic acids and esters suggests that tunneling makes this process favorable.

Friedreich ataxia is an autosomal recessive, inherited neuro- and cardio-degenerative disorder characterized by progressive ataxia of all four limbs, dysarthria, areflexia, sensory loss, skeletal deformities, and hypertrophic cardiomyopathy. Most disease alleles have a trinucleotide repeat expansion in the first intron of the FXN gene, which decreases expression of the encoded protein frataxin. Frataxin is involved in iron-sulfur-cluster (ISC) assembly in the mitochondrial matrix, and decreased frataxin is associated with ISC-enzyme and mitochondrial dysfunction, mitochondrial iron accumulation, and increased oxidative stress. To assess the role of oxidative stress in lipid peroxidation in Friedreich ataxia we used the novel approach of treating Friedreich ataxia cell models with polyunsaturated fatty acids (PUFAs) deuterated at bis-allylic sites. In ROS-driven oxidation of PUFAs, the rate-limiting step is hydrogen abstraction from a bis-allylic site; isotopic reinforcement (deuteration) of bis-allylic sites slows down their peroxidation. We show that linoleic and α-linolenic acids deuterated at the peroxidation-prone bis-allylic positions actively rescue oxidative-stress-challenged Friedreich ataxia cells. The protective effect of the deuterated PUFAs is additive in our models with the protective effect of the CoQ10 analog idebenone, which is thought to decrease the production of free radicals. Moreover, the administration of deuterated PUFAs resulted in decreased lipid peroxidation as measured by the fluorescence of the fatty acid analog C11-BODIPY (581/591) probe. Our results are consistent with a role for lipid peroxidation in Friedreich ataxia pathology, and suggest that the novel approach of oral delivery of isotope-reinforced PUFAs may have therapeutic potential in Friedreich ataxia and other disorders involving oxidative stress and lipid peroxidation., (© 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

Polyunsaturated fatty acids (PUFAs) undergo autoxidation and generate reactive carbonyl compounds that are toxic to cells and associated with apoptotic cell death, age-related neurodegenerative diseases, and atherosclerosis. PUFA autoxidation is initiated by the abstraction of bis-allylic hydrogen atoms. Replacement of the bis-allylic hydrogen atoms with deuterium atoms (termed site-specific isotope-reinforcement) arrests PUFA autoxidation due to the isotope effect. Kinetic competition experiments show that the kinetic isotope effect for the propagation rate constant of Lin autoxidation compared to that of 11,11-D(2)-Lin is 12.8 ± 0.6. We investigate the effects of different isotope-reinforced PUFAs and natural PUFAs on the viability of coenzyme Q-deficient Saccharomyces cerevisiae coq mutants and wild-type yeast subjected to copper stress. Cells treated with a C11-BODIPY fluorescent probe to monitor lipid oxidation products show that lipid peroxidation precedes the loss of viability due to H-PUFA toxicity. We show that replacement of just one bis-allylic hydrogen atom with deuterium is sufficient to arrest lipid autoxidation. In contrast, PUFAs reinforced with two deuterium atoms at mono-allylic sites remain susceptible to autoxidation. Surprisingly, yeast treated with a mixture of approximately 20%:80% isotope-reinforced D-PUFA:natural H-PUFA are protected from lipid autoxidation-mediated cell killing. The findings reported here show that inclusion of only a small fraction of PUFAs deuterated at the bis-allylic sites is sufficient to profoundly inhibit the chain reaction of nondeuterated PUFAs in yeast., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Oxidative damage of membrane polyunsaturated fatty acids (PUFA) is thought to play a major role in mitochondrial dysfunction related to Parkinson's disease (PD). The toxic products formed by PUFA oxidation inflict further damage on cellular components and contribute to neuronal degeneration. Here, we tested the hypothesis that isotopic reinforcement, by deuteration of the bisallylic sites most susceptible to oxidation in PUFA may provide at least partial protection against nigrostriatal injury in a mouse model of oxidative stress and cell death, the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model. Mice were fed a fat-free diet supplemented with saturated acids, oleic acid and essential PUFA: either normal, hydrogenated linoleic (LA, 18:2n-6) and α-linolenic (ALA, 18:3n-3) or deuterated 11,11-D2-LA and 11,11,14,14-D4-ALA in a ratio of 1:1 (to a total of 10% mass fat) for 6 days; each group was divided into two cohorts receiving either MPTP or saline and then continued on respective diets for 6 days. Brain homogenates from mice receiving deuterated PUFA (D-PUFA) vs. hydrogenated PUFA (H-PUFA) demonstrated a significant incorporation of deuterium as measured by isotope ratio mass-spectrometry. Following MPTP exposure, mice fed H-PUFA revealed 78.7% striatal dopamine (DA) depletion compared to a 46.8% reduction in the D-PUFA cohort (as compared to their respective saline-treated controls), indicating a significant improvement in DA concentration with D-PUFA. Similarly, higher levels of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were detected in MPTP-exposure mice administered D-PUFA; however, saline-treated mice revealed no change in DA or DOPAC levels. Western blot analyses of tyrosine hydroxylase (TH) confirmed neuroprotection with D-PUFA, as striatal homogenates showed higher levels of TH immunoreactivity in D-PUFA (88.5% control) vs. H-PUFA (50.4% control) in the MPTP-treated cohorts. In the substantia nigra, a significant improvement was noted in the number of nigral dopaminergic neurons following MPTP exposure in the D-PUFA (79.5% control) vs. H-PUFA (58.8% control) mice using unbiased stereological cell counting. Taken together, these findings indicate that dietary isotopic reinforcement with D-PUFA partially protects against nigrostriatal damage from oxidative injury elicited by MPTP in mice., (Published by Elsevier Ireland Ltd.)

The facile abstraction of bis-allylic hydrogens from polyunsaturated fatty acids (PUFAs) is the hallmark chemistry responsible for initiation and propagation of autoxidation reactions. The products of these autoxidation reactions can form cross-links to other membrane components and damage proteins and nucleic acids. We report that PUFAs deuterated at bis-allylic sites are much more resistant to autoxidation reactions, because of the isotope effect. This is shown using coenzyme Q-deficient Saccharomyces cerevisiae coq mutants with defects in the biosynthesis of coenzyme Q (Q). Q functions in respiratory energy metabolism and also functions as a lipid-soluble antioxidant. Yeast coq mutants incubated in the presence of the PUFA α-linolenic or linoleic acid exhibit 99% loss of colony formation after 4h, demonstrating a profound loss of viability. In contrast, coq mutants treated with monounsaturated oleic acid or with one of the deuterated PUFAs, 11,11-D(2)-linoleic or 11,11,14,14-D(4)-α-linolenic acid, retain viability similar to wild-type yeast. Deuterated PUFAs also confer protection to wild-type yeast subjected to heat stress. These results indicate that isotope-reinforced PUFAs are stabilized compared to standard PUFAs, and they protect coq mutants and wild-type yeast cells against the toxic effects of lipid autoxidation products. These findings suggest new approaches to controlling ROS-inflicted cellular damage and oxidative stress., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Lysyl oxidase (LOX) is implicated in several extracellular matrix related disorders, including fibrosis and cancer. Methods of inhibition of LOX in vivo include antibodies, copper sequestration and toxic small molecules such as β-aminopropionitrile. Here, we propose a novel approach to modulation of LOX activity based on the kinetic isotope effect (KIE). We show that 6,6-d(2)-lysine is oxidised by LOX at substantially lower rate, with apparent deuterium effect on V(max)/K(m) as high as 4.35 ± 0.22. Lys is an essential nutrient, so dietary ingestion of D(2)Lys and its incorporation via normal Lys turnover suggests new approaches to mitigating LOX-associated pathologies., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A new method suitable for single nucleotide polymorphism (SNP) detection using differential oligonucleotide probe extension has been developed. Sulfur-linked laser-cleavable trityl labels are implemented in this protocol. The method is based on mass spectrometry and utilizes a single surface for affinity purification of extended probes and matrix-independent desorption-ionization of the cleavable labels. The usefulness of this method for SNP genotyping is demonstrated.

The rational design of novel triarylmethyl (trityl)-based mass tags (MT) for mass-spectrometric (MS) applications is described. We propose a "pK(R+) rule" to correlate the stability of trityl carbocations with their MS performance: trityls with higher pK(R+) values ionise and desorb better. Trityl blocks were synthesised that have high pK(R+) values and are stable in conditions of MS analysis; these MTs can be ionised by matrix as well as irradiation with a 337 nm nitrogen laser. (13)C-Labelled tags were prepared for MS quantitation applications. Moreover, the tags were equipped with a variety of functional groups allowing conjugation with different functionalities within (bio)molecules to enhance the MS characteristics of the latter. The MS behaviour of model polycationic trityl compounds with and without the matrix was studied to reveal that poly-trityl clusters are always singly charged under the (MA)LDI-TOF conditions. Several peptide-trityl conjugates were prepared and comparisons revealed a beneficial effect of trityl tags on the conjugate detection in MS. Trityl compounds containing para-methoxy- and dimethylamine groups, as well as a xanthene fragment, showed considerable enhancement in MS detection of model peptides; thus they are promising tools for proteomic applications. Dimethoxytrityl derivatives allow one to distinguish between Arg- and Lys-containing peptides. Maleimido trityl derivatives are suitable for the efficient derivatisation of thiol-containing peptides in pyridine.

A new method suitable for single nucleotide polymorphism detection and other applications based on oligonucleotide probe extension has been developed. The method is based on mass spectrometry and utilizes a single surface for affinity purification of extended probes and matrix-independent desorption/ionization of the cleavable labels. A new family of sulfur-linked laser-cleavable trityl labels with vastly improved flying abilities is implemented in this study. Corresponding reagents compatible with automated oligonucleotide synthesis are presented. Utility of this method for SNP genotyping is demonstrated.

A new hypothesis is put forward, linking cellular endurance with dietary consumption of stable heavy isotopes. Due to the isotope effect, biomolecules that incorporate heavier isotopes give rise to more stable molecular structures with increased resistance to damages associated with aging and age-related disease. The inclusion of heavy isotopes might be either active (selection for heavier isotopes) or passive (incorporation reflecting the existing abundance). The hypothesis links consumption of foods relatively rich in heavy isotopes (such as (13)C and D, derived from C4-plants), especially at the early stages of the organism's development, with enhanced longevity. Implications of diets and intestinal microflora are also discussed., (2007 Wiley Periodicals, Inc)

A method is proposed that has the potential to lessen detrimental damages caused by reactive oxygen species (ROS) to proteins, nucleic acids, lipids, and other components in living cells. Typically, ROS oxidize substrates by a mechanism involving hydrogen abstraction in a rate-limiting step. The sites within these (bio)molecules susceptible to oxidation by ROS can thus be "protected " using heavier isotopes such as (2)H (D, deuterium) and (13)C (carbon-13). Ingestion of isotopically reinforced building blocks such as amino acids, lipids and components of nucleic acids and their subsequent incorporation into macromolecules would make these more stable to ROS courtesy of an isotope effect. The implications may include enhanced longevity and increased resistance to cancer and age-related diseases.