Congenital adrenal hyperplasia (CAH) comprises a group of autosomal recessive inherited disorders that arise due to defects in one of the enzymes of steroidogenesis pathway in the adrenal glands. Ninety-five percent of the cases occur due to deficiency in 21-hydroxylase (21-OH). Clinically, CAH due to 21-OH deficiency presents in two distinct forms, classic CAH and non-classic CAH. Females with classical forms present with genial ambiguity while the presentation in males is more subtle with severe electrolyte disturbances being the initial manifestation in many cases. Arrhythmias are a rare manifestation of CAH. We report the case of an 18-day-old male child who presented with pulseless ventricular tachycardia and was later diagnosed with congenital adrenal hyperplasia based on the laboratory findings of elevated 17-hydroxyprogesterone (17-OHP) levels. Our case reveals that fatal arrhythmias such as a pulseless ventricular tachycardia can be the primary manifestation of the adrenal insufficiency of CAH even in the absence of any physical findings and hence clinicians should always maintain a strong suspicion for CAH in any child presenting with unexplained arrhythmia. Furthermore, this case also highlights the need for CAH screening in neonates so that the appropriate hormone replacement can be initiated before the development of life-threatening adrenal crisis. Congenital adrenal hyperplasia (CAH) comprises a group of autosomal recessive inherited disorders that arise due to defects in one of the enzymes of steroidogenesis pathway in the adrenal glands. More than 95% of cases of CAH are due to the deficiency of 21-hydroxylase, the enzyme responsible for the conversion of progesterone into deoxycorticosterone and 17-hydroxyprogesterone to 11-deoxycortisol . This deIn males with classic CAH, the electrolyte imbalances may present before the recognition of genital abnormalities. The subtle virilizing features in males make CAH difficult to diagnose until the electrolyte imbalance is severe.Patients with CAH are prone to develop cardiac arrhythmias due to electrolyte imbalances particularly due to hyperkalemia. Some rare cases of CAH have presented in the form of cardiac arrest  and ventAn 18-day-old male baby, first product of a non-consanguineous marriage, born at full term through normal vaginal delivery was brought to the emergency department in an unresponsive state. According to the parents, the child had been vomiting and eating poorly for the past two days. Birth history was unremarkable with no antenatal and postnatal complications.On admission, his blood pressure and peripheral pulses were undetectable. He was bradycardiac (heart rate 40/minute) and moderately dehydrated. He was unresponsive with shallow breathing the respiratory rate being 33 breaths per minute. His oxygen saturation was 92% and the temperature was 37°C. Capillary refill time was found to be four seconds. Random blood sugar came out to be 32 mg/dl. Cardiovascular examination revealed muffled heart sounds but no murmurs. Central nervous system examination revealed normal tone, reactive pupils and normal fontanelles. The remainder of the systemic examination was also unremarkable.An electrocardiogram (ECG) was instantly obtained which revealed ventricular tachycardia Figure . Other E3 with neutrophils 56.3%, lymphocytes 31.8%, platelets 652,000/mm3 and C-reactive protein (CRP) 1.9.Laboratory test results showed hemoglobin 15.1 g/dl, mean cell volume 90 fL, total leukocyte count 26,600/mmElectrolyte report revealed sodium 123 mEq/dl, potassium 6.0 mEq/dl, chloride 80 mEq/dl, calcium 9.6 mg/dl and magnesium 1.9 mg/dl while creatinine and blood urea nitrogen (BUN) were 1.4 mg/dl and 37 mg/dl, respectively.His labs revealed significant hyperkalemia which was most likely the underlying cause of ventricular tachycardia. The high potassium and low sodium levels made congenital adrenal hyperplasia a plausible diagnosis.A laboratory test for the detection of 17-hydroxyprogesterone was sent. The test revealed high levels of 17-hydroxyprogesterone (320 ng/ml). Renal ultrasound was done to check for adrenal hyperplasia but it came out to be normal. The genital examination was unremarkable with no ambiguity, the penis was of normal length and no skin hyperpigmentation was noted on the axilla, neck, and genitals. The child was diagnosed with CAH based on the laboratory results of increased levels of 17-hydroxyprogesterone. To determine the type of CAH, tests for plasma renin and aldosterone were also performed. Plasma renin came out to be elevated . Serum aldosterone was also high .2/24 hours in three divided doses) and fludrocortisone (0.2 mg daily in two divided doses) along with supplementary NaCl (8 mmol/kg). Parents were advised to consult the doctor in case the child fell ill, as stress requires increment in the dose of glucocorticoids to prevent adrenal crisis. The dose was also increased prior to circumcision. Follow-ups in the outpatient department have shown normal electrolytes and ECG and optimal growth and development of the child.The infant was discharged on hydrocortisone . The simple virilizing form has no features of salt-losing crisis and usually manifests late in childhood with precocious puberty or with clitoral or penile enlargement secondary to excess adrenal androgens .Our patient presented with life-threatening salt crisis and electrolyte imbalances, features of classic CAH-salt wasting form. Patients with classical CAH are prone to develop cardiac arrhythmias secondary to electrolyte imbalances particularly due to hyperkalemia. However, in most cases of CAH, hyperkalemia resulted in sudden cardiac death and arrhythmias have rarely been documented .Hyperkalemia is defined as a potassium level of >5.5 mEq/l. The ECG findings of hyperkalemia are progressive from tall peaked T waves, a shortened QT interval to prolongation of PR interval and loss of P waves and then to diffuse widening of QRS complex and if untreated, death .Shockable rhythms, like pulseless ventricular tachycardia or PVT, are a rare event in neonates -13. The Deterioration of VT into pulseless ventricular tachycardia and ventricular fibrillation may occur. In pulseless VT, the patient is apneic, unresponsive with undetectable peripheral pulses, as was documented in our case. The management of ventricular tachycardia and pulseless ventricular tachycardia is different. Pulseless VT is a shockable rhythm and early recognition is necessary for prompt management  as was sA similar case of ventricular tachycardia in a 20-day-old male child with CAH was reported by Virdi et al. . The corSalt-wasting form of classical CAH is characterized by low levels of serum aldosterone . SurprisThe adrenal ultrasound of this child was also unexpectedly normal; although unusual, this does not rule out the diagnosis of CAH and normal adrenal ultrasound in untreated children with CAH has been documented by Al-Alwan et al. .21-hydroxylase deficiency, in its classic SW form, is characterized by markedly elevated levels of 17-OHP usually >600 nmol/ml or >2000 ng/ml. Corticosteroids are the mainstay of treatment of classical CAH . TreatmeNewborn screening programs using 17-OHP assays have been initiated in many countries resulting in a considerable decrease in morbidity and mortality that was previously attributable to the severe adrenal crisis in salt-wasting forms of CAH . ReviewsOur case reveals that rare fatal arrhythmias, such as a pulseless ventricular tachycardia, can be the primary manifestation of the adrenal insufficiency of CAH in a neonate even in the absence of any physical findings. Electrolyte testing should be sought in any infant who presents with significant unexplained arrhythmias without any evidence of congenital heart disease. Moreover, our case also highlights the need for CAH screening in newborns in developing countries like Pakistan so that affected neonates can be identified before the development of the life-threatening salt-losing crisis and appropriate hormone replacement be initiated.


Fast food restaurant-fried potato chip serving (FFRPCS) aldehyde contents were also monitored. Substantially lower levels of aldehydes were generated in the MRAFO product than those observed in PUFA-richer oils during LSSFEs. Toxicologically-significant concentrations of aldehydes were detected in FFRPCSs, and potato chips exposed to DBRDFEs when using a PUFA-laden sunflower oil frying medium: these contents increased with augmented deep-frying episode repetition. FFRPCS aldehyde contents were 10–25 ppm for each class monitored. In conclusion, the MRAFO product generated markedly lower levels of food-penetrative, toxic aldehydes than PUFA-rich ones during LSSFEs. Since FFRPCS and DBRDFE potato chip aldehydes are predominantly frying oil-derived, PUFA-deplete MRAFOs potentially offer health-friendly advantages.Human ingestion of cytotoxic and genotoxic aldehydes potentially induces deleterious health effects, and high concentrations of these secondary lipid oxidation products (LOPs) are generated in polyunsaturated fatty acid (PUFA)-rich culinary oils during high temperature frying practices. Here, we explored the peroxidative resistance of a novel monounsaturate-rich algae frying oil (MRAFO) during laboratory-simulated shallow- and domestically-based repetitive deep-frying episodes (LSSFEs and DBRDFEs respectively), the latter featuring potato chip fryings. Culinary frying oils underwent LSSFEs at 180 °C, and DBRDFEs at 170 °C: aldehydes were determined by  Mechanisms available for this process primarily involve the oxidative conversion of such UFAs to primary lipid oxidation products (LOPs), commonly described as lipid hydroperoxides  sources, abbreviated HPMs and CHPDs respectively), a process sequentially followed by their fragmentation to secondary ones, the latter including extremely toxic aldehydes in particular2. Further HPM and CHPD degradation products include epoxy acids, alcohols, ketones, oxoacids, alkanes and alkenes, in addition to further toxic oxidation and fragmentation products5.The peroxidation of unsaturated fatty acids (UFAs) at temperatures commonly used for standard frying or cooking episodes  are much more resistant to oxidation than polyunsaturated ones (PUFAs), and hence they give rise to lower levels of only particular LOPs when heated in this manner, and generally only after exposure to prolonged thermal stressing episodes at standard frying temperatures. Therefore, the order and extent of toxic LOP production in culinary oils is PUFAs > MUFAs >>> saturated fatty acids (SFAs), and the relative oxidative susceptibilities of 18-carbon chain length fatty acids (FAs) containing 0, 1, 2 and 3 carbon-carbon double bonds (i.e. >C=C< functions) are 1:100:1,200:2,500 respectively8, and these results have been available to the scientific, food and public health research communities since 19946. Indeed, samples of repeatedly-used oils collected from domestic kitchens, fast-food retail outlets and restaurants, have confirmed the generation of these aldehydes at high concentrations during ‘on-site’ frying practices. Such results have been repeated, replicated and ratified by many other research laboratories worldwide (most notably9). We can also employ these NMR techniques to monitor the corresponding degradation of culinary oil PUFAs and MUFAs during such standard frying/cooking practices7, and also to detect and quantify a range of further LOPS, i.e. CHPDs and HPMs, ketones and alcohols, together with toxic epoxy acids, the latter including leukotoxin and its derivatives such as isoleukotoxin and leukotoxindiol9.Previous NMR-based investigations focused on the peroxidative degradation of culinary oil UFAs during standard frying practices, or corresponding laboratory-simulated thermal stressing episodes, have demonstrated the thermally-promoted generation of very high levels of highly toxic aldehydes and their hydroperoxide precursors in such products (particularly those rich in PUFAs)in vivo10 following oral ingestion, where they have access and may cause damage to cells, tissues and essential organs. Indeed, these agents have been demonstrated to promote a broad spectrum of concentration-dependent cellular stresses, and their adverse health properties include effects on critical metabolic pathways ; the promotion and perpetuation of atherosclerosis and cardiovascular diseases14; mutagenic and carcinogenic properties19; teratogenic actions ; the exertion of striking pro-inflammatory effects22; the induction of gastropathic properties (peptic ulcers) following dietary ingestion23; neurotoxic actions, particularly for 4-hydroxy-trans-2-nonenal (HNE) and -hexenal (HHE)24; and impaired vasorelaxation coupled with the adverse stimulation of significant increases in systolic blood pressure25. Further deleterious health effects include chromosomal aberrations, which are reflective of their clastogenic potential, sister chromatid exchanges and point mutations, in addition to cell damage and death27.Of critical importance to their public health risks as food-borne toxins, typical chemically-reactive α,β-unsaturated aldehydes produced during the thermal stressing of culinary oils according to standard frying practices are absorbed from the gut into the systemic circulation 18. Moreover, these secondary LOPs are also able to form covalent adducts with many proteins via Schiff base or Michael addition reactions10, and these can induce significant structural and conformational changes in these macromolecules, which serve to impair their biocatalytic functions. However, until recently, such toxicological considerations have generally continued to elude interest or focus from many food industry and public health researchers.The toxicity of these aldehydes, particularly the α,β-unsaturated ones, is ascribable to their aggressive chemical reactivity. Indeed, they cause damage to critical biomolecules such as DNA: since they are powerful electrophilic alkylating agents, α,β-unsaturated aldehydes readily alkylate DNA base adducts, and this serves to explain their mutagenic and carcinogenic properties2-fueled peroxidation during common frying cycles, this predominantly MUFA-containing [i.e. >90% (w/w)] culinary oil offers further major advantages, including a very high smoke-point of 252 °C , and also a neutral taste contribution.The recent development of algae-derived cooking oils has provided much scope and benefits regarding the effective bioengineering of their triacylglycerol FA contents, in particular for their valuable uses as cooking oils. Indeed, the novel MUFA-rich algae frying oil (MRAFO) tested in this work  represents the first ever such algal product available to consumers in the USA. In addition to its potential resistance to thermally-induced, O1H NMR analysis to determine the concentrations of a series of highly toxic classes of aldehydic LOPs therein as a function of heating time, i.e. from 0–90 min. for LSSFEs, and 8 × 10 min. DBRDFEs (the latter featuring a 30 min. oil cooling rest period between each frying cycle). The time-dependent production of epoxy acid LOP toxins was simultaneously monitored in all oils investigated. Such experiments serve to provide valuable information and insights regarding the possible health-threatening effects of these aldehydes when ingested in human diets featuring fried food sources of these toxins, e.g. potato chips, fish fillets, battered chicken, chicken strips, etc., and here we have also demonstrated, for the first time, the availability for human consumption of high, toxicologically-significant (up to 25 ppm) levels of two major classes of α,β-unsaturated, and one major class of saturated aldehydes in servings of fried foods collected directly from fast food retail outlets/restaurants, and also in potato chips subjected to DBRDFEs when PUFA-rich sunflower oil is used as the frying medium. The potential deleterious health effects presented by these oils when employed as frying media, particularly those associated with PUFA-rich frying oil sources of dietary aldehydes, are discussed in detail.Therefore, in view of the much lowered susceptibility of MUFAs to oxidation than PUFAs, in this study we have explored the oxidative resistance of the above high-stability MRAFO product during laboratory-simulated standard shallow frying practices, i.e. one of its major culinary applications, and also during DBRDFEs. For this purpose, we exposed this MRAFO, in addition to commonly-utilised sunflower, corn, canola and extra-virgin olive oils, to these episodes at 180 °C and 170 °C respectively, and have employed multicomponent high-resolution 1H NMR analysis demonstrated the thermally-induced generation of aldehydic LOPs in all oils investigated, and Fig. 1H NMR profiles demonstrating the time-dependent production of -CHO function resonances ascribable to a range of these toxins when culinary oil products were heated according to our LSSFEs . Spectra of heated sunflower, corn and canola oils also contained resonances assignable to aldehydic precursors, in particular cis,trans- and trans,trans-CHPDs , and cis,trans-conjugated hydroxydienes (δ = 5.40–6.50 ppm range), as previously reported8; broad -OOH resonances were also visible in spectra acquired on thermally-stressed extra-virgin olive oil and MRAFO products, although presumably they largely arise from HPMs rather than CHPDs in these cases. The CHPDs detectable are produced during recycling O2- and heat-stimulated peroxidative bursts throughout the whole simulated frying process, especially since these LOPs remain detectable in spectra acquired on the PUFA-rich oils after a 90 min. LSSFE period. Moreover, relatively low concentrations of these aldehydes and their CHPD precursors were also detectable in unheated sunflower and corn oil products [Fig. cts Fig. .Figure 1trans-2-alkenals [(E)-2-alkenals], trans,trans- and cis,trans-alka-2,4-dienals , along with 4-hydroperoxy-/4-hydroxy-, and 4,5-epoxy-trans-2-alkenals [the latter three all substituted (E)-2-alkenal derivatives]  confirmed that it was assignable to an α,β-unsaturated aldehyde, since it was directly connected to two vinylic proton multiplet signals located at δ = 6.59 and 5.95 ppm -2-butenal , which has corresponding signals at δ = 10.10 ppm , 6.70 (dq) and 5.97 ppm (ddq) .Also notable was the diminishing intensity of the omega-3 FA (linolenoylglycerol) 1H NMR spectra acquired on potato chip samples purchased from fast-food restaurants (data not shown).The above epoxy acid resonances were also observed in 10, where they have the potential to exert a wide range of adverse health effects in humans. In particular, HNE and HHE represent highly toxic and carcinogenic secondary LOPs derived from the thermally-induced degradation of culinary oil PUFAs29; indeed, HNE is also a toxic second messenger30. However, since these α,β-unsaturated aldehydes predominantly arise from glycerol-bonded linoleate (HNE) and omega-3 FAs (HHE), little or none of them were detectable in thermally-stressed MRAFO and olive oil products (or other PUFA-deplete ones), since these contain only 4 and 5–10% (w/w) linoleoylglycerols respectively, and both products contain ≤1% (w/w) linolenoylglycerols. This also explains why much lower levels of similarly toxic trans,trans-alka-2,4-dienals are generated in such MUFA-rich oils when subjected to thermal stressing episodes.Heating of linoleoylglycerol- and linolenoylglycerol-rich culinary oil products according to LSSFEs generates very high levels of a range of extremely toxic aldehydic LOPs. These established toxins have been proven to be absorbed from the gut into the systemic circulation following their dietary ingestionn-alkanals and trans-2-alkenals, the former of which are arguably of a lower toxicity than the latter), whereas a much broader pattern of these agents are produced from PUFA-derived CHPDs 9. Intriguingly, the total unsaturated aldehyde concentration determined in PUFA-rich sunflower oil heated for a period of 90 min. according to our LSSFEs was very close to or exceeded a staggeringly high figure of 0.05 mol.kg−1.Although significant amounts of aldehydic LOPs also arise from MUFAs, these were only generated at prolonged heating times, i.e. significant lag phases preceded their evolution Figs . Moreove2-driven peroxidation processes occurring during standard frying practices than frequently employed PUFA-laden ones. Indeed, significantly lower levels of trans-2-alkenals and n-alkanals were generated in this product at each sampling time-point following exposure to LSSFEs at 180 °C. Furthermore, markedly lower concentrations of PUFA-derived aldehydes, such as trans,trans-alka-2,4-dienals and 4,5-epoxy-trans-2-alkenals, were detected in this product when thermally-stressed in this manner, as expected. Indeed, samples of this oil collected at the 10 and extreme pan-frying 20 min. LSSFE time-points contained little or no toxic aldehydes, whereas substantially higher concentrations of these toxins were found in corresponding samples of PUFA-rich corn and sunflower oils.Our results therefore clearly demonstrate that the predominant, >90% (w/w) MUFA source of the MRAFO product renders it much more resistant to thermally-induced, autocatalytic, OTherefore, when used as a medium for shallow frying purposes, this MUFA-rich algae oil serves to offer a high level of protection to human consumers, i.e. it is anticipated that much lower quantities of toxic aldehydes will permeate into food matrices during frying episodes performed with it.1H NMR investigations which compared the extent of aldehyde generation within fixed volumes of oils heated according to standard frying practices in vessels of increasing diameter7. Concentrations of trans-2-alkenals, trans,trans-alka-2,4-dienals and n-alkanals for the LSSFEs performed were found to be ca. 6, 3.5 and 3 mmol./mol. FA respectively for sunflower oil at the 60 min. heating time-point, whereas these values were only ca. 0.13, 1.0 and 0.5 mmol./mol. FA respectively following the 6th repetitive frying episode (i.e. 6 × 10 min. sessions with 30 min. cooling periods between each one) performed with this oil according to our domestic deep-frying protocol. Moreover, at the 60 min. heating time-point, although LSSFE levels of trans-2-alkenals, trans,trans-alka-2,4-dienals and n-alkanals were ca. 5, 1.7 and 2 mmol./mol. FA respectively for extra-virgin olive oil, and ca. 1, 0.3 and 0.5 respectively for the MRAFO product, little or none of these were found in either of these oils when exposed to our DBDRFEs; low concentrations were only detectable in these oils at the later DRDRFE episodes.The higher levels of aldehydes found in sunflower, extra virgin olive and MRAFO oils when exposed to our LSSFEs than those found in the ‘at-home’ domestic frying ones (DBDRFEs) are predominantly explicable by the fact that the former processes were performed under shallow frying conditions, whereas the latter domestic ones involved deep-frying, albeit repetitive sessions. Indeed, these results are fully consistent with our previously conducted 2 required for the peroxidation process, and also the subsequent dilution of surface-formed aldehydic LOPs into the larger volume frying medium available in the deep-frying experiments conducted.Specifically, these results arise from the greater surface area of the frying oil medium during shallow frying practices, and hence a greater exposure of it to atmospheric Oet al.31 was followed. A further, albeit less significant, explanation is that chain-breaking antioxidants such as tocopherols in the oil products tested are likely to be more effective at suppressing the peroxidation process under deep- rather than shallow-frying conditions in view of the much lower levels of aldehydic LOPs formed during the former process, i.e. such antioxidants will have an enhanced ability to compete for lipid peroxyl radicals. Additionally, conceivably there will be less volatilisation and thermally-induced degradation of these antioxidants at the lower temperature employed for these domestic deep-frying experiments.However, these results are, of course, also explicable by the lower temperature employed for the domestic deep-frying experiments, i.e. 170 rather than 180 °C used for the LSSFEs. A temperature of 170 °C was used for the former experiments since the method of Boskou However, the lower levels of aldehydes found in culinary oils exposed to deep-frying processes may, at least in part, be compromised by a greater extent of oil absorption by the fried food available for human consumption.32.The marked susceptibility of omega-3 FAs to thermo-oxidation is also very likely to exert a major effect on the omega-6 to omega-3 FA concentration ratio health indices of the cooking oils evaluated here, and this is discussed in more detail in section S6. In view of the very high susceptibility of omega-3 FAs to thermoxidative deterioration, both Belgium and France have sensibly adopted regulations which limit the linolenate content of frying oils to 2% (w/w)remaining therein8. Indeed, a large number of these secondary LOPs generated are volatilised at standard frying temperatures, and this also presents austere health hazards in view of their inhalation by humans, especially those working in fast-food retail outlets or restaurants with insufficient or inadequate ventilation precautions. This is especially the case for secondary LOPs arising from the oxidation of linoleoyl- and linolenoyglycerols. Indeed, a substantial fraction of such aldehydes have boiling-points (b.pts) < 180 °C , notable examples being trans-2-heptenal, trans,trans-deca-2,4-dienal and n-hexanal  from peroxidation of the former source33, and acrolein, trans,trans-2,4-heptadienal and propanal  from peroxidation of the latter acylglycerol. Information regarding the toxicological concerns and actions of inhaled or ingested trans,trans-deca-2,4-dienal are provided in section S7.Intriguingly, the above concentrations of aldehydes arising from the thermal stressing of commercially-available culinary oils is a value representing only that trans-2-undecenal and trans-2-decenal, which have b.pt values of 194, 213, 234 and 230 °C respectively33, and hence this consideration amply, albeit indirectly, serves to reinforce the hypothesis that MUFA-rich cooking oils are much less susceptible to thermally-induced oxidation than PUFA-rich ones, since despite their low b.pts and hence greater volatilities, residual oil trans-2-alkenal and n-alkanal concentrations in post-heated PUFA-rich oils are always greater or much greater than those observed in corresponding MUFA-rich ones such as MRAFO and, to a lesser extent, canola and olive oils. However, the heating/frying time-dependence of these differences observed is also a critical factor for consideration. Furthermore, an additional aldehyde arising from the fragmentation of HPMs is n-octanal, which has a b.pt of 173 °C33; hence, presumably somewhat higher concentrations of this LOP would be expected to be found in the gaseous (volatile emission) phase during standard frying practices, whereas higher levels of the above alternative HPM-sourced aldehydes may be anticipated to remain in the thermally-stressed oil medium, together with accessible foods fried therein.Contrastingly, major aldehydes derived from the peroxidation of oleoylglycerols include nonanal, decanal, 33 found that the predominant trans-2-alkenals and n-alkanals detected in the headspace of extra-virgin olive oil heated at a temperature of 190 °C for 20 hr. in a stainless steel frying tank were n-nonanal, and a combination of trans-2-decenal and -undecenal, respectively . Similar results were found by Fullana et al. in 200434, who also observed much lower headspace concentrations of peroxidised linoleoylglycerol-derived trans,trans-alka-2,4-dienals than those of trans-2-alkenals and n-alkanals for olive oil when thermally-stressed at 180 °C for a period of 15 hr. in a closed Pyrex Instatherm reaction flask and head.However, Guillen and Uriate (2012)1H NMR detection of cis-2-alkenals in thermally-stressed culinary oils (section S1). Such cis- isomers, including cis-2-butenal, are also known to be secondary LOPs which arise from the peroxidation of PUFA sources 4. However, it is conceivable that they may also arise from the thermally-induced isomerisation of their corresponding trans-2-alkenals, and this may explain their generation at only the later LSSFE time-points. Indeed, cis -2-heptenal may arise from cis-trans isomerism of its trans-isomer, which is a β-homolysis product of linoleate-12-hydroperoxide35. PCA of our oil dataset confirmed a correlation between cis- and trans-2-alkenal resonances, i.e. they were both found to load significantly and positively on the second orthogonal PC 37. However, we found no major multivariate statistical evidence for associations between the cis-2-alkenal 1H NMR resonance intensities and those of either cis,trans- or trans,trans-alka-2,4-dienals. Indeed, this possible precursor may also decompose to 2,3- or 4,5-epoxyaldehydes, which are then further degraded to combinations of either isomeric 2-octenals and acetaldehyde, or glyoxal and 2-octene38. Although these mechanistic pathways appear to be consistent with Boskou et al.’s results31, our observations revealed that oil trans,trans-alka-2,4-dienal concentrations continued to increase from the 60 to 90 min. time-points for all products explored, with the exception of those for extra virgin olive oil in which it saturated at 60 min.Here, we have also demonstrated, for the first time, the  section . Such cicis-2-alkenal levels and those of all other aldehydic LOPs (r = 0.86–0.93), the most significant one was that with trans-2-alkenals , frying time and frying temperature, these values broadly range from 6–38% (w/w)41. Moreover, Naseri et al.42 have reported that the deep frying of fish (silver carp) gave rise to a substantial exchange of acylglycerol (predominantly triacylglycerol) FAs between the food and the culinary oils employed for this purpose, and as expected, the frying oil FA composition substantially altered that of these silver carp fillets on completion of these frying episodes. Comparable results have been observed in similar investigations focused on lipid uptake by potato chips during standard frying practices . For example, the total lipid content of different varieties of fresh, unfried potato tubers is only ca. 0.10% (w/w), of which the total PUFA content is 70–76%44, but escalates to values exceeding 30% (w/w) in chipped potatoes after frying45. Hence, frying oil acylglycerol-normalised (proportionate) concentrations of LOPs will also be expected to migrate into foods fried in such media, and in 2012 Csallany et al.46 found that HNE was readily detectable in French fry samples collected from n = 6 fast-food restaurants at concentrations of 8–32 µg/100 g portion .Although the lipid content of fried products is dependent on the types of food, class of frying episode . These observations are consistent with previous investigations33, in which HNE was detectable in sunflower oil at concentrations of ca. 350 and 430 μmol.L−1 when thermally-stressed at 190 °C for prolonged 17.5 and 20.0 hr. episodes respectively, whereas neither of these LOPs were observed in extra-virgin olive oil at either of these time-points. Similarly, levels of trans-4,5-epoxy-trans-2-decenal and 4-oxo-trans-2-nonenal were found to be much greater in heated, PUFA-rich sunflower oil than those in a correspondingly-heated extra-virgin olive oil product tested, as expected29. Further information regarding the levels of HNE and other hydoxyaldehydes detectable in thermally-stressed culinary oils, and their evaporative loss therefrom, is available in section S8, along with that relating to their availability for human consumption in fried food sources.However, PUFA-derived HNE is always detectable in thermally-stressed PUFA-containing oils at much lower levels than those of similarly health-threatening 1H NMR analysis results clearly demonstrate that much greater levels of trans-2-alkenals, trans,trans-alka-2,4-dienals, and arguably somewhat less toxic n-alkanals, are present in FFRPCSs purchased from fast food retail outlets. Indeed, for peroxidised linoleoylglycerols, the predominant compounds featured within the three major aldehydic LOP classes detectable are trans-2-octenal, trans,trans-deca-2,4-dienal and n-hexanal respectively33, and assuming that these represent 100% of the above 3 classes of aldehydes, our estimated mean μmol.kg−1 values would constitute as much as 1.53, 2.44 and 1.25 mg aldehyde/100 g portions of FFRPCSs, equivalent to 1.1, 1.7 and 0.9 mg per small (71 g), and 2.4, 3.8 and 1.9 mg per large (154 g) servings, respectively  than those of the oils in which they are fried (ca. ≤ 25%). This may reflect the lower reactivities of n-alkanals than α,β-unsaturated aldehydes towards free, and/or protein-incorporated amino acids with selected ‘target’ side-chain amino or thiol functions, processes involving Maillard and/or Michael addition reactions47 (section S9).Also notable is the higher mean molar percentage levels of 48. This alone is a critical toxicological concern, especially since we have found here, for the first time, substantially greater contents of trans-2-alkenals, trans,trans-alka-2,4-dienals and n-alkanals present in FFRPCSs available to consumers for purchase in fast food restaurants. Indeed, assuming that linoleoylglycerol CHPD-derived trans-2-octenal represents the total trans-2-alkenal content therein, this estimate for a 154 g ‘large’ serving portion of this fried food (ca. 2.4 mg) is 68-fold greater than that of this acceptable daily intake limit for its lower homologue acrolein . Strikingly, this 2.4 mg value is that of only one of the 3 major classes of such cytotoxic/genotoxic aldehydes detectable at similar levels, together with at least several minor ones. Corresponding estimates for the major trans,trans-alka-2,4-dienal  and n-alkanal  species arising from linoleoylglycerol peroxidation were > 3.7 and 1.9 mg , respectively, per 154 g serving. Moreover, these estimates correspond to only one potato chip portion of a single fried meal!In view of these observations, very recently the Australian Government Department of Health (AGDH) specified that the acceptable daily intake of the similarly-toxic, simplest α,β-unsaturated aldehyde acrolein, i.e. that which is considered to be a level of intake of this molecule that can be ingested daily over an entire lifetime without any appreciable risk to health, to be only 0.5 µg per kg of body weight, i.e. a total of only 35 μg for an assumed (average) human body weight of 70 kg49. This tolerable intake level is ca. 4.5-fold less than that estimated for only the total trans-2-alkenal content of the above 154 g single FFRPCS, although this is only one of the 7 or more classes of aldehydes detected and monitored in this work. Moreover, the estimated mean molar % of this aldehyde was only an estimated 30% of the total aldehyde concentration found in FFRPCSs; that for similarly-toxic trans,trans-alka-2,4-dienals was 39 molar %  over that of acrolein (56.06), the estimated tolerable daily intake level for an average human would be 1.18 mg, and therefore the content of the former still exceeds this tolerable intake value > 2-fold. Moreover, the ‘acrolein-mass equivalent’ content of total α,β-unsaturated (i.e. both mono- and di-unsaturated) aldehydes would be approximately 3-fold higher than this maximum tolerable intake value.The health-threatening significance of these estimated LOP intake values are further exemplified by the World Health Organisation (WHO)’s tolerable intake level of acrolein, which in 2002 was specified as a higher 7.5 μg 0.13 μmol) per day per kg of body weight  μmol pertrans,trans-deca-2,4-dienals present in FFRPCS samples pre-fried in a hypothetical oil containing a 100% (w/w) linoleic acid content (24 ppm) is comparable to that determined in French fries exposed to repeated frying episodes using sunflower oil in a domestic deep-fryer (up to 11 ppm after 3–4 fryings)31. As expected, similar levels of this α,β-unsaturated aldehyde were found when this food was fried in PUFA-rich vegetable shortening, but intermediate ones were observed when cottonseed oil was employed in place of sunflower oil, and lower values still were found for palm and olive oils, with olive oil giving rise to the lowest ones . Our higher estimated total trans,trans-alka-2,4-dienal level of 157 ± 43 µmol.kg−1 (mean ± SEM) is not dissimilar to Boskou et al.’s31 maximal deep-frying value of ca. 65 µmol.kg−1 (10–11 ppm) for trans,trans-deca-2,4-dienal in French fries. However, our higher estimated mean value will, of course, also include contributions from alternative trans,trans-alka-2,4-dienals such as linolenate hydroperoxide-derived trans,trans-nona-2,4-dienal, and trans,trans-hepta-2,4-dienal (the latter arising from scission of linolenate’s 12-hydroperoxide), in the FFRPCS samples investigated, although the linolenate content of sunflower oil is negligibly small. Moreover, this difference observed may also reflect continued sequential reuse of UFA-rich frying oils in the fast-food outlets from which they were purchased. Our estimated FFRPCS total mean trans,trans-alka-2,4-dienal content value is also similar to that found in a further report50.Our estimated mean level of total et al.31 i.e. 61–88 µg/g of absorbed oil from the 2nd to the 8th frying sessions, equivalent to 401–578 µmol.kg−1 oil; assuming an overall 15% (w/w) absorption uptake of sunflower oil frying medium into this fried food31 would yield potato chip mass-normalised contents of 60–87 µmol.kg−1. Our results also confirmed that MUFA-rich oils such as extra virgin olive and especially MRAFO oils offer little or no toxicological threats to human health when employed for such deep-frying practices.Results acquired from our domestic deep-frying experiments clearly show that only PUFA-laden sunflower oil gave rise to the availability of significant levels of aldehydes for human consumption in repeatedly-fried potato chips, and these levels were similar to those found for 2,4-decadienal by Boskou trans-2-alkenals, 1-2 for trans,trans-alka-2,4-dienals, and 2 for n-alkanals respectively, and hence for each of these aldehyde classes, these values provide an indication of the maximum levels of repetitive use for sunflower oil when used for frying purposes according to our domestic deep-frying protocol.The oil reuse lag periods observed in Fig. 51] in potatoes.However, it appears that the use of sunflower oil for a maximum of 2 episodes under our deep-frying experimental conditions would not give rise to any significant levels of each aldehyde in fried potato chips, and therefore would not present any adverse toxicological or health concerns. However, our data provide evidence that the additional reuse of this and perhaps other PUFA-rich oils does indeed give rise to toxicologically-relevant concentrations of aldehydes in this commonly consumed fried food. Although significant levels of lipid hydroperoxides were found to be generated in this culinary oil, which sequentially increased with increasing number of repetitive frying episodes performed, they were not detectable in any of the DBRDFE potato chip samples collected, an observation which indicates that they are rapidly degraded to secondary LOPs such as aldehydes and/or epoxy fatty acids, or further products, when uptaken by this food during the frying practice employed. Indeed, such decomposition is likely to be promoted by the availability of catalytic trace levels of transition metal ions , of which 3 g represents SFAs. Therefore, 4 portions of these fries consumed per week (mean 0.57 portion per day) would constitute an overall fried food fat intake of ca. 19% of the reported 77 g mean total daily human UK fat consumption54 (14.3 g/day), which would predominantly comprise thermally-peroxidisable UFAs for this particular food service outlet, i.e. ca. 16%, corresponding to 12.6 g/day.The total FA contents of French fries varies widely, and generally ranges from 5 to >15% by weightet al.55 found that CHD patients reported a much elevated, highly statistically significant daily consumption of both deep- and shallow-fried foods (15 ± 25 and 24 ± 60 g respectively) than that of an age-matched healthy control group (1 ± 5 and 3 ± 17 g respectively).Strikingly, Panwar et al.56, ingestion of a 150 g serving of French fries which have been deep-dried in sunflower oil and therefore which contain a maximal aldehyde amount of 1.65 mg is sufficient to induce 97% oxidative conversion of LDL in vitro.According to Kaliora Previously reported epidemiological, meta-analysis, animal model and laboratory experimental investigations which connect the ingestion of fried foods and/or more specifically, aldehydes themselves  to the pathogenesis and/or incidence of further human diseases , are outlined in section S10 of the Supplementary Materials section. This section includes information relating to the mechanisms of the toxicities of aldehydes and/or lipid oxidation products (LOPs) present in pre-heated frying oils.33.Our results further demonstrate that the shallow- or deep-frying of foods in MUFA-rich, PUFA-deplete cooking oils such as MRAFO, which generate much lower thermally-inducible aldehyde levels in frying oils than those produced in PUFA-rich ones, gives rise to the passage of proportionately much lower concentrations of these toxins into fried food matrices available for human consumption, and therefore such servings offer less potential adverse dietary threats to human health. However, as noted above, a further major factor for consideration is that the b.pts of aldehydes derived from linoleoylglycerol CHPD fragmentation are predominantly lower than those arising from the scission of oleoylglycerol HPMsn-hexanal (2.5–9.5 mg) and malondialdehyde (0.24–0.66 mg)57. Moreover, the acrolein content of such a cigarette allocation to humans has been estimated to be 0.62–3.5 mg 58.Intriguingly, the above FFRPCS quantities of aldehydes available for human ingestion are not dissimilar to those arising from the smoking of a mean daily allocation of 25 cigarettes, i.e. mg quantities of crotonaldehyde (1.8–5.7 mg), butyraldehyde (2.2–23.2 mg), Of course, PUFAs localised within or originating from the food sources themselves will also be expected to undergo thermally-induced oxidative deterioration during frying practices. The conceivable consumption of aldehydic LOPs by amino acids and proteins present in fried foods is outlined in section S11.10. This is attributable to the poor capacities of such low antioxidant concentrations to combat the aggressive, recycling autocatalytic oxidative assaults upon highly-susceptible PUFAs induced by their exposure to such high temperatures. Along with their chemical consumption by thermally-inducible lipid peroxyl radicals during frying practices, the loss of these antioxidants during such episodes is also ascribable to (1) their volatilisation at such temperatures , and (2) their thermal instability when exposed to these temperatures (details available in supplementary section S11).An additional consideration is that the concentrations of natural or oil-supplemented lipid-soluble, chain-breaking dietary antioxidants such as α-tocopherol (vitamin E) and DTBHQ, molecules which are known to terminate the autocatalytic lipid peroxidation process, unfortunately appear to be only poorly effective at suppressing the adverse generation of toxic LOPs produced during standard frying practicesThe possible therapeutic intervention of L-cysteine, especially in relation to attenuating acetaldehyde toxicity, in both humans and experimental animals, is available in section S12. Further previously documented interventional and preventative/prophylactic strategies for guarding against oxidative stress induced by the consumption of diets containing peroxidised culinary oils are also documented in this Supplementary Materials section.1H NMR signals assignable to a range of toxic epoxy acid LOPs were also detectable in samples of culinary oils exposed to LSSFEs. As expected, a multicomponent pattern of these resonances was observed in PUFA-rich sunflower oil when heated according to our LSSFEs at 180 °C for ≥30 min. periods, and these included those assigned to leukotoxin, isoleukotoxin and leukotoxindiol. However, in view of its predominant (>90%) MUFA content, the only 1H NMR-detectable epoxy acids found in the MRAFO product evaluated were trans- and cis-9,10-epoxystearates, although these only evolved at the 60 and 90 min. heating time-points, which are very lengthy and hence irrelevant to those of shallow frying practices . Leukotoxin  and its diol derivative, which can also be generated in vivo, are known to give rise to the degeneration and necrosis of leukocytes, and have been implicated in the pathogenesis of multiple organ failure, breast cancer, and perturbations to the reproductive functions of rats59. Leukotoxins also exert disruptive effects on cell proliferation and the respiratory burst of neutrophils in vitro60.A series of trans-fatty acid (TFA) intake to coronary heart diseases (CHDs) remains widespread , and their potential health risks in this context are currently considered to be greater than those presented by SFAs62 (section S13). However, in view of these estimates, it should be stressed that, on a mole-for-mole basis, aldehydes arising from PUFA and MUFA peroxidation are clearly very much more toxic than TFAs, although estimated human intakes of the latter are, of course, much greater than those of the former. However, such investigations focused on the roles of TFAs in promoting CHDs, e.g.62, have failed to also consider the myriad of adverse health effects presented by aldehydes ingested in fried food sources, which include atherosclerosis and its pathological sequelae14. Therefore, without any efficient control for such potentially confounding effects, and those also offered by further toxic LOPs , along with the quantities of each of these LOP toxins available in human diets, then such public health studies targeting TFAs as CHD ‘malefactor’ molecules may indeed be compromised.Documented evidence which relates cis-configuration FAs to their corresponding TFA derivatives, although one study has reported a marginal increase in levels of the latter in corn oil following its exposure to stir-frying episodes63.Moreover, in principle TFAs may themselves also be susceptible to peroxidative damage, followed by the possible sequential fragmentation of their corresponding hydroperoxides to toxic secondary LOPs. Despite some major conjecture in the literature available, the heating of oils according to frying practices does not appear to transform natural 65 (further details are available in section S14).In addition to their greater resistance than PUFAs to thermo-oxidative damage, particularly that induced by high temperature frying practices, dietary MUFAs offer many additional further potential health benefitsUnless exposed to such frying practices (single or repeated), or alternatively stored and/or exposed to light for prolonged periods of time at ambient temperature, the authors accept that PUFA-rich culinary oils offer little or no threats to human health. Indeed, unperoxidised, intact essential FAs therein such as linoleoyl- and especially α-linolenoylglycerols offer valuable protective health benefits. However, the presence of only trace concentrations of LOP processing contaminants, aldehydic or otherwise, in these products may substantially negate such benefits.Therefore, a full investigation of all factors exerting an influence on the nature and levels of LOP toxins available in fried foods and hence their roles in the development of NCDs, particularly fried food and CO types used for frying, frying practices , temperatures and durations, and oil reuse status, is required. Further considerations should include the extent of fried food consumption prepared at home or at commercial food service outlets, and also the overall dietary patterns of populations surveyed.trans-2-alkenals, trans,trans-alka-2,4-dienals and n-alkanals in FFRPCSs , in principle this MUFA-laden algae oil should present a lower level of health hazards to human consumers than those associated with PUFA-rich oils when employed for this purpose. Indeed, experiments involving the analysis of fried potato chip samples collected during repetitive domestic deep-frying episodes clearly demonstrated that the use of PUFA-rich sunflower oil gave rise to significant reuse-dependent levels of each class of these aldehydes in this regularly consumed food source, whereas only negligible amounts were found in these when MUFA-rich extra virgin olive and MRAFO oils were employed as frying media. Clearly, these results have a high level of public health significance in view of a wealth of evidence available for a myriad of toxicological effects exerted by these secondary LOPs.Exposure of PUFA-rich culinary oils to LSSFEs for periods of up to 90 min. generates extremely high levels of hazardous aldehydic LOPs, which may present both serious and chronic threats to human health. Contrastingly, results acquired here also clearly demonstrated that the predominantly MUFA-containing, PUFA-deplete MRAFO oil explored was particularly resistant to LSSFE-induced thermo-oxidation, i.e. much more so than PUFA-rich sunflower and corn oils, and also more so than other MUFA-rich oils tested; the PSI value and [MUFA]:[PUFA] % content ratio of this oil were significantly lower and greater, respectively, than those of the other MUFA-rich oils investigated here. Indeed, little or no toxic aldehydes, nor epoxystearoyl species, were generated in the MRAFO oil at recommended shallow-frying time-points of 5–20 min. Since we have also, for the first time, demonstrated the availability of potentially health-threatening levels of cytotoxic and genotoxic 1H NMR method66, which involves expression of the intensity (I) of the intelligently-bucketed omega-3 FA chain terminal-CH3 function resonance  chemical shift bucket to that of the total FA chain terminal-CH3 signals [i.e. I0.97/(I0.90 + I0.97)], the δ = 0.90 ppm one representing that for all non-omega-3 FAs. In this manner, the molar percentage omega-3 FA (predominantly linolenic acid) contents of these oils was found to be 0.20, 1.89, 10.61, 1.37 and 0.92 molar % for the sunflower, corn, canola, extra-virgin olive and MRAFO oil products tested, respectively. Where appropriate, correction was made for the interfering 13C satellite resonance of the major terminal-CH3 one, i.e. especially for COs with low or very low omega-3 PUFA FA contents.Sunflower, corn, canola, extra-virgin olive and MRAFO oils were all purchased from UK or USA retail stores. Each oil was then de-identified in the laboratory via its transference to coded but unlabelled storage containers. The specified SFA, MUFA and PUFA contents of these oils were 11.0, 28.0 and 61.0% for sunflower oil; 14.4, 23.3 and 61.4% for corn oil; 7.0, 64.4 and 28.5% for canola oil; 13.0, 77.4 and 9.4% for extra-virgin olive oil; and 4.0, 91.2 and 4.2% (w/w) respectively for MRAFO. [MUFA]:[PUFA] % content ratios for these oils were 0.46, 0.38, 2.26, 8.23 and 21.71 for sunflower, corn, canola, extra-virgin olive and the MRAFO products investigated respectively. The molar percentage of omega-3 FAs in these samples was estimated by a previously reported −1) of the antioxidant product Fortium® brand MT70 IP liquid, which contained a mixture of α-, β-, γ- and δ-tocopherols  in sunflower oil .The MRAFO oil purchased was supplemented with 1,000 ppm (1.00 g.kgca. 0.25 ml) of oil samples were collected at the 0, 5, 10, 20, 30, 60 and 90 min. heating time-points for 1H NMR analysis. Immediately following collection, the lipid-soluble chain-terminating antioxidant 2,5-di-tert-butylhydroquinone (DTBHQ) was added to each oil sample  in order to block or retard the further generation of aldehydes and their CHPD and HPM precursors during periods of storage and sample preparation at ambient temperature. Samples were prepared for 1H NMR analysis within 2 hr. after collection, and were stored in sealed containers within a light-excluded zone whilst awaiting analysis.All oils were heated at 180 °C for periods of up to 90 min. according to a LSSFE, and these experiments were conducted by a ‘blinded’ laboratory researcher. Each 90 min. heating cycle was completed n = 6 replicated sessions for all oils investigated. This shallow frying simulation involved the heating of a 6.00 ml volume of culinary oil in an air-dried 250 ml glass beaker within a thermostated silicon oil bath maintained at a temperature of 180 °C throughout the total heating period. Aliquots  operating at a frequency of 399.94 MHz and a probe temperature of 293 K. All spectra were acquired as described in8. Typically, a 0.20 ml aliquot of each oil sample was diluted to a final volume of 0.60 ml with deuterated chloroform (C2HCl3) containing 3.67 mmol.L−1 tetramethylsilane (TMS) and 15.00 mmol.L−1 1,3,5-trichlorobenzene : the C2HCl3 diluent provided a field frequency lock, the TMS acted as an internal chemical shift reference (δ = 0.00 ppm), and 1,3,5-TCB  served as an internal concentration reference standard. These solutions were then placed in 5-mm diameter NMR tubes. Typical pulsing conditions were: 128 or 256 free induction decays (FIDs) using 65,536 data points and a 4.5 s pulse repetition rate, the latter to allow full spin-lattice (T1) relaxation of protons in the samples investigated. Resonances present in each spectrum were routinely assigned by a consideration of chemical shifts, coupling patterns and coupling constants. One- and two-dimensional COSY and TOCSY spectra were acquired to confirm 1H NMR assignments as previously described8.1H NMR spectral profiles for the determination of seven classes of aldehyde, and selected epoxy acids, is described in section S15 of the supplementary material section.Preprocessing of these 67, i.e. PSI = [0.025(% monoenoic FA)] + [1.00(% dienoic FA)] + [2.00(% trienoic FA)] + [4.00(% tetraenoic FA)] + [6.00(% pentaenoic FA)] + [8.00(% hexaenoic FA)]. However, for all oils investigated here, contributions to the PSI from tetraenoic, pentaenoic and hexaenoic FA sources were negligible.From the FA compositions of these oils, their intrinsic peroxidative susceptibility indices (PSIs) were computed as previously described1H NMR analyses of culinary oil and potato chip determinations of aldehydic LOPs are provided in section S12 of the supplementary materials section. These include evaluations of the accuracies and precision of each of these assay determinations, together with corresponding lower limits of detection (LLOD) and quantification (LLOQ) values for trans-2-alkenals, trans,trans-alka-2,4-dienals and n-alkanals. These parameters were evaluated in both neat C2HCl3 solutions, and also unheated (control) culinary oils either ‘spiked’ with trans-2-octenal and n-hexanal, or unspiked. ‘Between-frying cycle’ and repeat determination coefficients of variation (CV) for these LOP assays are also provided.Details regarding the quality assurance/quality control monitoring of our trans-2-alkenals and n-alkanals (0–600 µmol.L−1 and 0.20–50.00 mmol.L−1) were linear, with R2 values ≥ 0.993 for neat C2HCl3 solutions, and ≥0.987 for aldehyde-‘spiked’ C2HCl3-diluted oil media prepared as described above.Calibration curves for typical 2HCl3 were added (predominantly ca. 1.0 ml per g), and these mixtures were then mechanically homogenised using an electric pestle rotor . Subsequently, the homogenates were then centrifuged at 10,000 × g for a period of 10.0 min. at 4 °C. Exactly 0.60 ml volumes of each clear supernatant were removed and then treated with either 0.06 or 0.12 ml aliquots of a 1.00 × 10−2 mol./L stock solution of the 1,3,5-TCB internal standard  in C2HCl3, and 0.06 ml of a 10.0 mmol.L−1 solution of the chain-breaking antioxidant DTBHQ , also in C2HCl3, the mixtures thoroughly rotamixed, and then transferred to 5-mm diameter NMR tubes for analysis. C2HCl3 extractions of FFRPCSs were performed either singly, or as 3 or 4 replicates of these samples as indicated in Table 1H NMR spectra of these extracts were acquired in duplicate.FFRPCSs were purchased from a total of 12 local fast-food restaurants. To accurately weighed quantities of each sample (1.00–6.30 g), 1.00–8.00 ml volumes of C1H NMR detection of the DTBHQ antioxidant in 1H NMR spectra of these FFRPCS sample extracts  confirmed that it had been predominantly retained and not consumed via its chain-terminating antioxidant actions during the sample preparation stages of our experiments.The bis-allylic-CH2-, -CH2-CH = CH- and α-CH2-CO2- function resonances , i.e. an adaption of the method described in9.The SFA, MUFA and PUFA contents of these fried food samples was estimated via integration of ISBs featuring their acylglycerol et al.31. The deep-frying facility employed was a domestic model  equipped with a variable thermostat and an inert cross-lined steel mesh for the purpose of lowering the chips into the oil without contacting the fryer’s inner surface. This deep fryer was filled with 3.00 litres of oil according to the manufacturer’s instructions, and 400 ± 10 g of potato chips then deep fried at a temperature of 170 °C for a period of 10.0 min.Eight batches of hand-cut chips of lengths and widths of 87.00 ± 1.15 and 12.70 ± 0.36 mm (mean ± SEM) respectively were consecutively fried 8 times using sunflower, extra virgin olive and the MRAFO oils. For this purpose, we used a modification of the approach utilised by Boscou The % (w/w) total PUFA, MUFA and SFA contents of the frying oils employed for these deep-frying studies were 61.0, 28.0 and 11.0%, respectively for sunflower oil, and 30.8, 62.3 and 6.9% respectively for extra-virgin olive oil.Each frying oil used was allowed to cool for a period of exactly 30 min. between each repetitive frying episode . Following each 10 min. frying episode, chips were thoroughly shaken in their wire basket for 15 s, and then allowed to drain therein for 30 s in order to remove excess oil. Subsequently, these chips were transferred to a steel mesh draining board.1H NMR analysis. Two samples of the unfried (raw) potatoes were also collected and subjected to storage in this manner.For each of the 8 consecutive frying episodes, 2 randomly-selected samples of chips were transferred to plastic-stoppered sample tubes and immediately frozen at a temperature of −20 °C until transported to the laboratory where they were then stored at −80 °C for a maximum duration of 18 hr. prior to On completion of each frying episode, duplicate samples of each oil were also collected for analysis, and these were also stored prior to analysis in the same manner as the potato chip samples, as were duplicate unheated (control) frying oil samples. Further samples of these oils were collected at a time-point of 10.0 hr. following completion of each repeated frying session.1H NMR reference purposes, including n-hexanal, n-octanal, trans-2-octenal, trans-2-nonenal trans,trans-deca-2,4-dienal, etc. were obtained from the Sigma-Aldrich Chemical Co. (UK). Crotonaldehyde  was also purchased from Sigma-Aldrich as a 20:1 molar ratio of its trans-(E-):cis-(Z-) isomers in order to permit assignments of resonances for cis-2-alkenal LOPs in thermally-stressed culinary oils.Authentic aldehydes employed for 1H NMR aldehydic class ISB intensity datasets involved an analysis-of covariance (ANCOVA) model, which incorporated 2 prime factors and a total of 3 sources of variation: (1) that ‘between-culinary oils’, qualitative fixed effect (Oi); (2) ‘between-sampling time-points’ quantitative fixed effect ‘nested’ within ‘culinary oils’ (Tj); and (3) the culinary oil x time-point first-order interaction effect (OTij). This experimental design is represented by equation ijk represents the (univariate) aldehyde ISB dependent variable values observed, μ its overall population mean value in the absence of any significant, influential sources of variation, and eijk the unexplained error  contribution.For experiments featuring the exposure of culinary oils to LSSFEs, the experimental design for univariate analysis of the total acylglycerol-normalised XLSTAT2016 software. Datasets were generalised logarithmically (glog)-transformed, mean-centred and standardised prior to analysis in order to satisfy assumptions of normality and homoscedasticity. Post-hoc analysis of significant differences observed between individual culinary oils and sampling time-points were performed using the Bonferroni method which corrected for false discovery rates.ANCOVA was conducted with Metaboanalyst 3.0 software module options. Datasets  were analysed either untransformed and unscaled, or alternatively after cube root- or glog-transformations, and Pareto scaling. AHC dendograms were generated employing Euclidean distance and Ward’s linkage clustering algorithm. Heatmap and correlation feature diagrams were also obtained using this software module.Agglomerative hierarchal clustering (AHC), principal component analysis (PCA) and Pearson (linear) correlation analysis of the extensive culinary oil NMR-based aldehyde concentration dataset was performed using ijk represents FA content-normalised sample aldehyde dependent variable values, Si and Fj the ‘between-sample’ (i.e. potato chip vs oil levels) and ‘between-sequential frying episode number’ sources of variation (both fixed effects), μ the overall population mean value in the absence of any significant, influential sources of variation, and eijk the unexplained error  contribution.For experiments featuring the domestic deep-frying of potato chips in sunflower oil only, statistical analysis of experimental data was performed according to the ANOVA mathematical model described by equation SUPPLEMENTARY INFORMATION