The molecular nature, toxicities and health hazards potentially presented by aldehydic LOP toxins have been previously explored in some detail, as have analytical strategies available for their determination and monitoring, e.g. , in fried food sources, and human/animal biofluids and tissues, for probing their in vivo absorption, biodistribution, metabolism and urinary excretion (an example of the 1
H NMR analysis of aldehydes, specifically LOPs and vanillin, in a typical non-fried food product is shown in Figure 4
). Indeed, the toxicological and pathogenic properties conceivably arising from the ingestion of aldehydic LOP-containing COs heated according to standard frying practices (in the form of CO-absorbing fried foods for humans), and also aldehyde model systems, include their potential roles in the development and perpetuation of cardiovascular diseases [54
], their carcinogenic [57
], gastropathic [62
], pro-inflammatory [63
], and teratogenic properties [64
], contributions towards neurodegenerative disorders [65
], their hypertensive effects [66
]; the development and perpetuation of diabetes [67
];and respiratory and pulmonary complications, the latter especially for acrolein [68
]; this list is inexhaustive. The inhalation of volatile aldehydes and other carbonyl compounds by workers employed in poorly-ventilated fast-food/restaurant retail outlets is also considered to pose a major threat to human health [69
], particularly with reference to established links between an increased incidence of lung cancer and cooking oil fume inhalation amongst such personnel [13
]. Indeed, since a wide range of aldehydic LOPs such as acrolein (the lowest homologue trans
-2-alkenal) have boiling-points (b.pts) below or far below standard frying temperatures (ca.
180 °C), cooking oil fumes are very rich indoor air pollutant sources of these toxins.
3.2.1. In Vivo Absorption of and Metabolic/Biotransformation Routes for Aldehydic LOPs
The GI tract is continually exposed to toxic aldehydes, and subsequent to digestion they are absorbed into the lymphatic system, or directly into the systemic circulation [73
]. Indeed, in 1998, our laboratory demonstrated that typical trans
-2-alkenals generated during the thermal stressing of PUFA-containing frying oils (trans
-2-pentenal and -nonenal) are indeed absorbed from the gut into the systemic circulation in vivo, then metabolised by a process involving the primary addition of GSH across their electrophilic carbon-carbon double bonds, and finally excreted in the urine as C-3 mercapturate alcohol derivatives, i.e., as N
-cysteine and -(3-hydroxy-nonyl)-L-cysteine derivatives, respectively, in experimental rats [73
]. However, the administered levels of these aldehydes were as high as 10 and 100 mg/kg. Generation of these metabolites also involves reduction of their chemically-reactive aldehyde/aldehyde hydrate (-CHO/-CH(OH)2
) functions to primary alcohol species via the actions of hepatic alcohol dehydrogenase. These results were consistent with the findings made in [50
], which provided evidence for the at least partial absorption of such aldehydes into the circulation.
However, it should also be noted that this study found that at a 16 hr. post-dosing time-point, approximately 15% of the orally-administered dose of trans
-2-nonenal was oxidatively transformed to its corresponding carboxylic acid metabolite within the stomach [73
Consistently, following the subcutaneous injection of the simplest trans
-2-alkenal acrolein to rats, N
-cysteine was detected and isolated as a key urinary excretion product [74
], and these results ae also fully consistent with our 1
H NMR-based urinary metabolic screening investigations [73
], including the hepatic metabolic reduction of the aldehyde functions to alcohol derivatives. However, for these experiments, acrolein was administered by the subcutaneous injection of a 1% (v/v) solution in arachis (peanut) oil into the lumbar region; the vehicle may itself have served as a source of aldehydic LOPs, especially if allowed to peroxidise during periods of storage or solution preparation.
In a scientifically elegant and highly informative early study published in 1985, McGirr et al. [75
] found that a significantly high proportion of dietary MDA is covalently linked to dietary proteins, and an acid-labile urinary metabolite (the Nα
-acetyl derivative of the lysine-MDA enaminal Nε
-(2-propenal) lysine) was detectable in experimental rats following oral administration of a serum albumin-MDA adduct at a level of 2 mg MDA equivalents/kg BW. Furthermore, this compound was also demonstrated to be a major urinary metabolite of this dialdehyde administered as its sodium enolate salt via stomach intubation. Elevated concentrations of this metabolite were excreted by rats fed a diet rich in highly-peroxidisable cod liver oil. However, these researchers were also able to identify low levels of this metabolite in the urine of fasted rats, and this observation provided evidence that it is also formed as a product derived from the in vivo peroxidation of PUFAs, in addition to its ingestion as a dietary LOP (such as those formed during high temperature frying practices in the human diet), or alternatively, through the prolonged storage of PUFA-containing foods. Injection of MDA as its sodium enolate salt to fasted animals markedly increased its urinary concentration, as expected. In view of the acid lability of Nε
-(2-propenal) lysine, it is possible that free MDA may be liberated from this primary Schiff base product, and perhaps also from more prevalent dietary protein lysyl residue adducts, in the GI tract (particularly the stomach), so that it may be ingested into the systemic circulation as a free (non-adducted) agent.
One recent key investigation appears to have resolved the longstanding critical question regarding whether there is some clinically-significant in vivo absorption of 4-hydroxy-trans
-2-alkenals, potentially one of the most toxic classes of α,β-unsaturated aldehydes available in human dietary sources [76
]. Details of this study are provided in Section S5 (Supplementary Materials)
Since HNE is universally considered to represent a very important secondary LOP, its metabolic fate has been extensively investigated. An exhaustive review of the roles of 4-HNE in health and disease is provided in [77
], including a detailed evaluation of its metabolic and biotransformation products. However, important examples of studies of its metabolic fate both in vivo and in vitro
are also provided in Section S5 of the Supplementary Materials
. Interestingly. HNE-modified proteins also appear to be key features of metabolic diseases, and hence offer potential to serve as effective biomarkers for such conditions [78
3.2.2. Associations between Dietary Fried Food Aldehyde Concentration Patterns and Those of Human Blood Plasma: Potential Tracking of Dietary LOPS In Vivo?
In 2000, Mak et al. [79
] determined a total of 22 individual aldehydes in circulating arterial blood plasma samples collected from n
= 8 patients with congestive heart failure (CHF), along with those from an equivalent number of age-matched participants with normal left ventricle (LV) function, i.e., non-CHF controls. Aldehydes were determined via
a GC/MS bioanalytical strategy, and these included long- and short-chain n
-2,4-alkadienals, MDA and the dietary flavouring agent furfural. Mean plasma concentrations, or ranges for the mean aldehyde concentration values of specific structural homologues within each class, are provided in Table 3
for both control and CHF groups, as are full ranges for the individual sampling values found in n
= 36 samples of potato chips collected from fast-food/take-away restaurant outlets.
The blood plasma results acquired in [79
] demonstrated that CHF patients had significantly higher levels of total aldehydes, together with a range of unsaturated ones (specifically, trans
-2-alkenals and 4-hydroxy-trans
-2-alkenals, the latter including HHE and HNE), and furfural. Conversely, the normal LV function control group involved had significantly higher levels of n
-alkanals over those of the CHF patients. Furthermore, the dietary flavouring agent furfural was by far the most predominant aldehyde present, i.e., 37 and 44% of the total aldehydes determined in control subjects and CHF participants respectively) and was found be significantly upregulated in the latter. Furfural is not a LOP, but in addition to its potential genotoxic and carcinogenic properties [73
], this food flavourant has been shown to give rise to the accumulation of ROS and cellular damage in Saccharomyces cerevisiae
However, aldehydes of the 2,4-alkadienal class monitored in these samples only featured trans,trans-hepta- and trans,trans-2,4-nonadienals, and the only other di-unsaturated aldehyde monitored was trans,trans-2,6-nonadienal. Moreover, cis- and trans-deca-4-enals were measured as a combined sum. Additionally, this study was complicated by (1) the very high incidences of comorbidities in the male participants recruited to it (mean within-group ages ca. 60 years), specifically diabetes, hypertension, and hyperchloesterolemia in both groupings, and (2) medical therapies received by them, i.e., β-blockers, nitrates, ACE inhibitors and calcium channel blockers in both groups, and additionally diuretics in the CHF one. Notably, all vitamin supplements were withheld from participants for a minimum duration of 7 days prior the study, and all oral medications were withheld on the morning of the investigation.
From these results, we therefore elected to perform a comparative statistical evaluation of these blood plasma LOP profiles in terms of the mean molar levels of different classes of aldehydes determined therein expressed as a proportion of the total LOP-relevant aldehyde concentration found in the samples analysed, i.e., those within the above control and CHF groups, to those of the same mean molar ratios of the aldehyde classification contents found in frequently-consumed fried potato chip samples collected from fast-food restaurants (Table 2
and Table 3
), specifically those fried in commonly-utilized vegetable oil frying media, as noted in Section 2.3
above. The use of molecular ratio variables for this exercise is, however, quite fortuitous, since they are expected to be less sensitive to the potential influences of a range of latent generic variables such as participant BMIs, ages, etc.
For this purpose, blood plasma levels of furfural were excluded from the computation of proportionate aldehyde contents since it is not a LOP, and nor was it detectable in any of the fried potato chip samples analysed by 1
H-NMR analysis Unfortunately, it was also not possible to compute the relative proportions of alka-2,4-dienals in the above two blood plasma groups, since trans,trans
-2,4-decadienal, the major trans,trans
-2,4-alkadienal arising from the peroxidative deterioration of linoleoylglycerols (Section S2
), was not determined in [79
], and neither was HHE, the major 4-hydroxy-trans
-2-alkenal derived from the decomposition of CHPDs generated from the oxidation of ω-3 FAs, e.g. , α-linolenoylglycerols. Short-chain aldehyde concentrations provided in this report were those for n
-butanal only – since this was the only such analyte included, these values were also removed from the dataset prior to statistical analysis, although they do remain valuable, since such aldehydes predominantly arise from the peroxidation of ω-3 FAs [12
]. Therefore, each proportionate aldehyde class considered comprised those of n
-2-alkenals and MDA only, and all proportions computed represented the concentrations of each of these LOPs divided by the sum total of them, plus those of all possible alkadienals found. In view of these limitations, results obtained from these comparative evaluations should be treated with some caution.
The mean relative proportions (ratios) of the concentrations of long-chain n-alkanals:trans-2-alkenals:4-hydroxy-trans-2-alkenals:MDA in these three groups of samples were compared and statistically tested for any significant differences between them. Expressed as percentages of the total aldehydes detectable (minus contributions from furfural), these ratios were: 40:31:0.20:0.60 for fried potato chips (mean percentages for a newly-acquired 1H-NMR dataset, n = 36); 46:30:9:2 for normal LV function (control) subject blood plasma; and 26:39:17:2 for CHF patient blood plasma. Direct comparison of these proportions for the potato chip sample profiles with those of the control blood plasma group showed that although the trans-2-alkenal and, to a lesser extent, long-chain n-alkanal values were quite similar for this comparison, those of 4-hydroxy-trans-2-alkenals were much elevated in the latter, and these data indicate that, in addition to post-ingestional, aldehyde class-dependent modifying factors such as differential rates and extents of their absorption, metabolism, chemical reactivity, protein adduct formation and biodistribution, etc. between each aldehyde class considered, this aldehyde classification appears to arise from in vivo peroxidation processes. Moreover, although the proportionate MDA levels remained small for both these groups, such mean values were elevated approximately 4-fold in the normal LV function blood plasma one.
However, a further major consideration is the dietary availability of all aldehydes considered, i.e., what proportion of them are ‘free’ and what are constituted as adducts with food proteins (as noted for MDA [75
]), alternative biomacromolecules, or low-molecular-mass nutrient metabolites such as free amino acids?; such adducts may represent latent sources of these toxins, which may be liberated within the GI system, for example. Notably, our laboratory determines the ‘free’, non-adducted form of these aldehydes in fried food products, and hence our estimated values (Table 2
and Table 3
) will presumably represent underestimates of the total taken up from COs during frying practices.
A permutation testing strategy performed via partial redundancy analysis (PRDA) on the log10-transformed proportionate aldehyde level dataset (involving 104 permutations) revealed that aldehyde classification-conditioned differences observed between the three sample groups were statistically significant (p = 0.049), as indeed were those ‘between-aldehyde classifications’ (p = 0.009), the latter being expected, of course (the log10-transformation was required to counteract within-sample negative correlations between proportionate/percentage variables). These significant differences were clearly manifested by 4-hydroxy-trans-2-alkenals and MDA being much greater in the normal LV function (control) blood plasma profiles over those of fried potato chips. However, they also arise from the CHF blood plasma group having upregulated proportionate trans-2-alkenal and 4-hydroxy-trans-2-alkenal levels (over both the control plasma and potato chip serving groups), and significantly higher proportionate MDA concentrations than the fried potato chip group. This significant ‘’between-sample group’ effect observed is also explicable by the large differences observed between the proportionate levels of total n-alkanals between the CHF group and the two others compared.
Therefore, the observation of very similar fractional aldehyde contents of both n
-alkanals and trans
-2-alkenals in the large potato chip and smaller control blood plasma sampling groups may serve to indicate that such LOPs are dietary-derived. If this is the case, then the in vivo ‘conservation’ of their proportionate levels may also reflect the overall lower, albeit differential chemical/biochemical reactivities of these classes of aldehydes than those of 4-hydroxy-trans
-2-alkenals and MDA, following their ingestion by humans. Of particular note, in vivo, n
-alkanals serve as substrates for pyruvate dehydrogenase, but α,β-unsaturated aldehydes are not affected by this enzyme [81
]. Moreover, as noted above, unsaturated aldehydes readily take part in Michael addition reactions with GSH to form their primary detoxification GSH conjugate products [82
], but n
-alkanals clearly do not (although they may form Schiff base adducts with the terminal amino function of this tripeptide). However, the rather substantial differences observed between these two groups’ proportionate 4-hydroxy-trans
-2-alkenal and MDA concentrations certainly indicate, but do not confirm, that such toxins may be generated from in vivo lipid peroxidation processes.
Proportionate total 4-hydroxy-trans
-2-alkenal levels in the CHF blood plasma group were also significantly greater than those of the normal LV function control group (ca.
2-fold), but this observation was reversed for long-chain n
-alkanal concentrations, the latter results being consistent with our proportionate levels estimated in fried potato chips. The markedly elevated proportionate value of CHF blood plasma levels of the former class of aldehydes may have been expected in view of an enhanced level of in vivo oxidative stress associated with this condition. However, if this was the case, why was it that the mean level of furfural, a non-LOP dietary flavouring agent, was also significantly increased from 2.45 µmol/L in the control group to 4.06 µmol/L in the CHF one (p
< 0.01)? Possibly these differences are also partially explicable by differing dietary regimens between these two groups, perhaps an increased level of aldehyde-loaded fried food consumption and/or an enhanced furfural intake in the latter (this flavouring agent is readily absorbed subsequent to administration by any route [84
])? Study participants were not fasted for a minimum required period prior to the collection of blood samples in this study, so it certainly appears that such aldehydes may at least partially arise from such dietary, or perhaps alternative exogenous sources. Further possible limitations of the study reported in [79
] are detailed in Section S6 (Supplementary Materials)
However, the authors of [79
] also suggested that differences in the aldehyde profiles between their two groups may arise from those between the FA compositions of their diets [73
], i.e., with a possible higher peroxidised PUFA and therefore aldehyde content of those received by the CHF one, but they also indicated that such systematic dietary variations between them were unlikely.
In the control group of participants, the order of decreasing total blood plasma total trans
-2-alkenal concentrations were (peroxidised FA source(s) in brackets, with L, α-Ln, γ-Ln, O, Po and Ar representing predominant linoleoyl-, α-linolenoyl-, γ-linolenoyl-, oleoyl-, palmitoleoyl- and arachidonylglycerols respectively) trans
-2-octenal (L) > trans
-2-hexenal (α-Ln - minor aldehydic LOP, but also a major dietary flavouring agent (Section 3.1
)) > trans
-2-heptenal (O and L) > trans
-2-nonenal (Po, Ar and γ-Ln, but also a food flavouring agent); that for n
-alkanals was n
-heptanal (L) > -nonanal (O) > -octanal (O) > -hexanal (Ar/L, but also derived from the decomposition of trans,trans
]); and that for 4-hydroxy-trans
-2-alkenals was HHE (α-Ln) > HNE (L and Ar) >>> 4-hydroxy-trans
-2-decenal (L) ≈ 4-hydroxy-trans
-2-octenal (unknown peroxidised FA sources). As expected, for the 4-hydroxy-trans
-2-alkenals determined, the most predominant ones were those arising from the sequential peroxidation of α-linolenoyl- (HHE) and linoleoyl-/arachidonoylglycerols (HNE).
Using the somewhat broad assumption that a highly significant fraction of at least some of these blood plasma aldehyde levels arise from dietary sources, it should be considered that those found therein represent only residual concentrations, i.e., what remains following their in vivo consumption through their metabolic fate in the GI system, in vivo absorption and then further metabolism thereafter in organs such as the liver, along with any biotransformation of them in human blood plasma and other environments, e.g. , the generation of protein carbonyl species from the reaction of α,β-unsaturated aldehydes with plasma proteins such as human serum albumin and gamma-globulins, and additional Schiff base products arising from the reactions of all possible aldehydes with free primary and secondary amine functions present in selected biomolecules, for example. ‘Between-aldehyde class’ differences in the rates and extents of their consumption will also account for those observed between their relative blood plasma concentrations. In principle, since α,β-unsaturated aldehydes are more chemically-reactive than saturated ones [82
], should we perhaps expect higher n
-2-alkenal ratios in human blood plasma than what is found in fried food products? Indeed, this ratio is already significantly > 1 in fried potato chip samples (mean ± SEM 1.39 ± 0.10 for our dataset (Table 2
); 95% confidence intervals 1.18–1.61, p
< 0.01) than it is in the culinary oil sources of these aldehydes, and this has been attributed to their differential levels of reactivity with potato proteins, amino acids and further biomolecules between these two classes of LOP toxins [3
]. This was indeed the case in the above normal LV function group plasma profiles explored, the ratio being 1.53; however, this difference observed was found not to be statistically significant from that found in fried potato chips (one sample t-test). Notwithstanding, it may be conjectured that these proportionately lower control group blood plasma trans
-2-alkenal levels may also arise from a higher level of reactivity of them in vivo. Interestingly, the fractional blood plasma MDA aldehydic LOP content in these control participants (only 2%) was found to be ca.
3-fold greater than that observed in potato chip samples (0.60%). Nevertheless, this observation again confirms that MDA represents only a minor secondary LOP.
Overall, also important is the observation that the total unsaturated aldehyde content of normal LV function patient blood plasma is significantly greater than that of saturated aldehydes, and this is indeed also the case for estimated weight percentage human dietary intakes of these toxins by humans in Refs. [19
], i.e., an unsaturated:saturated aldehyde ratio of 5:2 in mg/kg units [85
]. Corresponding weight percentage (ppm) values for fried potato chip samples were found to be a very similar value of ca.
A further study performed by Ogihara et al. in 1999 [87
], which determined the blood plasma concentrations of secondary aldehydic LOPs in premature infants with and without chronic lung disease (CLD), was, however, limited to only 3 long-chain n
-alkanals and 4 trans
-2-alkenals, together with HNE. Full details of this study are available in Section S7 of the Supplementary Materials
However, it is clear that further research investigations targeted on dietary patterns, human intake, GI fate, absorption, biodistribution and further metabolism of such dietary LOPs are required in order to ratify potential relationships between their dietary availabilities and those detected in human biofluids and tissues. Although not simply conceivable for all patient and age-matched control groups investigated in human trials, it is also thoroughly recommended that for future clinical studies focused on explorations of oxidative stress in vivo (particularly the in vivo generation of LOPs such as reactive aldehydes), all participants involved should be fasted for a sufficient minimal time period prior to the collection of biofluid or biopsy samples for analysis. Such an approach will presumably overcome any interferences or confounding effects arising from dietary LOP sources.