Human Second Window Pre-Conditioning and Post-Conditioning by Nitrite Is Influenced by a Common Polymorphism in Mitochondrial Aldehyde Dehydrogenase

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SUMMARY
Pre-conditioning is an exciting physiological phenomenon that, despite great efforts, has so far resisted translation to mainstream clinical medicine. Many potential triggers (e.g., ischemia of the organ in question or a remote organ, many different drugs) have been investigated, but recent work has implicated activation of mitochondrial aldehyde dehydrogenase (ALDH2) as central to the process. A genetic polymorphism, known as ALDH2*2, is common worldwide (present in up to 40% of Han Chinese people) and produces a functionally different enzyme. The authors used a variety of protocols in the human ischemic forearm model, in participants with both enzyme types, to assess cytoprotection with low-dose sodium nitrite and attempt to further in recent years (1)(2)(3). Nitrite may have a role in hypoxic vasodilation (4) and has been shown to protect renal tissue (5,6), liver (7), and myocardium (8,9)  Mitochondrial aldehyde dehydrogenase (ALDH2) is a member of the 19-strong human aldehyde dehydrogenase family of NAD(P) þ -dependent enzymes (12). A common polymorphism in exon 12 (Glu487Lys, or Glu504Lys in the unspliced protein), known as the ALDH2*2 allele, is present in up to 50% of individuals of East Asian descent (13). Heterozygosity at this allele results in a near inactive enzyme and produces the "Asian Flushing" phenotype, a phenomenon linked to the accumulation of acetaldehyde following alcohol ingestion; mutation of a single subunit destabilizes the cofactor binding site and dimer interface such that heterozygotes are functionally similar to homozygotes with the variant allele (14). Individuals possessing 1 or 2 copies of the ALDH2*2 allele may be at greater risk of coronary artery disease (15) and myocardial infarction (16). ALDH2 activation by phosphorylation has been postulated to be central to protection conferred against myocardial ischemia reperfusion injury (17). Pre-conditioning was induced by activation of PKCε (which phosphorylates ALDH2) and subsequently by a direct activator of ALDH2, alda-1. In a later study, the volatile anesthetic isoflurane induced cardioprotection in a rat model (18).
Protection was associated with activation of ALDH2 and was abolished by an inhibitor of PKCε. On the basis of these data, combined with the observation that ALDH2 also exhibits intrinsic nitrite reductase activity (19), we hypothesized that an interaction between ALDH2 and nitrite might contribute to IR protection in humans.
We hypothesized that nitrite would be protective in the human forearm, either when administered 24 h before ischemia reperfusion ("second-window preconditioning") or when administered during ischemia (with the primary effect in the "post-conditioning" window), and that its protective effects would be modified by variations in ALDH2 activity. We used a combination of genetic and pharmacological tools in an established model of IR injury in the human forearm (20), to investigate protection by nitrite and the role of ALDH2.

METHODS
This study was approved by the local research ethics committee. All participants gave written informed consent. All studies were performed in a dedicated   (20). The study design is summarized in Figure 1A.  Values are mean AE SD or n.
ALDH2 ¼ mitochondrial aldehyde dehydrogenase; BMI ¼ body mass index; HR ¼ heart rate; MABP ¼ mean arterial blood pressure. there was a washout period of >1 week between any given studies in the same   Expression and purification of ALDH2 was performed as previously described (22). Dehydrogenase activity was measured by monitoring the formation of NADH as an increase in light absorbance at 340 nm in 50 mmol/l sodium pyrophosphate buffer (pH 7.5) containing 0.4 mmol/l acetaldehyde, 10 mmol/l MgCl 2 , and 5 mmol/l NAD. After 2 min of equilibration, the reactions were started by the addition of ALDH2*1 or ALDH2*2 (19 and 111 mg/ml final concentrations, respectively) and monitored for w3 min to obtain initial reaction rates. Activities were subsequently measured after the addition of 10 mmol/l NaNO 2 and 1 mmol/l dithiothreitol.
TREATMENT PROTOCOLS. Treatment protocols are summarized in Figure 1B.

RESULTS
All subjects tolerated the procedures with no complications. Baseline characteristics of volunteers are Ormerod et al.
given in Table 1. All Caucasian participants were homozygous for the wild-type ALDH2 (ALDH2*1/*1) allele. Of the East Asian participants, 11 were homozygous for wild-type ALDH2 (ALDH2*1/*1) and 11 were heterozygotes (ALDH2*1/*2). One was homozygous for the variant ALDH2 (ALDH2*2/*2) and was excluded from the analysis.  The IR protocol did not affect heart rate or blood pressure in any group; resting heart rate and blood pressure were similar on each study day (data not shown). Changes in endothelium-independent FBF were similar between groups (data not shown).
Twenty minutes of forearm ischemia induced significant endothelial dysfunction in individuals with (p < 0.0001, n ¼ 9) or without (p ¼ 0.0001, n ¼ 9) the ALDH2*2 allele ( Figures 3A and 3B).  The NIAMI study in acute ST-segment elevation myocardial infarction was also negative (11). Our results in those homozygous for wild-type ALDH2*1 are consistent with these previous studies in demonstrating no reduction in ischemia reperfusion injury when nitrite is administered during ischemia.   (25); however, the relevance of this mechanism in humans is unclear.
Because the study involves puncture of the brachial artery, it was felt that it was not reasonable to require more than 2 runs from each participant.
Ideally, the effect of disulfiram and delayed protection would have been studied in the East Asian population, but insufficient participants were recruited.
It was not possible to analyze as a crossover study, because not all participants completed both arms of the study. The data from placebo and nitrite runs were analyzed separately in the same manner as previously discussed (20,26  activation by nitrite was investigated using the homozygous variant enzyme because it is a dominantnegative polymorphic variant (22).
Clinical translation in the area of pre-conditioning and post-conditioning, despite ongoing efforts, has been slow. Direct activation of the ALDH2 pathway remains an attractive area of investigation (27); however, the negative NIAMI study (11) has somewhat reduced enthusiasm for further trials of exogenous nitrite in ischemia. More recent work has focused on a potential role of endogenous nitrite in cytoprotection, for example as a mediator of remote ischemic pre-conditioning (28). In a mouse model of myocardial infarction, this study confirmed the role of nitrite by use of a nitrite scavenger to abolish the effect of RIPC, and then recapitulated this protection with nitrite-supplemented plasma. In their editorial accompanying this study, Corti and Gladwin (29) argued that reliance on animal models may be limiting our ability to translate this undoubtedly exciting physiological process to mainstream medicine. Equally, human ischemic models (such as the one used here) may not translate to other tissues or organs, or to pathological states, though they may well provide insight into the mechanisms involved.
It is generally agreed that cytoprotection proceeds down a final common pathway (30), so it seems likely that protection by nitrite seen in the forearm may be achieved in other tissues under the correct conditions. However, as Corti and Gladwin (29) argue, multiple factors-the nitrite dose, the plasma levels achieved, and the timing of interventioncreate a complex system and are all critical to the successful translation of cytoprotection to human patients.

CONCLUSIONS
This proof-of-concept study of endothelial protection in healthy volunteers provides evidence that inorganic nitrite may have therapeutic use to prevent IR injury in man. Specifically, we demonstrate nitrite-induced second-window pre-conditioning in humans and identify the important impact of a common global polymorphism on post-conditioning with nitrite.