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Published in final edited form as: Amino Acids. 2018 Sep 6;51(1):97–102. doi: 10.1007/s00726-018-2647-y

Biochemical characterization of the catecholaldehyde reactivity of L-carnosine and its therapeutic potential in human myocardium

Margaret Ann M Nelson 1, Zachariah J Builta 2, T Blake Monroe 2, Jonathan A Doorn 2, Ethan J Anderson 2,3,§
PMCID: PMC6924506  NIHMSID: NIHMS1506009  PMID: 30191330

Abstract

Oxidative deamination of norepinephrine (NE) and dopamine (DA) by monoamine oxidase (MAO) generates the catecholaldehydes 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL) and 3,4-dihydroxyphenylacetaldehyde (DOPAL), respectively, and H2O2. Catecholaldehydes are highly reactive electrophiles that have been implicated as causal factors in the etiology of neurodegenerative diseases and cardiac injury from ischemia and diabetes. The reactivity of both catechol and aldehyde groups enables the catecholaldehdyes to cross-link proteins and other biological molecules. Carnosine is a β-alanyl-histidine dipeptide found in millimolar concentrations in brain and myocardium.It is well-known to detoxify aldehydes formed from oxidized lipids and sugars, yet the reactivity of carnosine with catecholaldehydes has never been reported. Here we investigated the ability of carnosine to form conjugates with DOPAL and DOPEGAL. Both catecholaldehydes were highly reactive toward L-cysteine (L-Cys), as well as carnosine; however, glutathione (GSH) showed essentially no reactivity towards DOPAL. In contrast, GSH readily reacted with the lipid peroxidation product 4-hydroxy-2-nonenal (4HNE), while carnosine showed low reactivity to 4HNE by comparison. To determine whether carnosine mitigates catecholaldehyde toxicity, samples of atrial myocardium were collected from patients undergoing elective cardiac surgery. Using permeabilized myofibers prepared from this tissue, mitochondrial respiration analysis revealed a concentration-dependent decrease in ADP-stimulated respiration with DOPAL. Pre-incubation with carnosine, but not GSH or L-Cys, significantly reduced this effect (p<0.05). Carnosine was also able to block formation of catecholaldehyde protein adducts in isolated human cardiac mitochondria treated with NE. These findings demonstrate the unique reactivity of carnosine toward catecholaldehydes, and therefore suggest a novel and distinct biological role for histidine dipeptides in this detoxification reaction. The therapeutic potential of carnosine in diseases associated with catecholamine-related toxicity is worthy of further examination.

Keywords: Catecholamines, aldehydes, carnosine, glutathione, human heart, mitochondria

Introduction

Catabolism of norepinephrine (NE) and dopamine (DA) by monoamine oxidase (MAO) constitutes the principal route of neurotransmitter metabolism in oxidative tissues. MAO metabolizes norepinephrine (NE) and dopamine (DA) to produce the catecholaldehydes 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL) and 3,4-dihydroxyphenylacetaldehyde (DOPAL), respectively, and H2O2 (Eisenhofer, Kopin et al. 2004). Products of oxidative stress inactivate the enzymes responsible for catecholaldehyde metabolism, amplifying their levels and toxicity Florang, Rees et al. 2007, Rees, Florang et al. 2007, Jinsmaa, Florang et al. 2009). Both DOPEGAL and DOPAL have been demonstrated to be far more cytotoxic and reactive than the parent catecholamines (i.e., NE and DA) or any other known metabolites (Burke, Li et al. 2004). The surprisingly high toxicity appears to stem from the reactivity of both catechol and aldehyde groups. Due to their electrophilic nature, catecholaldehydes form covalent and very stable adducts with protein amines (e.g., Lys) and other biological molecules, permanently modifying their structure and function, as previously reported (Kristal, Conway et al. 2001, Mexas, Florang et al. 2011) (Rees, Florang et al. 2009, Jinsmaa, Florang et al. 2011).

Carnosine is an endogenous histidyl dipeptide found in millimolar concentrations in skeletal muscle, heart and brain (Boldyrev, Aldini et al. 2013). The dipeptide has therapeutic effects, exhibiting pH buffering, metal chelating, and antioxidant properties. Numerous groups have documented the ability of carnosine to detoxify and form stable conjugates with oxidized sugar- and lipid-derived aldehydes, such as acrolein and 4-hydroxy-2-nonenal (4HNE) (Hipkiss, Michaelis et al. 1995, Zhou and Decker 1999, Aldini, Carini et al. 2002, Aldini, Granata et al. 2002, Carini, Aldini et al. 2003, Colzani, De Maddis et al. 2016), yet the ability of carnosine to conjugate catecholaldehydes has, to our knowledge, never been examined. Therefore, the aim of this study was to investigate if carnosine can attenuate reactivity and toxicity of catecholaldehydes through the formation of catecholaldehyde-carnosine conjugates. In addition, we sought to compare reactivity of carnosine with other endogenous nucleophiles and antioxidants, such as the tripeptide glutathione (GSH), and L-Cys, to determine if carnosine is a novel scavenger of catecholaldehydes.

Materials and Methods

Chemicals

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise described. DOPAL was purchased from Cayman Chemicals or synthesized via the method of Fellman (Fellman 1958, Anderson, Mariappan et al. 2011). DOPEGAL was biosynthesized using commercially available MAO (Nilsson and Tottmar 1987).

Reactivity of Biogenic Aldehydes

DOPAL (100 μM) was incubated with 1 mM carnosine, 1 mM GSH or 1 mM L-Cys at 37 °C in 50 mM sodium phosphate buffer, pH 7.4. At time points (0, 30, 60, 90 and 120 min), aliquots were taken from the reaction mixture and the reaction stopped by diluting 1:10 with 0.1% trifluoroacetic acid (TFA). Samples were stored at −20°C in freezer or 4°C in auto-sampler prior to analysis. The concentration of DOPAL was measured via a 1200 Series Agilent Capillary HPLC system with a photodiode array detector (202 and 280 nm). Separation was achieved using a Phenomenex C18 Luna column (1×150 mm) with flow rate of 50 μL/min and mobile phase of 0.1% TFA with 6% acetonitrile (isocratic). Dilution of the DOPAL-conjugates in 0.1% TFA and incubation for up to 2hrs at 37°C did not result in appreciable decomposition of the DOPAL-carnosine conjugate. 4HNE (100 μM) was incubated with 1 mM GSH or 1 mM carnosine and time-dependent change in concentration of 4HNE was measured spectrophotometrically at 224 nm. The reaction of 100 μM 4HNE with 1 mM carnosine was allowed to equilibrate for 20 min before recording the time-dependent change in [4HNE] to be used in the rate calculation. Given the rapid reaction of 100 μM 4HNE with 1 mM GSH, the time-point at 60 min (>7 half-lives) was used as the blank for the reaction. Kinetics for the reaction of DOPAL and 4HNE with nucleophiles (e.g., carnosine, L-Cys) were pseudo-first order given the nucleophile (e.g., 1mM carnosine) was in 10-fold excess of the electrophile (e.g., 100 μM DOPAL). During the course of the reaction, the change in [carnosine] would be very little compared to the change in [DOPAL].

Human research participants, informed consent and collection of myocardial samples

This study was reviewed and approved by the Institutional Review Board of the Brody School of Medicine at East Carolina University. Enrolled in the study were patients undergoing elective cardiac surgery who were between the ages of 50 to 70 and presented without a prior history of cardiovascular surgery or arrhythmias. Informed consent for participation in the study was obtained by a member of the research team who was not involved in data collection or analysis. Prior to the initiation of cardiopulmonary bypass, a purse-string suture was made around the portion of the right atrial appendage (RAA) for insertion of the venous cannula. A section of the RAA was then excised and immediately rinsed in phosphate buffered saline. Myocardium was dissected from the endocardial side of the right atrial biopsy and placed in ice-cold Buffer X (7.23mM K2EGTA, 2.77mM CaK2EGTA, 20mM Imidazole, 0.5mM DTT, 20mM Taurine, 5.7mM ATP, 14.3mM PCr, 6.56mM MgCl2-6H2O, 50mM K-MES) and immediately processed for mitochondrial measurements. A small portion was frozen in liquid N2 and maintained at −80°C for protein analysis.

Preparation of permeabilized myofiber and mitochondrial O2 measurements

Techniques used for permeabilization of human cardiac myocardium have been previously described (Anderson, Kypson et al. 2009, Anderson, Efird et al. 2014).

Briefly, myocardial samples were placed in ice-cold Buffer X containing 3mg/mL Collagenase Type I on a rocker at 4°C for 30 minutes. Myocardial tissue was cleared of connective tissue and separated into fibers along the longitudinal axis followed by permeabilization in Buffer X containing 50μg/mL saponin for 30 minutes at 4°C. The fibers were washed in Buffer Z (in mM: 105 K-MES, 30 KCl, 1 EGTA, 10 KH2PO4, 5 MgCl2–6H2O, 5mg/mL BSA) containing 20μM blebbistatin and placed on a rotator at 4°C until analysis (< 1 hour).

Mitochondrial O2 consumption was measured using the O2K oxygraph system (Oroboros Instruments) at 30°C with continuous stirring. Permeabilized fiber bundles were placed in a chamber with Buffer Z Lite (in mM: 105 K-MES, 30 KCl, 1 EGTA, 10 KH2PO4, 5 MgCl2-6H2O, 0.5mg/mL BSA, 0.05 palmitoyl-L-carnitine, 2 malate). State 3 respiration was initiated by the addition of 5 mM ADP and followed by titrations of DOPAL. A second fiber from each patient was pretreated with 10 mM L-carnosine, 1 mM GSH or 1 mM L-Cys for 5 minutes prior to measuring state 3 respiration.

Analysis of catechol-modified proteins in isolated mitochondria

Mitochondria from myocardial atrial samples were isolated with methods adapted from previous studies (Gostimskaya and Galkin 2010). In brief, fresh myocardium was minced on ice for 4 minutes and underwent a 2-minute trypsin digestion. To halt trypsin activity, trypsin inhibitor was added and the mixture was placed in a 50mL conical tube and allowed to settle. The supernatant was removed and the minced tissue was resuspended in mitochondrial isolation medium-MIM (in mM: 300 sucrose, 10 Na-HEPES, 0.2 EDTA) and homogenized using a pre-chilled Dounce homogenizer. The mixture was homogenized slowly with 10-12 strokes. The homogenate underwent differential centrifugation steps and the pellet of mitochondria were resuspended in MIM + BSA. Isolated mitochondria (50 μg) were incubated for 3 hours at 37°C with 75 μM NE and increasing concentrations of carnosine (1, 2.5, 5, 10, 25 mM). Mitochondrial proteins were subject to SDS-PAGE under reducing conditions followed by transfer to a nitrocellulose membrane.

Nitro-blue tetrazolium (NBT) was used to detect catechol-modified protein adducts. NBT, a redox-cycling dye, stains nitrocellulose blue in the presence of catechol functional group (Paz, Fluckiger et al. 1991, Rees, Florang et al. 2009). The nitrocellulose membrane was incubated with 0.24 mM NBT in 2 M potassium glycine buffer and rinsed in distilled water. The membrane was imaged using a ChemiDoc Imaging System (Bio-rad).

Statistical analysis

Data from this study are presented as means ± SEM or means ± SD, as noted. A student’s paired t-test was used to define differences between ADP-stimulated respiration +/− carnosine within patient samples. Statistical significance was denoted at p<0.05 and statistical tests and linear regression were performed with GraphPad Prism (GraphPad Prism, La Jolla, CA).

Results

Carnosine rapidly conjugates catecholaldehydes.

Carnosine (1 mM) reacted with DOPAL (100 μM) and demonstrated pseudo-1st order kinetics with time-dependent loss of the catecholaldehyde in the presence of the dipeptide; however, much less reactivity was observed following incubation of carnosine (1 mM) with the lipid peroxidation product 4HNE (100 μM) (Figure 1A). The pseudo-1st order kinetics were anticipated given that [carnosine] >> [DOPAL], such that there was little change in [carnosine] with depletion of [DOPAL]. In addition, we have shown using HPLC with electrochemical detection (ESA Biosciences coulometric, E1 = −150mV; E2 = +200mV) analysis that carnosine reacted with DOPAL produces a unique product of DOPAL-carnosine (data not shown).

Figure 1.

Figure 1.

Figure 1.

Figure 1.

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The reaction of (a) 1 mM carnosine with 100 μM DOPAL or 100 μM 4HNE; (b) 1 mM GSH with 100 μM DOPAL or 100 μM 4HNE; (c) 100 μM DOPAL with 1 mM carnosine or 1 mM L-Cys. Data are reported as mean ± SD (n=3, carnosine with DOPAL or 4HNE; L-Cys with DOPAL), (n=4, GSH with 4HNE) or (n=2, GSH with DOPAL). Measured slopes for (a) and (b) were significantly non-zero (p<0.01) except for GSH with DOPAL (p=0.97).

In contrast, GSH (1 mM) rapidly scavenged 4HNE (100 μM) with a half-life of approximately 3 min for 4HNE but exhibited no reactivity towards DOPAL (100 μM) over the course of 120 min (Figure 1B).

Like carnosine, L-Cys (1 mM) exhibited scavenging activity towards DOPAL (100 μM); however, the kinetics may not be pseudo-1st order and there appeared to be reversibility for conjugate formation (Figure 1C).

DOPEGAL was labile under ambient conditions (37 °C in 50 mM sodium phosphate buffer, pH 7.4) as a time-dependent loss of the compound was observed (data not shown). Reactivity towards carnosine was rapid, with conjugation occurring over the course of minutes (data not shown). Work is in progress to characterize the rapid kinetics of DOPEGAL reactivity towards carnosine and protein nucleophiles.

Carnosine attenuates DOPAL toxicity in human cardiac mitochondria.

Permeabilized myofibers exposed to titrations of DOPAL revealed a dose-dependent decrease in state 3 respiration, even exhibiting a significant drop with the initial addition of 1μM DOPAL (p<0.05). Simultaneous incubation of DOPAL with 10 mM carnosine significantly attenuated the decrease in respiration across all concentrations of DOPAL (Figure 2A), while incubation with GSH and L-Cys under similar conditions did not mitigate the effect of DOPAL on respiration (Figure 2B, C).

Figure 2.

Figure 2.

Figure 2.

Figure 2.

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Effect of DOPAL titration on state 3 respiration in permeabilized myofibers isolated from human RAA, either alone or following a pre-incubation with L-carnosine (a), GSH (b) and L-cysteine (c). Respiration was supported by palmitoyl-carnitine & malate. Data are reported as mean ± SEM, *p<0.05 vs. DOPAL, Ψ p<0.05 vs vehicle, εp<0.05 L-Cys+DOPAL vs vehicle.

Carnosine mitigates catechol-protein modifications in human cardiac mitochondria.

Isolated mitochondria incubated with NE showed a greater degree of NBT-staining (Figure 3) compared to the control lane (first lane), indicating increased presence of catechol-modified protein adducts. Pre-treatment of mitochondria with carnosine showed a concentration dependent decrease in catechol-modified adducts, with efficacy even at the lowest concentration of carnosine (2.5mM).

Figure 3.

Figure 3.

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Representative blot showing catechol-adduct protein modifications in isolated human cardiac mitochondria when incubated with norepinephrine (NE) and/or increasing concentrations of L-carnosine.

Discussion

The results from this study reveal, for the first time to our knowledge, the ability of carnosine to conjugate and sequester catecholaldehydes, the aldehyde byproduct of catecholamine deamination via MAO. Recent work has shown the rapid reactivity of DOPAL with protein amines, yielding stable adducts predicted to be a rearranged indole; however, the scavenging of DOPAL by carnosine was previously not known. Interestingly, GSH is not an effective scavenger of catecholaldehydes (Anderson, Florang et al. 2016); however, GSH quickly reacts with the lipid peroxidation product 4HNE, while carnosine’s reactivity is much slower. Such findings demonstrate the novel ability of carnosine, but not GSH, to scavenge a unique class of biogenic aldehydes (i.e., catecholaldehydes), and suggest a biological role for carnosine in cytoprotection against endogenous electrophiles formed via neurotransmitter metabolism. In addition, while L-Cys and carnosine readily reacted with DOPAL, carnosine but not L-Cys appeared to generate a stable conjugate that was not reversible during the reaction time-frame. It is known that L-Cys conjugates with aldehydes (e.g., acetaldehyde) to form thiazolidine products, and previous reports have noted this reaction to be reversible (Wlodek, Rommelspacher et al. 1993).

Our findings also indicate that carnosine mitigates catecholaldehyde-mediated disruptions in mitochondrial respiration, while GSH and L-Cys do not. Owing to the study design, our mitochondrial experiments cannot provide mechanistic insight as to how DOPAL decreases state 3 respiration in permeabilized myofibers, or perhaps more importantly, how carnosine is able to prevent the decrease in respiration. Never-the-less, the decrease in respiration observed in the presence of DOPAL signifies an interruption in enzymatic processes central to the efficiency of oxidative phosphorylation. Therefore, additional studies are necessary to investigate which specific enzymes within the electron transport and/or phosphorylation systems are modified by catecholaldehydes, and the broad effect of these modifications.

Protein modification by catecholaldehyde adducts has been shown in work from our laboratory (Jinsmaa, Florang et al. 2011, Doorn, Florang et al. 2014, Vanle, Florang et al. 2017) and others (Burke, Li et al. 2003, Goldstein, Sullivan et al. 2012) to cause toxicity in neuronal cell cultures and to contribute to the pathophysiology of neurological disease. Our finding that carnosine dose-dependently mitigates catecholaldehyde protein adduct formation following norepinephrine exposure in isolated human cardiac mitochondria indicates that 1) MAO content and activity is more than sufficient in human heart to generate catecholaldehydes when catecholamines are present; 2) there are numerous mitochondrial protein targets for these catecholaldehydes, as indicated by the large number of bands within each well of our NBT stain (Figure 3); and 3) carnosine is capable of blunting formation of catecholaldehyde protein adducts, even at physiologically relevant concentrations. In future studies, targeted proteomic- and metabolomic- based approaches will be necessary to elucidate the extent to which catecholaldehyde modifications alter cellular metabolism and mitochondrial energetics, and the potential for carnosine to mitigate these effects.

In summary, our results highlight the capacity of carnosine to conjugate and scavenge catecholaldehydes, thus expanding the repertoire of carnosine reactivity beyond those formed by lipid- and sugar-derived aldehydes. Such findings indicate carnosine to be a unique scavenger for catecholaldehydes, a property not seen for GSH. There are many clinical implications for these findings. The carnosine-DOPAL conjugate may be a unique biomarker for elevated levels of DOPAL, and could be used in a variety of clinical applications in patients at risk for neurological and cardiovascular diseases. Furthermore, these findings illustrate the therapeutic potential of carnosine in pathological states where increased MAO activity and sympathetic tone (i.e., catecholamine overload), and compromised aldehyde detoxification are known to be involved (Lamensdorf, Eisenhofer et al. 2000, Burke 2003, Goldstein 2013).

Acknowledgments

This study was supported by funding from the National Institutes of Health grants R21AG057006 (J.A.D. and E.J.A) and R01HL122863 (E.J.A).

Footnotes

Disclosure

Authors have no conflicts of interest to declare.

Statement of Human Rights

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed Consent

As described above in Methods section, informed consent was obtained from all participants involved in this study.

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