Role of aldose reductase in the metabolism and detoxification of carnosine-acrolein conjugates

Abstract

Oxidation of unsaturated lipids generates reactive aldehydes that accumulate in tissues during inflammation, ischemia, or aging. These aldehydes form covalent adducts with histidine-containing dipeptides such as carnosine and anserine, which are present in high concentration in skeletal muscle, heart, and brain. The metabolic pathways involved in the detoxification and elimination of these conjugates are, however, poorly defined, and their significance in regulating oxidative stress is unclear. Here we report that conjugates of carnosine with aldehydes such as acrolein are produced during normal metabolism and excreted in the urine of mice and adult human non-smokers as carnosine-propanols. Our studies show that the reduction of carnosine-propanals is catalyzed by the enzyme aldose reductase (AR). Carnosine-propanals were converted to carnosine-propanols in the lysates of heart, skeletal muscle, and brain tissue from wild-type (WT) but not AR-null mice. In comparison with WT mice, the urinary excretion of carnosine-propanols was decreased in AR-null mice. Carnosine-propanals formed covalent adducts with nucleophilic amino acids leading to the generation of carnosinylated proteins. Deletion of AR increased the abundance of proteins bound to carnosine in skeletal muscle, brain, and heart of aged mice and promoted the accumulation of carnosinylated proteins in hearts subjected to global ischemia ex vivo. Perfusion with carnosine promoted post-ischemic functional recovery in WT but not in AR-null mouse hearts. Collectively, these findings reveal a previously unknown metabolic pathway for the removal of carnosine-propanal conjugates and suggest a new role of AR as a critical regulator of protein carnosinylation and carnosine-mediated tissue protection.

Keywords: Brain Metabolism; Cardiac Metabolism; Ischemia; Metabolism; Post Translational Modification; Protein Chemical Modification; Protein Cross-linking; Skeletal Muscle.

FIGURE 1.

Histidine and carnosine conjugates in human urine. A, Orbitrap mass spectra of human urine samples collected from healthy, non-smoking adults. The urine samples were de-proteinized, and the carnosine and histidine conjugates were separated by HPLC as described under “Experimental Procedures.” The spectra show prominent ions that were assigned to carnosine-propanal and carnosine-propanol (m/z = 283.139 and 285.155) (i), histidine propanal and histidine-propanol (m/z = 212.103 and 214.118) (ii), and histidine-HNE and histidine-DHN (m/z = 312.191 and 314.207), respectively (iii). B, the concentrations of urinary conjugates calculated using tyrosine-histidine (m/z 319) as an internal standard. Data are the mean ± S.E. (n = 7).

FIGURE 2.

Identification of the carnosine conjugates of acrolein. A, ESI⁺/MS spectra of carnosine-propanal conjugates generated by incubating acrolein with carnosine as described under “Experimental Procedures.” Putative structures of the conjugates identified by mass spectrometry are shown in the figure for each m/z value. The fragmentation patterns of the major ions with m/z values of 283 (B), 321(C), and 339 (D) are shown.

FIGURE 3.

Aldose reductase catalyzes the reduction of carnosine-propanal conjugates. A, the ESI⁺/MS spectra of carnosine-propanal conjugates after incubation with AR. The carnosine-propanal conjugates were isolated by HPLC and incubated with recombinant AR and NADPH for 18–24 h. The ions generated in the presence of AR are indicated by an asterisk (*). Fragmentation patterns of the major ions with m/z values of 285 (B), 323 (C), and 341 (D) are shown.

FIGURE 4.

Metabolic conversion of carnosine-propanal to carnosine-propanol via aldose reductase. A, ESI⁺/MS spectra of the skeletal muscle homogenates (2–4 mg of protein) from WT (solid line) and AR-null (dotted line) mice incubated with acrolein for 18–24 h. B, the relative abundance of reduced versus non-reduced carnosine conjugates calculated as intensity ratio (reduced/non-reduced) in lysates of WT and AR-null skeletal muscle. Values are the mean ± S.E.; n = 3; *, p < 0.05, WT versus AR-null.

FIGURE 5.

Reduction of carnosine-propanal conjugates in murine tissues. A, ESI⁺/MS spectra of tissue lysates prepared from mouse heart incubated with NADPH and carnosine-propanal conjugates. Tissue lysates (1–2 mg of protein) from WT (i and ii) AR-null (iii and iv) and FR-1-transgenic (FR-1-TG; v and vi) mice were incubated with HPLC-purified carnosine-propanal conjugates in the presence (solid line) and the absence (dotted line) of NADPH. B, Orbitrap analysis of WT (i and ii) and AR-null (iii and iv) heart homogenates. The ratio of ion intensities at m/z 285/283 and 323/321 are shown at different time intervals in lysates prepared from WT and AR-null skeletal muscle (C and D), WT AR-null and FR-1-transgenic hearts (E) and WT and AR-null brains (F). Data are the mean ± S.E.; n = 4; *, p < 0.05 versus WT + carnosine-propanal; #, p < 0.05 versus WT + carnosine-propanal + NADPH; $, p < 0.05 versus WT + carnosine-propanal + NADPH (2 h).

FIGURE 6.

Identification of aldose reductase-generated carnosine metabolites in mouse urine. A, Orbitrap mass spectra of reagent histidine-propanal conjugate (i) and histidine [¹³C]propanal (ii) and the spectra of the m/z 210–218 region of urine collected from WT (iii) and AR-null (iv) mice. The urine samples were spiked with histidine-[¹³C]propanal (m/z = 215.1133) as indicated. B, the abundance of endogenous histidine-propanal conjugate was calculated as a ratio of its intensity to that of the internal standard in the urine of WT and AR-null mice. C, Orbitrap mass spectra of urine from WT (i) and AR-null (ii) mice fed octyl-d-carnosine. Mice were fed 50 μmol of octyl-d-carnosine by oral gavage, and urine samples were collected over 24 h, purified by HPLC, and analyzed by Orbitrap mass spectrometer. D, relative abundance of carnosine-propanol was calculated as a ratio of its intensity to that of the internal standard (Tyr-His, m/z 319). Data are the mean ± S.E. *, p < 0.05 versus WT (n = 4–5).

FIGURE 7.

Formation of protein-carnosine adducts. A, representative Western blots of albumin incubated with the carnosine-propanal. The blots were developed with either the anti-protein-carnosine or the anti-protein-propanal antibodies (Ab). B, ESI⁺/MS spectra of Ac-RVCAKH before (i) and after (ii) incubation with carnosine-propanal for 30 min or with carnosine-propanol for 60 min (iii) and percent modification of the model peptide by the carnosine-propanal conjugate (iv). C, Western blot of skeletal muscle lysates prepared from WT and AR-null mice incubated with 200–300 μm carnosine-propanal and 1 mm NADPH for 18 h developed using the anti-protein-carnosine antibody (i) and the relative intensity of bands of apparent molecular masses 130 (ii), 45 (iii), and 30 (iv) kDa normalized to total protein in the gel measured by Amido Black staining. Data are the mean ± S.E.; *, p < 0.01 versus WT + carnosine-propanal; #, p < 0.01 versus AR-null + carnosine propanal + NADPH (n = 3–4). D, accumulation of carnosinylated proteins in aged tissue from WT and AR-null mice. Total tissue lysates were prepared from the brain (i), heart (ii), and skeletal muscle (iii), and Western blots were developed using anti-protein-carnosine antibody. The relative intensity in specific immune-positive bands is shown in corresponding bar graphs (iv, v, and vi, respectively). vii, a representative Western blot developed using anti-propanal-protein antibody of skeletal muscle lysates immunoprecipitated (IP) with anti-protein-carnosine antibody. Data are expressed as the mean ± S.E. *, p < 0.01 versus WT tissues (n = 3–4). E, accumulation of carnosine-propanal-protein adducts in WT mouse hearts perfused with 10 μm acrolein (10 min). Blots were developed with anti-protein-carnosine antibody (inset). Data are expressed as mean ± S.E. *, p < 0.01 versus untreated hearts (n = 3–4).

FIGURE 8.

Aldose reductase-dependent protection of the ischemic heart by carnosine. A, changes in developed pressure in isolated mouse hearts subjected to 30 min of ischemia followed by 45 min of reperfusion. In the carnosine-treated group the hearts were continuously perfused with 1 mm carnosine before induction of ischemia and during reperfusion. Data are the mean ± S.E.; n = 4–7 hearts/group; *, p < 0.01 versus WT without carnosine or AR-null hearts with and without carnosine. B, levels of total released lactate dehydrogenase (LDH) (i) and CK (ii) in post-ischemic myocardial effluent collected from WT and AR-null hearts treated with and without carnosine. Data are expressed as the mean ± S.E.; n = 4–7 hearts/group. *, p < 0.01 versus WT without carnosine. C, Western blots from Langendorff-perfused hearts from WT and AR-null mice subjected to either 30 min of perfusion (P) or perfusion followed by 30 min of ischemia (i). At the end of the protocol, proteins from the heart homogenates were separated by SDS-PAGE and immunoblotted with anti-protein-carnosine antibody. Relative intensity of immuno-reactive bands of 45 (ii), 37 (iii), and 30 (iv) kDa was measured and normalized to total protein in the gel measured by Amido Black staining. Data are expressed as the mean ± S.E.; *, p < 0.01 versus perfused hearts; #, p < 0.01 versus WT ischemic hearts (n = 3–4 mice).

FIGURE 9.

Mass spectrometric identification of carnosine-propanal-modified paired basic amino acid-cleaving system protein. Representative HCD fragmentation spectrum for +3-charged ion with a monoisotopic m/z 545.63898 Da was observed at +4.99 millimass unit (mmu)/9.14 ppm mass error using a 1D-LC-LTQ-Oribitrap-ELITE mass spectrometer. Analysis of the collected data by Sequest and Mascot (v1.20) identified a peptide (XCorr, 1.70; probability, 0.00) with the sequence WLQQQEVkRR, K₈-C₁₂H₁₆N₄O₃ (264.1224 Da), and a MH⁺ of 1634.90238 Da. The fragmentation of the peptide yielded four c-ion and three z-ion series with sub 2.5 ppm mass accuracy. The inset shows the putative structures of the adducts that could be due to the formation of lysine-propanal-β-alanyl-histidine (i) or β-alanyl-histidine-propanal-lysine (ii) adducts.