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Association between hydrogen peroxide-dependent byproducts of ascorbic acid and increased hepatic acetyl-CoA carboxylase activity.

Persons with an impaired ability to be fed by mouth for long periods of time require intravenous nutrient support or total parenteral nutrition (TPN), [3] but hepatic complications such as steatosis are common with this life-saving mode of parenteral nutrition (1). The etiology of TPN-related steatosis is multifactorial (2), and many nutritional elements have been suspected, such as an amino acid imbalance (3-6), an excess of glucose or lipids (7), or a carbohydrate /nitrogen ratio imbalance (8). Oxidative stress (1, 9-11) and products derived from photooxidation of amino acids (9,12) have also been proposed. In a previous study (13), we reported that in a neonatal animal model on TPN, fatty liver was associated with the infusion of parenteral multivitamin preparations (MVPs), a component of TPN. We suspected an ingredient of MVP that might be generated during light exposure. A mass spectroscopy (MS) study (14) led us to document the light-dependent formation of byproducts of ascorbic acid (AA), the most abundant of which had molecular masses of 136 and 208 (hereafter named molecule-136 and molecule-208). The antioxidant property of AA resides in its ability to lose 2 electrons, generating dehydroascorbate (DHA) in a reversible reaction. The subsequent oxidation of DHA causes opening of the lactam ring, leading to 2,3-dike-togulonic acid (DKG) (15-18). The degradation of DKG produces threonic acid (19), which also has a molecular mass of 136 (Fig. 1). As molecule-136 found in photoexposed MVP solutions derives from the byproduct molecule-208 and not from DKG (14), we suspected that it would be different from threonic acid. On the basis of previous reports (14, 20), we hypothesized that the difference in reactions leading to the generation of molecule136 and threonic acid resides in the presence of [H.sub.2][O.sub.2]. In photo-exposed MVP, photo-excited riboflavin catalyzes transfer of electrons from a donor, such as AA, to oxygen, thereby generating hydrogen peroxide ([H.sub.2][O.sub.2]) (21).

Because our previous study suggested that [H.sub.2][O.sub.2] was an important factor in the formation of molecules-136 and -208, we hypothesized that in the presence of [H.sub.2][O.sub.2], a peroxidative pathway of AA leading to formation of molecules-136 and -208 competes with its classic degradation to DKG. The first objective of the present study, therefore, was to assess the role of [H.sub.2][O.sub.2] in the generation of these molecules. The second aim was to differentiate molecule-136 from threonic acid. The third aim was to propose a chemical structure for molecules-136 and -208. The last, final aim was to test their biochemical activity by measuring their effect on hepatic lipid metabolism.


Materials and Methods

To fulfill the objectives of this study, we designed both in vitro and in vivo studies


The concentrations of L-ascorbic acid (AA; 1.8 mmol/L) and riboflavin (vitamin [B.sub.2]; 30 [micro]mol/L) used in the study are equivalent to those reported by the manufacturer for a 2% (by volume) multivitamin preparation (Multi12 pediatric, Sabex Boucherville). This concentration and the exposure to ambient light (75 foot candles) correspond to clinical conditions found in neonatal TPN solutions. The cocktail of AA and riboflavin, in water, generates a mean (SE) of ~660 (28) [micro]mol/L peroxides [according to the FOX method (22-24)] when exposed to ambient light for 24 h. The mass spectrum of this solution is shown in Fig. 2.

The predominant byproducts formed by oxidation or light degradation of AA (14,15,18) are DHA, DKG, molecule-136, and molecule-208; we therefore limited monitoring to AA, DKG, and molecules-136 and -208. Because the mass spectrometer was operated in negative mode, DHA was not detectable. We used DKG to monitor the classic oxidation of AA and used molecule-136 and its parent, molecule-208 (14), to monitor the peroxidation of AA. In the negative mode, AA, DKG, and molecules-136 and -208 have m/z of 175, 191, 135, and 207, respectively.

Protocol 1. We tested the effect of photo-exposed riboflavin on the generation of ascorbate byproducts by measuring the relative abundances of ions m/z 175, 191, 135, and 207 in solutions, prepared in water, containing 1.8 mmol/L AA (Sigma) in the presence or absence of 30 [micro]mol/L riboflavin (Sigma), exposed or not exposed to ambient light, and incubated at room temperature (22[degrees]C) for up to 24 h.

Protocol 2. We tested the effect of [H.sub.2][O.sub.2] on the generation of ascorbate byproducts by measuring the relative abundances of ions m/z 175, 191, 135, and 207 in the following solutions, prepared in water, exposed to ambient light and incubated for up to 24 h at room temperature: (a) 1.8 mmol/L AA + 30 [micro]mol/L riboflavin in the presence or absence of 0.4 mmol/L sodium bisulfite, a peroxide scavenger (20, 22), which was added first to quench the peroxides formed; (b) 1.8 mmol/L AA with increasing concentrations of [H.sub.2][O.sub.2] up to 0.8 mmol/L, in the presence or absence of 30 [micro]mol/L riboflavin.


Protocol 3. We tested the hypothesis that molecules-136 and -208 have a peroxide function by measuring the relative abundances of ions m/z 135 and 207 in solutions containing 1.8 mmol/L AA + 30 [micro]mol/L riboflavin treated or not treated for 15 min with 0.4 mmol/L sodium bisulfite, added after exposure to ambient light for 24 h.

Protocol 4. To test whether molecule-136 is different from threonic acid, we compared fragmentation spectra of commercial threonic acid (Aldrich) and molecule-136. Molecule-136 was derived from a solution containing 1.8 mmol/L AA + 30 [micro]mol/L riboflavin and exposed to ambient light for 24 h at room temperature.


As described previously (13, 25, 26), 3-day-old Hartley guinea pig pups (Charles River) were fed exclusively, via a fixed jugular catheter, with an intravenous solution (50 g/L dextrose + 4.5 g/L NaCl + 1 kIU/L heparin) at a continuous rate of 240 mL * [(kg of body weight).sup.-1] * [day-.sup.1]. After 4 days, the animals were anesthetized, and their livers were minced and frozen at -80[degrees]C until measurement of acetyl-CoA carboxylase (ACC; EC activity. ACC is a known key enzyme involved in hepatic triglyceride synthesis and accumulation. Animals were housed in an institutional vivarium with a 12 h/12 h dark/light cycle. The protocols were carried out in accordance with the Canadian Council of Animal Care guidelines.

Protocol 5. The effects of molecules-136 and -208 on hepatic ACC activity were assessed in 16 animals. Four groups of 4 animals receiving 2 concentrations of test or control solutions for 4 days were compared. These 2 concentrations correspond to the amounts of AA and riboflavin reported by the manufacturer for 1% and 5% MVP and are at the extremes of what is used in TPN solutions prepared for premature newborns. To confirm that the animals received test or control solutions, we measured the abundances of ions m/z 191,135, and 207 by MS in the urine collected on the fourth day. These ions were also measured in a fifth group receiving the base solution devoid of vitamins. Results were normalized for creatinine as followed: abundances in 20 [micro]L of urine/[micro]g of creatinine.


The test solutions were a 1% solution (1.8 mmol/L AA + 30 [micro]mol/L riboflavin + 800 [micro]mol/L [H.sub.2][O.sub.2] in 15 mL of water) and a 5% solution (9.0 mmol/L AA + 150 [micro]mol/L riboflavin + 800 [micro]mol/L [H.sub.2][O.sub.2] in 15 mL of water).

These solutions were (a) exposed to ambient light for 24 h (75 foot candles) to generate molecules-136 and -208, (b) treated with catalase (100 U/mL for 15 min) to eliminate [H.sub.2][O.sub.2], (c) centrifuge-filtered (4000g for 15 min) with a Centricon Plus-20 (Millipore; 10 000 nominal molecular weight limit) to eliminate the enzyme catalase and sterilize the solutions, and (d) diluted 1:1 with basal solution (100 g/L dextrose + 9.0 g/L NaCl + 2 kIU/L heparin).


The control solution was the same as the test solution except that AA and riboflavin were added in water containing 1 mmol/L sodium bisulfite, [H.sub.2][O.sub.2] was not added, and the solutions remained photo-protected for 24 h before catalase treatment.


For MS analyses, 5 [micro]L of each in vitro solution was injected without preparation. Urine samples were acidified by the 1:1 addition of perchloric acid and centrifuged for 20 min at 4000g and 4[degrees]C. After centrifugation, 20 [micro]L of the supernatant was injected on a C1, column [Zorbax Eclipse XDB [C.sub.18]; 25 cm x 4.6 mm (i.d.)] and eluted with a 15-min gradient of 40% to 60% acetonitrile in water. The retention time was 5.17 [+ or -] 0.05 min, and the ions were not separated.

Samples were injected into an Agilent simple quadrupole LC/MS 1100 mass spectrometer. The electrospray ionization mode was used, and the mass spectrometer was operated in the negative-ion mode. The ions were monitored by either the selected-ion monitoring (SIM) mode for the selected ions or the SCAN mode in the m/z 50 to 250 range. Conditions were as follows: fragmentor voltage, 70V; capillary voltage, 3000V; nitrogen flow, 13 L/min; nebulizer pressure, 50 psi; gas temperature, 300[degrees]C. The direct injections were done with a constant flow of H20-acetonitrile (70:30 by volume) at a rate of 0.4 mL/min. Tandem MS was performed on an Agilent ion-trap mass analyzer to compare the fragmentation spectrum of threonic acid with that of molecule-136 produced from the mixtures of AA and riboflavin exposed to ambient light for 24 h. The samples were introduced by infusion with the same ionization and detection modes used above.


ACC activity was measured in liver extracts as described by Kudo et al. (27). The 6% PEG 8000 fraction in buffer (60.6 mmol/L Tris acetate, 1 g/L bovine serum albumin, 1.3 [micro]mol/L 2-mercaptoethanol, 5 mmol/L magnesium acetate, 2.1 mmol/L ATP, 1.1 mmol/L acetyl-CoA, pH 7.5) was pre-incubated for 20 min at 37[degrees]C in the presence or absence of 1.1 mmol/L glutamate. This amino acid stimulates ACC activity by favoring the action of protein phosphatase A2 (28, 29). The ACC activity was measured by the (3 x [10.sup.6] dpm) [sup.14]C-bicarbonate fixation assay (total volume, 165 p,L) (28). After a 5-min incubation, 100 mL/L perchloric acid was added to stop the reaction. The supernatant obtained by centrifugation for 20 min at 1000g was transferred to vials, dried overnight, and counted in a beta counter. The ACC activity was expressed as nmol of malonyl-CoA produced * [min.sup.-1] * [(mg protein).sup.-1]. The protein concentration was measured by the Bradford assay with reagents from Bio-Rad Laboratories.


Data, expressed as the mean (SE), were compared by factorial ANOVA after verification of their homoscedasticity by Bartlett [chi square] test. The threshold of significance was set at P <0.05.



IN VITRO STUDIES The scan in Fig. 2 (m/z 50 to 250) confirms the presence of the 3 targeted species in this study: m/z 135 (molecule136),191 (DKG), and 207 (molecule-208). Ion m/z 175 (AA) does not appear because it was completely degraded after prolonged exposure to light (14).

The rapid degradation of AA and the generation of molecules-136 and -208 were a function of time (P <0.01), presence of riboflavin (P <0.01), and light exposure (P <0.01; Fig. 3). In all situations, the relative abundance of molecule-136 was higher than that of molecule-208. Concomitantly, without riboflavin or light, the generation of ion m/z 191 (DKG) was dependent on time (P <0.01). In the presence of riboflavin and light, DKG formation was accelerated but reached a plateau after 3 h.


The addition of bisulfite (Fig. 4) during the initial step of mixing the components decreased (P <0.01) the loss of AA and the generation of molecules-136 and -208. DKG generation was not affected by the presence of bisulfite. However, when added at the end of the 24-h incubation, bisulfite lowered (P <0.01) the relative abundances of ions m/z 135 and 207 by 65% but not the intensity of m/z 191 (data not shown).

The influence of [H.sub.2][O.sub.2] on the degradation kinetics of AA is shown in Fig. 5. The disappearance of AA in the reaction mixture and the production of molecules-136 and -208 were proportional to the [H.sub.2][O.sub.2] concentration (P <0.01), incubation time (P <0.01), and the presence of photo-excited riboflavin (P <0.01). The effect of peroxides did not appear by 24 h of incubation in the presence of riboflavin, as the abundances of ions m/z 135,175, and 207 reached a plateau. The effect of peroxide on the formation of DKG differed. Without riboflavin, in the first 3 h, the amount of DKG formed was independent of peroxide concentrations, whereas after 24 h, DKG concentrations decreased as a function of increasing peroxide concentration. This negative effect of peroxides was also observed in the presence of riboflavin. Without peroxide, the presence of photo-exposed riboflavin enhanced DKG production, which was proportionally lower in the presence of peroxides. The negative effect of peroxides on the abundance of DKG may suggest degradation or lower generation of the molecule. The concomitant increasing abundances of molecules-136 and -208 suggest competitive utilization of the substrate, leading to lower DKG production.

To differentiate molecule-136 from threonic acid, we compared a commercial threonic acid with the m/z 135 ion present in the test mixture (1.8 mmol/L AA + 30 [micro]mol/L riboflavin + light at room temperature for 24 h) by tandem MS. The m/z 135 ion from each solution was trapped and fragmented. The fragmentation spectra are shown in Fig. 6. The most abundant ions, m/z 75, 89, 117, and 135, were common to both solutions, but the m/z 133 ion was specific to the mixture of AA + riboflavin.


The mean (SE) initial body weight differed (P <0.01) between groups receiving control or test solutions [112 (2) vs 98 (3) g, respectively], but not between the groups receiving the 1% and 5% doses [107 (3) vs 104 (5) g, respectively]. Statistical analysis of hepatic ACC activities showed a significant interaction (P <0.05) between variables, leading us to analyze data according to the presence of glutamate (Fig. 7). There was a significant dose effect (P <0.01) only in animals receiving test solutions and only on activated ACC (in presence of glutamate). Abundances of m/z 135 and 207, but not of 191, were higher (P <0.01) in urine of animals receiving test solutions (Fig. 8). The 3 ions were higher (P <0.05) in the 5% solution. Abundances of ions in urine from animals receiving 1% control solution were not different from those from animals receiving the base solution devoid of vitamin.


The phosphorylated form of ACC is inactive. Because glutamate is reported to facilitate the action of phosphatase on ACC (28, 29), we quantified its activation by measuring the ratio of activities in the presence or absence of glutamate. This ratio was influenced more by the concentration of vitamin used (P <0.01; Fig. 9B) than by exposure of the solutions to light (control vs test). However, the correlation between the activation of ACC and the abundance of the m/z 207 ion in urine (Fig. 9A) was significant (y = 2 x [10.sup.-5]x +1.07; [r.sup.2] = 0.67; P <0.01), whereas correlations with other ions were not ([r.sup.2] <0.17).


The main finding of the present study is that [H.sub.2][O.sub.2] at concentrations found in TPN solutions induces the transformation of DHA into new, biologically active compounds. We suggest that these molecules have a peroxide and aldehyde function, and we demonstrate that they stimulate the activity of hepatic ACC, a key enzyme in lipid metabolism.

Principal actors in this new pathway of degradation of DHA are photo-excited riboflavin, [H.sub.2][O.sub.2], and time. All of these are present in the clinical setting of TPN, particularly in neonatal care. MVP containing AA and riboflavin is an essential constituent of TPN solutions. Knowing that premature newborn infants have immature antioxidant defenses, physicians are prompted to prescribe antioxidant vitamins, such as those found in MVP. Because vitamins are light sensitive, the TPN bag is frequently covered by an opaque shield, but the tubing is rarely protected from light (30). The lack of photoprotection of tubing coupled with the up to 4-h transit time between the TPN bag and the site of infusion is long enough to generate peroxides (30, 31). Photo-excited riboflavin catalyzes the electron transfer between a donor, such as AA, and dissolved oxygen to form [H.sub.2][O.sub.2], (21). When 1% MVP is present in the TPN solution, peroxide concentrations measured in the tubing close to the infusion site are >0.2 mmol/L (30, 31). Peroxide generation in the TPN solution is proportional to the MVP concentration (31). In the present study, protocols were designed to be close to clinical conditions.


Shown in Fig. 10 are the proposed chemical reactions leading to the production of molecules-136 and -208. We previously reported that molecule-136 is derived from molecule-208 (14). On the basis of its mass, the parent molecule, molecule-208, can only be DHA. This is in accordance with the observation of Deutsch et al. (16) that formation of the m/z 207 ion derives from peroxidation of DHA. Because hydrolysis of the DHA lactam ring leads to DKG production (Fig. 1), we propose that a double hydroxyl radical attack is required to generate molecule-208. The intervention of hydroxyl radicals in the generation of molecule-208 has been demonstrated previously (14). Indeed, the short incubation time (30 min) and low peroxide concentration (40 [micro]mol/L) in the AA solution led to generation of molecules-136 and -208 only in the presence of [Fe.sup.2+], suggesting a Fenton-like reaction. Similarly to the degradation of DKG (Fig. 1), decarboxylation of molecule-208 leads to the generation of the m/z 163 ion and, subsequently, the m/z 135 ion, both observed in the mass spectrum of the AA + riboflavin solution exposed to ambient light for 24 h (Fig. 2).

This pathway suggests that molecule-136 (2-hydroperoxyl-3,4-dihydroxybutanal) and molecule-208 (2,3-diketo4-hydroxyperoxyl-5,6-dihydroxyhexanoic acid) are peroxides. This is supported by the fact that the addition of bisulfite, a general peroxide scavenger (20,22), at the end of the 24-h incubation period lowered their abundances by 65%. That is also in accordance with our previous study (14) in which catalase-resistant peroxides were decreased by the addition of bisulfite. However, this is in contrast with findings by Deutsch et al. (15,16), who suggested that the m/z 207 ion, observed after the incubation of DHA with [H.sub.2][O.sub.2], is rather 2,3-diketo-4,5,5,6-tetrahydroxyhexanoic acid.

Although they have the same molecular mass, the small difference in chemical structure between molecule136, generated by the [H.sub.2][O.sub.2] dependent oxidation of DHA, and threonate, derived from DKG, is confirmed by their mass fragmentation spectra (Fig. 6) and is explained in Fig. 11. The mass spectrum patterns presented in Figs. 3-5 support the fact that molecule-136 derives from molecule208 (14) rather than from DKG. The similarity of the m/z 135 spectrum to that of m/z 207, but not m/z 191, suggests that molecule-136 is linked to molecule-208 and not to DKG.


The presence of reactive peroxide and aldehyde functions on molecules-136 and -208 suggests that they have potential biochemical effects. This was tested with the same animal model used to induce fatty liver in response to the infusion of MVP solution exposed to ambient light (13). The infusion for 4 days of a mixture containing high concentrations of molecules-136 and -208 to neonatal guinea pig pups induced stimulation of hepatic ACC activity in the presence of glutamate. As for our previous results, in which shielding TPN from light prevented the induction of fatty livers (13), protection of the solution (control) from light prevented ACC stimulation. The fact that the animals presenting with higher ACC activity had also higher urinary abundance of ions m/z 135 and 207, but not 191 (DKG), points to the association between molecules-136 and -208 and a disturbance of hepatic lipid metabolism.


Regulation of the hepatic triglyceride pool is multifactorial; it depends on the balance between synthesis, export, and mitochondrial [beta]-oxidation of fatty acids. ACC is a key enzyme in the accumulation of triglycerides as it regulates fatty acid synthesis and mitochondrial oxidation. Indeed, malonyl-CoA, produced by ACC, is a substrate for fatty acid synthesis and an inhibitor of mitochondrial (3-oxidation. Malonyl-CoA inhibits carnitine palmitoyltransferase (32, 33), an enzyme that catalyzes the transfer of fatty acids into the mitochondrion (32, 33). The regulation of ACC activity is dependent of its phosphorylation. The inhibitory effect of AMP-dependent protein kinase (32-34) is counteracted by protein phosphatase 2A activity (35-37). Glutamate is known to activate ACC by facilitating the action of protein phosphatase A2 (28, 29). The fact that infusion of a mixture containing high concentrations of molecules-136 and -208 stimulated ACC activity only in the presence of glutamate suggests that these molecules interfere with the phosphatase activity. The ratio of activities measured in the presence or absence of glutamate would confirm this mode of action of molecules generated in test solutions. However, the ratio was not different between the control and test solutions; it varied as a function of vitamin concentrations (Fig. 9B). Because the urinary concentrations of molecules-136 and -208 also increased as a function of vitamin concentrations received by the animals, we searched for a correlation with each ion. Only molecule-208 correlated with the activation of ACC, suggesting that this molecule may interfere with lipid metabolism.


Further studies will be required to confirm the proposed structures of molecules-136 and -208 and to document whether the biochemical activity of molecule-208 and/or -136 occurs via AMP-activated protein kinase, protein phosphatase A2, or gene expression. However, we hypothesize that molecule-208 interferes with the phosphorylated state of ACC, leading to its activation.


This work was supported in part by a grant from the AACC Van Slyke Society and a grant from the Canadian Institutes of Health Research (MOP 53270).

Received March 2, 2005; accepted May 12, 2005.

Previously published online at DOI: 10.1373/clinchem.2005.050427


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[1] Research Centre and Paediatric Department, CHU Sainte-Justine, University of Montreal, Montreal, Quebec, Canada.

[2] Division of Neonatology, Children's and Women's Health Centre of British Columbia, Vancouver, British Columbia, Canada.

[3] Nonstandard abbreviations: TPN, total parenteral nutrition; MVP, parenteral multivitamin preparation; MS mass spectrometry; AA, ascorbic acid; DHA, dehydroascorbate; DKG, 2,3-diketogulonic acid; and ACC, acetyl-CoA carboxylase.

* Address correspondence to this author at: Research Centre, CHU Sainte-Justine, 3175 Chemin Cote Ste-Catherine, Montreal, Qc, Canada H3T 1C5. Fax 514-345-4801; e-mail
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Title Annotation:Drug Monitoring and Toxicolgy
Author:Knafo, Laurent; Chessex, Philippe; Rouleau, Therese; Lavoie, Jean-Claude
Publication:Clinical Chemistry
Date:Aug 1, 2005
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