Methylglyoxal and pulmonary hypertension in broiler chickens

Methylglyoxal and pulmonary hypertension in broiler chickens F. Khajali,*1 R. Liyanage,† and R. F. Wideman‡ *Department of Animal Science, Shahrekord University, Shahrekord, Iran 88186-34141; †Department of Chemistry/Biochemistry; and ‡Department of Poultry Science, University of Arkansas, Fayetteville 72701 ABSTRACT Methylglyoxal (MG) is a dicarbonyl molecule that forms during glycolysis and normally is detoxified via the glyoxalase system. Methylglyoxal is highly reactive with various amino acid residues in proteins, leading to oxidative stress and irreversible protein damage. Increased levels of MG have been associated with endothelial damage and vascular remodeling contributing to the development of systemic arterial hypertension in mammals. This study was conducted to determine whether administering exogenous MG can trigger pulmonary hypertension (increased pulmonary arterial pressure) in broilers. Hematological assays and preliminary mass spectrometric analyses also were conducted using blood samples from broilers that had been injected intramuscularly with either saline or MG to determine whether MG triggers either a toxic response or oxidative posttranslational modification of hemoglobin within 24 h postinjection. Clinically healthy male broilers received 100-μL intravenous injections of sa-

line and then MG, followed by a 500-μL intramuscular injection. Neither intravenous nor intramuscular injections of saline altered the pulmonary arterial pressure, whereas both intravenous and intramuscular MG injections triggered pulmonary hypertension attributable to increased pulmonary vascular resistance. The precise mode of action by which MG triggers pulmonary vasoconstriction remains to be determined. Pulse oximetry, hematology, and matrix-assisted laser desorption ionization–time-of-flight spectra data did not provide evidence of an overt toxic response to MG, nor was modification of hemoglobin detected, although increased heterophil:lymphocyte ratios did demonstrate that MG caused a stress response. To the best of our knowledge the present results constitute the first demonstration in any vertebrate species that exogenously administered MG rapidly initiates pulmonary hypertension attributable to pulmonary vasoconstriction.

Key words: methylglyoxal, pulmonary arterial pressure, ascites, oximetry, chicken 2011 Poultry Science 90:1287–1294 doi:10.3382/ps.2010-01120

INTRODUCTION Methylglyoxal (MG; C3H4O2), also known as pyruvaldehyde or 2-oxo-propanal, is formed from carbohydrates, fatty acids, and proteins by enzymatic and nonenzymatic metabolic pathways (Kalapos, 1999). Endogenous MG primarily is formed spontaneously during glycolysis as a result of elimination of phosphate from glyceraldehyde-3-phosphate and dihydroxyacetonephosphate (Thornalley, 1996). Methylglyoxal is a dicarbonyl molecule having both aldehyde and ketone moieties that are highly reactive with various amino acid residues in proteins, leading to oxidative stress and irreversible protein damage through the formation of advanced glycation end products (Shinohara et al., 1998; Bourajjaj et al., 2003; Kilhovd et al., 2003; ©2011 Poultry Science Association Inc. Received September 11, 2010. Accepted February 11, 2011. 1 Corresponding author: [email protected] or [email protected]

Wang et al., 2005, 2007, 2008; Wu, 2006). Methylglyoxal is highly cytotoxic and normally is detoxified via the glyoxalase system, which comprises 2 enzymes, glyoxalase I and II. Methylglyoxal reacts with glutathione forming a hemithioacetal, which is converted into S-dlactoyl-glutathione by glyoxalase I, and then further metabolized to d-lactate by glyoxalase II (Thornalley, 1996; Shinohara et al., 1998; Kalapos, 1999). Increased tissue levels of MG have been associated with endothelial damage and vascular remodeling contributing to the development of systemic arterial hypertension in laboratory animals and humans (Shinohara et al., 1998; Wang et al., 2005, 2007; Chang and Wu, 2006; Wu, 2006; Sankaralingam et al., 2009), yet the potential role of MG in the onset of pulmonary hypertension (increased blood pressure in the pulmonary circulation) apparently has not been evaluated. Rapidly growing broiler chickens (broilers) develop pulmonary hypertension leading to terminal pulmonary hypertension syndrome (PHS; also known as ascites syndrome) when the right ventricle is forced to increase

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the pulmonary arterial pressure to propel the cardiac output through lungs having an inadequate pulmonary vascular capacity (Wideman and Bottje, 1993; Wideman, 2000, 2001). Pulmonary vascular capacity is broadly defined to encompass anatomical constraints related to the compliance and effective volume of blood vessels as well as functional limitations related to the tone (degree of constriction) maintained by the primary resistance vessels (arterioles) within the lungs. Comparisons of clinically healthy PHS-susceptible and PHS-resistant broilers failed to demonstrate consistent differences in cardiac output, whereas both the pulmonary vascular resistance and pulmonary arterial pressure were consistently higher in susceptible than in resistant broilers (Bowen et al., 2006; Chapman and Wideman, 2006; Wideman et al., 2007, 2010; Lorenzoni et al., 2008). Increases in pulmonary vascular resistance and pulmonary arterial pressure have been attributed to the functional predominance of pulmonary vasoconstrictors over vasodilators (Wideman, 2000, 2001; Wideman et al., 2004, 2007) and to oxidative stress associated with antioxidant depletion or activation of the innate immune system in broilers (Enkvetchakul et al., 1993; Bottje and Wideman, 1995; Iqbal et al., 2001a,b, 2002; Wideman et al., 2004; Lorenzoni and Ruiz-Feria, 2006; Khajali and Fahimi, 2010). Two factors suggest that MG may contribute to the pathogenesis of PHS in broiler chickens. First, unlike mammals that use the enzymes of their urea cycle to synthesize l-Arg, the chicken lacks the key enzyme carbamoyl phosphate synthase I (EC 6.3.5.5) and has low activities of hepatic arginase (EC 3.5.3.1, 2) and ornithine transcarbamoylase (EC 4.4.4.17). Therefore, chickens are unable to synthesize Arg and are highly dependent on dietary Arg. Arginine is the precursor of nitric oxide, the key vasodilator that inhibits PH (Khajali, and Wideman, 2010). Second, chickens maintain a blood sugar concentration that is at least twice as high as that of mammals (Braun and Sweazea, 2008). In mammals, high blood sugar concentrations are associated with high MG concentration so that plasma levels of MG increase 3- to 5-fold in diabetic patients. In mammalian species MG damages the vascular endothelium, leading to systemic vasoconstriction and hypertension, potentially by inhibiting the production of the vasodilator nitric oxide or by causing the release of vasoconstrictors (Kalapos, 1999). Therefore, one objective of the present study was to determine whether exogenous MG administration can elicit pulmonary vasoconstriction and pulmonary hypertension in broilers. Methylglyoxal also triggers oxidative damage to hemoglobin and thus potentially can indirectly reduce the oxygen-carrying capacity of blood in mammals (Gao and Wang, 2006). Hypoxia and hypoxemia are key components of the onset and pathogenesis of PHS in broilers. Accordingly, a second objective of the present study was to assess blood oxygenation, hematological parameters, and oxidative damage to hemoglobin after MG administration to broilers. We anticipated that if

these initial studies demonstrated that exogenous MG can elicit pulmonary hypertension or relevant hematological derangement, then subsequent studies could be conducted to more closely assess the potential contribution of endogenous MG during the spontaneous pathogenesis of PHS.

MATERIALS AND METHODS Animal procedures were approved by the University of Arkansas Institutional Animal Care and Use Committee (protocol no. 08036). One hundred male broiler chickens were reared on wood shavings litter in environmental chambers (dimensions: 3.7 m long × 2.5 m wide × 2.5 m high) within the Poultry Environmental Research Laboratory at the University of Arkansas Poultry Research Farm (Fayetteville). Single-pass ventilation was maintained at a constant rate of 6 m3/min per chamber. The photoperiod was set for 23 h light:1 h dark for the first 4 d and 18 h light:6 h dark thereafter. Thermoneutral temperatures were maintained throughout: 32°C for d 1 to 3, 31°C for d 4 to 6, 29°C for d 7 to 10, 25°C for d 11 to 14, and 24°C for d 22 to 56. A corn and soybean meal-based feed [23% (230 g/ kg) CP and 3,200 kcal/kg of ME] formulated to meet minimum NRC (1994) standards for all ingredients was provided to ensure rapid growth throughout the trial. All chicks were wing banded on d 10. Feed and water (via nipple waterers) were provided ad libitum throughout the trial.

Experiment 1 Between 6 and 7 wk of age, 8 clinically healthy birds were weighed (2,708 ± 92 g of BW; mean ± SEM) and anesthetized to a surgical plane with intramuscular injections of allobarbitol (5,5-diallylbarbituric acid, 3.0 mL, 25 mg/mL; Sigma-Aldrich Chemical Co., St. Louis, MO) and ketamine HCl (1.0 to 2.5 mL of 100 mg/mL; Henry Schein Inc., Melville, NY). The anesthetized broilers were fastened in dorsal recumbency on a surgical board. Lidocaine HCl (2%; Interstate Drug Exchange Inc., Amityville, NY) was injected subcutaneously around the left basilica vein (wing vein), then the proximal end of a Silastic catheter (0.030 cm i.d., 0.063 cm o.d.; Dow Corning Corp., Midland, MI) filled with heparinized saline (200 IU of heparin/mL of 0.9% NaCl) was inserted into the vein. The distal end of the catheter was attached to a blood pressure transducer (World Precision Instruments, Sarasota, FL) interfaced through a Transbridge preamplifier (World Precision Instruments) to a Biopac MP100 data acquisition system using Acqknowledge software (Biopac Systems Inc., Goleta, CA). Characteristic pulse pressures (Owen et al., 1995; Wideman et al., 1996, 1999) were monitored to identify the catheter’s location as it was slowly advanced through the right atrium, right ventricle, and into the main trunk of the pulmonary artery to continuously record pulmonary arterial pressures. For intra-

METHYLGLYOXAL AND PULMONARY HYPERTENSION IN BROILER CHICKENS

venous injections PE-50 polyethylene tubing filled with heparinized saline was advanced through the basilica vein to a position near the inferior vena cava. Ongoing intravenous infusions were not administered. Three additional birds were used to simultaneously record the pulmonary arterial pressure and cardiac output (2,646 ± 153 g of BW; mean ± SEM). They were anesthetized and fastened in dorsal recumbency on a surgical board. An incision was made to open the thoracic inlet and a Transonic 3SB ultrasonic flowprobe (Transonic Systems Inc., Ithaca, NY) was positioned on the left pulmonary artery. The probe was connected to a Transonic T206 blood flow meter (Transonic Systems Inc.) to confirm signal acquisition and record blood flow, and then the skin of the thoracic inlet was sealed with surgical wound clips (Wideman et al., 1996; Wideman, 1999). Silastic tubing filled with heparinized saline was advanced through the basilica vein into the right pulmonary artery to record pulmonary arterial pressure, and PE-50 polyethylene tubing filled with heparinized saline was inserted into the basilica vein for intravenous injections. Preliminary studies indicated that 100-µL intravenous injections of MG 40% solution (Sigma-Aldrich Chemical Co.) and 500-µL intramuscular injections of MG were the lowest MG doses that reliably led to increases in the pulmonary arterial pressure. In humans, plasma levels of MG are normally in the range 100 to 200 nM and in tissues 2 to 4 µM (Thornalley, 1996). Plasma and tissue levels of MG were not measured in the present study. After surgical preparations were complete and a stabilization period of 10 min had elapsed, control pulmonary arterial pressures were recorded for 4 min and then the broilers were injected intravenously with 100 µL of saline (0.9% NaCl; intravenous volume control). Five minutes later, the birds were injected intravenously with 100 µL of MG and the responses were recorded for 12 min. Saline (500 µL) then was injected intramuscularly (intramuscular injection control; pectoralis major muscle) and 3 min later the birds were injected intramuscularly with 500 µL of MG. Data were recorded for 20 min following the final MG injection, and the experiments were terminated with a 10-mL intravenous injection of 0.1 M KCl. The 3 birds used for cardiac output measurements also were injected intravenously with 100 µL/mL of MG.

Experiment 2 Clinically healthy broilers (7 wk of age) were injected intramuscularly with 500 µL of 0.9% saline (injection control, n = 5) or with 500 µL of MG (n = 5). Blood samples (3 mL) were collected from the wing vein in heparinized syringes immediately before the MG or saline injections and again 3 and 24 h postinjection. Before collecting each blood sample the percentage saturation of hemoglobin with oxygen and the heart rate were measured using a universal “C” sensor attached to a Vet/ Ox 4403 pulse oximeter (Sensor Devices Inc., Wauke-

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sha, WI) as described previously (Wideman and Kirby, 1995, 1996; Wideman et al., 1998, 2000). After the final blood sample was collected the birds were killed with CO2 gas, the heart was removed, and the ventricles were dissected and weighed to calculate the right ventricular weight:total ventricular weight (RV:TV) ratio, which is positively correlated with right ventricular work and the pulmonary arterial pressure (Julian et al., 1987; Peacock et al., 1989; Julian, 1993; Wideman, 2000). The blood samples were used for hematological assays and for mass spectrometer analyses. An automated hemotology analyzer (Cell-Dyn 3500 system, Abbott Laboratories, Abbott Park, IL), which was standardized for analysis of chicken blood, was used to determine the concentrations of white blood cells and red blood cells, the heterophil:lymphocyte ratio, the hematocrit, and the hemoglobin concentration. Mass spectrometry was used to identify possible modifications in the structure of hemoglobin. Chicken blood (2 μL) and an equal volume of 1 M 2,5-dihydroxybenzoic acid in 90% methanol containing 0.1% formic acid were mixed and spotted onto a Bruker MTP 384 stainless steel matrixassisted laser desorption ionization (MALDI) target. The MALDI time-of-flight spectra were acquired over the m/z range of 2 to 0 kDa in the positive ion linear mode using a Bruker Reflex III MALDI time-of-flight mass spectrometer (Bruker Daltonik GMBH, Bremen, Germany). The protocol used to average the pulmonary arterial pressure and blood flow during consecutive sampling intervals accommodates the influences of pulse pressure and respiratory cycles (Wideman et al., 1996). Based on the assumption that cardiac output (mL/min) normally is divided approximately equally between the lungs, the cardiac output was calculated as twice the blood flow through the left pulmonary artery. Assuming the left and right atrial pressures remain close to 0 as assessed by direct cannulation (right atrium) or wedge pressure (left atrium) measurements (Chapman and Wideman, 2001; Lorenzoni et al., 2008; Wideman et al., 2010), the pressure gradient across the pulmonary circulation is essentially equal to the pulmonary arterial pressure. Accordingly the relationship between pressure, flow, and resistance can be summarized by the following equation: pulmonary arterial pressure = cardiac output × pulmonary vascular resistance (Wideman et al., 1996). Thus, pulmonary vascular resistance was calculated in relative resistance units as pulmonary arterial pressure divided by cardiac output (Wideman et al., 1996). In experiment 1 the individual bird was used as the experimental unit and the data were analyzed over time (across sample intervals) using the SigmaStat one-way repeated measures ANOVA procedure (Jandel Scientific, 1994). Means were separated by the Tukey test when the F-test from the ANOVA was declared significant. In experiment 2 the data were analyzed by t-test using JMP software (SAS Inst. Inc., Cary, NC). The threshold for significance for all statistical comparisons was P ≤ 0.05.

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Figure 1. Pulmonary arterial pressure (mm of mercury) recorded in 8-wk-old male broilers before, during, and after injections of 100 µL of saline intravenously, 100 µL of methylglyoxal intravenously, 500 µL of saline intramuscularly, and 500 µL of methylglyoxal intramuscularly (mean ± SEM; n = 8). Data were recorded for 20 min following the final methylglyoxal injection. Letters represent differences (P ≤ 0.05) between sample intervals.

RESULTS AND DISCUSSION Experiment 1 The responses of the pulmonary arterial pressure to intravenous and intramuscular injections of saline and MG are shown in Figure 1. The intravenous injection of 100 µL of saline did not alter the pulmonary arterial pressure during the ensuing 5 min; however, the intravenous injection of 100 µL of MG triggered a rapid increase in the pulmonary arterial pressure that peaked approximately 2 min postinjection and then subsided to the preinjection (baseline) level during the next 4 min. The pulmonary arterial pressure did not change after the 500-µL intramuscular injection of saline; however, the 500-µL intramuscular injection of MG caused the pulmonary arterial pressure to increase and reach a peak approximately 3 to 4 min postinjection. During the ensuing 7 to 8 min the pulmonary arterial pressure returned again toward the baseline level (Figure 1). The contemporaneous values for pulmonary arterial pressure, cardiac output, and pulmonary vascular resistance at preinjection baseline levels, during the peak response to an intravenous injection of 100 µL of MG, and after the response to MG had subsided are shown in Figure 2. The peak increase in pulmonary arterial pressure (P = 0.019 compared with pre- and postinjection values) coincided with a decrease in cardiac output (P = 0.005) and an increase in pulmonary vascular resistance; P = 0.006). The baseline levels for

all parameters were restored within 6 min postinjection (Figure 2). The reduction in cardiac output during the peak response to MG indicates that the relatively weak right ventricle in these clinically healthy broilers was incapable of developing sufficiently increased pumping pressure to fully overcome the intense vasoconstriction triggered by MG. Increases in pulmonary vascular resistance sufficient to reduce the cardiac output despite concurrent increases in pulmonary arterial pressure have been observed previously in clinically healthy broilers in response to injections of serotonin (Chapman and Wideman, 2002, 2006), thromboxane (Wideman et al., 1999, 2005a; Chapman and Wideman, 2006), endotoxin (Wideman et al., 2001), epinephrine (Wideman, 1999), or acid (Wideman et al., 1998, 1999); inhalation of low atmospheric oxygen (Owen et al., 1995; Ruiz-Feria and Wideman, 2001); or physical occlusion (Wideman et al., 1996, 2005b; Wideman and Erf, 2002). The precise mode of action by which MG triggers pulmonary vasoconstriction remains to be determined. Methylglyoxal reacts with free Arg (Bourajjaj et al., 2003) and potentially can deplete the Arg reserves that are required for the synthesis of the potent vasodilator nitric oxide. In mammals MG damages the endothelium and interferes with the synthesis of nitric oxide by arginase (Wang et al., 2007, 2008; Sankaralingam et al., 2009). Nitric oxide also is an important vasodilator in the pulmonary vasculature of broilers, but acute inhibition of nitric oxide production typically only modestly increases the pulmonary arterial pressure by ≤2mm of Hg above


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Table 1. Pulse oximetry values for percentage saturation of hemoglobin with oxygen (Hb-O2) and heart rate recorded before and after broiler chickens were injected intravenously with 100 µL of either saline or methylglyoxal (MG)1 Hb-O2 (% saturation) Sample interval Preinjection 3 h postinjection 24 h postinjection 1Values

Saline

MG

93.8 ± 0.8 93.2 ± 0.8 93.8 ± 0.8

93.8 ± 0.8 92.0 ± 0.8 92.0 ± 0.8

Heart rate (beats/min)

     

Saline

MG

284 ± 7 284 ± 7 284 ± 7

284 ± 7 287 ± 7 281 ± 7

represent the mean ± pooled SE; n = 5 birds/treatment.

baseline levels (Wideman et al., 1996, 2005b, 2006; Wideman and Chapman, 2004; Bowen et al., 2006). Methylglyoxal is metabolized to lactic acid (Thornalley, 1996; Shinohara et al., 1998; Kalapos, 1999) and intra-

venous acid injections trigger pulmonary hypertension in broilers, presumably by stimulating thrombocytes to produce thromboxane or release serotonin (Wideman et al., 1998, 1999). Evidence that MG can enhance adrenergic receptor mediated responses in mammals (Rowan and Davies, 1991) may be significant in view of the potent pulmonary vasoconstriction elicited by epinephrine in broilers (Wideman, 1999). To the best of our knowledge the present results constitute the first demonstration in any vertebrate species that exogenously administered MG rapidly initiates pulmonary hypertension attributable to pulmonary vasoconstriction.

Experiment 2

Figure 2. Three birds were used to simultaneously assess contemporaneous values for pulmonary arterial pressure (mm of mercury), cardiac output (mL/min), and pulmonary vascular resistance (relative resistance units) during 4 min of preinjection baseline recordings (−4 min), during the peak response approximately 2 min after an intravenous injection of 100 µL of methylglyoxal, and 10 min postinjection after the response to methylglyoxal had subsided (mean ± SEM; n = 3). Letters represent differences (P ≤ 0.05) between sample intervals.

The pulse oximetry values for percentage saturation of hemoglobin with oxygen and heart rate are shown in Table 1. Preinjection pulse oximetry readings of 93% saturation of hemoglobin with oxygen indicated the birds in both groups were clinically healthy. The numerical reduction to 92% saturation in MG-injected birds was not significant (P > 0.10), nor were there significant changes in the heart rate. Figure 3 illustrates typical MALDI mass spectra of chicken blood from saline-injected control broilers (upper panel) and from broilers that had been injected with MG 3 h (middle panel) or 24 h (lower panel) before blood sample collection. Direct MALDI-MS spectra of hemoglobin typically show 2 separate peaks at 15,289.0 and 16,316.7 m/z attributable to the α and β subunits, respectively, as illustrated in Figure 3. These spectra are similar, suggesting no significant modifications occurred in the structure of hemoglobin, which concurs with the absence of significant change in the pulse oximetry readings. In vitro investigations of human hemoglobin demonstrated that MG can modify Arg residues in both α and β subunits (Gao and Wang, 2006). Methylglyoxal reacts with Arg residues in proteins or with free Arg to form the fluorescent product argpyrimidine and nonfluorescent product 5-hydro 5-methylimidazolone (Bourajjaj et al., 2003). The possibility exists that the high selectivity of MG for Arg residues in hemoglobin may have been neutralized under the in vivo condition of our experiments. The values for the hematological variables before (preinjection) and after (3 or 24 h postinjection) broilers were injected with saline or MG are shown in Table 2. Total white blood

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Table 2. Hematology values for white blood cell (WBC) and red blood cell (RBC) counts, heterophils, and lymphocyte percentages, heterophil to lymphocyte (H:L) ratio, hematocrit percentage, and hemoglobin concentration before and 3 or 24 h after broiler chickens were injected intramuscularly with 500 µL of either saline or methylglyoxal (MG)1 Preinjection Variable

Saline

WBC (×103/µL) RBC (×106/µL) Heterophil (%) Lymphocyte (%) H:L ratio Hematocrit (%) Hemoglobin (g/dL)

36.3 2.30 9.4 88.7 0.11 30.8 7.39

± ± ± ± ± ± ±

2.18 0.10 1.7 2 0.02 1.3 0.29

3 h postinjection MG

33.3 2.34 12.1 84.8 0.13 31.4 7.43

± ± ± ± ± ± ±

2.18 0.10 1.7 2 0.02 1.3 0.29

Saline 36.2 2.31 10.2 88.0 0.12 30.8 7.38

± ± ± ± ± ± ±

2.18 0.10 1.7 2 0.02 1.3 0.29

24 h postinjection

MG 37.7 2.24 9.6 88.3 0.11 30.4 7.10

± ± ± ± ± ± ±

2.18 0.10 1.7 2 0.02 1.3 0.29

Saline 37.1 2.20 9.7b 89.1b 0.11b 29.3 7.10

± ± ± ± ± ± ±

2.18 0.10 1.7 2 0.02 1.3 0.29

MG 35.6 2.09 16.5a 79.7a 0.22a 28.0 6.69

± ± ± ± ± ± ±

2.18 0.10 1.7 2 0.02 1.3 0.29

a,bValues 1Values

within a row and age group comparison are different (P < 0.05). represent the mean ± pooled SE; n = 5 birds/treatment.

cell and red blood cell concentrations, hematocrit, and hemoglobin concentrations were unaffected by the injections (P > 0.05). The percentages of heterophils and lymphocytes remained unchanged at 3 h postinjection; however, at 24 h after the MG injection the percentage of heterophils increased and the percentage of lymphocytes decreased when compared with the respective 24 h postinjection values for the saline group. Accordingly, the heterophil:lymphocyte ratio increased 2-fold in the MG group when compared with the saline group at 24 h postinjection (P < 0.05). The increased heterophil:lymphocyte ratio indicates the MG injections triggered a chronic stress response that was

evident at 24 h but not 3 h postinjection (Gross and Siegel, 1983; Vleck, 2001; Khajali et al., 2007). At 24 h postinjection the RV:TV ratios for the MG group (0.25 ± 0.01) were not higher than the RV:TV ratios for the saline group (0.23 ± 0.01). Nevertheless 1 bird in the MG group had an RV:TV value of 0.29 and spontaneously had developed ascites. The increased pulmonary arterial pressure triggered by intramuscular injection of MG (Figure 2) increased the right ventricular workload for less than 1 h before the response dissipated and the pulmonary vascular resistance returned to baseline values. Overall these pulse oximetry, hematology, and MALDI-MS spectra data do not provide evidence of an

Figure 3. Matrix-assisted laser desorption ionization–time-of-flight mass spectra of chicken blood from a saline-injected control broiler (upper panel) and from broilers that had been injected with methylglyoxal (MG) 3 h (middle panel) or 24 h (lower panel) before blood sample collection in experiment 2.


overt toxic response to the MG injections, although the increased heterophil:lymphocyte ratios do demonstrate that MG caused a chronic stress response. Broilers are susceptible to PHS when their pulmonary vascular capacity is anatomically or functionally inadequate to accommodate the requisite cardiac output without an excessive increase of the pulmonary arterial pressure. The consequences of an inadequate pulmonary vascular capacity have been demonstrated to include increased pulmonary vascular resistance attributable to noncompliant, fully engorged vascular channels. All factors contributing to an overall increase in pulmonary vascular resistance theoretically can initiate or accelerate the pathophysiological progression leading to terminal PHS (Wideman, 2000, 2001; Wideman et al., 2007). The present study demonstrates for the first time that exogenously administered MG rapidly initiates pulmonary hypertension attributable to pulmonary vasoconstriction. Methylglyoxal may contribute to the pathogenesis of PHS by initiating oxidative damage to the endothelium and by interfering with the production of the potent vasodilator nitric oxide. Finally, plasma and tissue levels of MG may be related to the high feed intake and primary metabolism of carbohydrates in ad libitum fed broilers (Fedde et al., 1998). In humans, MG levels are normally in the range 100 to 200 nM in the plasma and 2 to 4 µM in the tissues (Thornalley, 1996). In diabetic patients plasma levels of MG increase 3- to 5-fold, suggesting increased blood glucose levels lead to increased nonenzymatic dismutation of phosphotrioses into MG (Bourajjaj et al., 2003). Limited evidence also has associated diabetes mellitus with pulmonary hypertension and vascular damage in human patients (Movahed et al., 2005). Chickens normally have plasma glucose concentrations that are at least twice as high as those of mammals (Braun and Sweazea, 2008), but it remains to be determined whether increased blood sugar leads to increased production of MG in broiler chickens and the extent to which MG contributes.

ACKNOWLEDGMENTS This research was supported by NIH/National Heart Lung Blood Institute (Bethesda, MD) grant 1R15HL092517 01.

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