Per Jynge

High performance liquid chromatography: a rapid isocratic method for determination of creatine compounds and adenine nucleotides in myocardial tissue

Previous methods for high performance liquid chromatography (HPLC) measurements of myocardial high energy phosphate (HEP) compounds have used either two different assays or a gradient technique in order to determine both creatine compounds (creatine (Cr), creatine phosphate (CrP)), and adenine nucleotides (ATP, ADP, AMP). An isocratic ion-pair reversed-phase assay has been developed which allows for the rapid (single run within 10 min) measurement of these compounds in extracts from freeze-clamped and freeze-dried myocardial tissue. The HPLC system made use of a RP18 column and a spectrophotometer, set at 206 nm. The mobile phase consisted of KH2PO4 (215 mM), tetrabutylammonium hydrogen sulphate (2.3 mM) and acetonitrile (3.5%) at pH 6.25. A distinct separation (confirmed by enzymatic and other methods) of HEP compounds was achieved. Standard curves were linear and passed through the origo for all examined concentrations. In isolated rat hearts subjected to control aerobic perfusion the following values were obtained (mumol/g dry wt, mean +/- S.E.M.): ATP 26.6 +/- 0.4, ADP 4.6 +/- 0.1, AMP 1.4 +/- 0.1, CrP 35.5 +/- 1.0, Cr 46.2 +/- 1.1. Values for HEP compounds of hearts undergoing anoxia +/- reoxygenation, and ischemia +/- reperfusion are presented. The importance of the procedure for extraction of HEP from freeze-dried cardiac tissue is highlighted.

Cardiovascular safety of MnDPDP and MnCl2

Purpose: To investigate the apparent discrepancy between expected basic physiological responses at the cellular level and the in vivo behaviour of both MnDPDP and MnCl2 adminstered i.v. prompted parallel investigations of these substances. Material and Methods: Studies were performed in isolated perfused rat hearts, isolated bovine mesenteric arteries, conscious dogs, and dogs with acute ischaemic heart failure. Results: These studies confirmed that Mn++ at high concentrations acted as a calcium antagonist inducing negative inotropy. Mn++ at low concentrations was an effective su-peroxide scavenger, conserving nitric oxide and facilitating vasodilation. Mn++ maintained or elevated heart rate (HR) and blood pressure (BP), and did not worsen existing cardiac failure. MnDPDP was about 10 times less potent than MnCl2 in eliciting these cardiovascular responses. Conclusion: The ex vivo properties of Mn++, inducing vasodilation and negative inotropy, are counter-balanced in vivo through the action of 2 mechanisms: extensive plasma protein binding reducing active M++, and the release of catecholamines which maintain or even raise HR and BP. Taken together with pharmacokinetic factors, including maximal plasma concentrations in humans given the recommended 5 μmol/kg dose, it is concluded that MnDPDP in normal clinical use represents no safety risk to the cardiovascular system.

Intracellular manganese ions provide strong T1 relaxation in rat myocardium.

The efficacy of manganese ions (Mn2+) as intracellular (ic) contrast agents was assessed in rat myocardium. T1 and T2 and Mn content were measured in ventricular tissue excised from isolated perfused hearts in which a 5‐min wash‐in with 0, 30, 100, 300, or 1000 μM of Mn dipyridoxyl diphosphate (MnDPDP) was followed by a 15‐min wash‐out to remove extracellular (ec) Mn2+. An inversion recovery (IR) analysis at 20 MHz revealed two T1 components: an ic and short T1‐1 (650–251 ms), and an ec and longer T1‐2 (2712–1042 ms). Intensities were about 68% and 32%, respectively. Tissue Mn content correlated particularly well with ic R1‐1. A two‐site water‐exchange analysis of T1 data documented slow water exchange with ic and ec lifetimes of 11.3 s and 7.5 s, respectively, and no differences between apparent and intrinsic relaxation parameters. Ic relaxivity induced by Mn2+ ions in ic water was as high as 56 (s mM)−1, about 8 times and 36 times higher than with Mn2+ aqua ions and MnDPDP, respectively, in vitro. This value is as high as any reported to date for any synthetic protein‐bound metal chelate. The increased rotational correlation time (τR) between proton and electron (Mn2+) spins, and maintained inner‐sphere water access, might make ic Mn2+ ions and Mn2+‐ion‐releasing contrast media surprisingly effective for T1‐weighted imaging. Magn Reson Med 52:506–514, 2004.

Gut luminal microdialysis of glycerol as a marker of intestinal ischemic injury and recovery

Objective:To evaluate microdialysis as a method to assess different degrees of intestinal damage and recovery during ischemia and reperfusion; to evaluate information obtained from microdialysis catheters in the peritoneum, the gut wall, and the gut lumen. Design:Randomized, controlled animal experiment. Setting:University laboratory animal center. Subjects:Twenty-seven domestic pigs. Interventions:The superior mesenteric artery was cross-clamped for 60 mins (n = 14) or 120 mins (n = 10) followed by 2 or 4 hrs of reperfusion. Three pigs served as controls. Measurements and Main Results:Intestinal mucosal integrity was assessed by morphometry, adenosine triphosphate in the gut wall, and permeability of 14C-polyethylene glycol. Lactate, glycerol, pyruvate, and glucose were measured by microdialysis. Changes in adenosine triphosphate, permeability, or lactate did not correlate to different extents of intestinal damage caused by 60 or 120 mins of ischemia. During the reperfusion period, pigs with 60 mins of intestinal ischemia showed a faster recovery of these variables than pigs with 120 mins of intestinal ischemia. Glycerol increased with increasing duration of the ischemic insult. After 60 mins of intestinal ischemia, glycerol in the gut lumen decreased toward baseline but remained high after 120 mins of intestinal ischemia. There was a good correlation between gut luminal glycerol and recovery of mucosal damage throughout the reperfusion period. In the peritoneal cavity, both glycerol and lactate decreased to baseline relatively shortly after onset of reperfusion independent of the duration of intestinal ischemia. Conclusions:Microdialysis of glycerol provides information about the extent and severity of intestinal damage after ischemia and about the ensuing recovery. The gut lumen is to be preferred as a site for placement of microdialysis catheters.

Manganese dipyridoxyl-diphosphate (MnDPDP) as a viability marker in patients with myocardial infarction

To evaluate contrast accumulation in left ventricular (LV) myocardium after manganese dipyridoxyl‐diphosphate (MnDPDP) administration in patients with recent first time myocardial infarction.

Effects of manganese dipyridoxyl diphosphate, dipyridoxyl diphosphate--, and manganese chloride on cardiac function. An experimental study in the Langendorff perfused rat heart.

RATIONALE AND OBJECTIVES.Manganese dipyridoxyl diphosphate (MnDPDP) is a promising contrast agent for magnetic resonance imaging of the liver. The authors explored the possibility that high concentrations of MnDPDP may cause manganese ion (Mn++)-induced side effects on cardiac function. METHODS.Potential cardiodepression by MnDPDP, DPDP– –, and manganese chloride (MnCl2) (100-3,0000 µmol/ L) was investigated in the isolated rat heart, with left ventricular developed (systolic—end-diastolic) pressure and heart rate as the primary indices of cardiac function. RESULTS.During 5-minute exposures, 10% and 50% decreases in left ventricular developed pressure were observed for MnDPDP, 250 µmol/L and 1580 /µmoI/L; DPDP– –, less than 100 µmol/L and 1000 /µmoI/L; MnCl2, 30 /µmol/L and 250 /µmol/L. Heart rate changes were not observed with MnDPDP. Cardiodepression was reversed within 2 minutes during a 14- minute recovery period for all investigated concentrations of MnDPDP and was less rapid for the highest concentrations of MnCl2. CONCLUSIONS.Manganese dipyridoxyl diphosphate is well tolerated in the rat heart at concentrations as high as 200 to 250 limol/L and is approximately 10 times less cardiotoxic than MnCl2. Cardiodepressive effects of MnDPDP in the present rat heart model, perfused in the absence of blood and proteins, are related primarily to the release of free Mn++ions and in part to the simultaneous release of DPDP¯ ¯.

Myocardial manganese elevation and proton relaxivity enhancement with manganese dipyridoxyl diphosphate. Ex vivo assessments in normally perfused and ischemic guinea pig hearts.

Manganese (Mn) dipyridoxyl diphosphate (MnDPDP) is the active component of a contrast medium for liver MRI. By being metabolized, MnDPDP releases Mn2+, which is taken up and retained in hepatocytes. The study examined whether MnDPDP elevates Mn content and enhances proton relaxivity in normal myocardium, but not in ischemic myocardium with reduced coronary flow and impaired metabolism. Isolated guinea pig hearts were perfused at normal flow or low flow, inducing global subtotal ischemia. Ventricular ATP and Mn contents, T1 and T2 were measured. At normal flow tissue Mn content increased from the control level of 4.1 to 70.4 µmol/100g dry wt with MnDPDP (3000 µM), while low‐flow perfusion with MnDPDP (3000 µM) resulted in a Mn content of 16.6 µmol/100 g dry wt. Prolonged ischemia (35 and 90 min) reduced tissue Mn down to the control level. T1 shortening closely paralleled myocardial Mn elevations during both normal and low‐flow perfusion. The use of a Mn2+‐releasing contrast agent like MnDPDP may be a promising principle in MRI assessments of myocardial function and viability in coronary heart disease by revealing a differential pattern of changes in T1 relative to coronary flow, cell Mn uptake and retention, ion channel function and metabolism. Copyright

Oxidative stress and inflammatory response during and following coronary interventions for acute myocardial infarction

Background: In acute myocardial infarction (AMI) treated with percutaneous coronary intervention (PCI), myocardial injury results from complex processes during both ischemia and reperfusion. Release of reactive oxygen species (ROS) may contribute to the accumulated myocardial damage. Aims: To examine by frequent sampling of peripheral blood oxidative stress and early inflammation in patients undergoing primary PCI for AMI. Secondly, to assess whether a correlation exists between these parameters and the extent of myocardial damage. Methods: Sixteen patients undergoing primary PCI within 6 h of AMI onset were included. Peripheral blood was sampled at start of procedure (t0) and repeatedly over 24 h following reperfusion. Main plasma analyses were: 8-iso-PGF2α (oxidative stress), 15-keto-dihydro-PGF2α (cyclooxygenase-mediated inflammation); and troponin-T (myocardial injury). Additional analyses included: total antioxidant status (TAS); vitamins; hsCRP and lipids. Results: 8-Iso-PGF2α increased following restoration of blood flow, returned to t0 values after 3 h and was reduced below t0 the following day. TAS decreased significantly from t0 to the next day. There was no significant correlation between 8-iso-PGF2α and troponin T values. 15-Keto-dihydro-PGF2α was elevated during the first hour. There was a major rise in hsCRP after 24 h. Conclusion: Following reperfusion by primary PCI in AMI, oxidative stress and an inflammatory response are induced immediately. A rise in 8-iso-PGF2α during ischemia indicate that ROS generation may also take place during severely reduced coronary blood flow and hypoxia. No direct relationship between 8-iso-PGF2α or 15-keto-dihydro-PGF2α and troponin T was evident. The present study adds to the increasingly complex pathophysiological roles of ROS acting both as signal molecules and as mediators of tissue injury.

Oxidative stress and myocardial damage during elective percutaneous coronary interventions and coronary angiography. A comparison of blood-borne isoprostane and troponin release.

The role of oxidative stress in clinical cardiology is still controversial. The aims of the present study were to examine if minor ischaemic episodes as may occur during elective percutaneous coronary intervention (PCI) induce oxidative stress and, eventually, if oxygen stress correlates with myocardial injury. Thirty eight and nine patients underwent PCI and diagnostic coronary angiography, respectively. Peripheral blood was sampled at different time points for plasma analyses of: 8-iso-PGF2α (free radical-mediated oxidative stress); 15-keto-dihydro-PGF2α (cyclooxygenase-mediated inflammation); troponin-T (myocardial injury); hsCRP, vitamin A and vitamin E; and, total antioxidants status (TAS). In both groups 8-iso-PGF2α increased transiently by approximately 80% (p<0.001) during the procedure. There was a minor troponin-T release (p<0.001) after PCI, but no correlation with 8-iso-PGF2α. Troponin-T did not increase after angiography. 15-keto-dihydro-PGF2α decreased by 50% after ended procedure, but increased by 100% after 24 h compared to baseline. hsCRP increased significantly (p<0.001) from baseline to the next day in the PCI-group, but not in the angiography group. Vitamins and TAS decreased slightly after the procedures. It is concluded that a moderate oxidative stress was induced by both elective PCI and coronary angiography but that no correlation was found between oxidative stress and myocardial injury in this setting. This indicates that other mechanisms than ischaemia–reperfusion episodes caused an elevation in plasma isoprostane such like the injury at a vascular site mutual for both procedures. A secondary finding from the study was elevated markers of early inflammatory response, not only after PCI, but also after angiography.

Sodium-calcium balance and cardiac function with isotonic iodixanol. An experimental study in the isolated rat heart.

RATIONALE AND OBJECTIVES.Three formulations of the nonionic dimer iodixanol (150/200/300 mg I/mL), made isotonic by the addition of NaCl (70/53/24 mM), were investigated regarding their potential for depressing cardiac contractility during coronary angiography. To maintain a stable cardiac function, the authors sought the requirements for the addition of a balanced amount of calcium (Ca). METHODS.Iodixanol 150, 200, and 300 mg I/mL were applied as a short-lasting bolus in isolated perfused rat hearts in the absence and presence of added Ca. The contractile function was assessed by measurement of changes in left ventricular developed pressure (LVDP). RESULTS.A transient LVDP depression was markedly alleviated by adding 0.2 to 0.4 mM Ca and almost abolished by 0.4 to 0.9 mM. Ca higher than 0.9 mM led to unstable hearts and too-extensive Ca loading. CONCLUSIONS.Caution should be used when adding Ca to iodixanol, particularly with sodium-calcium (Na–Ca) relationships. Appropriate Ca concentrations are probably 0.6 mM for iodixanol (150 mg I/mL); 0.4 to 0.6 mM for iodixanol (200 mg I/mL; and 0.4 mM for iodixanol (300 mg I/mL).