Fast, affordable and eco-friendly enzyme kinetic method for the assay of α-ketoglutaric acid in medical product and sports supplements.

A novel kinetic method was developed for the quantitation of α-ketoglutaric acid (AKG) in cardioplegic solution and athletic supplements. The assay relied on an enzymatic transamination of AKG and d-4-hydroxyphenylglycine to form 4-hydroxybenzoylformic acid and l-glutamic acid using d-phenylglycine aminotransferase. Since 4-hydroxybenzoylformic acid absorbed UV strongly at 334 nm, the initial rate of the reaction was determined by the increasing absorbance at this wavelength without the need for colorimetric probes or coupling reactions, and this information was used for the construction of a standard curve against AKG concentration. The method showed good linearity (r2 = 0.9994) over an AKG concentration range of 20-160 µM. The limits of detection and quantitation were 4.09 and 13.62 µM respectively. It was simple, inexpensive, accurate and precise, as well as repeatable, and was not interfered with by excipients in the samples. Regarding the environmental friendliness, the method was free from the use of organic solvents or hazardous reagents and required no chemical pre-treatment of samples. The proposed method gave assay results tested in real samples in agreement with the HPLC method and commercial assay kits, therefore being suitable for routine analysis of AKG in quality control laboratories.

Haemodynamic and metabolic effects of gallopamil as additive to calcium-containing and calcium-free cardioplegic solutions in mature pig hearts.

Myocardial haemodynamic and metabolic effects of the calcium-channel blocker gallopamil as additive to calcium-containing (St Thomas Hospital, STH) and calcium-free (Bretschneider procaine-containing, BRT) crystalloid cardioplegic solutions were evaluated. Adult pig hearts (weight 0.033 kg) were randomized to four groups and perfused with 1 litre of cold (4 degrees C) cardioplegic solution; group A: BRT without gallopamil, n = 9, group B: BRT with gallopamil (0.4 microM), n = 8, group C: gallopamil-free STH, n = 8, and group D: STH with gallopamil (0.4 microM), n = 8. After storage at 4 degrees C for 6 hours the hearts were reperfused with blood/Ringer solution in a modified Langendorff model for 60 min. Developed left ventricular pressure, rate-pressure product and +dP/dt were lower in gallopamil-treated hearts during reperfusion (p < 0.05), as were oxygen extraction and oxygen uptake (p < 0.05) and lactate release (p < 0.05). Myocardial blood flow was greater in gallopamil-treated hearts (p < 0.05). In hearts comparable in size and anatomy to the human heart, gallopamil added to both cardioplegic solutions reduced cardiac function and oxygen uptake despite increased myocardial blood flow. The findings suggest reduced myocardial protection after addition of gallopamil to cardioplegic solutions.

Substrate enhancement of cardioplegic solution: experimental studies and clinical evaluation.

BACKGROUND: The development of myocardial protective strategies depends on a complete understanding of the pathophysiology of myocardial ischemia and reperfusion. This article reviews the rationale for inclusion of metabolic substrates in cardioplegic solutions on the basis of our current understanding of the underlying pathophysiologic pathways and speculates on the inclusion of future additives that await further investigation. METHODS: The pathophysiology of myocardial ischemia and reperfusion was evaluated from an extensive review of the pertinent literature. Experimental and clinical studies supporting the inclusion of metabolic substrates in clinical cardioplegic solutions were reviewed and summarized. Speculation on possible future additives to these formulas was made on the basis of encouraging, albeit preliminary, experimental data. RESULTS: Sound experimental and clinical evidence supports the inclusion of glucose, amino acids, calcium chelators, and oxygen as fundamental substrate additives to current cardioplegic solutions. Antioxidants, calcium-channel blockers, and tricarboxylic acid cycle intermediates may be of value. Adenosine, potassium-adenosine triphosphate channel modulators, and nitric oxide may join these lists after further research. CONCLUSIONS: Substrate enhancement of clinical cardioplegic solutions is based on physiologic principles that have been confirmed in the clinical setting. Further definition of the intricacies of myocardial ischemia and reperfusion promises to expand the current list of additives.

Effect of hemoglobin concentration on oxyhemoglobin dissociation during hypothermic blood cardioplegic arrest.

BACKGROUND: This study compares oxyhemoglobin dissociation during the nonperfused periods of hypothermic cardioplegic arrest in two blood cardioplegic solutions with different hemoglobin concentrations. The hypothesis is that more oxygen will dissociate from hemoglobin in a blood cardioplegic solution with a higher hemoglobin content than from a cardioplegic solution with a lower hemoglobin content. However, the increment in the volume of oxygen that dissociates from hemoglobin will be less than anticipated by a ratio of hemoglobin concentrations in the cardioplegic solution. METHODS AND RESULTS: Pigs (n = 22) were supported by bypass and subjected to 60 minutes of hypothermic cardioplegic arrest with either a high-hemoglobin (n = 10) or low-hemoglobin (n = 12) blood cardioplegic solution. Aortic root and coronary sinus blood samples were obtained before bypass and 5 seconds after the start of cardioplegic infusions at 20, 40, and 60 minutes of cardioplegic arrest. Oxyhemoglobin dissociation occurred in both experimental groups during the ischemic intervals of cardioplegic arrest. However, there were no significant differences between the high- and low-hemoglobin groups in the arterial-venous oxygen content differences for samples taken after each of the three ischemic intervals (p values: control = 0.78; cardioplegia interval 1 = 0.95; interval 2 = 0.56; and interval 3 = 0.12). CONCLUSIONS: The present study emphasizes the inherent limitations of unmodified erythrocyte hemoglobin as an oxygen source in hypothermic alkalotic cardioplegic solutions. These limitations may be obviated by methods that increase the dissolved oxygen content of the cardioplegic solution or methods that decrease the affinity of hemoglobin for oxygen under conditions of hypothermia and alkalosis.

Inhomogeneous and complementary antegrade and retrograde delivery of cardioplegic solution in the absence of coronary artery obstruction.

Inhomogeneous delivery of cardioplegic solution may result in postischemic myocardial injury. This study compares the distribution of warm blood antegrade and retrograde cardioplegia to multiple discrete left ventricular myocardial regions in pigs with unobstructed coronary arteries. Cardioplegic solution was delivered antegradely and retrogradely at 150 ml/min, and flows to 1152 individual myocardial regions were determined twice for each route with four different radiolabeled microspheres. The antegrade system delivered greater flow to each gram of myocardium than did the retrograde system (1.37 +/- 0.31 versus 0.39 +/- 0.09 ml/gm per minute, p < 0.001). Flow to individual myocardial regions was significantly inhomogeneous for both antegrade and retrograde cardioplegia, but much more so for retrograde cardioplegia (coefficient of variation was 48% +/- 17% for antegrade cardioplegia and 106% +/- 16% for retrograde cardioplegia; p < 0.001). The pattern of flow to individual myocardial regions was highly reproducible for a given route of delivery as confirmed by repeated measurements with different radioactive microsphere isotopes (correlation coefficients 0.88 +/- 0.12 for AC1-AC2 and 0.84 +/- 0.10 RC1-RC2), but antegrade cardioplegia and retrograde cardioplegia patterns were significantly different and therefore complementary (correlation coefficients 0.03 +/- 0.04, p < 0.001). These findings support the routine combined use of antegrade cardioplegia and retrograde cardioplegia to enhance delivery of cardioplegic solution to all regions of the heart and minimize the potential risk of postischemic myocardial dysfunction.

[Practical aspects of cardioplegia]. / Nekotorye prakticheskie voprosy kardioplegii.

Concentrations of the ingredients have been analysed in a nonstandard cardioplegia solution (CS) and CS from St. Thomas Hospital at the outset before CS introduction into the coronary vessels of patients under cardioplegia. It has been shown that the use of nonstandard CS was accompanied by undesirable variability in ingredient concentration, which to some extent may be accounted for by the use of incompletely unfrozen CS. The use of CS from St. Thomas Hospital could be accompanied by moderate hypermagnesiumemia, which did not lead to the onset of vascular insufficiency. It is stressed that plasma magnesium content should be controlled when this CS is used.

Cardiac-derived neutrophil chemotactic factors: detection in coronary sinus effluents of patients undergoing myocardial revascularization.

Recent studies from our laboratory have demonstrated the release of neutrophil chemotactic factors from isolated rabbit hearts perfused with cardioplegic solutions and from ischemic dog hearts after coronary artery occlusion for 1 hour. On the basis of these animal studies, a test is now made of the hypothesis that neutrophil chemotactic factors are released by myocardial tissues of patients who undergo surgical myocardial revascularization. By means of modified Boyden chambers, the levels of neutrophil chemotactic factors were measured in effluent collected from the coronary sinuses of six patients undergoing cardiopulmonary bypass during periods of cold cardioplegia. Plain cardioplegic solutions were also analyzed. The standard formyl-methionyl-leucyl-phenylalanine, a stimulant of neutrophil recruitment, was used as a positive control solution. Results indicated the recovery of significantly high levels of neutrophil chemotactic factors in patient samples (i.e., 128% +/- 19% of formyl-methionyl-leucyl-phenylalanine) compared with control plain cardioplegic solution (less than 5% of formyl-methionyl-leucyl-phenylalanine) (p less than 0.0001). A standard checkerboard analysis indicated that the observed activity is chemotactic (i.e., directed migration) and not chemokinetic (i.e., random migration). This study also showed that these factors are proteins of a molecular weight in excess of 300 kd and exhibit in vivo activity by recruiting neutrophils into rabbit skin. The absence of immune cell-derived chemoattractants such as interleukin-1 and leukotriene B4 in these coronary sinus effluents suggests that the observed chemotactic activity is cardiac derived. Results of this investigation therefore demonstrate the release of neutrophil chemotactic factors by ischemic human hearts during cardiopulmonary bypass.

Initial experience with low-potassium cold blood cardioplegia: a clinical comparative study.

This study presents the results of bypass grafting in 96 patients operated on for triple-vessel coronary artery disease between May 1988 and September 1990. In the first 54 patients a cold crystalloid solution was employed, and in the 42 more recent patients cold blood low-potassium cardioplegia was employed. There were no differences in postoperative cardiac index or left ventricular stroke work index. Yet, in patients with impaired prebypass left ventricular stroke work index, postbypass left ventricular performance correlated negatively with duration of aortic cross-clamping in the cold crystalloid group (r = -0.441, p = 0.045). In contrast, no correlation was found in the cold blood low-potassium group (r = 0.125, p = 0.587). The incidence of myocardial infarction, need for inotropic support, and need for intraaortic balloon counterpulsation were similar among the groups. Release of the myocardial isoenzyme creatine kinase-MB from 12 to 30 hours after operation was significantly less in the low-potassium blood cardioplegia group. The use of low-potassium blood cardioplegia resulted in a marked reduction in the operative administration of fluids (1,527 +/- 87 versus 3,511 +/- 148 mL; p less than 0.001). In conclusion, low-potassium cold blood cardioplegia is a simple and effective method of myocardial protection. The fact that left ventricular stroke work index recovery was not dependent on the duration of aortic occlusion and that release of the MB isoenzyme of creatine kinase was reduced in the low-potassium blood cardioplegia group implies better myocardial protection.

[The effects of calcium concentration of reperfusion solution upon myocardial protection].

The effects of several calcium concentrations in reperfusion solution were studied. Experimental time course was as followed: 20 min working perfusion, 3 min cardioplegic infusion with St. Thomas cardioplegic solution (STS) followed by global ischemia for 30 min at 37.5 degrees C, 15 min early Langendorff reperfusion with reperfusion solution and 5 min late reperfusion with Krebs Henseleit bicarbonate buffer [( Calcium] = 2.5 mM), followed by 20 min working perfusion. Percent recoveries of aortic flow at the Ca concentration of 0, 0.1, 0.6, 1.2, 1.8, 2.5 mM were 0, 14 +/- 1, 43 +/- 4, 64 +/- 3, 55 +/- 2, 59 +/- 1 (%), respectively. Our data indicated that reperfusion solution with less than 1.2 mM calcium reduced the protective properties of STS.

Purines, lactate and phosphate release from child and adult heart during cardioplegic arrest.

The release of lactate, phosphate and purine catabolites from the heart in adult and children undergoing cardiac surgery was recorded. The compounds were determined in the coronary effluent collected during subsequent infusions of cardioplegic solution into the coronary root. As compared to the infusion just after onset of ischemia, both in adults and children manifold increase of the release was observed during subsequent infusions. The rates of release of lactate, phosphate and purines (adenosine + inosine + hypoxanthine) were 1.5 to 2.5 times higher in children than in adult hearts during the second cardioplegic infusion and 3 to 7 times higher during the third cardioplegic infusion in spite of a more frequent infusion of cardioplegic solution in children. A much greater increase of the release of lactate, phosphate and purines provides evidence for more severe metabolic injury during cardioplegic arrest to the heart in children than in adults.

The concentration of calcium in neonatal cardioplegia.

The optimal calcium concentration in cardioplegia for the newborn has not been determined. Therefore, the effect of 0, 0.6, 1.2, 1.8, and 2.4 mmol/L calcium in modified St. Thomas cardioplegia was evaluated in isolated working hearts of 7- to 10-day-old rabbits. Functional recovery was determined by comparing aortic flow, developed pressure, and first derivative of left ventricular pressure (dP/dt) before and after 1 hour of normothermic (37 degrees C) ischemia. As percentages of baseline values, recovery of developed pressure and dP/dt averaged 10% +/- 1% (mean +/- standard error of the mean) and 10% +/- 1% with 0 mmol/L, 46% +/- 7% and 44% +/- 8% with 0.6 mmol/L, 79% +/- 2% and 76% +/- 2% with 1.2 mmol/L, 67% +/- 2% and 61% +/- 5% with 1.8 mmol/L, and 65% +/- 5% and 65% +/- 7% with 2.4 mmol/L calcium, respectively. Significant improvement in recovery of developed pressure and dP/dt was detected when the calcium concentration was increased from 0 to 0.6 mmol/L and from 0.6 to 1.2 mmol/L, but the groups with 1.2, 1.8, and 2.4 mmol/L did not differ from one another significantly in terms of developed pressure and dP/dt recovery. There was no recovery of aortic flow when 0 mmol/L calcium was used; at calcium concentrations of 0.6, 1.2, 1.8, and 2.4 mmol/L, recovery of aortic flow averaged 16% +/- 7%, 63% +/- 10%, 23% +/- 10%, and 36% +/- 11% of baseline values, respectively. Recovery of aortic flow with 1.2 mmol/L calcium was significantly higher than at concentrations of 0.6 and 1.8 mmol/L.(ABSTRACT TRUNCATED AT 250 WORDS)

[Optimal potassium concentration in cardioplegic solutions].

A model of perfused cardiac papillary muscle from guinea pig was set up in our lab. The procaine-free St. Thomas' Hospital solution was used as the basic cardioplegic solution. The potassium concentration of the solution was designed by optimization (14.6, 22.8, 28.5, 32.6, 57.8 mmol/L). The papillary muscle was undergone anoxic arrest for 60 min in 32 degrees C. The effect of myocardium preservation was assessed with cardiac action potential, contractility and quantitative analysis of ultrastructure. We concluded that; (1) in this research the proper potassium concentration is at the range of 20.7-26.0 mmol/L and the optimal one is 22.8 mmol/L; (2) despite any deviation from the range, phases II and III of the action potential changes first. [K+] greater than or equal to 32.6 mmol/L slows conductivity, weakens contractility and damages the subcellular structure severely; (3) if [K+] is 57.8 mmol/L, anoxic arrest for 60 minutes in 32 degrees C damages of both structure and function of myocardium is irreversible; (4) following the hyperkalemic cardioplegia, there is a secondary change of cardiac action potential in reperfusion period, which shows longer phase II and shorter phase III, smaller Vmax and APA; (5) reperfusion arrhythmia after anoxic hyperkalemic asystole is likely caused by all kinds of conductive disorders resulting from the secondary changes of action potential; (6) arrest time, AT (Y, s) has the negative relativity to the potassium concentration (X, mmol/L) of cardioplegic solution, which can regress to curve 1/Y = 0.10-1.38/X(14.6 less than or equal to X less than or equal to 57.8). The minimum potassium concentration which can guarantee AT within 30 seconds is 20.7 mmol/L.

Estudio comparativo de tres soluciones cardiopléjicas en pacientes sometidos a cirugía cardíaca / Comparative study of three cardioplegic solutions in patients submitted to cardiac surgery

Veinte pacientes operadores de cirugía de by pass aortocoronario fueron divididos en grupo I (n=7), que recibió como protección miocárdica una solución cardiopléjica standard; grupo II (n=6), que recibió una solución cardiopléjica conteniendo manitol; y grupo III (n=7) que recibió solución cardiopléjica con deferoxamina. Se tomaron biopsias de miocardio, previo al período isquémico (muestras A o preisquémicas) y a los 10 minutos de la reperfusión (muestras B o de reperfusión) las cuales fueron procesadas para quimioluminiscencia para detectar actividad de radicales libres y para microscopía electrónica con el objeto de evaluar lesión miocárdica a nivel de ultraestructura. Se observó que en el grupo I se producía un significativo aumento de los valores de quimioluminiscencia en las muestras B y ésto se asociaba con la presencia de áreas con franco edema mitocondrial. En los grupos II y III no había diferencia significativa entre las muestras A y B. Los resultados sugieren que durante la reperfusión se produce un daño miocárdico que en parte es causado por la citotoxicidad de los radicales libres del oxígeno. Se concluye además que la deferoxamina y más el manitol reducen las lesiones de reperfusión y que su mecanismo de acción sería por la capacidad antioxidante que poseen ambas sustancias

Adenylate degradation products release from the human myocardium during open heart surgery.

Purine degradation products were determined in the human heart coronary sinus effluent collected from patients undergoing cardiac surgery, during infusion of a cardioplegic solution. At the onset of cardiopulmonary bypass the mean concentrations of adenosine, inosine and hypoxanthine were 0.1, 0.5 and 0.3 mumol/l, respectively. Ischemic arrest leads to a progressive increase of the respective levels to 1.4 17.8 and 9.6 mumol/l after 60-80 min of ischemia. Xanthine concentration was undetectable (less than 0.2 mumol/l) throughout. A substantial urate release (20 mumol/l) was observed which decreased with the duration of ischemia. Xanthine oxidoreductase activity in human myocardium was found to be below the detection limit (0.1 mU/g wet weight). Thus, urate release represented wash out of urate which had accumulated in the tissue.

Free radicals and cardioplegia: the absence of an additive effect with allopurinol pretreatment and the use of antioxidant enzymes in the rat.

The isolated perfused working rat heart model of cardiopulmonary bypass was used to assess whether (a) allopurinol pretreatment enhances resistance to normothermic (30 min) or hypothermic (4 h) ischemia; (b) addition of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) to cardioplegic and/or reperfusion solutions are protective; (c) any protective effects are additive. With normothermic ischemia, allopurinol pretreatment improved recovery of aortic flow from its control value of 25 +/- 3% to 48 +/- 6% (P less than 0.05). Similarly, SOD plus CAT used during both ischemia and reperfusion improved recovery of aortic flow from a control value of 28 +/- 4% to 48 +/- 6% (P less than 0.05). However, various combinations of the two types of intervention afforded no additive protection. Under hypothermic (21 degrees C) conditions, allopurinol pretreatment was not effective, whereas SOD and CAT added during ischemia and reperfusion improved recovery of aortic flow from its control value of 53 +/- 4% to 69 +/- 5% (P less than 0.05). This value was similar to allopurinol pretreatment and SOD plus CAT added during ischemia and reperfusion (69 +/- 6%: P less than 0.05). These results provide further evidence that reperfusion-induced free radical formation may adversely affect postischemic recovery of function. The absence of an additive effect suggests a common mechanism of action, which is likely to involve the free radical-generating enzyme xanthine oxidase; however, other mechanisms may exist. Our results further support the use of antifree radical intervention in conjunction with cardioplegia to protect the heart during ischemia and reperfusion.