Nicotinamide Adenine Dinucleotide Fluorescence Research Articles

Mechanism of action of hyaluronidase in decreasing myocardial ischemia post coronary occlusion in the isolated perfused rabbit heart

The influence of hyaluronidase (H) on subacute experimental myocardial ischemia was studied in isolated perfused rabbit hearts. Changes in ischemic area were assessed by epicardial nicotinamide adenine dinucleotide (NADH) fluorescence photography, an intrinsic high-resolution display of myocardial ischemia. Computerized determination of ischemic area was made from standardized photographs. Hyaluronidase was begun 20 minutes after coronary artery occlusion at 4 units/ml perfusate. NADH fluorophotographs were taken at 10-minute intervals up to 60 minutes of ischemia. Coronary sinus oxygen tension (P csO 2), myocardial oxygen consumption (MV̇O 2), and coronary flow were determined. After 70 minutes, the hearts were perfused with rhodamine solution to identify areas of myocardial perfusion. In 13 H-treated hearts 54.3% ± 3.7% (mean ± SEM) of the nonperfused area (rhodamine stained) was ischemic (NADH fluorescent). In 14 untreated hearts 79.8% ± 3.2% of the nonperfused area was ischemic ( p < 0.0001) and the ischemic areas were uniform. The distance between perfused and ischemic tissue was 952 ± 78 μm in the H hearts and 504 ± 35 μm in the untreated hearts ( p < 0.0001). In the H treated hearts P csO 2 increased to 155% of the post-ligation control while it decreased to 79% in the untreated hearts ( p < 0.0001). MV̇O 2 decreased in the H-treated hearts to 62%; the untreated hearts had no further change. In the H-treated hearts, coronary flow increased to 146% of the post-ligation control while it fell to 91% in the untreated group ( p < 0.0001). We conclude that H increases coronary flow while decreasing MV̇O 2 during subacute ischemia. In H-treated hearts, significant amounts of myocardium remain normoxic within the nonperfused areas, and may potentially be salvaged after prolonged myocardial ischemia.

Intracellular oxidation-reduction state measured in situ by a multichannel fiber-optic surface fluorometer.

The principles of the measurement in vivo of the oxidation-reduction state of intramitochrondrial pyridine nucleotides were used in establishing a multichannel fluorometer-reflectometer. This approach made possible the study of changes of mitochrondrial redox states in four different organs (brain, liver, kidney, and testis) of the same animal, as well as the monitoring of four different cortical areas of the same brain hemisphere. In the measurement of reduced nicotinamide adenine dinucleotide fluorescence, oximetric and movement artifacts are negligible, but blood volume changes and tissue absorption properties are a source of error. The corrected fluorescence is obtained by subtracting the reflectance from the fluorescence signed in 1:1 ratio., During graded hypoxia, the corrected fluorescence showed a gradual increase and was maximal during anoxia in all four organs tested.

Regional myocardial function and metabolism during acute coronary artery occlusion

Regional changes in myocardial function and oxidative metabolism during acute coronary artery occlusion were recorded spectrophotometrically by incorporating fiber optics in the isolated rat heart perfused by Langendorff's procedure. Oxygen saturation of myoglobin, reduction of cytochrome aa3, and the dynamic wall thickness of the left ventricle were continuously and concurrently measured from absorbancy increments at 581-592 nm, 605-630 nm, and 568-592 nm, respectively. In contrast to a gradual decrease in the extent of systolic wall thickening in anoxia, observed decreases in both the extent and the duration of systolic wall thickening and the appearance of a late systolic bulge occurred within 5 s after the onset of regional ischemia. After 10 s of both anoxia and regional ischemia, oxygen saturation of myoglobin decreased by 50%, but fluorescence of nicotinamide adenine dinucleotide remained at aerobic level which indicated that mitochondrial oxidative energy production might still be maintained. Thus early and pronounced dysfunction of the ischemic region appeared to precede a substantial loss of ATP production.

Visualization of the distance between perfusion and anoxia along an ischemic border.

The distance between perfusion and anoxia was measured on the border of an experimental ischemic area in the rabbit heart. Reduced nicotinamide adenine dinucleotide (NADH) fluorescence photography was used to detect myocardial anoxia. Fluorescein angiography marked areas of myocardial perfusion. The hearts were isolated, perfused with a hemoglobin-free solution and performed no external work. In all hearts there was a narrow band between areas of perfusion and anoxia that measured 329 +/- 42 mu (mean +/- SD). The transition from minimal to full NADH fluorescence was abrupt, less than 80 mu. We conclude that the normoxic/anoxic transition is sharp, and the gap between perfusion and anoxia is narrow along an ischemic border in the isolated heart performing no external work. These data suggest that in the vivo working heart the gap between perfusion and anoxia would be even narrower.

Epicardial ischemia as delineated with epicardial S-T segment mapping and nicotinamide adenine dinucleotide (NADH) fluorescence photography

In isolated rabbit hearts with an experimental coronary arterial occlusion, epicardial ischemia was identified by reduced nicotinamide adenine dinucleotide (NADH) fluorescence photography, a technique that detects areas of myocardial anoxia. Epicardial S-T segment mapping was performed to evaluate the S-T segment changes across an ischemic border defined by NADH fluorescence. After S-T segment mapping and perfusion with a fluorescein dye, serial selections of the hearts revealed that the ischemic area was transmural and the border was nearly perpendicular to the epicardial surface. As the epicardial ischemic border was approached, S-T segment elevation was first detected 3.3 mm outside the ischemic border, and increased over a transition zone 7 mm wide. S-T segment negativity was not detected immediately outside the ischemic border. It is concluded from these studies that S-T segment changes give relatively imprecise definition of an ischemic border, and that S-T segment changes across an ischemic border are not consistent with those predicted by solid angle analysis.

Two and three dimensional display of myocardial ischemic “border zone” in dogs

It is intuitively apparent that the ultimate fate of reversibly damaged, peri-ischemic “border zone” tissue should relate to individual patient survival. The purpose of this study was: (1) to describe a technique of assessing the adequacy of epicardial and myocardial oxygenation, and (2) to examine the extent and character of the peri-ischemic “border zone” in the dog after coronary arterial ligation. A diagonal branch of the left anterior descending coronary artery was ligated in four anesthetized open chest, neurohormonally intact dogs. The same diagonal coronary artery was ligated in six isolated perfused canine heart preparations in which coronary partial pressures of oxygen and carbon dioxide, pH, blood flow and temperature were fixed. The ischemic zones were rapidly frozen and reduced nicotinamide adenine dinucleotide (NADH) fluorescence photographs were taken of the epicardium and at 0.5 mm depths into the myocardium. The distinction between perfused and ischemic myocardium is not apparent with the naked eye or natural light photography. Epicardial and myocardial oxidation-reduction status is well seen with NADH fluorophotography. In both the intact and perfused heart preparations the NADH-fluorescent (ischemic) border is jagged along all edges. Islands of perfused nonfluorescent tissue appear within the ischemic border. The transition between NADH-fluorescent ischemic cells and adjacent nonfluorescent tissue is less than 0.1 mm. The ischemic border is narrow. The distance between homogeneously NADH-fluorescent tissue and homogeneously nonfluorescent tissue (across the zone of island normoxia or microheterogeneity) may be as wide as 6 to 8 mm.

A fluorometric study of oxidative metabolism in the in vivo canine heart during acute ischemia and hypoxia.

Optical techniques for monitoring the fluorescence of intramitochondrial nicotinamide adenine dinucleotide (NADH) were employed to allow an on-line, non-invasive study of the metabolic state of healthy and ischemic cardiac tissue in the intact dog. Acute interruption of blood flow to the area of myocardium studied resulted in an immediate rise in NADH, indicating impairment of oxidative metabolism due to limitation of oxygen availability. Release of the coronary artery occlusion resulted in an oxidation of NADH to preocclusion baseline and signaled repayment of incurred O2 debt. Collateral flow produced a spontaneous fall in NADH levels towards preocclusion baseline. Hypoxic tissue was recognized by an oxidation of NADH on ventilation with 100 per cent O2, while in normal hearts no oxidation indicated the O2 was not rate limiting. Ischemic NADH increases preceded the onset of epicardial EKG changes, suggesting that an inability to rephosphorylate ADP to ATP is the primary cause of the EKG changes. Fluorometry of the in vivo heart provides a sensitive, continuous technique of monitoring cardiac cellular mitochondrial NAD;NADH redox levels and, therefore, the adequacy of high energy phosphate production and allows localization of cardiac tissue with marginal oxygen supply.

Effect of hypocapnia, hypercapnia, and blood pressure on NADH fluorescence, electrical activity, and blood flow in normal and partially ischemic monkey cortex.

Nicotinamide adenine dinucleotide fluorescence, cortical reflectance, cortical blood flow, and electroencephalograms were recorded from squirrel monkey brains before, during, and after focal transient cerebral ischemia produced by the temporary clipping of the middle cerebral artery. After release of the occluding clip, the monkeys were followed through an N2-breathing cycle and then to death from anoxia. The effects of controlled variations in arterial carbon dioxide tensions (PaCO2) and mean arterial blood pressures (MABP) were investigated in normal and in ischemic brain. In normal brain, with preserved autoregulation, NADH fluorescence was constant through a wide range in Paco2, MABP, and cortical blood flow. In ischemic brain, NADH levels increased, correlated closely with decreased cortical blood flow and EEG abnormalities, and became dependent on MABP. Artifacts in fluorescent measurements were reduced by: monochromators for excitation, emission, and reflected light; low intensity vertical excitation energy and high sensitivity recording instrumentation; and a small avascular (123 microns) field.

Application of a new method for detecting streptococcal nicotinamide adenine dinucleotide glycohydrolase to various M types of Streptoccus pyogenes

The production of extracellular nicotinamide adenine dinucleotide glycohydrolase (NADase) by Streptococcus pyogenes can easily be demonstrated using the fluorescence of nicotinamide adenine dinucleotide in ultraviolet light occurring on addition of strong alkali. The new method described here uses microtiter plates and can be read with the naked eye. It permits the screening of large numbers of strains in a short time and with minimal amounts of reagents. The sensitivity of the new method proved to be good in comparison with the bisulfite spectrophotometric method. Culture supernatants of 177 group A streptococci were tested for NADase production by the microtiter fluorescence method. We could confirm former findings that strains within a certain M type of S. pyogenes are usually either producers or nonproducers of the enzyme. The usefulness of the test for screening of streptococcal NADase production is discussed.

Reduced Nicotinamide Adenine Dinucleotide Fluorescence and Cortical Blood Flow in Ischemic and Nonischemic Squirrel Monkey Cortex. 1. Animal Preparation, Instrumentation, and Validity of Model

Reduce nicotinamide adenine dinucleotide (NADH) fluorescence was recorded from an avascular area on the squirrel monkey cortex prior to, during, and after focal incomplete ischemia. By using the instrumentation described, stable recordings were obtained free from hemoglobin artifact and with only minimal photodecomposition. Pentobarital was compared to urethane and halothane as the anesthetic agent and was found acceptable for these types of studies in the dosages used. NADH levels were constant prior to ischemia, increased during ischemia, returned to pre-ischemic levels after restoration of blood flow, and then increased greatly at death produced by anoxia. The use of the infrared microscope for semiquantitative measurements of cortical blood flow throughout the duration of these acute studies was investigated and found to the reliable.

Reduced Nicotinamide Adenine Dinucleotide Fluorescence and Cortical Blood Flow in Ischemic and Nonischemic Squirrel Monkey Cortex. 2. Effects of Alterations in Arterial Carbon Dioxide Tension, Blood Pressure, and Blood Volume

The fluorescence of reduced nicotinamide adenine dinucleotide (NADH) from cerebral cortex was measured before, during, and after middle cerebral artery (MCA) occlusion and then at death of the animal. In normal cortex, NADH remained constant throughout a wide range of variations in blood pressure and Paco2. In ischemic cortex, NADH levels were higher in hypovolemic hypotensive animals than in normotensive normovolemic animals. Neither hypercapnia nor hypocapnia was effective in decreasing NADH in regions of ischemia, but the latter was associated with a degree of hypotension that interfered with interpretation of data. NADH returned to normal with restoration of flow, supporting the reversibility of this degree of ischemia. The high levels of NADH at death, compared to those during ischemia, are consistent with incomplete ischemia in this model of cerebral infarction.

Intracellular redox changes in functioning cerebral cortex. II. Effects of direct cortical stimulation.

Intracellular redox changes in functioning cerebral cortex. II. Effects of direct cortical stimulation.

Reduced nicotinamide adenine dinucleotide-linked analysis of 2,3-diphosphoglyceric acid: spectrophotometric and fluorometric procedures.

Abstract A rapid and relatively specific assay method for 2,3-diphosphoglyceric acid (DPG) is described. The procedure involves the stoichiometric conversion of 2,3-DPG to glyceraldehyde-3-phosphate with measurement of the resulting change in reduced nicotinamide adenine dinucleotide (NADH) absorbance or fluorescence. 2-Phosphoglycolate is utilized to activate the 2,3-DPG phosphatase activity present in muscle monophosphoglycerate mutase.

59. Studies on the isoenzyme pattern of fetal and adult red cells

Quantitative differences in the enzyme activities of fetal and adult red cells are well known, but few data on differences in the isoenzyme patterns between these cells have been published. In this study isoenzymes of phosphopyruvatee hydratase (EC.4.2.1.11) and pyruvate kinase (EC.2.7.1.40) were investigated in hemolysates of isolated fetal and adult erythrocytes. Electrophoresis was performed on cellulose acctate foils. Sites of enzyme activity were detected utilizing the fluorescence of nicotinamide adenine dinucleotide by illuminating the foils with ultraviolet light after incubation with specific identification-reaction mixtures. For both phosphopyruvate hydratase and pyruvate kinase differences in number, intensity, and/or clectrophorectic mobility of isoenzyme bands between fetal and adult erythrocytes could be demonstrated. These findings suggest further evidence for the biochemical distinction of fetal and adult red cells.