Biomedical subjects
Publications and source records attributed to D K Das.
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The ideal temperature and hematocrit level of blood cardioplegia has not been clearly established. This study was undertaken (a) to determine the optimal temperature of blood cardioplegia and (b) to study the effect of hematocrit levels in blood cardioplegia. A comparison of myocardial preservation was done among seven groups of animals on the basis of variations in hematocrit levels and temperature of oxygenated cardioplegic solution. The experimental protocol consisted of a 2-hour hypothermic cardioplegic arrest followed by 1 hour of normothermic reperfusion. Group 1 received oxygenated crystalloid cardioplegic solution at 10 degrees C. Groups 2 through 7 received oxygenated blood cardioplegic solution with the following hematocrit values and temperatures: (2) 10%, 10 degrees C; (3) 10%, 20 degrees C; (4) 10%, 30 degrees C; (5) 20%, 10 degrees C; (6) 20%, 20 degrees C; and (7) 20%, 30 degrees C. Parameters studied include coronary blood flow, myocardial oxygen extraction, myocardial oxygen consumption, and myocardial high-energy phosphate levels of adenosine triphosphate and creatine phosphate during control (prearrest), arrest, and reperfusion. Myocardial oxygen consumption at 30 degrees C during arrest was significantly higher than at 10 degrees C and 20 degrees C, which indicates continued aerobic metabolic activity at higher temperature. Myocardial oxygen consumption and the levels of adenosine triphosphate and creatine phosphate during reperfusion were similar in all seven groups. Myocardial oxygen extraction (a measure of metabolic function after ischemia) during initial reperfusion was significantly lower in the 30 degrees C blood group than in the 10 degrees C blood group at either hematocrit level and in the oxygenated crystalloid group, which suggests inferior preservation. The hematocrit level of blood cardioplegia did not affect adenosine triphosphate or myocardial oxygen consumption or extraction. It appears from this study that blood cardioplegia at 10 degrees C and oxygenated crystalloid cardioplegia at 10 degrees C are equally effective. Elevating blood cardioplegia temperature to 30 degrees C, however, reduces the ability of the solution to preserve metabolic function regardless of hematocrit level. Therefore, the level of hypothermia is important in blood cardioplegia, whereas hematocrit level has no detectable impact, and cold oxygenated crystalloid cardioplegia is as effective as hypothermic blood cardioplegia.
The effect of nicotinic acid, an antilipolytic drug, on myocardial preservation was studied on the basis of cardiac performance after 2 hours of cardioplegic arrest. Isolated in situ pig hearts were subjected to 120 minutes of hypothermic potassium (35 mEq) crystalloid cardioplegic arrest followed by 60 minutes of reperfusion. The experimental group received nicotinic acid 0.08 mmol/L 15 minutes before cardioplegic arrest, whereas the control group received 15 minutes of unmodified perfusion. There was a marked decline in myocardial creatine phosphate levels during cardioplegic arrest in both groups that returned to the baseline level during reperfusion without a significant intergroup difference, and adenosine triphosphate levels remained stable throughout the experiment in both groups. Myocardial oxygen consumption during reperfusion was significantly higher in hearts treated with nicotinic acid, which was consistent with a significantly greater cardiac contractile force as evaluated by isovolumetric left ventricular pressure measurements. There appeared to be less cardiac membrane damage as measured by creatine kinase release during reperfusion, which was significantly inhibited by treatment with nicotinic acid. The present study supports the conclusion that nicotinic acid improves cardiac performance after hypothermic cardioplegic arrest.
Xanthine oxidase (XO) has been hypothesized to be a potential source of oxygen-derived free radicals during reperfusion of ischemic myocardium based on the fact that allopurinol, a XO-inhibitor, can reduce reperfusion injury. In this communication we report that both allopurinol and oxypurinol, the principle metabolite of allopurinol, prevent the reperfusion injury in isolated pig heart. However, we found that neither pig heart nor pig blood contain any XO activity. Our study showed a direct free radical scavenging action of these XO-inhibitors during ischemia and reperfusion, as judged by the reduction of free radical signals when compared using an Electron Paramagnetic Resonance Spectrometer. Using a Luminometer, we also confirmed that both allopurinol and oxypurinol can scavenge ClO2, HOCl, and significantly inhibit free radical signals generated by activated neutrophils. These XO-inhibitors, however, failed to scavenge O2. and OH. radicals. Our results suggest that these XO-inhibitors salvaged the ischemic-reperfused myocardium by scavenging free radicals, and not by inhibiting XO in the pig heart.
A new ion-pair reversed-phase high-performance liquid chromatography method was applied to simultaneous measurements of porcine myocardial adenine nucleotides and creatine phosphate. The homogeneity of each desired peak was tested by the retention times of standards, chromatography of spiked samples, the absorbance ratios at various wavelengths, and the differing retention times for a number of other compounds found in porcine myocardial extracts.
Fine-needle aspiration cytology of lymph nodes and extranodal swellings in 160 cases showed granulomatous reaction with or without caseation necrosis in 83%. The material was acellular or predominantly composed of necrotic material, polymorphs, and lymphocytes in 17%. The age of the patient ranged from 1.5 to 72 yr. The male to female ratio was 1:1.3. Acid-fast bacilli (AFB) could be demonstrated in 40.6% of cases. In cases associated with cellular reaction and necrosis. AFB positivity was 50.0%, while it was 66.7% in cases with acellular necrotic material.
Pure polymorphonuclear leukocytes (PMNs) have been isolated from a small amount of human blood by a single-step density gradient centrifugation method using a commercially available Ficoll-Hypaque mixture of density 1.114. The cells were labelled with [111In]tropolone in both buffer and plasma. Cell viability, ability to generate superoxide anion, and chemotaxis were found to be unaltered both before and after labeling. The optimum tropolone concentration for labeling was found to be 1 X 10(-4) M. Labeling efficiency was higher at 37 degrees C than at room temperature. Compared to [111In]oxine, tropolone preparation both in buffer and plasma resulted in consistently higher yields. Preliminary experiments of in vivo cell viability of the labeled PMNs were carried out in rabbits. The ability of the cells to localize in experimentally produced inflammatory lesions was found to be intact. The method of cell separation and labeling described has been found to be simple and rapid and could easily be incorporated in routine nuclear medicine laboratory practice.
The developmental profiles of the antioxidative defense system and the peroxidative status of the heart during growth and development were studied in pigs of three different age groups. A unique age-specific myocardial lipid peroxidation expressed in terms of malonaldehyde formation occurred after incubation of neonatal and adult pig heart homogenates in the absence of any added factors. Very little malonaldehyde release was noticed in the 0- to 2-day age group, while considerably higher activity was found in the 8- to 10-day-old animals. The 2-month-old pig heart again formed very little malonaldehyde. Myocardial injury from lipid peroxidation was highest in the 0- to 2-day age group, as evidenced by the release of oxidized glutathione, lactate dehydrogenase (LDH) and creatine kinase (CK) activities. Release of glutathione, LDH and CK decreased with age and was minimal in the adult group. The antioxidative enzymes, superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, increased during the first 10 days of neonatal growth and then levelled off. Glucose-6-phosphate dehydrogenase was present in appreciably lower amounts in adult hearts compared to neonatal hearts. Heart weight increased with aging, but myocardial water content decreased. Protein and DNA contents of hearts increased with age, such that the protein/DNA ratio almost doubled from the newborn to adult age. The results indicate that the newborn pig hearts are equipped with the antioxidative defense system, which undergoes significant development during the initial phase of neonatal growth and does not change appreciably thereafter. The results further suggest that the change in activity profile with aging is different for different enzymes, and the peroxidative status of the myocardium is not a function of these enzyme activities.
Tumor-promoting phorbol esters may alter alpha 1-adrenoceptor-mediated cardiac response by stimulating protein kinase C activity. We investigated the effect of phorbol-12,13-dibutyrate (PDBu) on the positive inotropic effect (PIE) in rat left ventricular papillary muscle. PDBu (1-100 nM) potentiated the phenylephrine (PE)-induced PIE in a dose- and time-dependent manner. The PIE induced by PE and PDBu was abolished by pretreatment with 3 x 10(-7) M prazosin. PDBu also enhanced PE-induced slow responses 2- to 3-fold. These results suggest that PDBu enhances alpha 1-adrenoceptor-mediated PIE by potentiating slow Ca2+ channels, presumably through the activation of protein kinase C.
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Anaerobic metabolism in heart muscle plays a role in maintenance of myocardial preservation only during ischemia or hypoxia. In an ischemic state, such as during a heart attack or even during the induced ischemia of open heart surgery, there is impairment of blood flow to the myocardium. The major energy-yielding process in the heart is through the metabolism of glucose and lipids by oxidative reactions. Under anaerobic conditions, oxygen is not available to accept the electrons in the metabolic degradation of substrates and anaerobic glycolysis becomes important in the preservation of myocardial viability during the ischemic process. Unfortunately, the accumulated products of glycolysis, namely protons and lactate, work to inhibit glycolysis, ultimately resulting in a depression of anaerobic metabolism. Cardioplegia, as utilized during open heart surgery, has the effect of inducing instantaneous induction of myocardial mechanical and electrical arrest with a maximal inhibition of the energy utilizing metabolic processes. This effectively reduces substrate utilization and prevents the deleterious consequences of the ischemic process. Cardioplegia is most effective when combined with the additive properties of hypothermia, which plays a significant role in decreasing myocardial metabolism. However, during prolonged hypothermic cardioplegic arrest, sufficient ATP cannot be maintained for cellular integrity and anaerobic glycolysis becomes of increasing importance for maintenance of myocardial preservation. This presentation deals with the mechanics of aerobic versus anaerobic metabolism during the ischemic process of open heart surgery.
Oxygen free radicals and phospholipid degradation have been implicated in the pathogenesis of ischemia and reperfusion injury. The present study examines the involvement of such mechanisms in myocardial reperfusion injury in neonatal hearts. The isolated neonatal pig hearts from two different age groups, 0 to 2 days old (newborn) and 7 to 9 days old (week-old), were subjected to 60 min of normothermic global ischemia followed by 60 min of reperfusion. Although myocardial ischemia reduced superoxide dismutase, catalase, and glutathione peroxidase activities in both age groups, superoxide dismutase and catalase activities remained significantly lower in the newborn pig heart during ischemia and reperfusion. Oxidized glutathione release from the neonatal pig hearts was at minimum levels before ischemia, but it increased 10-fold at the onset of reperfusion and was significantly higher in the newborn heart. This indicates that generation of oxygen free radicals was enhanced in the newborn compared with that in the week-old heart. The increase in phospholipase A2 activity and decrease in acyl CoA synthetase and lysophosphatidylcholine acyl transferase activities during ischemia and reperfusion were associated with comparable loss of membrane phospholipids and accumulation of lysophosphatidylcholine and free fatty acids in both age groups, except that oleic acid content was significantly higher in the newborn heart during reperfusion. Myocardial damage appears to be potentiated in the newborn heart during reperfusion, as evidenced by higher release of creatine kinase and a lower content of high-energy phosphates. These results indicate that oxygen free radicals may play a crucial role in the occurrence of reperfusion injury in immature hearts.
Phosphoinositide metabolism is known to be associated with neuronal or humoral stimulation of excitable cells. The present study examined whether the phosphoinositide response is involved in such events using isolated rat papillary muscles labeled with [3H]inositol. It was found that neither increase in the stimulation frequencies (0-2 Hz) nor prolongation of the pulse duration (10-70 msec) altered the labeling of phosphoinositides and the accumulation of [3H]inositol phosphates in this preparation. However, phenylephrine, a known alpha 1-agonist, was capable of provoking the breakdown of phosphoinositides associated with a positive inotropic effect in this preparation. We report the evidence that phosphoinositide response is mediated by alpha 1-adrenoceptor stimulation, but not linked with excitation-contraction coupling in cardiac muscle.
Nine cases of plasma cell tumors were diagnosed by fine-needle aspiration cytology, the sites of aspiration being soft-tissue swellings in five cases, lymph nodes in three cases, and both soft-tissue swelling and lymph node in one case. The age of the patients ranged from 33 to 74 yrs, with a median of 52 yrs. Only three of these nine cases were clinically diagnosed as multiple myeloma. Unequivocal demonstration of myeloma cells was possible in eight cases. The smear in ninth case showed mostly undifferentiated blast cells with a few cells showing plasmacytoid differentiation. Giant myeloma cells with monstrous segmented nuclei were encountered in two cases. M-component could be demonstrated in serum and/or urine samples in all seven cases in which it was sought.
Twenty-five cases of cytologically and histologically confirmed hepatocellular carcinoma (HCC) were cytomorphologically analyzed using May-Grünwald-Giemsa (MGG)-stained aspiration smears and supplemented with special stains. The cell types were categorized as well differentiated (18 cases), vacuolated (four cases), giant cell (one case), and poorly differentiated (two cases). Glycogen staining was positive in 80% of the cases and hence served as a reliable parameter of diagnostic importance in HCC. Cytoplasmic hyaline bodies (14.6%) and bile pigment (17%), when present, were other important features supporting the diagnosis of HCC. Vacuolation of the cell cytoplasm (80%) was possibly related to glycogen accumulation. The cause of nuclear vacuolation (60%) and the significance of nuclear argyrophilia as markers of abnormal cell growth remain to be studied.
The technique of fine-needle aspiration cytology (FNAC) was utilized in the diagnosis of 31 cases of malignant lymphoma of convoluted lymphocytes. The age of the patients ranged from 5 to 70 yr (median, 24 yr). Clinical history was available in 29 cases. All patients had peripheral lymphadenopathy. There was radiological evidence of mediastinal adenopathy in 62% of the cases, and 41% had pleural effusion; 52% of the patients presented with mediastinal compression syndrome. The diagnostically crucial convoluted cells comprised 47.3 +/- 23.73% of all lymphoid cells. Nuclear convolutions were appreciated in a significant number of cells in paraffin sections of 17 of 21 cases (85.7%) having subsequent histopathology. Cytochemical and E-rosette tests performed in 17 cases confirmed the T cell nature of the neoplasms. On an average, 72.3% of cells showed focal positive reaction for acid phosphatase, and 73.8% of cells formed E-rosettes.
The effects of the inhibition of phospholipid degradation and superoxide radical generation on prostaglandin synthesis associated with myocardial ischemia and reperfusion were studied in the isolated, in-situ pig heart model subjected to 60 mins of regional ischemia and a further 60 mins of hypothermic potassium cardioplegic arrest, followed by 60 mins of reperfusion. Myocardial biopsies were taken from the ischemic and non-ischemic regions of the myocardium for measurement of phospholipids, and samples of the perfusate were drawn for estimation of the end-products of arachidonic acid metabolism, 6-keto-prostaglandin-F1 alpha and thromboxane B2. A significant amount of 6-keto-prostaglandin F1 alpha and thromboxane B2 appeared during reperfusion, corresponding with the loss of membrane phospholipids in control animals. Mepacrine, a phospholipase inhibitor, protected the depletion of membrane phospholipids and inhibited the products of arachidonate metabolism. Superoxide dismutase (SOD) and catalase, on the other hand, enhanced the formation of 6-keto-prostaglandin F1 alpha and thromboxane B2. The effects of both mepacrine and the free radical scavengers were pronounced during the reperfusion phase when the most significant depletion in membrane phospholipids occurred. These results suggest that the arachidonate cascade is activated during reperfusion of ischemic myocardium as a consequence of phospholipid breakdown, and this activation can be attenuated by inhibiting phospholipases or enhanced by scavenging oxygen-free radicals generated during reperfusion.
Depletion of membrane phospholipids is known to be associated with myocardial ischemia, but its relationship to the injury involved with the reperfusion of ischemic myocardium is not known. The present study was designed to relate phospholipid degradation with reperfusion injury. The isolated in situ pig heart was subjected to 60 min of regional ischemia induced by occluding the left anterior descending (LAD) coronary artery and 60 min of global ischemia by hypothermic cardioplegic arrest followed by 60 min of reperfusion. The pigs were divided into two groups. In the treatment group, the heart was preperfused with mepacrine (0.05 mM), a known phospholipase inhibitor, for 15 min prior to LAD occlusion. In the control group, the total phospholipid content was not significantly decreased during LAD occlusion and arrest, but was reduced appreciably after reperfusion. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol followed a similar pattern. The lowering of these phospholipids during reperfusion was accompanied by enhancement of lysophosphatidylcholine. Mepacrine restored the normal levels of these phospholipids. During reperfusion, fatty acyl CoA synthetase, lysophospholipase, and lysophosphatidylcholine acyltransferase were depressed, whereas phospholipase A2 was enhanced. Mepacrine inhibited phospholipase A2, but had no effects on the other enzymes. Mepacrine also provided significant protection against reperfusion injury, as documented by the preservation of high-energy phosphate compounds and inhibition of the appearance of creatine kinase activity in the perfusate. These results suggest that membrane phospholipids play an important role in myocardial injury associated with ischemia and reperfusion, primarily because the deacylation-reacylation cycle of phospholipid biosynthesis becomes defective.