Anwar S. Abd-Elfattah

Superiority of HPLC to Assay for Enzymes Regulating Adenine Nucleotide Pool Intermediates Metabolism: 5'-Nucleotidase, Adenylate Deaminase, Adenosine Deaminase, and Adenylosuccinate Lyase-A Simple and Rapid Determination of Adenosine

Abstract A new application of HPLC analysis to assay for all the enzymes involved in regulation of adenine nucleotide pool metabolism is described. In vitro, enzymatic reactions were carried out in buffered reaction media containing appropriate concentrations of metal ions, specific substrate, inhibitors and enzymes. Following an incubation period, the enzymatic reactions were terminated and extracted with cold trichloroacetic acid. The soluble acid extracts were neutralized and injected in a HPLC system. Using a C18-Nova Pak reverse phase column, we were able to separate, identify and quantify the substrate, products and their possible catabolites and UV-detectable inhibitors. A complete separation and quantitation of metabolites was accomplished within 16-18 minutes. However, rapid and simple HPLC runs were also developed which can be routinely used to determine adenosine levels within 3-4 minutes, using a single solvent HPLC system. This procedure is extremely reproducible and very reliable as demonstr...

Embryonic versus adult myocardium: Adenine nucleotide degradation during ischemia

Neonatal myocardium demonstrates better recovery from ischemia than does adult tissue. We tested the hypothesis that developmental differences in adenine nucleotide degradation might facilitate recovery by quantitating depletion of high-energy phosphates in nine-day-old embryonic (n = 9) and 15-month-old adult (n = 14) chicken hearts at 15-, 30-, 45-, and 60-minute intervals of normothermic ischemia in vitro. Nucleotides adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate and nucleosides adenosine, inosine, hypoxanthine, and xanthine were determined by high-performance liquid chromatography. Several observations in metabolite degradative response to ischemia were noted. The embryonic myocardium maintained higher adenosine triphosphate and adenosine monophosphate levels over the course of the investigation than did mature myocardium. Moreover, the adult group showed an increase in diffusible nucleoside pool metabolites. Relative immaturity of enzymes responsible for nucleotide degradation may facilitate postischemic recovery by preserving nondiffusible high-energy phosphate precursors to participate in salvage resynthesis of adenosine triphosphate.

Preload Dependence of Right Ventricular Blood How: I. The Normal Right Ventricle

Right ventricular (RV) failure is commonly treated with intravascular volume expansion to increase the RV-left atrial pressure gradient and improve left-sided filling. As RV pressure rises, chamber distention occurs and wall tension increases. These studies were designed to determine if increased wall tension might impede RV myocardial blood flow in the normal canine right ventricle and thus contribute to RV failure. Hemodynamic data, the septal-RV free wall dimension, and RV myocardial blood flow were obtained at low and high levels of preload and in both the autoregulated and vasodilated (adenosine, 2 mg per kilogram of body weight per minute) states. Elevated filling pressure decreased RV myocardial blood flow in both the autoregulated (0.85 +/- 0.18 to 0.67 +/- 0.15 ml/min/gm; p less than .05) and vasodilated (2.25 +/- 0.50 to 0.85 +/- 0.25 ml/min/gm; p less than .05) states but did not change the transmural distribution of blood flow to the right ventricle. Vasodilator reserve was markedly impaired in the high-preload state. These observations suggest that preload is an important determinant of RV myocardial blood flow. Volume loading to treat RV dysfunction may be limited by impairment of RV myocardial blood flow.

Myocardial protection: what the surgeon does.

With changing patient demographics resulting in greater risk of myocardial ischemia, avoidance of low‐output states must begin with patient selection. From that point, a variety of well‐established surgical techniques can be used to provide myocardial protection. Hypothermia and cardioplegia are fundamental among these; however, it should be recognized that alternate approaches must be considered.

70 BIOCHEMICAL BASES FOR TOLERANCE OF THE NEWBORN HEART TO ISCHEMIC INJURY: DEVELOPMENTAL DIFFERENCES IN ADENINE NUCLEOTIDE DEGRADATION BETWEEN ISCHEMIC IMMATURE AND ADULT MYOCARDIUM. A POSSIBLE ROLE OF SARCOLEMMAL 5|[prime]|-NUCLEOTIDASE

We determined the rate of ATP depletion and catabolism in 8 canine immature hearts (IM-2 weeks old) and 5 adult (AM) hearts in response to normothermic global ischemia in vitro. Transmural serial biopsies were obtained just before ischemia and at 2, 5, 10, 15, 20, 25, 30, 40, 50 and 60 min of ischemia. Myocardlal adenine nucleotides, nucleosides, purines and NAD+ were determined by HPLC.IM retained more AMP than AM during ischemia. Purines accumulated more in AM than IM. IM may have either less 5′-nucleotidase and/or the enzyme may be kinetically less responsive to AMP accumulation. AMP reserve in ischemic IM may enhance the tolerance of newborn hearts to ischemia and hypoxia by increasing the rate of ATP repletion bypassing both the salvage and the de novo pathways. Inhibition of 5′-nucleotidase may provide increased metabolic protection in mature hearts during ischemia and reperfusion.

Dilated cardiomyopathy created by chronic rapid pacing induces heat shock proteins and limits myocardial infarction in dogs

It is not known whether chronic rapid pacing preconditions the myocardium against infar~on. Twenty seven dogs underwent chronic rapid pacing (240- 250 beat par minute) for 4-5 weeks. Eeshocardingraphy revealed progrss. slve decline in ejection fraction, wall thinning and increase in ventdcular d!!ation. Four pafrs crystals were sutured ontO the anterior and posterior segment of the left ventricle to monitor systolic segmental shorting and wall thickening and ventriculer balloon.tippad catheter pressure Iraneducer (Mil- lax). The loft anterior descending (LAD) coronanj artery was occluded for 45, 60 or 90 min and reperfused for 4 hours. Normal dogs were used as control and subjected to the same isshemic and repedusion protocols. Color mtcrosphoros were used to determine collateral blood flow during ischemia. Myocardial biopsies were obtained to determine adenine nuciaotide and nu- daoside. Tissue samples were also obtained at baseline and at the end of reparfesion from normal, dilated hearts to datermine the induction of heat shock protein expression using Western blot. Myocardial infarction and area at risk wore measured using Monastral blue and TIC stain at postmortem. Myocardial Infarction Alter 90 rain isdnemia and 4 hrs Repadusion