Extraction and assay of creatine phosphate, purine, and pyridine nucleotides in cardiac tissue by reversed-phase high-performance liquid chromatography.

The levels of creatine phosphate, purine, and pyridine nucleotides in tissues provide important information on energetic and oxidative cellular states. Nevertheless, technical, theoretical, and methodological difficulties in extraction and quantification procedures have so far limited our understanding of the exact role that these substances play in metabolic processes which take place in cells. The objective of our study was to find an easy and rapid method for extracting, separating, and quantifying creatine phosphate, purine, and pyridine nucleotides in solid tissues. We adapted the classic acid-extraction procedure with HClO4 for purine and oxidized pyridine nucleotides and then developed a new alkaline extraction with phenol in a phosphate buffer solution (pH 7.8) for reduced pyridine nucleotides. Biopsies of myocardial tissue were frozen and ground at -180 degrees C using the appropriate extraction procedure. The separation and quantification of the metabolites were performed using a reversed-phase 3-microns Supelchem C18 column, with the addition of tetrabutylammonium as an ion-pair agent to the buffer solution, by ultraviolet detection. The recovery of the external and internal standards always exceeded 90%. The autooxidation or interconversion processes were almost insignificant for each reduced form. This technique allowed us to avoid complex enzymatic procedures and difficulties in the selective assay of pyridine nucleotides with chemiluminescence and surface spectroscopy.

Simultaneous determination of creatine compounds and adenine nucleotides in myocardial tissue by high-performance liquid chromatography.

A rapid high-performance liquid chromatography method was developed for the determination of creatine phosphate, creatine, adenine nucleotides, and related compounds in myocardial tissue. Analysis was performed by reversed-phase chromatography on a C18 column containing 3-microns particles, employing gradient elution and uv detection at 210 nm. Separation was achieved in less than 5 min. Total analysis time, including equilibration of the column after return of the gradient to starting conditions, was 8 min. The high reproducibility and short analysis time make this method suitable for the routine analysis of large series of samples.

Rapid separation of creatine, phosphocreatine and adenosine metabolites by ion-pair reversed-phase high-performance liquid chromatography in plasma and cardiac tissue.

A rapid ion-pair reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous detection of creatine, phosphocreatine, hypoxanthine, inosine, adenosine, AMP, ADP, ATP, 8-azaguanine, 2-chloroadenosine, and 2'-O-methyladenosine. This method has proven useful for measuring changes in nucleotide concentrations in both heart tissue and plasma samples. Separation of the compounds of interest is achieved in less than 8 min with re-equilibration in 7 min, making the total run time 15 min. Separation is performed on a 3-microns Ultrasphere ODS column employing tetrabutylammonium phosphate as the ion-pair agent and dipotassium hydrogenphosphate as the counter ion. The accuracy, rapid separation, and re-equilibration time make this method particularly useful for the routine analysis of a large number of samples.

Differential extractability of creatine phosphate and ATP from cardiac muscle with ethanol and perchloric acid solution.

To compare the extractability of creatine phosphate with that of ATP by alcohol extraction, both compounds were extracted from normal perfused rat heart tissues by using various stepwise concentrations of ethanol and 0.4 M HClO4. Powdered samples (6-15 mg wet wt) from the freeze-clamped tissues were homogenized in 2 ml of the ethanol solutions. After centrifugation, the supernatant was removed; each centrifuged sediment was rehomogenized with 2 ml of 0.4 M HClO4 and centrifuged. The supernatant was neutralized with 0.4 m KHCO3. The same powdered samples were directly homogenized with 2 ml of 0.4 M HClO4 and treated in the same manner. Only a small amount of ATP in the tissues was extracted by an 85% or higher concentration of ethanol. Further, about 13% of the tissue ATP was not extractable by the subsequent perchloric acid extraction. In contrast to ATP, creatine phosphate in the tissues was partially extracted by 95% ethanol and nearly all of the tissue creatine phosphate was extracted by 70% ethanol. The total creatine phosphate obtained by 70% ethanol and by subsequent perchloric acid extraction was significantly higher than that obtained by direct perchloric acid extraction. From these results, it was concluded that the extractability of creatine phosphate in the tissue by alcohol extraction is clearly different from that of ATP. Additionally, the stepwise extraction is recommended as a useful method for the extraction of energy metabolites in perfused rat heart tissue.

Ammonia metabolism during intense dynamic exercise and recovery in humans.

This study examined the dynamics for ammonia (NH3) metabolism in human skeletal muscle during and after intense one-legged exercise. Subjects (n = 8) performed dynamic leg extensor exercise to exhaustion (3.2 min). Muscle NH3 release increased rapidly to a maximum of 314 +/- 42 mumol/min and declined immediately on cessation of exercise. Recovery was complete in approximately 20 min. Arterial [NH3] increased less rapidly and reached its maximum 2-3 min into recovery. These data demonstrate that NH3 clearance is more sensitive to the cessation of exercise than is NH3 release from skeletal muscle. Muscle [NH3] increased three to fourfold during exercise and represented 74 +/- 8% of the total net NH3 formation. Thus the change in muscle [NH3] alone underestimates the NH3 production. There was no evidence that the muscle-to-venous blood NH3 ratio shifts in accordance with the H+ data. Thus other factors must contribute to the NH3 release from active muscle. The total net NH3 formed corresponded with the intramuscular inosine 5'-monophosphate accumulation, suggesting that the NH3 was derived from AMP deamination. Changes in the known modulators of AMP deaminase (ATP, ADP, H+) were moderate, so the mechanisms initiating the deamination remain obscure.

Phosphocreatinine, a high-energy phosphate in muscle, spontaneously forms phosphocreatine and creatinine under physiological conditions.

Phosphocreatinine undergoes the following spontaneous simultaneous reactions at pH 7.4 (0.02 M sodium phosphate and 120 mM KCl) and 38 degrees C. (Formula: see text) The first order rate constants are 0.046 h-1 (ka) and 0.048 h-1 (kb). There is a major effect of pH on the reactions such that at pH values higher than 7.4 phosphocreatine production predominates, while at pH values less than 7.4 creatinine is the major product. This along with titration data showing apparent pK values of about 3.0 and 7.5 for phosphocreatinine suggest that the dianionic form of phosphocreatinine is involved in the conversion to phosphocreatine, whereas the monoanionic form is exclusively converted to creatinine. Possible mechanisms to account for the reactivity of phosphocreatinine are discussed. Several lines of evidence suggest that the apparent Keq for phosphocreatine formation from phosphocreatinine is about 300 at pH 9.0 and about 70 at pH 7.0, and the delta G0' (pH 7.0) is-2.6 kcal/mol. The delta G0' (pH 7.0) for the hydrolysis of the phosphoryl bond in phosphocreatinine is-12.8 kcal/mol. The phosphocreatinine content of rabbit white skeletal muscle was measured to be 0.05 mumol/g, which is 0.4% of the phosphocreatine content. The in vitro experiments suggest that phosphohydrolysis of phosphocreatinine can account for a creatinine formation equal to 0.5% of the phosphocreatine content/day. We conclude that it is likely that a substantial fraction of the in vivo creatinine production from phosphocreatine goes through the novel high energy phosphate, phosphocreatinine, as an intermediate.

Phosphocreatine does not inhibit rabbit muscle phosphofructokinase or pyruvate kinase.

Certain phosphocreatine preparations contain a contaminant that inhibits phosphofructokinase and pyruvate kinase assays. The contaminant can be separated from phosphocreatine by anion exchange chromatography. After appropriate purification, phosphocreatine has no effect on phosphofructokinase or pyruvate kinase; thus, there is no evidence that it serves muscle as a regulator of these enzymes. Although the inhibitory preparations of phosphocreatine contain inorganic phosphate and trace amounts of more negatively charged phosphorylated contaminants, the inhibitor is not inorganic phosphate or pyrophosphate. The nature of the inhibitor remains to be determined.