2',3'-cGMP exists in vivo and comprises a 2',3'-cGMP-guanosine pathway.

The discovery in 2009 that 2',3'-cAMP exists in biological systems was rapidly followed by identification of 2',3'-cGMP in cell and tissue extracts. To determine whether 2',3'-cGMP exists in mammals under physiological conditions, we used ultraperformance LC-MS/MS to measure 2',3'-cAMP and 2',3'-cGMP in timed urine collections (via direct bladder cannulation) from 25 anesthetized mice. Urinary excretion rates (means ± SE) of 2',3'-cAMP (15.5 ± 1.8 ng/30 min) and 2',3'-cGMP (17.9 ± 1.9 ng/30 min) were similar. Mice also excreted 2'-AMP (3.6 ± 1.1 ng/20 min) and 3'-AMP (9.5 ± 1.2 ng/min), hydrolysis products of 2',3'-cAMP, and 2'-GMP (4.7 ± 1.7 ng/30 min) and 3'-GMP (12.5 ± 1.8 ng/30 min), hydrolysis products of 2',3'-cGMP. To validate that the chromatographic signals were from these endogenous noncanonical nucleotides, we repeated these experiments in mice (n = 18) lacking 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), an enzyme known to convert 2',3'-cyclic nucleotides to their corresponding 2'-nucleotides. In CNPase-knockout mice, urinary excretions of 2',3'-cAMP, 3'-AMP, 2',3'-cGMP, and 3'-GMP were increased, while urinary excretions of 2'-AMP and 2'-GMP were decreased. Infusions of exogenous 2',3'-cAMP increased urinary excretion of 2',3'-cAMP, 2'-AMP, 3'-AMP, and adenosine, whereas infusions of exogenous 2',3'-cGMP increased excretion of 2',3'-cGMP, 2'-GMP, 3'-GMP, and guanosine. Together, these data suggest the endogenous existence of not only a 2',3'-cAMP-adenosine pathway (2',3'-cAMP → 2'-AMP/3'-AMP → adenosine), which was previously identified, but also a 2',3'-cGMP-guanosine pathway (2',3'-cGMP → 2'-GMP/3'-GMP → guanosine), observed here for the first time. Because it is well known that adenosine and guanosine protect tissues from injury, our data support the concept that both pathways may work together to protect tissues from injury.

Renal excretion of clofarabine: assessment of dose-linearity and role of renal transport systems on drug excretion.

The renal excretion of clofarabine was studied in vitro in the isolated perfused rat kidney (IPK) model and in vivo in rats. Clofarabine excretion was studied at four doses (160, 800, 2000 and 4000microg) in the IPK, targeting perfusate levels of 2, 10, 25, 50microg/mL, respectively. Clofarabine (2microg/mL) was also co-perfused with known inhibitors of the, organic cation (cimetidine, ranitidine and tyramine) and organic anion (probenecid, ellagic acid) transport systems. Additionally, the effect of medications including, itraconazole, digoxin, fludarabine and cytarabine (Ara-C) on clofarabine excretion was, evaluated. Based on IPK results, in vivo studies were performed to assess the plasma, pharmacokinetics and urinary recovery of clofarabine (6.5mg/kg, IV) pretreatment, with cimetidine (250mg/kg, IV). Clofarabine clearance was non-linear in the IPK, although at concentrations that were approximately 10- to 100-fold higher than maximum concentrations observed in humans. Excretion ratio data indicated a shift from net, secretion (XR=1.2+/-0.33) to net reabsorption (XR=0.78+/-0.40) at the highest dose, tested. Clofarabine clearance was significantly reduced upon co-administration with, cimetidine (0.83+/-0.22-->0.32+/-0.058mL/min). In vivo data correlated with IPK results as clofarabine clearance decreased 61% (20.2-7.80mL/min/kg), upon co-administration with cimetidine in rats. The results suggest that clofarabine is a substrate for a cimetidine-sensitive organic cation transporter system in the kidney, presumably OCT2. The magnitude of the cimetidine-clofarabine interaction was similar, in IPK and in vivo, demonstrating the utility of the IPK model in characterizing renal, drug excretion and assessing potential drug-drug interactions.

Noninvasive methods for measuring DNA alkylation in experimental animals and humans.

Alkylpurines are liberated from alkylated DNA by glycosylase repair enzymes and, in most cases, excreted in urine without further metabolism. This phenomenon forms the basis of noninvasive methods to measure DNA alkylation in vivo. In the case of methyl adducts, such as 7-methylguanine (7-MeGua), natural backgrounds exist due to RNA turnover. However, deuterated (d3) methylating agents or precursors give rise to d3-7-MeGua and d3-3-methyladenine (3-MeAde), which can be readily quantitated using gas chromatography-mass spectrometry (GC-MS). A deuterated probe drug, such as d6-aminopyrine, can be used to measure endogenous nitrosation levels in experimental animals. In contrast, for higher alkyl homologues of alkylpurines, natural backgrounds are low or nonexistent and can be directly measured by GC-MS using stable isotope labeled internal standards. For example, increased levels of urinary 3-ethyladenine were observed in cigarette smokers. Due to recent advances in analytical methodology, notably immunoaffinity cleanup of urine, measurements of excreted DNA adducts can be used in studies in human populations exposed to low levels of alkylating carcinogens.

McArdle's disease and gout.

We report the first case of McArdle's disease (muscle phosphorylase deficiency) and tophaceous gout. To examine the contribution of adenine nucleotide degradation to the disturbance of uric acid metabolism, we labeled the adenine nucleotide pool with [8-14C]adenine, and measured plasma and urine purines following vigorous exercise tests. Plasma and urinary hypoxanthine and xanthine concentrations and the specific radioactivity of urinary purines increased markedly, but plasma urate levels and uric acid excretion were not substantially modified. We suggest that, in this patient, the association of McArcle's disease with gout is coincidental.

Increased excretion of modified adenine nucleosides by children with adenosine deaminase deficiency.

We have identified seven adenine nucleosides in urines of untreated adenosine deaminase (ADA) deficient patients, four of which (adenosine, 2'-deoxyadenosine, 1-methyladenosine and N6-methyladenosine) have been previously identified in urines of normals and/or ADA deficient patients. We confirm that ADA deficient patients excrete markedly increased amounts of 2'-deoxyadenosine (582 +/- 363 versus normal of less than 0.1 nmoles/mg creatinine) and increased amounts of adenosine (29.4 +/- 5.7 versus normal of 4.12 +/- 1.0 nmoles/mg creatinine). We have found three modified adenine nucleosides previously undetected in human urine. These three compounds are 2'-O-methyladenosine, N6, 2'-O-dimethyladenosine and an as yet incompletely characterized modified adenine nucleoside, R-adenosine. Only ADA deficient patients excrete detectable amounts of 2'-O-methyladenosine (2.1 +/- 1.1 versus normal of less than 0.1 nmoles/ mg creatinine), whereas both normals and ADA deficient children excrete N6, 2'-O-dimethyladenosine and R-adenosine. However, ADA deficient patients do excrete increased amounts of R-adenosine (5.5 +/- 1.0 versus normal of 1.4 +/- 0.4 nmoles/mg creatinine).

Urinary adenosine 3',5'-monophosphate in the switch process from depression to mania.

Marked elevations of urinary adenosine 3',5'-monophosphate occurred on the day of rapid switch from a depressed into a manic state in patients with manic-depressive illness. It is suggested that this increase might serve a trigger function for the process by which catecholamines are elevated during the manic phase of the illness.

Kinetic parameters and renal clearances of plasma adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate in man.

Kinetic parameters and the renal clearances of plasma adenosine 3',5'-monophosphate (cyclic AMP) and guanosine 3',5'-monophosphate (cyclic GMP) were evaluated in normal subjects using tritium-labeled cyclic nucleotides. Each tracer was administered both by single, rapid intravenous injection and by constant intravenous infusion, and the specific activities of the cyclic nucleotides in plasma and urine were determined. Both cyclic AMP and cyclic GMP were cleared from plasma by glomerular filtration. The kidney was found to add a variable quantity of endogenous cyclic AMP to the tubular urine, amounting to an average of approximately one-third of the total level of cyclic AMP excreted. Plasma was the source of virtually all of the cyclic GMP excreted. Plasma levels of the cyclic nucleotides appeared to be in dynamic steady state. The apparent volumes of distribution of both nucleotides exceeded extracellular fluid volume, averaging 27 and 38% of body weight for cyclic AMP and cyclic GMP, respectively. Plasma production rates ranged from 9 to 17 nmoles/min for cyclic AMP and from 7 to 13 nmoles/min for cyclic GMP. Plasma clearance rates averaged 668 ml/min for cyclic AMP and 855 ml/min for cyclic GMP. Approximately 85% of the elimination of the cyclic nucleotides from the circulation was due to extrarenal clearance.

Effects of glucagon on adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate in human plasma and urine.

Glucagon, infused intravenously into fasting, well-hydrated, normal men in doses of 25-200 ng/kg per min, induced up to 30-fold increases in both plasma and urinary cyclic AMP. Cyclic GMP levels were unaffected by glucagon. Simultaneous cyclic AMP and inulin clearance studies demonstrated that the glucagon-induced increase in urinary cyclic AMP was entirely due to glomerular filtration of the elevated plasma levels of the nucleotide. The cyclic AMP response to glucagon was not mediated by parathyroid hormone or epinephrine, and trypsintreated glucagon was completely inactive. The perfused rat liver released cyclic AMP into the perfusate in response to glucagon, indicating that the liver is a possible source of the cyclic AMP entering the extracellular fluids in response to glucagon in vivo.

Effects of parathyroid hormone on plasma and urinary adenosine 3',5'-monophosphate in man.

The effects of parathyroid hormone (PTH) on plasma and urinary adenosine 3',5'-monophosphate (cyclic AMP) levels were studied in normal subjects. Under basal conditions normal adults have plasma concentrations of cyclic AMP ranging from 10 to 25 nmoles/liter and excrete from 1.5 to 5 mumoles of cyclic AMP per g of urinary creatinine. About one-half to two-thirds of the cyclic AMP excreted in the urine is derived from the plasma by glomerular filtration, and the remainder is produced by the kidney. Renal production of cyclic AMP is partly under the control of PTH. It can be suppressed by infusions of calcium and stimulated by infusions of the calcium chelating agent, EDTA. Infusions of PTH in doses up to 10 mU/kg per min were associated with dose-related increases both in urinary cyclic AMP and phosphate. Infusions of PTH in doses ranging from 20 to 80 mU/kg per min did not lead to any further increase in phosphaturia but did lead to further marked increases in urinary cyclic AMP. A modest increase in plasma cyclic AMP was noted when PTH was infused at 40 mU/kg per min. Anephric patients failed to show appreciable increases in plasma cyclic AMP in response to large doses of PTH but did show expected increases in response to glucagon. Surgical removal of parathyroid adenomas from nine patients with primary hyperparathyroidism was invariably followed by a decrease in urinary cyclic AMP, PTH, in large doses, and calcium infusion produced up to 2-fold increases in the other known naturally occurring cyclic nucleotide, guanosine 3',5'-monophosphate (cyclic GMP).