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Deuterium oxide (D2O) appearance in blood is a marker of fluid bioavailability. However, whether biomarker robustness (e.g., relative fluid delivery speed) is consistent across analytical methods (e.g., cavity ring-down spectroscopy) remains unclear. Fourteen men ingested fluid (6 ml/kg body mass) containing 0.15 g/kg D2O followed by 45 min blood sampling. Plasma (D2O) was detected (n = 8) by the following: isotope-ratio mass spectrometry after vapor equilibration (IRMS-equilibrated water) or distillation (IRMS-plasma) and cavity ring-down spectroscopy. Two models calculated D2O halftime to peak (t1/2max): sigmoid curve fit versus asymmetric triangle (TRI). Background (D2O) differed (p 2 = .98) among IRMS-equilibrated water, IRMS-plasma, and cavity ring-down spectroscopy (152.2 ± 0.8, 147.2 ± 1.5, and 137.7 ± 2.2 ppm), but did not influence (p > .05) D2O appearance (Δppm), time to peak, or t1/2max. Stratifying participants based on mean t1/2max (12 min) into “slow” versus “fast” subgroups resulted in a 5.8 min difference (p 2 = .73). Significant t1/2max model (p = .01, η2 = .44) and Model × Speed Subgroup interaction (p = .005, η2 = .50) effects were observed. Bias between TRI and sigmoid curve fit increased with t1/2max speed: no difference (p = .75) for fast (9.0 min vs. 9.2 min, respectively) but greater t1/2max (p = .001) with TRI for the slow subgroup (16.1 min vs. 13.7 min). Fluid bioavailability markers are less influenced by which laboratory method is used to measure D2O as compared with the individual variability effects that influence models for calculating t1/2max. Thus, TRI model may not be appropriate for individuals with slow fluid delivery speeds.

S-Adenosyl-L-methionine (SAM) was used to probe the functional effects exerted via the cardiac RyR isoform (RyR2) adenine nucleotide binding site. Single channel experiments revealed that SAM applied to the cytoplasmic face of RyR2 had complex voltage dependent effects on channel gating and conductance. At positive transmembrane holding potentials, SAM caused a striking reduction in channel openings and a reduced channel conductance. In contrast, at negative potentials, SAM promoted a clearly resolved subconductance state. At membrane potentials between −75 and −25 mV, the open probability of the subconductance state was independent of voltage. ATP, but not the non-adenosine-based ryanodine receptor (RyR) activator 4-chloro-m-cresol, interfered with the effects of SAM at both negative and positive potentials. This suggests that ATP and SAM interact with a common binding site. Molecular docking showed SAM bound to the adenine nucleotide binding site and formed a hydrogen bond to Glu4886 in the C-terminal end of the S6 alpha helix. In this configuration, SAM may alter the conformation of the RyR2 ion conduction pathway. This work provides novel insight into potential functional outcomes of ligand binding to the RyR adenine nucleotide binding site.

INTRODUCTION Because impaired excitation-contraction coupling and reduced sarcoplasmic reticulum (SR) Ca2+ release may contribute to the age-associated decline in skeletal muscle strength, we investigated the effect of aging on regulation of the skeletal muscle isoform of the ryanodine receptor (RyR1) by physiological channel ligands. METHODS [3 H]Ryanodine binding to membranes from 8- and 26-month-old Fischer 344 extensor digitorum longus (EDL) and soleus muscles was used to investigate the effects of age on RyR1 modulation by Ca2+ and calmodulin (CaM). RESULTS Aging reduced maximal Ca2+ -stimulated binding to EDL membranes. In 0.3 μM Ca2+ , age reduced binding and CaM increased binding to EDL membranes. In 300 μM Ca2+ , CaM reduced binding, but the age effect was not significant. Aging did not affect Ca2+ or CaM regulation of soleus RyR1. DISCUSSION In aged fast-twitch muscle, impaired RyR1 Ca2+ regulation may contribute to lower SR Ca2+ release and reduced muscle function. Muscle Nerve 57: 1022-1025, 2018.