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A Novel Nicotinamide Adenine Dinucleotide Correction Method for Intracellular Ca<sup>2+</sup> Measurement with Fura-2-Analog in Live Cells

To measure [Ca2+] quantitatively, fura-2 analogs, which are ratiometric fluoroprobes, are frequently used. However, dye usage is intrinsically limited in ...

University of Ulsan College of Medicine/Asan Medical Center

Asan Medical Center

Asan Medical Institute of Convergence Science and Technology

Fura-2 analogs are ratiometric fluoroprobes that are widely used for the quantitative measurement of [Ca(2+)]. However, the dye usage is intrinsically limited, as the dyes require ultraviolet (UV) excitation, which can also generate great interference, mainly from nicotinamide adenine dinucleotide (NADH) autofluorescence. Specifically, this limitation causes serious problems for the quantitative measurement of mitochondrial [Ca(2+)], as no available ratiometric dyes are excited in the visible range. Thus, NADH interference cannot be avoided during quantitative measurement of [Ca(2+)] because the majority of NADH is located in the mitochondria. The emission intensity ratio of two different excitation wavelengths must be constant when the fluorescent dye concentration is the same. In accordance with this principle, we developed a novel online method that corrected NADH and Fura-2-FF interference. We simultaneously measured multiple parameters, including NADH, [Ca(2+)], and pH/mitochondrial membrane potential; Fura-2-FF for mitochondrial [Ca(2+)] and TMRE for Ψm or carboxy-SNARF-1 for pH were used. With this novel method, we found that the resting mitochondrial [Ca(2+)] concentration was 1.03 µM. This 1 µM cytosolic Ca(2+) could theoretically increase to more than 100 mM in mitochondria. However, the mitochondrial [Ca(2+)] increase was limited to ~30 µM in the presence of 1 µM cytosolic Ca(2+). Our method solved the problem of NADH signal contamination during the use of Fura-2 analogs, and therefore the method may be useful when NADH interference is expected. 

A Novel Nicotinamide Adenine Dinucleotide Correction Method for Mitochondrial Ca(2+) Measurement with FURA-2-FF in Single Permeabilized Ventricular Myocytes of Rat.

Seasonal variation in resting energy expenditure (REE) is still under debate. This study investigated seasonal changes in REE and relevant factors among Korean adults. A total of 867 healthy volunteers (385 men and 482 women) aged 20-69 years were divided into four seasonal groups and subgroups based on age, body mass index (BMI), and percent body fat (PBF) quartiles. REE, body composition, glucose metabolism, thyroid hormones, and catecholamines were measured. The seasonal factor contributed to REE independent of anthropometric indices, with additional variation decreasing from 6% to 2% among younger and older persons, respectively. The adjusted REE in the winter was 5.4-13.9%, 7.8-14.3%, and 8.6-11.9% higher than that in the summer in the age, BMI, and PBF subgroups, respectively. T3 and log-transformed norepinephrine (NE) were higher, whereas log-transformed epinephrine (EPI) was lower in the winter compared to the summer. The magnitude of the winter-summer difference in REE and T3 and of the summer-winter difference in EPI were reduced three-fold between the lowest and highest intervals of age and PBF, whereas the difference in NE was constant across all age and PBF intervals. There was no obvious change in seasonal differences in REE or its relevant biomarkers across BMI intervals. In summary, season is an independent predictor of REE and its effect is attenuated by the increment of age and PBF but not BMI.

Chae Hun Leem

Department of Physiology

University of Ulsan College of Medicine/Asan Medical Center

A Novel Nicotinamide Adenine Dinucleotide Correction Method for Intracellular Ca²⁺ Measurement with Fura-2-Analog in Live Cells

Chae Hun Leem

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Physiology

University of Ulsan College of Medicine/Asan Medical Center

A Novel Nicotinamide Adenine Dinucleotide Correction Method for Intracellular Ca2+ Measurement with Fura-2-Analog in Live Cells

Department of Physiology

A Novel Nicotinamide Adenine Dinucleotide Correction Method for Intracellular Ca²⁺ Measurement with Fura-2-Analog in Live Cells