Analysis of Mitochondrial Calcium Retention Capacity in Cultured Cells: Permeabilized Cells Versus Isolated Mitochondria

In response to various pathological stimuli, such as oxidative and energy stress accompanied by high Ca2+, mitochondria undergo permeability transition (PT) leading to the opening of the non-selective PT pores (PTP) in the inner mitochondrial membrane. Opening of the pores at high conductance allows the passage of ions and solutes <1.5 kD across the membrane, that increases colloid osmotic pressure in the matrix leading to excessive mitochondrial swelling. Calcium retention capacity (CRC) reflects maximum Ca2+ overload of mitochondria that occurs just before PTP opening. Quantification of CRC is important for elucidating the effects of different pathological stimuli and the efficacy of pharmacological agents on the mitochondria. Here, we performed a comparative analysis of CRC in mitochondria isolated from H9c2 cardioblasts, and in permeabilized H9c2 cells in situ to highlight the strengths and weaknesses of the CRC technique in isolated cell mitochondria vs. permeabilized cells. The cells were permeabilized by digitonin or saponin, and the Ca2+-sensitive fluorescence probe Calcium Green-5N was used in both preparations. Results demonstrated the interference of dye-associated fluorescence signals with saponin and the adverse effects of digitonin on mitochondria at high concentrations. Analysis of the CRC in permeabilized cells revealed a higher CRC in the saponin-permeabilized cells in comparison with the digitonin-permeabilized cells. In addition, the mitochondrial CRC in saponin-permeabilized cells was higher than in isolated mitochondria. Altogether, these data demonstrate that the quantification of the mitochondrial CRC in cultured cells permeabilized by saponin has more advantages compared to the isolated mitochondria.

Low-intensity treadmill exercise protects cognitive impairment by enhancing cerebellar mitochondrial calcium retention capacity in a rat model of chronic cerebral hypoperfusion

Chronic cerebral hypoperfusion (CCH) is caused by reduced blood flow to the brain representing gradually cognitive impairment. CCH induces mitochondrial dysfunction and neuronal cell death in the brain. Exercise is known to have a neuroprotective effect on brain damage and cognitive dysfunction. This study aimed to clarify the neuroprotective effect of low-intensity treadmill exercise (LITE) by enhancing cerebellar mitochondrial calcium retention capacity in an animal model of CCH. Wistar rats were divided into the sham group, the bilateral common carotid arteries occlusion (BCCAO) group, and the BCCAO and treadmill exercise (BCCAO+Ex) group. BCCAO+Ex group engaged the LITE on a treadmill for 30 min once a day for 8 weeks before the BCCAO surgery to investigate the protective effect of LITE on cognitive impairment. CCH induced by BCCAO resulted in mitochondrial dysfunction in the cerebellum, including impaired calcium homeostasis. CCH also decreased cerebellar Purkinje cells including of calbindin D28k and parvalbumin, resulting in cognitive impairment. The impairment of mitochondrial function, loss of cerebellar Purkinje cells, and cognitive dysfunction ameliorated by exercise. The present study showed that LITE hindered the deficit of spatial working memory and loss of Purkinje cell in the cerebellum induced by CCH. We confirmed that the protective effect of LITE on Purkinje cell by enhanced the mitochondrial calcium retention capacity. We suggest that LITE may protect against cognitive impairment, and further studies are needed to develop the intervention for patients who suffered from CCH.

Reviewing the Analeptic Activity of Calcium Citrate Malate

Calcium Citrate Malate’s (CCM) health benefits were patented over three decades ago. It is involved in calcium retention, in children and adolescents. In adulthood, it is seen to promote the maintenance of bone health. In conjunction with vitamin D, CCM also decreases the risk of bone fracture in aged people and ensures that postmenopausal women’s health is made better. CCM, unlike other supplements, does not need to be taken along with a meal and is seen to deliver benefits to individuals of any age group. CCM is usually used when the person has achlorhydria where the gastric acid secretion is reduced and there is an absence of an acidic environment (required by calcium carbonate). Calcium citrate Malate possesses outstanding bioavailability properties and is a useful extension to the calcium available presently, either for direct supplementation or for food fortification.

Calcium from Finger Millet—A Systematic Review and Meta-Analysis on Calcium Retention, Bone Resorption, and In Vitro Bioavailability

Calcium deficiency during child growth leads to osteoporosis in later stages of life. Finger millet is one of the calcium dense foods, with three times the level of calcium than milk, and the only cereal that contains high calcium content which is consistent across different varieties (364 ± 58 mg/100 g). Thus, finger millet has potential for addressing calcium deficiency naturally. This study aimed to determine the retention and impact of finger millet calcium on bone turnover through a systematic review and meta-analysis. Three human studies were eligible for systematic review. Of these, only two were eligible for meta-analysis to assess the retention of calcium in children of 9 to 12 years. One study on bone turnover markers was not used in the meta-analysis as at least two studies are required to conduct meta-analysis. Due to the lack of complete data only four studies were eligible for meta-analysis to assess the in vitro bioavailability of calcium from unprocessed and a range of different types of processed finger millet. The result shows that there was significant retention (p < 0.05) of 23.4 ± 2.9% calcium from finger-millet-based diet which could help bone accretion during child growth if finger-millet-based diet is consumed. The bone turnover marker study shows that the resorption of calcium reduced by 28% and 47% among peri and post-menopausal women respectively after feeding the nutria mixed grain ball. However, there is no significant change in bone formation marker. Depending on the type of processing, calcium bioavailability either increased or decreased. One in vitro study showed that calcium bioavailability from finger millet was 28.6% when boiled, whereas three studies on processing show that certain processing can double the calcium bioavailability to 61.4%. Irrespective of the type of processing, finger millets contribute to high calcium retention and extremely high bioavailable calcium and could be useful for healthy growth and in dealing with complications related to calcium deficiency.

Calcium fixation on fortified rice made with various rice varieties

The aim of this study was to evaluate calcium fixation and calcium binding in calciumfortified rice. Calcium-fortified rice was made by soaking (infusing) rice in a Ca-lactate or Ca-gluconate solution at temperature of 80oC for 20 mins. The rice types used in this study were low-, medium- and high amylose rice, represented by Memberamo, Ciherang and IR-42 rice varieties. Calcium retention in rice was tested by washing and dialysis, and calcium levels in the rice were determined by Atomic Absorption Spectroscopy. Calcium fixation was determined by using an FT-IR infrared spectrometer based on the changes in infrared spectra of the functional groups of -OH and C-O. The research showed that calcium retention in rice after washing was between 86.23% - 94.38% (Ca-lactate) and 89.37% - 90.15% (Ca-gluconate), Ca retention after dialysis was between 37.49% to 44.13% (Ca-lactate) and 37.40% to 42.86% (Ca-gluconate). The addition of Ca-lactate or Ca-gluconate to rice caused a decrease in the absorbance value and the absorption band area square at a wave numbers 3425 cm-1 (-OH group) and 1300-1000 cm-1 (C-O stretching vibration). Based on these data and the retention of Ca2+ after washing and dialysis, the Ca2+ in fortified-rice was bound by hydrogen bonding to form Ca-hydrate and by ionicdipole bonding with –OH group of starch molecules, and/or trapping in gelatinized starch.

Factors Affecting the Function of the Mitochondrial Membrane Permeability Transition Pore and Their Role in Evaluation of Calcium Retention Capacity Values

Values of the calcium retention capacity (CRC) of rat liver mitochondria are highly dependent on the experimental conditions used. When increasing amounts of added calcium chloride are used (1.25-10 nmol), the values of the CRC increase 3-fold. When calcium is added in 75 s intervals, the CRC values increase by 30 % compared with 150 s interval additions. CRC values are not dependent on the calcium/protein ratio in the measured sample in our experimental design. We also show that a more detailed evaluation of the fluorescence curves can provide new information about mitochondrial permeability transition pore opening after calcium is added.

Moderate Consumption of Freeze-dried Blueberry Powder Increased Net Bone Calcium Retention in Healthy Postmenopausal Women: A Randomized Crossover Trial

Objectives Preclinical studies suggest that blueberry consumption is associated with improved bone health. This study quantified changes in urinary excretion of the long-lived calcium radioisotope 41Ca to assess the dose-response effect of blueberries on bone loss in postmenopausal women. We hypothesized that blueberry consumption would reduce bone loss in a dose-dependent manner. Methods Healthy women at least 4 years past menopause were dosed with 50 nCi of 41Ca and completed a 5-mo equilibration period for 41Ca deposition in bone followed by a 6-wk control period. Subsequently, participants were assigned to a random sequence of 3 intervention periods, each corresponding to a low (17.5 g/d), medium (35 g/d), or high (70 g/d) dose of freeze-dried blueberry powder equivalent to 0.75, 1.5, or 3 cups of fresh blueberries. The blueberry powder was incorporated into granola bars, spread, and drinks consumed daily for 6 wk, followed by a 6-wk washout period. Urinary 41Ca: Ca ratio was measured by accelerator mass spectrometry in 24-h urine collected weekly during intervention and every 3 wk during washout. Serum 25-hydroxyvitamin D and calcium concentrations were assessed prior to each intervention. Serum bone resorption biomarkers and urinary polyphenols were measured at the end of each control and intervention period. Data were analyzed using a linear mixed model and repeated measures ANOVA. Results Fourteen healthy, non-osteoporotic (mean bone mineral density t-score: −1.33) women completed the study. Net bone calcium retention increased by 6% in the low (P &lt; 0.01) and 4% in the medium (P &lt; 0.05) dose intervention compared with the control and washout periods. The high dose had no significant effect on net bone calcium retention (P = 0.19). Urinary excretion of hippuric acid increased dose-dependently with blueberry consumption. No significant relationships were found between bone resorption biomarkers, 25-hydroxyvitamin D, or calcium and any of the interventions. Conclusions A hormetic response was observed to blueberry intake, whereby daily consumption of foods containing 17.5–35 g, but not 70 g, of freeze-dried blueberry powder increased net bone calcium retention. Moderate consumption of blueberries may be an effective strategy to attenuate bone loss in healthy postmenopausal women. Funding Sources NIH/NCCIH, Project Development Team within the ICTSI NIH/NCRR.

Effect of Chronic Continuous Normobaric Hypoxia on Functional State of Cardiac Mitochondria and Tolerance of Isolated Rat Heart to Ischemia and Reperfusion: Role of µ and δ2 Opioid Receptors

Chronic continuous normobaric hypoxia (CNH) increases cardiac tolerance to ischemia/reperfusion injury in vivo and this effect is mediated via µ and δ2 opioid receptors (ORs) activation. CNH has also been shown to be cardioprotective in isolated rat heart. In this study, we hypothesize that this cardioprotective effect of CNH is mediated by activation of µ and δ2 ORs and preservation of mitochondrial function. Hearts from rats adapted to CNH (12 % oxygen) for 3 weeks were extracted, perfused in the Langendorff mode and subjected to 45 min of global ischemia and 30 min of reperfusion. Intervention groups were pretreated for 10 min with antagonists for different OR types: naloxone (300 nmol/l), the selective δ OR antagonist TIPP(ψ) (30 nmol/l), the selective δ1 OR antagonist BNTX (1 nmol/l), the selective δ2 OR antagonist naltriben (1 nmol/l), the selective peptide μ OR antagonist CTAP (100 nmol/l) and the selective κ OR antagonist nor-binaltorphimine (3 nmol/l). Creatine kinase activity in coronary effluent and cardiac contractile function were monitored to assess cardiac injury and functional impairment. Additionally, cardiac tissue was collected to measure ATP and to isolate mitochondria to measure respiration rate and calcium retention capacity. Adaptation to CNH decreased myocardial creatine kinase release during reperfusion and improved the postischemic recovery of contractile function. Additionally, CNH improved mitochondrial state 3 and uncoupled respiration rates, ADP/O, mitochondrial transmembrane potential and calcium retention capacity and myocardial ATP level during reperfusion compared to the normoxic group. These protective effects were completely abolished by naloxone, TIPP(ψ), naltriben, CTAP but not BNTX or nor-binaltorphimine. These results suggest that cardioprotection associated with adaptation to CNH is mediated by µ and δ2 opioid receptors activation and preservation of mitochondrial function.