Ashing of bone: errors due to loss of CO2 and their correction.

INTRODUCTION: Ashing is widely used to determine weight fraction of water-free bone that is mineral, but no standard procedure exists and the range of techniques used spans a range of temperatures and times over which the amount of weight loss is variable. We show that variability is largely due to progressive loss of CO2 from CO3 2- ions in the apatite crystal lattice, beginning at 600 ℃, typically used for ashing. We test the effect of varying temperature, time, and weight of sample and develop a reliable method, using small samples. MATERIALS AND METHODS: Replicate samples of bovine cortical bone were tested at 500 ℃, 600 ℃, and 700 ℃ for times ranging up to 24 h. We also tested samples of multiple humans at what we concluded to be the optimal conditions. RESULTS: Varying conditions of ashing resulted in variations in apparent ash weight % by up to 7%. Samples between 5 and 20 mg heated to 600 ℃ for 1 h gave results agreeing with generally accepted values, but with much smaller variability. Ash wt% values for multiple human bone samples differed by up to 4.8%, but replicate data for individuals agree to ± 1 wt%. DISCUSSION: In conclusion, a satisfactory method is given for ash weight determination using small samples, and yielding highly reproducible data. If accepted widely, ash weight values between laboratories could be used to study variations due to diet, age, drug treatment, and disease.

Mitochondrial bioenergetics of astrocytes in Fragile X syndrome: new perspectives on culture conditions and sex effects.

Fragile X syndrome (FXS) is a genetic disorder that is characterized by a range of cognitive and behavioral deficits, including mild-moderate intellectual disability. The disease is characterized by an X-linked mutation of the Fmr1 gene, which causes silencing of the gene coding for fragile X mental retardation protein (FMRP), a translational regulator integral for neurodevelopment. Mitochondrial dysfunction has been recently associated with FXS, with reports of increases in oxidative stress markers, reactive oxygen species, and lipid peroxidation being present in the brain tissue. Astrocytes, a prominent glial cell within the central nervous system (CNS), play a large role in regulating oxidative homeostasis within the developing brain and dysregulation of astrocyte redox balance in FXS, which may contribute to oxidative stress. Astrocyte function and mitochondrial bioenergetics are significantly influenced by oxygen availability and circulating sex hormones; yet, these parameters are rarely considered during in vitro experimentation. Given that the brain normally develops in a range of hypoxic conditions and FXS is a sex-linked genetic disorder, we investigated how different oxygen levels (normoxic vs. hypoxic) and biological sex affected mitochondrial bioenergetics of astrocytes in FXS. Our results demonstrate that both mitochondrial respiration capacity and reactive oxygen species emission are altered with Fmr1 deletion in astrocytes and these changes were dependent upon both sexual dimorphism and oxygen availability.