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

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Abstract

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.

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Acknowledgements

The authors acknowledge the assistance of the parents of AT for the use of their house for carrying out this research.

Funding

This research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada, grant number RGPIN-3669–2016, and by support from the Work-Study program at McMaster University.

Author information

Authors and Affiliations

  1. Dept. of Biochemistry, McMaster University, Hamilton, ON, Canada

    Aasritha Thotakura

  2. Dept. of Mechanical Engineering, McMaster University, Hamilton, ON, Canada

    Cheryl Quenneville

  3. School of Biomedical Engineering, McMaster University, Hamilton, ON, Canada

    Cheryl Quenneville & Henry P. Schwarcz

  4. School of Earth, Environment and Society, McMaster University, Hamilton, ON, Canada

    Henry P. Schwarcz

Authors
  1. Aasritha Thotakura
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  2. Cheryl Quenneville
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  3. Henry P. Schwarcz
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Contributions

The analyses were carried out by AT. Human samples donated by CQ. Writing by HPS, followed by editing by AT and CQ.

Corresponding author

Correspondence to Henry P. Schwarcz.

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Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

Ethics approval for use of human samples was obtained from HiREB (Hamilton Integrated Research Ethics Board) as a human tissue research study, approval 2016–2346-T.

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Thotakura, A., Quenneville, C. & Schwarcz, H.P. Ashing of bone: errors due to loss of CO2 and their correction. J Bone Miner Metab 40, 594–601 (2022). https://doi.org/10.1007/s00774-022-01327-5

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  • DOI: https://doi.org/10.1007/s00774-022-01327-5

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