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Impact of preanalytical freezing delay time on the stability of metabolites in oral squamous cell carcinoma tissue samples

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Abstract

Introduction

Metabolite stability is critical for tissue metabolomics. However, changes in metabolites in tissues over time from the operating room to the laboratory remain underexplored.

Objectives

In this study, we evaluated the effect of postoperative freezing delay time on the stability of metabolites in normal and oral squamous cell carcinoma (OSCC) tissues.

Methods

Tumor and paired normal tissues from five OSCC patients were collected after surgical resection, and samples was sequentially quenched in liquid nitrogen at 30, 40, 50, 60, 70, 80, 90 and 120 min (80 samples). Untargeted metabolic analysis by liquid chromatography–mass spectrometry/mass spectrometry in positive and negative ion modes was used to identify metabolic changes associated with delayed freezing time. The trends of metabolite changes at 30–120 and 30–60 min of delayed freezing were analyzed.

Results

190 metabolites in 36 chemical classes were detected. After delayed freezing for 120 min, approximately 20% of the metabolites changed significantly in normal and tumor tissues, and differences in the metabolites were found in normal and tumor tissues. After a delay of 60 min, 29 metabolites had changed significantly in normal tissues, and 84 metabolites had changed significantly in tumor tissues. In addition, we constructed three tissue freezing schemes based on the observed variation trends in the metabolites.

Conclusion

Delayed freezing of tissue samples has a certain impact on the stability of metabolites. For metabolites with significant changes, we suggest that the freezing time of tissues be reasonably selected according to the freezing schemes and the actual clinical situation.

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Data Availability

The data that support the findings of this study is available from the corresponding author upon reasonable request.

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Acknowledgements

The authors gratefully acknowledge Guowen Sun and Shu Xia for kind assistance in providing residual patient samples.

Funding

This research was funded by National Natural Science Foundation of China (82173159, 81902759, 82002865, 82173380), the Key Research and Development Projects in Jiangsu Province (BE2020628), Doctoral Program for Entrepreneurship and Innovation of Jiangsu Province (JSSCBS20211596), and Nanjing Medical Science and Technique Development Foundation (YKK19091, YKK20151).

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Author notes

  1. Shuai Wang, Yawei Sun and Tao Zeng contributed equally to this work.

Authors and Affiliations

  1. Department of Oral and Maxillofacial Surgery, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing, 210008, Jiangsu, China

    Shuai Wang, Yawei Sun & Qingang Hu

  2. State Key Lab of Pharmaceutical Biotechnology, College of Life Sciences, Nanjing University, Nanjing, 210008, Jiangsu, China

    Tao Zeng

  3. Department of Oral Pathology, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing, 210008, Jiangsu, China

    Yan Wu, Lei Zhang, Xiaofeng Huang & Huiling Li

  4. Central Laboratory of Stomatology, Nanjing Stomatological Hospital, Medical School of Nanjing University, Nanjing, 210008, Jiangsu, China

    Shuai Wang, Yan Wu, Liang Ding, Xiaoxin Zhang, Yanhong Ni & Qingang Hu

  5. Department of Oral and Maxillofacial Surgery, Affiliated Hospital of Jiangsu University, Zhenjiang, 212001, Jiangsu, China

    Xihu Yang

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  1. Shuai Wang
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Contributions

In this study, Shuai Wang designed the study and wrote the manuscript; Yawei Sun and Yan Wu collected data; Tao Zeng analyzed data; Liang Ding, Xiaoxin Zhang and Xihu Yang reviewed the manuscript; Lei Zhang, Xiaofeng Huang and Huiling Li provided study materials; Yanhong Ni and Qingang Hu supervised the study and reviewed the manuscript.

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Correspondence to Yanhong Ni or Qingang Hu.

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This study was approved by the Medical Ethics Committee of Nanjing Stomatology Hospital, Medical School of Nanjing University (NJSH-2021NL-025).

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Wang, S., Sun, Y., Zeng, T. et al. Impact of preanalytical freezing delay time on the stability of metabolites in oral squamous cell carcinoma tissue samples. Metabolomics 18, 82 (2022). https://doi.org/10.1007/s11306-022-01943-2

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