Appropriate Handling, Processing and Analysis of Blood Samples Is Essential to Avoid Oxidation of Vitamin C to Dehydroascorbic Acid
Vitamin C (ascorbate) is the major water-soluble antioxidant in plasma and its oxidation to dehydroascorbic acid (DHA) has been proposed as a marker of oxidative stress in vivo. However, controversy exists in the literature around the amount of DHA detected in blood samples collected from various pa...
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| Format: | Article |
| Language: | English |
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MDPI AG
2018-02-01
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| Series: | Antioxidants |
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| Online Access: | http://www.mdpi.com/2076-3921/7/2/29 |
| _version_ | 1797763302203326464 |
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| author | Juliet M. Pullar Simone Bayer Anitra C. Carr |
| author_facet | Juliet M. Pullar Simone Bayer Anitra C. Carr |
| author_sort | Juliet M. Pullar |
| collection | DOAJ |
| description | Vitamin C (ascorbate) is the major water-soluble antioxidant in plasma and its oxidation to dehydroascorbic acid (DHA) has been proposed as a marker of oxidative stress in vivo. However, controversy exists in the literature around the amount of DHA detected in blood samples collected from various patient cohorts. In this study, we report on DHA concentrations in a selection of different clinical cohorts (diabetes, pneumonia, cancer, and critically ill). All clinical samples were collected into EDTA anticoagulant tubes and processed at 4 °C prior to storage at −80 °C for subsequent analysis by HPLC with electrochemical detection. We also investigated the effects of different handling and processing conditions on short-term and long-term ascorbate and DHA stability in vitro and in whole blood and plasma samples. These conditions included metal chelation, anticoagulants (EDTA and heparin), and processing temperatures (ice, 4 °C and room temperature). Analysis of our clinical cohorts indicated very low to negligible DHA concentrations. Samples exhibiting haemolysis contained significantly higher concentrations of DHA. Metal chelation inhibited oxidation of vitamin C in vitro, confirming the involvement of contaminating metal ions. Although EDTA is an effective metal chelator, complexes with transition metal ions are still redox active, thus its use as an anticoagulant can facilitate metal ion-dependent oxidation of vitamin C in whole blood and plasma. Handling and processing blood samples on ice (or at 4 °C) delayed oxidation of vitamin C by a number of hours. A review of the literature regarding DHA concentrations in clinical cohorts highlighted the fact that studies using colourimetric or fluorometric assays reported significantly higher concentrations of DHA compared to those using HPLC with electrochemical detection. In conclusion, careful handling and processing of samples, combined with appropriate analysis, is crucial for accurate determination of ascorbate and DHA in clinical samples. |
| first_indexed | 2024-03-12T19:40:36Z |
| format | Article |
| id | doaj.art-aa1da13cde9c4929a8c737c3c3a898c1 |
| institution | Directory Open Access Journal |
| issn | 2076-3921 |
| language | English |
| last_indexed | 2024-03-12T19:40:36Z |
| publishDate | 2018-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Antioxidants |
| spelling | doaj.art-aa1da13cde9c4929a8c737c3c3a898c12023-08-02T03:54:40ZengMDPI AGAntioxidants2076-39212018-02-01722910.3390/antiox7020029antiox7020029Appropriate Handling, Processing and Analysis of Blood Samples Is Essential to Avoid Oxidation of Vitamin C to Dehydroascorbic AcidJuliet M. Pullar0Simone Bayer1Anitra C. Carr2Department of Pathology and Biomedical Science, University of Otago, Christchurch, P.O. Box 4345, Christchurch 8140, New ZealandDepartment of Pathology and Biomedical Science, University of Otago, Christchurch, P.O. Box 4345, Christchurch 8140, New ZealandDepartment of Pathology and Biomedical Science, University of Otago, Christchurch, P.O. Box 4345, Christchurch 8140, New ZealandVitamin C (ascorbate) is the major water-soluble antioxidant in plasma and its oxidation to dehydroascorbic acid (DHA) has been proposed as a marker of oxidative stress in vivo. However, controversy exists in the literature around the amount of DHA detected in blood samples collected from various patient cohorts. In this study, we report on DHA concentrations in a selection of different clinical cohorts (diabetes, pneumonia, cancer, and critically ill). All clinical samples were collected into EDTA anticoagulant tubes and processed at 4 °C prior to storage at −80 °C for subsequent analysis by HPLC with electrochemical detection. We also investigated the effects of different handling and processing conditions on short-term and long-term ascorbate and DHA stability in vitro and in whole blood and plasma samples. These conditions included metal chelation, anticoagulants (EDTA and heparin), and processing temperatures (ice, 4 °C and room temperature). Analysis of our clinical cohorts indicated very low to negligible DHA concentrations. Samples exhibiting haemolysis contained significantly higher concentrations of DHA. Metal chelation inhibited oxidation of vitamin C in vitro, confirming the involvement of contaminating metal ions. Although EDTA is an effective metal chelator, complexes with transition metal ions are still redox active, thus its use as an anticoagulant can facilitate metal ion-dependent oxidation of vitamin C in whole blood and plasma. Handling and processing blood samples on ice (or at 4 °C) delayed oxidation of vitamin C by a number of hours. A review of the literature regarding DHA concentrations in clinical cohorts highlighted the fact that studies using colourimetric or fluorometric assays reported significantly higher concentrations of DHA compared to those using HPLC with electrochemical detection. In conclusion, careful handling and processing of samples, combined with appropriate analysis, is crucial for accurate determination of ascorbate and DHA in clinical samples.http://www.mdpi.com/2076-3921/7/2/29vitamin Cascorbatedehydroascorbic acidwhole bloodplasmaEDTAheparinHPLC with electrochemical detectionpneumoniacancerdiabetescritically ill |
| spellingShingle | Juliet M. Pullar Simone Bayer Anitra C. Carr Appropriate Handling, Processing and Analysis of Blood Samples Is Essential to Avoid Oxidation of Vitamin C to Dehydroascorbic Acid Antioxidants vitamin C ascorbate dehydroascorbic acid whole blood plasma EDTA heparin HPLC with electrochemical detection pneumonia cancer diabetes critically ill |
| title | Appropriate Handling, Processing and Analysis of Blood Samples Is Essential to Avoid Oxidation of Vitamin C to Dehydroascorbic Acid |
| title_full | Appropriate Handling, Processing and Analysis of Blood Samples Is Essential to Avoid Oxidation of Vitamin C to Dehydroascorbic Acid |
| title_fullStr | Appropriate Handling, Processing and Analysis of Blood Samples Is Essential to Avoid Oxidation of Vitamin C to Dehydroascorbic Acid |
| title_full_unstemmed | Appropriate Handling, Processing and Analysis of Blood Samples Is Essential to Avoid Oxidation of Vitamin C to Dehydroascorbic Acid |
| title_short | Appropriate Handling, Processing and Analysis of Blood Samples Is Essential to Avoid Oxidation of Vitamin C to Dehydroascorbic Acid |
| title_sort | appropriate handling processing and analysis of blood samples is essential to avoid oxidation of vitamin c to dehydroascorbic acid |
| topic | vitamin C ascorbate dehydroascorbic acid whole blood plasma EDTA heparin HPLC with electrochemical detection pneumonia cancer diabetes critically ill |
| url | http://www.mdpi.com/2076-3921/7/2/29 |
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