Pullar Juliet M, Bayer Simone, Carr Anitra C
Department of Pathology and Biomedical Science, University of Otago, Christchurch, P.O. Box 4345, Christchurch 8140, New Zealand.
Antioxidants (Basel). 2018 Feb 11;7(2):29. doi: 10.3390/antiox7020029.
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.
维生素C（抗坏血酸盐）是血浆中主要的水溶性抗氧化剂，其氧化为脱氢抗坏血酸（DHA）被认为是体内氧化应激的一个指标。然而，关于从不同患者队列采集的血样中检测到的DHA量，文献中存在争议。在本研究中，我们报告了一系列不同临床队列（糖尿病、肺炎、癌症和重症患者）中的DHA浓度。所有临床样本均采集到EDTA抗凝管中，并在4℃下处理，然后在-80℃下储存，以便随后通过高效液相色谱-电化学检测进行分析。我们还研究了不同处理和加工条件对体外以及全血和血浆样本中抗坏血酸盐和DHA短期和长期稳定性的影响。这些条件包括金属螯合、抗凝剂（EDTA和肝素）以及加工温度（冰浴、4℃和室温）。对我们临床队列的分析表明，DHA浓度非常低或可忽略不计。出现溶血的样本中DHA浓度显著更高。金属螯合在体外抑制了维生素C的氧化，证实了污染金属离子的参与。尽管EDTA是一种有效的金属螯合剂，但与过渡金属离子形成的络合物仍具有氧化还原活性，因此其作为抗凝剂可促进全血和血浆中金属离子依赖性的维生素C氧化。在冰上（或4℃）处理和加工血样可将维生素C的氧化延迟数小时。对临床队列中DHA浓度相关文献的综述突出了这样一个事实，即与使用高效液相色谱-电化学检测的研究相比，使用比色法或荧光法测定的研究报告的DHA浓度显著更高。总之，对样本进行仔细处理和加工，并结合适当的分析，对于准确测定临床样本中的抗坏血酸盐和DHA至关重要。
