Evaluation of changes in plasma vitamin C levels in brain-dead organ donors

1Department of Anesthesia and Intensive Care, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran 2Surgical Oncology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran 3Department of Operating Room, Montaserieh Dialysis and Transplant Center, Mashhad University of Medical Sciences, Mashhad, Iran 4Department of Anesthesia and Intensive Care, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran Immunopathologia Persa http www.immunopathol.com


Introduction
Organs transplantation from deceased donors has become the definitive treatment for many patients with organ failure (1). Brain death (BD) is associated with physiological changes that may affect all organs acceptable for transplantation. BD may also cause systemic inflammation. BD-induced "Catecholamine storm" is a severe inflammatory reaction characterized by increased plasma levels of cytokines (2). In the last decade, significant progress has been made in identifying the pathophysiology of traumatic brain injury (TBI) which is the most common cause of BD. At present, TBI classifications consist of initial (primary) and delayed (secondary) brain injury (3). The main consequences of secondary brain injury are inflammation and increased reactive oxygen species (ROS), leading to apoptosis.
ROS were recognized as part of the most important components of the inflammatory reaction; they act as by-products, participate

Key point
Inflammatory events associated with brain death adversely affect donated organs and the outcome of transplant surgery. As a result, significant efforts have been conducted to protect the organs against inflammation. Ascorbic acid (AA) is of particular importance because of its antioxidant properties, which reduce oxidative damage in biological systems.
in the reaction and create a vicious cycle. Moderate to low levels of ROS are produced as a part of normal cellular metabolism and defense systems. In fact, there is a balance between ROS and the antioxidant system under physiological conditions. Therefore, ROS are regulated and kept low by the antioxidant system. However, during pathological conditions such as intracerebral hemorrhage, additional sources of ROS are formed. High concentrations of ROS can disturbance the dynamic balance, causing adverse changes to cellular components, DOI:10.34172/ipp.2022.06 lipids, proteins, and DNA (4). Therefore, increased free radicals and ROS production lead to oxidative stress (5). The human antioxidant defense system contains endogenous antioxidants such as glutathione (GSH). This can eliminate excess ROS and reduce oxidative stress. However, this defense system might be incomplete without the supplement of exogenous antioxidants, such as ascorbic acid (AA) (6).
AA is especially significant in restricting oxidative harm in biological systems. It may protect from the dysregulation of the immune and inflammatory response by its antioxidant properties (7). AA acts not only as an antioxidant but also as a pro-oxidant. The influence of exogenous antioxidants (such as AA) on oxidative metabolism and inflammatory processes may depend on their concentration (6). Therefore, for maintaining or reestablish the balance between oxidation and antioxidants (redox homeostasis, an essential function of biological systems), physiological doses of exogenous antioxidants are needed (8).

Objectives
This study aimed to evaluate the changes in serum AA levels in deceased solid organ donors between declaring BD and organ procurement.

Study design
This clinical research was performed between June 2014 and January 2015; plasma samples were obtained from 37 deceased organ donors in our procurement area. The standard management was based on the United Network for Organ Sharing (UNOS) recommendation for all BDDs. Methylprednisolone was given to all deceased donors. Blood samples were obtained from BDDs once in the intensive care unit (ICU) when they were accepted as organ donors and again in the operating room immediately before the organ procurement process. All specimens were obtained according to the standard procedure and stored at -20°C until the time of analysis.
AA was measured using a Human Vitamin C (VC) ELISA Kit (Cat No: E1538Hu), manufactured by the Bioassay Technology laboratory [China (Mainland)]. The practical technology of the ELISA kit is based on the double-antibody sandwich technology to detect human vitamin C. The assay range was 1-300 ng/mL with a sensitivity of 0.52 ng/mL. Donor information regarding gender, hemoglobin level, and cause of death were recorded for all cases.

Statistical analysis
Data were analyzed with IBM SPSS Statistics software version 20 (IBM Corp, Armonk, NY). To assess the normal distribution of variables, the Kolmogorov-Smirnov test was used. Data were described as frequency (percent) or mean ± standard deviation. Wilcoxon signed-rank test was used to compare values of serum vitamin C (ng/ mL) at admission (R1) and immediately before organ procurement (R2). Accordingly, P < 0.05 was defined as statistically significant.

Results
In the present study blood samples were obtained from 37 BD donors; 23 men and 14 women with a mean age of 26.48 ± 18.1 years. TBI was the most common cause of BD (40.5 %) among the studied cases. The mean hemoglobin level was 11.95 ± 3.21 g/dL and the mean time between samplings was 40.09 ± 12.10 hours. The most common blood group type among donors was B positive. Table 1 shows summary of demographic data of the deceased donors.
Differences between the mean R1-R2 values were analyzed according to the parameters of brain-dead donors (BDDs), which included gender, cause of BD, and blood group type.
In total, the mean (standard deviation) of serum AA level at admission (R1) and immediately before organ procurement (R2) was 40.0 (82.0) and 39.5 (80.9), respectively. There was a statistically significant difference between serum AA level on admission (R1) and immediately before organ procurement (R2) (P = 0.016). Table 2 shows plasma levels of AA in the deceased donors at admission (R1) and immediately before organ procurement (R2). Therefore, serum vitamin C levels were significantly reduced following BD.

Discussion
This study showed that the level of serum AA was significantly affected by the BD process during BD diagnosis and just before the procurement of donated organs (a median period range of 14-98 hours). TBI was the main cause of BD (40.5%) among the studied cases. It has been demonstrated that a significant reduction in plasma AA levels in brain-injured patients in comparison to healthy control subjects occurs on the first day. Interestingly, the plasma AA levels did not significantly change afterward (9). Nevertheless, our study showed that significant depletion of serum vitamin C occurs due to BD.
There are several mechanisms by which BDDs affect the graft function. These include significant changes in hemodynamics, hormonal changes, and systemic inflammation. Inflammatory responses in transplant surgery have been reported to be the main disorder caused by BD (10). Several clinical studies have shown that the anti-inflammatory plan of action for BDDs improves posttransplanted function. The impact of AA administration in BDDs has also been investigated; a beneficial effect for AA on the inflammatory response besides improvement of the primary allograft function has been reported in this regard (11,12). It is not clear whether anti-inflammatory strategies based on the combination of various antioxidants including vitamins C and E might be more efficient.
The present study showed a significant change in the serial measurement of serum vitamin C levels in braindead patients. This finding suggests that a standard dose of vitamin C should be considered in the management guidelines of BD cases.

Conclusion
Solid-organ transplantation is the best choice of treatment in many patients with end-stage organ failures. Obtaining organs from deceased donors is the only option for many of the patients. However, BD is a process that can trigger pathophysiological events and cause significant dysfunction in the donated organ. Thus, significant efforts have been made to ameliorate the function of donated organs by protecting them from the events. Vitamin C is an important nutrient that is especially significant in restricting oxidative harm in biological systems. It can protect against impaired immune and inflammatory responses. In our study, significant decreases in serum vitamin C levels were observed in BDDs, in addition to previous reports of early depletion of such levels in braininjured patients. Therefore, determining the most effective  a R1 -R2 shows the difference between values of serum vitamin C levels (ng/mL) at admission (R1) and immediately before organ procurement (R2). * Mean (SD) , ** Wilcoxon rank sign test.
dose of vitamin C supplementation and the best time to administer it to the patients is highly recommended for future studies.

Limitations of the study
The small sample size was the main limitation. Moreover, the exact relationship between plasma and brain levels of vitamin C in brain-death patients has not yet been considered.