Nutrition Support in Liver Transplantation and Postoperative Recovery: The Effects of Vitamin D Level and Vitamin D Supplementation in Liver Transplantation

Vitamin D plays an important role in the arena of liver transplantation. In addition to affecting skeletal health significantly, it also clinically exerts immune-modulatory properties. Vitamin D deficiency is one of the nutritional issues in the perioperative period of liver transplantation (LT). Although vitamin D deficiency is known to contribute to higher incidences of acute cellular rejection (ACR) and graft failure in other solid organ transplantation, such as kidneys and lungs, its role in LT is not well understood. The aim of this study was to investigate the clinical implication of vitamin D deficiency in LT. LT outcomes were reviewed in a retrospective cohort of 528 recipients during 2014–2019. In the pre-transplant period, 55% of patients were vitamin-D-deficient. The serum vitamin D level was correlated with the model for end-stage liver disease (MELD-Na) score. Vitamin D deficiency in the post-transplant period was associated with lower survival after LT, and the post-transplant supplementation of vitamin D was associated with a lower risk of ACR. The optimal vitamin D status and vitamin D supplementation in the post-transplant period may prolong survival and reduce ACR incidence.


Introduction
Vitamin D plays an important role in bone metabolism, regulating gene expression in multiple tissues, and increasing the intestinal absorption of calcium. Recently, in addition to the well-known effects on musculoskeletal metabolism, it has been reported that vitamin D has anti-inflammatory and immune-modulatory properties [1][2][3]. Clinically low serum levels of vitamin D have been associated with a higher prevalence of infections, cancer, cardiovascular, and autoimmune disorders [4,5]. Vitamin D deficiency is one of nutrition issues that is addressed in liver transplantation (LT) patients [6]. Due to the end-stage liver disease (ESLD) of the LT patients, malabsorption, inadequate dietary intake, and impairment in hepatic activation of vitamins are major issues [7,8]. While LT has been reported to have positive effects in increasing serum vitamin D concentrations as well as the percentage of patients with sufficient vitamin D levels, immunosuppression-related metabolic disturbances cause vitamin D

Demographical Characteristics of Patients
A total of 528 patients were included in the analytic cohort ( Table 1). The median recipient age at the time of LT was 58 years (IQR: 52-64), and more than half of the recipients were male (n = 350, 66.2%). Cause of cirrhosis was mainly alcohol (n = 154, 29.1%), NASH/Cryptogenic (n = 136, 25.9%), and viral hepatitis (n = 103, 19.5%). Half of the recipients received previous abdominal surgery (n = 266, 50.3%), and a minority of recipients had portal vein thrombosis at the time of LT (n = 104, 19.7%). The median laboratory MELD-Na score was 19 (IQR: 13-28). The median waiting time was 2.9 months (IQR: 0. 8-7.4). The majority of the donor graft type was donor after brain dead (DBD) (n = 458, 86.8%). The median cold ischemic time was 6.2 h (IQR: 5.3-7.4). Figure 1A shows the distribution of vitamin D status prior to LT, showing 55% were vitamin-D-deficient. The characteristics of vitamin-D-deficient and -sufficient patients were compared (Table 2). Recipient characteristics including age >60 years, presence of HCC, alcohol consumption rate, MELD-Na score, and serum albumin level had a significant difference (p < 0.05). The relationship between the MELD-Na score and serum levels of vitamin D in the pre-transplant period was analyzed ( Figure 1B). The correlation coefficient was −0.254 (p < 0.01; 95% CI: −0.34-−0.17).

Influence of Preoperative and Postoperative Serum Vitamin D Levels on Overall Survival
Differences in the long-term survival between patients who had vitamin D deficiency and sufficiency at pre-and post-transplant were compared (Figure 2A,B). There was no significant difference between the patients who had vitamin D deficiency and sufficiency in the pre-transplant period (p = 0.64) ( Figure 2A). However, there was a significant difference between the two groups in the post-transplant period ( Figure 2B).   A bivariate and multivariable Cox regression analysis was performed to assess the risk factors associated with the five-year OS (Table 3). Older age (>60 years old) (HR 3.47; 95% CI, 1.38-8.68, p < 0.01) and post-transplant vitamin D sufficiency (HR 0.31; 95% CI, 0.13-0.75, p < 0.01) were associated with five-year OS.

Influence of Preoperative and Postoperative Serum Vitamin D Levels on Acute Cellular Rejection
The incidence of ACR in LT recipients with pre-and post-transplant vitamin D deficiency was 19.1% and 22.0%, respectively. There was no significant difference in the cumulative incidence of ACR between vitamin D deficiency and sufficiency in both pre-and post-transplant periods ( Figure 3A,B).

Comparison of Patient Characteristics Based on Vitamin D Supplementation Status
In examining the effect of vitamin D supplementation, four groups were investigated: (1) patients who did not receive supplementation (No Supplement), (2) patients who received supplementation during only pre-transplant (Pre), (3) patients who received supplementation during only post-transplant (Post), and (4) patients who received supplementation during both pre-and post-transplant (Pre/Post). The characteristics of the four groups were compared (Table 4). Among the four groups, there was a significant difference in the ratio of sex and 25(OH)D level at pretransplant (p < 0.05).

Comparison of Patient Characteristics Based on Vitamin D Supplementation Status
In examining the effect of vitamin D supplementation, four groups were investigated: (1) patients who did not receive supplementation (No Supplement), (2) patients who received supplementation during only pre-transplant (Pre), (3) patients who received supplementation during only post-transplant (Post), and (4) patients who received supplementation during both pre-and post-transplant (Pre/Post). The characteristics of the four groups were compared (Table 4). Among the four groups, there was a significant difference in the ratio of sex and 25(OH)D level at pre-transplant (p < 0.05).

Effect of Vitamin D Supplementation on Overall Survival
Differences in the long-term survival among the four patient groups (No Supplement, Pre, Post, Pre/Post) were compared using the Kaplan-Meier curve ( Figure 4A). Regardless of the supplementation status, there were no significant differences in the OS (p = 0.60).

Effect of Vitamin D Supplementation on Overall Survival
Differences in the long-term survival among the four patient groups (No Supplement, Pre, Post, Pre/Post) were compared using the Kaplan-Meier curve ( Figure 4A). Regardless of the supplementation status, there were no significant differences in the OS (p = 0.60).

Effect of Vitamin D Supplementation on Acute Cellular Rejection
The cumulative incidence of ACR among the four groups (No Supplement, Pre, Post, Pre/Post) ( Figure 4B) was compared. Interestingly, the incidence rate of ACR showed a significant difference

Effect of Vitamin D Supplementation on Acute Cellular Rejection
The cumulative incidence of ACR among the four groups (No Supplement, Pre, Post, Pre/Post) ( Figure 4B) was compared. Interestingly, the incidence rate of ACR showed a significant difference based on the vitamin D supplementation status. The cumulative incidence was high in the Pre group and was low in the Post group. The proportional subdistribution hazard model of the Fine and Gray method was used for ACR in the No Supplement group and Pre group (Table 5). From the bivariate and multivariable analysis, age (>60 years) was the only variable that was significant (sHR 0.30; 95% CI, 0.12-0.77, p = 0.01). In the same manner, the Fine and Gray method was used for ACR in the No Supplement group and Post group (Table 6). Among all variables, calculated MELD-Na score >30 (sHR < 0.01; 95% CI, <0.01-<0.01, p < 0.01) at the time of LT and vitamin D supplementation during post-transplant (sHR 0.09; 95% CI, 0.01-0.72, p = 0.02) were significant.

Discussion
Although previous studies have reported that vitamin D plays an important role in solid organ transplantation including kidney and lung [12,13], the clinical impact of vitamin D on LT outcomes and vitamin D supplementation is still unknown. The current study included 528 patients which is larger than any other previous studies of vitamin D in LT. Our study investigated the correlation of the pre-transplant vitamin D level and the MELD-Na score. Moreover, the current study is important because we were able to reveal how the perioperative vitamin D levels and vitamin D supplementation status affect long-term outcomes, such as OS and ACR all in the same cohort. Using the cutoff of 20 ng/mL of 25(OH)D [18], 55% of the patients had vitamin D deficiency before LT. The MELD-Na score and serum levels of 25(OH)D before LT showed a negative linear relationship. While there was no survival difference based on the vitamin D level during pre-transplant, patients who had vitamin D deficiency at post-transplantation had worse survival compared with vitamin-D-sufficient patients. The cumulative incidence of ACR was not affected by the perioperative 25(OH)D level. There was no difference in the OS when it was assessed based on the vitamin D supplementation status. However, the accumulated incidence of ACR was high in the Pre group and low in the Post group, showing a significant difference. Importantly, the risk factor of ACR in the Pre group and Post group was younger age and no vitamin D supplementation and higher MELD-Na score at the time of LT, respectively.
Vitamin D3 is taken in the body by diet (20%) or is synthesized by the skin (80%) from 7-dihydrocholesterol following UVB exposure. Vitamin D3 becomes biologically active after hydroxylation in the liver by the enzymes cytochrome P450 2R1 and cytochrome P450 27 becoming 25-hydroxyvitamin D3. The fully active metabolite 1,25-dihydroxyvitamin D3 is hydroxylated in the kidney [21]. ESLD patients have both impaired liver and kidney function that can alter calcium and vitamin D homeostasis [22,23]. Even when there were patients taking supplemental vitamin D during pre-transplant, more than half of the patients had vitamin D deficiency ( Figure 1A). In addition, we found that there was a negative correlation between MELD-Na scores and serum levels of 25(OH)D in the pre-transplant period, which was compatible with the vitamin D physiology since the MELD-Na score incudes both hepatic and renal components [15].
Vitamin-D-sufficient status in the post-transplant period was associated with five-year survival after LT. The optimal vitamin D status prolonged survival. This indicated that post-transplant nutritional support including the correction of vitamin D deficiency will support better OS. These results are consistent with a study from Lowery et al., showing that the mortality of lung transplant recipients who remained vitamin-D-deficient at one-year post-transplant was higher than that of recipients who maintained normal vitamin D levels [13]. In the current study, the six-month mortality rate of vitamin-D-deficient patients in the post-transplant period was 8.0% while that in vitamin-D-sufficient patients was 0.63% (p < 0.01) ( Figure 2B). Post-transplant vitamin-D-deficient status had larger effects on mortality in the early period after LT compared with the late period. Even though early mortality after LT can be caused by different conditions such as early allograft dysfunction and infections [24], these complications might be related to decreased immune-modulatory properties because of the low vitamin D level [1][2][3]. On the other hand, low vitamin D can be a result of the malnourished condition of LT recipients. Malnutrition itself could also negatively affect mortality [25,26]. Thus, serum levels of 25(OH)D in the post-transplant period can be a prognostic factor or a predictive factor for survival. Further investigation to determine how the vitamin D status contributes to OS is needed.
Pre-transplant vitamin D status was not associated with the long-term accumulated incidence of ACR after LT in this study ( Figure 3A). Although focusing on the short term after LT, the vitamin D deficiency group had a higher rate of ACR compared to the sufficiency group. Similar findings were seen in a paper from Zhou et al. showing that high pre-transplant 25(OH)D level (>25 ng/mL) prior to LT significantly decreased the incidence of ACR in 30 days after LT [27]. Another report from Bitetto et al. also confirmed that low pre-transplant 25(OH)D level (<5 ng/mL) was independently associated with moderate to severe ACR episodes within two months after LT [28]. After the early period of LT, the incidence of ACR in the vitamin D sufficiency group tended to be higher than the deficiency group. This implies that the vitamin D sufficiency in the pre-transplant period contributed to reduced risks for ACR in the early post-transplant period and smoothly boosted the immune system of recipients through the recovery phase from LT-related surgical procedures in the late post-transplant period. The vitamin D status during the post-transplant period had no significant relationship with the incidence of ACR ( Figure 3B).
As such, pre-transplant vitamin D levels may be associated with ACR in the early period after LT, and it can be hypothesized that optimization of vitamin-D-deficient status by supplementation in the pre-transplant period may contribute to reducing the incidence of ACR.
The clinical effects of vitamin D supplementation on LT outcomes remained unclear. We demonstrated that vitamin D supplementation in the post-transplant period has positive effects in decreasing the incidence of ACR during one year after LT. A previous study showed similar findings that vitamin D supplementation for the first one month significantly decreased the incidence of ACR in 30 days after LT [27]. Vitamin D supplementation is assumed to increase the components of suppressor T cells/T memory cells, decrease the C3 co-stimulatory molecule expression (HLA-DR, CD28), and expand T naïve cells/cytotoxic T cells [27,29]. Our comparison between the No Supplement and the Post groups clarified the anti-rejection effects of the post-transplant vitamin D supplementation. From these results, we propose that vitamin D supplementation should be considered, especially to reduce the incidence of ACR. Yet, our result does not demonstrate that vitamin D supplementation in the pre-transplant period could reduce the incidence of ACR. Thus, prospective studies should be conducted to elucidate the importance of vitamin D supplementation in LT, including the relationship between vitamin D supplementation and modification of vitamin D levels.
This study had several limitations. Since this was a retrospective study, information bias was possible given that the data were manually abstracted from the medical records. Additionally, there was potential for unmeasured confounders of the relationship between vitamin D status and hepatic graft status. The multivariable analysis of all four groups (No Supplement, Pre, Post, Pre/Post) showed that sex is not a significant factor regulating the incidence of ACR (Tables 5 and 6) or five-year survival (data not shown), although there were gender differences among groups (Table 4). In this study, more female patients tended to be on vitamin D supplementation compared with male patients. We assume it was because of the higher incidence of bone-related disorders in female groups, such as osteoporosis. Future research is needed to elucidate the gender effects on outcomes after LT.
Finally, it should be noted that there are no guidelines for screening or supplementation of vitamin D in LT recipients. Further investigation is needed to clarify the importance of screening for vitamin D status and the effectiveness of vitamin D supplementation for patients in the peri-transplant period.

Conclusions
Vitamin D deficiency in the post-transplant period was associated with lower survival after LT, and the post-transplant supplementation of vitamin D was associated with a lower risk of ACR. Vitamin D levels in the pre-transplant period may be an important factor of ACR. Nutritional support with vitamin D supplementation might be contributing to improving LT outcomes.