Peritraumatic Vitamin D Levels Predict Chronic Pain Severity and Contribute to Racial Differences in Pain Outcomes Following Major Thermal Burn Injury

Abstract

Major thermal burn injuries result in approximately 40,000 hospitalizations in the United States each year. Chronic pain affects up to 60% of burn survivors, and Black Americans have worse chronic pain outcomes than White Americans. Mechanisms of chronic pain pathogenesis after burn injury, and accounting for these racial differences, remain poorly understood. Due to socioeconomic disadvantage and differences in skin absorption, Black Americans have an increased prevalence of Vitamin D deficiency. We hypothesized that peritraumatic Vitamin D levels predict chronic pain outcomes after burn injury and contribute to racial differences in pain outcomes. Among burn survivors (n = 77, 52% White, 48% Black, 77% male), peritraumatic Vitamin D levels were more likely to be deficient in Blacks vs Whites (27/37 [73%] vs 14/40 [35%], P < .001). Peritraumatic Vitamin D levels were inversely associated with chronic post-burn pain outcomes across all burn injury survivors, including those who were and were not Vitamin D deficient, and accounted for approximately one-third of racial differences in post-burn pain outcome. Future studies are needed to evaluate potential mechanisms mediating the effect of Vitamin D on post-burn pain outcomes and the potential efficacy of Vitamin D in improving pain outcomes and reducing racial differences.

Major thermal burn injuries result in approximately 40,000 hospitalizations in the United States each year.^1–3 Chronic pain affects up to 60% of burn survivors and results in substantial interference in life function.^4–8 Despite this high prevalence and morbidity, mechanisms of chronic pain pathogenesis after burn injury remain poorly understood, and no interventions are available to reduce chronic post-burn pain.

Vitamin D is a steroid hormone with potent anti-inflammatory and neuroprotective properties.^9–12 Several lines of evidence suggest that peritraumatic Vitamin D levels influence chronic pain outcomes after burn injury. These lines of evidence include data that low Vitamin D levels are associated with increased pain in other pain conditions,^13–18 and data that Vitamin D administration improves pain outcomes across a range of neuro/inflammatory disorders.^17,19–21

Due to reduced skin absorption²² and socioeconomic disadvantage^23,24, Black Americans have lower levels of Vitamin D than White Americans^22,25,26 and an increased prevalence of Vitamin D deficiency (<20 ng/ml, 86% vs 31%, respectively²⁷). In part related to socioeconomic disadvantage, Black Americans also experience disproportionally higher rates of burn injury compared to White Americans.^3,28,29 We have previously shown that Black Americans also have worse chronic pain outcomes after burn injury than White Americans, even after adjustment for sociodemographic factors.³⁰ Factors explaining racial differences in pain outcomes between Black and White Americans experiencing burn injury remain poorly understood.

In this study of burn survivors hospitalized for debridement and tissue autografting, we evaluated the association between peritraumatic levels of Vitamin D and pain outcomes over time. We hypothesized that peritraumatic Vitamin D levels would predict chronic post-burn pain severity. In addition, we also evaluated racial differences in Vitamin D levels and post-burn pain outcomes. We hypothesized that peritraumatic Vitamin D levels would be lower in Black Americans than White Americans, and that this difference would contribute to racial difference in pain outcomes.

METHODS

Design, Setting, and Participants’ Eligibility Criteria

Patients undergoing tissue autograft after major thermal burn injury between February 2012 through June 2015 at one of three burn centers (University of North Carolina, Chapel Hill, NC; MedStar Washington Hospital Center, Washington, DC; and University of South Florida, Tampa, FL) were enrolled in this study as previously reported.^8,30,31 All patients who had plasma samples collected that were available for analysis (n = 77) were included (Figure 1). Exclusion criteria included age <18 or >65, admission >72 hours after major thermal burn injury, estimated TBSA burn >30%, intentional injury, electrical or a chemical mechanism, autograft performed >14 days after admission to burn center or autograft decision made >7 days after admission, Child-Pugh liver failure stage B or C, end-stage renal disease, chronic opioid use >20 morphine milligram equivalents per day prior to burn, pre-burn skin disorder causing pruritus, substantial co-morbid injury (e.g., blast injury resulting in major trauma in addition to burn), pregnancy or breastfeeding, residing greater than 100 miles from enrolling site, and burn that required escharotomy. In addition, individuals unwilling to provide a blood sample, prisoners, and suicidal, homicidal, and/or psychotic individuals were excluded. Further, individuals who did not read and speak English were excluded.

Figure 1.

STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) flow diagram. Participants included in analysis were those who had available peritraumatic plasma samples for Vitamin D analysis.

Study Procedures

Study procedures have been previously reported.⁸ In brief, the institutional review board at each burn center approved the study protocol and each participant provided written informed consent. Study flow chart is shown in Figure 1. Structured, in-person interviews were conducted by research assistants at the time of enrollment. Follow-up telephone interviews were conducted at subsequent timepoints. Data regarding burn injury characteristics, including estimated burn TBSA and mechanism, were extracted from the medical record.

Pain Assessments

Verbal, Numeric Rating Scales (NRS) are strongly correlated with visual analogue scales^32–34 and are ideal for burn survivors who may have injury-related impairment in dexterity. A 0 to 10 verbal NRS score was used to evaluate burn-site/graft-site pain severity at the time of enrollment and at each subsequent timepoint. NRS scores were assessed at major timepoints including enrollment, week 6, month 3, month 6, and 1 year following burn injury.

Assessment of Plasma 25-Hydroxyvitamin D in Serum of Burn Survivors in the Immediate Aftermath of Burn

Plasma was collected from study participants (n = 77) using a previously described, standardized protocol³⁵ at the time of study enrollment. Plasma samples were stored at −80°C and thawed immediately before analysis. Total 25-hydroxyvitamin D (25OHD2 and 25OHD3) plasma concentration was analyzed by liquid chromatography-tandem mass spectroscopy (LC/MS/MS) (OmegaQuant, Sioux Falls, SD). An aliquot of plasma was transferred to a screw-cap glass vial which contained internal standard, 25OHD2-d3 and 25OHD3-d3 (IsoSciences, Ambler, PA). Methanol and water were added to precipitate proteins prior to extraction with hexane (Sigma-Aldrich, St. Louis, MO). An aliquot of the hexane layer was dried down and reconstituted in 50:50 methanol:water before injection was carried out using a Nextera LC/MS 8050 (Shimadzu Corporation, Columbia, MD) equipped with a Poroshell 120 Pentafluorophenyl, 2.1 × 50 mm, 1.9 µM column (Agilent, Palo Alto, CA).

The injection volume was 20 µl and the oven temperature was held at 45°C. The mobile phase consisted of 0.1% aqueous formic acid and methanol containing 0.1% formic acid and ramped from 50% to 100% methanol over 5 minutes. The mass spectrometer conditions were optimized for maximum signal intensity of 25OHD2 and 25OHD3 using atmospheric pressure chemical ionization source operated in positive-ion mode. Two mass transitions were monitored for each analyte and corresponding internal standard. The primary mass transitions were a mass/charge ratio (m/z) of 413.3 to 395.3 for 25OHD2 (secondary transition of 413.3 to 355.3) and an m/z of 401.4 to 383.2 for 25OHD3 (secondary transition 401.4 to 365.2).

Definition of Vitamin D Deficiency

Vitamin D deficiency is defined as plasma 25-hydroxyvitamin D concentrations less than 20 ng/ml. This definition of Vitamin D deficiency is consistent with the Endocrine Society Clinical Practice Guideline³⁶ and the Institute of Medicine.³⁷

Statistical Analysis

Patient characteristics and outcomes were summarized using standard descriptive statistics (SPSS Statistics version 23, IBM Corporation, Armonk, NY). Vitamin D concentrations were divided into tertiles and the computed means from the highest and lowest tertiles were used to generate Figure 2 (GraphPad Software, San Diego, CA). Repeated-measures linear regression models were generated to evaluate the association between the peritraumatic plasma concentration Vitamin D and pain severity over the 1 year following burn injury. This repeated-measures model was adjusted for age, sex, income, education, and days since injury, and was clustered by participant (Stata 14, College Station, TX). A P value less than .05 was considered statistically significant. To evaluate whether there was a cutoff value above which further increases in peritraumatic Vitamin D does not associate with reduced pain severity, we examined association above the established clinical cutoff for Vitamin D deficiency (20 ng/ml). We also examined the area under the receiver operator characteristic curve (ROC) to evaluate whether our data support a cutoff value to predict chronic pain. Linear regression models were also used to evaluate the relationship between Vitamin D concentration and pain interference 6 months following burn injury (Stata 14). Linear regression models were fit for each time point adjusting for age, sex, total body surface area burned, education status, and income. Marginal means were then calculated for Black Americans holding Vitamin D concentration at 25 ng/ml (Stata 14).

Figure 2.

Influence of peritraumatic, plasma Vitamin D concentration following major thermal burn injury on pain severity over 1 y. Pain severity was measured with a 0–10 Numeric Rating Scale (NRS). Mean pain severity over time is plotted among individuals in the highest tertile (dotted line, closed circles) vs those in the lowest tertile (solid line, closed circles) of 25-hydroxyvitamin D concentration (ng/ml). Note that 76% (19/25) of the lowest tertile were Black American, while 24% (5/26) were in the highest tertile.

RESULTS

Demographic Characteristics of the Sample

Three quarters of burn survivors (n = 77) were male, 52% were White and 48% were Black (Table 1). Most had some education beyond high school and earned less than $60,000 a year. Average hospital length of stay was 10 ± 3 days.

Table 1.

Patient characteristics (n = 77)

Characteristic	n (%)
Age
18–32	33 (43)
33–49	26 (34)
50–65	18 (23)
Gender
Male	59 (77)
Female	18 (23)
Race
White	40 (52)
Black	37 (48)
Education*
8–11 y	8 (11)
High school	27 (36)
Post high school	41 (54)
Income*
$0–$19,999	11 (15)
$20,000–$39,999	20 (26)
$40,000–$59,999	15 (20)
$60,000–$79,999	7 (9)
$80,000+	12 (16)
Do not know	10 (13)
Refused	1 (1)
Percent TBSA
Mean (SD)*	5 (3)

*Seventy-six of 77 participants had TBSA estimated, and reported education and income attainment.

Sample Burn Injury Characteristics

The majority of burn survivors sustained burn injuries that were <10% of their TBSA, with an average TBSA of 5 ± 3%. The most common mechanisms of thermal burn injury were scald (34/77, 44%) and flame (34/77, 45%). The most common locations of burn injury were the upper extremity (75%, 58/77) and the lower extremity (35%, 27/77).

25-Hydroxyvitamin D Concentrations in the Immediate Aftermath of Burn Injury

Mean peritraumatic plasma Vitamin D concentration in the sample was 20 ±10 ng/ml. Black burn survivors had lower peritraumatic levels of plasma Vitamin D than White burn survivors (15 ± 8 ng/ml vs 25 ± 10 ng/ml, t = 4.591, P < .001). More than half (40/77 [51%]) of the sample was Vitamin D deficient. Black burn survivors were more likely to be Vitamin D deficient than White burn survivors (27/37 [73%] vs 14/40 [35%]; unadjusted odds ratio = 6.6, 95% confidence interval [CI]: 2.17–20.1, P < .001). These racial differences persisted after adjustment for age, sex, income, education, and TBSA burned (Table 2). Based on ROC analysis, there was not an optimal cutoff (area under the ROC > 0.8) for Vitamin D that predicted chronic pain following burn injury (Supplementary Tables 1 and 2).

Table 2.

Logistic regression analysis demonstrating that Black Americans have greater odds of vitamin D deficiency (<20 ng/ml) compared to White Americans

Vitamin D < 20 ng/ml	OR	95% CI	P
Race (ref: White)	6.96	2.19, 22.12	.001
Gender (ref: male)	2.37	0.599, 9.41	.219
Age	1.00	1, 1	.150
Education	1.25	0.826, 1.896	.291
Income	1.05	0.736, 1.135	.549
% TBSA	1.05	0.001, 0.953	.047

CI, confidence interval; OR, odds ratio; ref, reference category. This analysis included 75 patients, as 2 patients did not report covariates.

Peritraumatic 25-Hydroxyvitamin D Predicts Pain Outcome Following Burn Injury

In the year following burn injury, individuals in the lowest tertile of peritraumatic Vitamin D levels experienced greater pain severity at each timepoint than those in the highest tertile (Figure 2). In repeated-measures linear regression adjusting for time, race, sex, income, education, and days since injury, low peritraumatic Vitamin D levels were associated with worse pain outcome after burn injury (Table 3). No interaction was observed between race and the influence of peritraumatic Vitamin D level and pain outcome after burn injury (β = 0.04, 95% CI: −0.0338552, 0.1222566, P = .263); therefore, this interaction term was not included in the final model (Table 3). When repeated-measures analyses were restricted to burn survivors who were not Vitamin D deficient (>20 ng/ml, n = 35), greater peritraumatic Vitamin D concentrations continued to predict reduced pain severity (β = −0.094, 95% CI: −0.164, −0.024, P = .010).

Table 3.

Vitamin D predicts pain severity over the year following major thermal burn injury

Dependent: Pain	β	P	95% CI
Vitamin D (ng/ml)	−0.042	0.018*	−0.076, −0.007
Race (ref: White)	0.059	0.913	−1.008, 1.126
Gender (ref: male)	1.966	0.007*	0.561, 3.371
Age	0.007	0.666	−0.026, 0.399
Education	−0.273	0.207	−0.702, 0.155
Income	0.063	0.553	−0.147, 0.273
% TBSA	0.022	0.722	−0.102, 0.147
Day since injury	−0.011	<0.001	−0.013, −0.009

CI, confidence interval. Repeated-measures linear regression models Vitamin D adjusted for time, race, sex, income, education, and days since injury were performed. This analysis included 75 patients, as 2 patients did not report covariates.

*Significance (P < .05).

Racial Differences in Peritraumatic Vitamin D Levels Contribute to Racial Differences in Pain Outcome After Burn Injury

For every 10 ng/ml increase in Vitamin D, average post-burn pain severity across time was reduced by 0.4 (0–10 NRS). We have previously found that Black burn survivors experienced an average of 1.2 higher pain severity across time than White burn survivors.³⁰ If Black burn survivors had the same peritraumatic vitamin D levels as White burn survivors, this difference would be reduced by 0.4 (33%). Supplementary Figure 1 illustrates the raw means of Black vs White Americans, and predicted model-estimated means of Black Americans if Vitamin D levels equaled that of White Americans (25 ng/ml).

DISCUSSION

In this multicenter cohort study of burn survivors hospitalized for debridement and tissue autografting, Vitamin D deficiency was common in Whites and the norm in Blacks. More than one-third of White burn survivors and nearly three quarters of Black burn survivors were Vitamin D deficient. Individuals with low peritraumatic levels of Vitamin D had worse pain outcomes, including among burn survivors who were Vitamin D sufficient. Further, even after accounting for other sociodemographic factors, racial differences in peritraumatic Vitamin D levels accounted for approximately one-third of racial differences in pain outcomes after burn injury.

Our finding of Vitamin D deficiency in over half of burn survivors is consistent with other studies of trauma survivors.^38,39 Our data suggest that lower peritraumatic Vitamin D levels have the same association with worse pain outcomes after burn injury in both Black and White burn survivors. This association is consistent with findings in other settings,^13,15,17 and documented anti-inflammatory,⁹ antidepressive,^21,40 and neuroprotective¹⁰ effects.

In addition to the association with worse chronic pain outcomes that were observed in this study, Vitamin D deficiency has been associated with increased intensive care and hospital length of stay; however, there was no association with other complications such as sepsis or mortality.³⁹ Given the paucity of data on the impact of Vitamin D deficiency on clinical outcomes (eg, mortality, sepsis), further prospective studies are needed. Our data do not support a clinical cutoff of Vitamin D that predicts chronic pain given that the inverse linear association of peritraumatic Vitamin D concentration and pain severity is maintained above sufficient Vitamin D levels (>20 ng/ml) and the ROC analyses performed do not support an optimal cut-off value.

While the specific mechanisms by which Vitamin D may improve post-burn pain outcomes are not known, current evidence suggests that beneficial effects of Vitamin D may be due to its influence on immune function. Increasing Vitamin D levels results in reducing effector T-cell activation⁴¹, reducing T-cell differentiation⁴², inducing anti-inflammatory T-regulatory responses^41,43, and reducing inflammatory Toll-like receptor 4 signaling.^44–48 Inflammatory pain, including pain experienced following burn injury, has been associated with inflammatory cytokines including interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNFα).^49,50 Interestingly, Vitamin D has been shown in other clinical and preclinical studies to attenuate pro-inflammatory cytokines including IL-1β, IL-6, and TNFα.^51–53 Further mechanistic studies are needed to determine whether supplementation of Vitamin D reduces pro-inflammatory immune signaling in burn survivors and whether this drives the association between Vitamin D status and chronic pain.

Black burn survivors had lower levels of Vitamin D than White burn survivors and were much more likely to be Vitamin D deficient. Importantly, this is a modifiable risk factor. Indeed, if peritraumatic Vitamin D levels were as high in Blacks as in Whites in the present cohort, racial differences in chronic post-burn pain outcomes are estimated to be reduced by one third. If this could be achieved by Vitamin D supplementation, this would be a substantial improvement in outcome for an intervention that is simple, inexpensive, and widely available. In addition, improvements in pain outcome were seen even among individuals who were Vitamin D deficient, suggesting potential improvements in pain outcomes for both Black and White burn survivors with exogenous Vitamin D supplementation. Further study is needed via randomized controlled trials into whether supplementation with Vitamin D is beneficial to burn survivors and can reduce clinical pain outcome disparity among Black and White Americans.

Limitations

There are a number of limitations that should be considered when interpreting the results of this study. First, our sample size of 77 participants is small. However, despite the small sample size, the study results were significant (Table 2). Second, the association of Vitamin D to pain outcomes may be confounded by unmeasured variables including other factors related to dietary composition or outdoor activity/sun exposure. However, this likelihood is reduced by substantial previous literature suggesting an effect of Vitamin D on neuropsychiatric outcomes in other settings,^17,19–21 including pain. Finally, there was no interrogation of inflammatory markers or other biomarkers which might yield further insights into mechanisms by which Vitamin D influences pain development following burn injury. Future prospective observational and interventional studies are needed to confirm results of this study and should rigorously address these limitations.

CONCLUSION

In this multicenter cohort study of major thermal burn injury survivors hospitalized for debridement and tissue autografting, peritraumatic Vitamin D deficiency was common in Whites and the norm in Blacks, and among both races low Vitamin D deficiency predicted worse chronic post-burn pain outcomes. Racial differences in peritraumatic Vitamin D levels were estimated to account for one-third of racial differences in post-burn pain outcomes. Further studies are needed to verify this association, determine mechanisms by which Vitamin D may improve post-burn pain outcomes, and to determine whether Vitamin D supplementation may improve pain outcomes and reduce racial disparities after burn injury.

Funding

Research reported in this publication was supported by the University of North Carolina Department of Anesthesiology, the Jaycee Burn Center, and DC Firefighters Burn Foundation. Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute Of Child Health & Human Development of the National Institutes of Health under Award Number K12HD001441.