Reference
Johny E, Jala A, Nath B, et al. Vitamin D supplementation modulates platelet-mediated inflammation in subjects with type 2 diabetes: a randomized, double-blind, placebo-controlled trial. Front Immunol. 2022;13:869591.
Study Objective
To determine whether vitamin D3 supplementation affects platelet activation and platelet-mediated systemic inflammation in vitamin D–deficient patients with uncontrolled (HbA1c ≥7%) noninsulin-dependent diabetes mellitus (type 2 diabetes)
Key Takeaway
Supplementing with vitamin D3 significantly decreased platelet aggregation, platelet activation, and platelet-mediated inflammatory cytokines/chemokines compared to placebo.
Design
Single-center, randomized, double-blind, placebo-controlled study
Participants
Participants were 59 patients with uncontrolled type 2 diabetes (HbA1c ≥7%) and serum 25-hydroxy (OH) vitamin D ≤20 ng/mL. Of those included in the study, 42 were male and 17 were female. The median age of the placebo group (n=29) was 55.06 ± 9.57 years, and it was 53.6 ± 9.6 years in the vitamin D3 group (n=30).
Excluded from the study were patients who had previously taken vitamin D and those who had evidence of liver or kidney failure, type 1 diabetes, cancer, or thyroid disease, and those who were pregnant.
Intervention
Vitamin D3 60,000 IU per week for 3 months followed by vitamin D3 60,000 IU per month for another 3 months; or placebo.
Study Parameters Assessed
-Serum total 25-OH vitamin D
-Serum vitamin D binding protein (VDBP)
-Fasting blood sugar (FBS)
-Hemoglobin A1c (HbA1c)
-Platelet activation markers:
- PAC-1 expression
- P-selectin expression
- Serum platelet factor 4 (PF-4)
- Urinary 11-dehydrothromboxane B2
-Serum inflammatory markers:
- Interleukin (IL) 1b, IL-2, IL- 4, IL-5, IL-6, IL-8, IL-12p70, IL-13, and IL-18
- Tumor necrosis factor alpha (TNFα)
- Interferon gamma (IFN-γ)
- Granulocyte-macrophage colony-stimulating factor (GM-CSF)
- Chemokines CXCL-1, CXCL-10, CXCL-12, CCL-2, CCL-3, CCL-4, CCL-5, and CCL-11
-Platelet–immune cell aggregation (%):
- Platelet–monocyte aggregates,
- Platelet–classical (CD14++ CD16-) monocyte aggregates
- Platelet–intermediate (CD14++ CD16+) monocyte aggregates
- Platelet-non-classical monocyte (CD14+ CD16++) aggregates
- Platelet–neutrophil aggregates
- Platelet–T cells CD4 (CD3+ CD4+) and CD8 (CD3+ CD8+) aggregates
- Platelet–NK cell (CD3- CD56+) aggregates
- Platelet-NKT cell (CD3+ CD56+) aggregates
- Platelet–plasmacytoid dendritic (HLA DR+ CD123+) cell aggregates
- Platelet-myeloid dendritic (HLA DR+ CD11C+) aggregates
-Oxidative stress markers:
- Serum superoxide dismutase (SOD)
- Glutathione (GSH)
- Total nitric oxide (TNO)
Primary Outcome
The effects of vitamin D3 supplementation on platelet activation and platelet-mediated systemic inflammatory markers in patients with type 2 diabetes and vitamin D deficiency.
Key Findings
Total 25-OH vitamin D significantly increased after 6 months of treatment (14.15 ± 5.8 ng/mL to 51.99 ± 16.56 ng/mL, P<0.0001).
25-OH vitamin D significantly increased after 6 months of treatment (14.02 ± 5.76 ng/mL to 53.12 ± 16.44 ng/mL, P<0.0001).
VDBP significantly increased after 6 months of treatment from 170.6 ± 60.24 µg/mL to 205.4 ± 88.30 µg/mL (P=0.0425).
No significant changes in FBS or HbA1c were observed after 6 months of treatment with vitamin D3.
Supplementation with vitamin D3 for 6 months significantly decreased percent PAC-1 and percent P-selectin expression compared to baseline. Results are expressed as the median value (25th–75th percentile).
PAC-1 expression (%) was 0.20 (0.07–0.57) at baseline and 0.10 (0.09–0.18) after 6 months (P=0.03), while percent P-selectin expression (%) decreased from 53.83 (42.51–59.76) to 34.10 (25.76–47.96) over the same period (P<0.001).
Serum PF-4 levels significantly decreased from baseline to 6 months with vitamin D3. Values not reported (P=0.0049).
Urinary 11-dehydrothromboxane B2 (ng/mL creatinine) also significantly decreased in volunteers taking vitamin D3 after 6 months compared to baseline. Values not reported (P=0.0390).
Six months of vitamin D3 supplementation significantly decreased platelet aggregation with the following innate immune cells:
- Platelet–monocyte aggregates (%): 80.0 (95% CI, 71.75–87.337) to 49.80 (95% CI, 36.80–67.88), P<0.001
- Platelet–classical monocyte aggregates (%): 84.33 (95% CI, 74.67–89.75) to 45.14 (95% CI, 33.11–65.03), P<0.001
- Platelet–intermediate monocyte aggregates (%): 94.80 (88.09–98.12) to 72.41 (42.69–87.38), P<0.001
- Platelet–non-classical monocyte aggregates (%): 91.39 (85.44–100) to 64.40 (52.73–81.04), P<0.001
- Platelet–neutrophil aggregates (%): 62.96 (54.53–69.92) to 54.10 (39.78–65.40), P=0.004
- Platelet–T cell aggregates (%): 28.55 (20.91–34.83) to 23.25 (19.96–28.66), P=0.001
- Platelet–NK cell aggregates (%): 26.80 (13.54–33.21) to 17.58 (13.24–25.56), P=0.03.
- Platelet–dendritic cell aggregates (%): 44.11 (34.0–55.89) to 31.49 (17.33–48.69), P=0.04
The following cytokines/chemokines significantly (P<0.05) decreased after 6 months of vitamin D3 supplementation compared to baseline: IL-18, TNF-α, IFN-γ, CXCL-10, CXCL-12, CCL-2, CCL-5, and CCL-11. The researchers did not report the baseline and follow-up values for the individual analytes.
SOD activity expressed as percent inhibition rate, serum GSH, and TNO (an inhibitor of platelet aggregation) all significantly increased in the treatment group after 6 months of supplementing with vitamin D3 compared to baseline (P<0.05).
The placebo group demonstrated no significant changes in any variable measured during the study.
Transparency
The authors declared that they had no commercial or financial relationships that would create a conflict of interest.
Practice Implications & Limitations
Chronic inflammation is a central mechanism in endothelial damage and end-organ failure in patients with diabetes, including the progression to diabetic retinopathy, diabetic kidney disease (DKD), and diabetic peripheral neuropathy.1-3 Patients with type 2 diabetes also have a higher incidence of coagulopathies than people without diabetes, mediated by increased inflammatory cytokines and platelet activation.4 Therefore, understanding ways to decrease inflammatory cytokines and platelet activation may benefit these patients.
In the current study, supplementing with vitamin D3 significantly attenuated platelet activation, reduced inflammatory cytokines/chemokines, and improved oxidative stress markers in patients with uncontrolled type 2 diabetes (HbA1c >7%) and vitamin D deficiency (25-OH vitamin D <20 ng/mL).
Prior studies have also demonstrated benefits, although they measured different—and fewer—markers of oxidative stress. A 2019 randomized clinical trial evaluated the ability of vitamin D to reduce oxidative DNA damage in vitamin D–deficient (<20 ng/mL) patients with type 2 diabetes. Baseline HbA1c was not reported. Supplementing with 2,000 IU vitamin D3 daily for 3 months raised serum 25-OH vitamin D levels to a mean of 31.8 ng/mL and significantly reduced oxidative DNA damage (P<0.05).5
Chronic inflammation is a central mechanism in endothelial damage and end-organ failure in patients with diabetes, including the progression to diabetic retinopathy, diabetic kidney disease (DKD), and diabetic peripheral neuropathy."
Similarly, a 2019 study evaluated the effects of 4,000 IU/day vitamin D3 for 8 weeks on oxidative stress and DNA damage in 75 patients (47 women, mean age 60.71 years; 28 men, mean age 65.24 years) with insulin-dependent type 2 diabetes and an HbA1c <9.5%. Eight weeks of treatment significantly increased serum 25-OH vitamin D and significantly decreased oxidative stress DNA damage (P<0.05 for both). Additionally, ALT, AST, and GGT all decreased as well (P<0.05 for all 3).6
In the current study, the vitamin D3 dose they used—60,000 IU per week for 3 months, followed by 60,000 IU per month for another 3 months—raised total serum 25-OH vitamin D to a median of 51.99 ng/mL. Clinically, it would be helpful to know if using a lower dose of vitamin D3 and obtaining a lower serum 25-OH vitamin D level also improves the same inflammatory markers. A future dose-response study could answer this question.
Multiple studies concluded that serum vitamin D increases by about 1 ng/mL for every 2.5 mcg (100 IU) of vitamin D3.7,8 Therefore, taking 125 mcg (5,000 IU) vitamin D per day may raise serum vitamin D levels above 50 ng/mL just as effectively as the higher, less-frequent dose used in the current study.
Finally, since the researchers prescribed only 1 dose of vitamin D and tested patients only at baseline and 6 months, we don’t know if lower serum vitamin D levels would produce similar results and if the results are evident earlier than 6 months.
