Dose-Dependent Influence of Vitamin D₃ on Blood Cell Indices and Clotting Function in Rats with Phenylhydrazine-Induced Haemolytic Anaemia
Main Article Content
Abstract
Background. Coagulation disturbances often coexist with anaemia but are rarely studied together. Vitamin D receptors on megakaryocytes and erythroid precursors suggest this hormone may influence both erythropoiesis and haemostasis simultaneously. Aim. To determine whether oral vitamin D₃ restores blood counts, iron status and clotting times in a rat model of phenylhydrazine-induced haemolytic anaemia, and how these outcomes relate to circulating 25-hydroxyvitamin D. Methods. Forty male Wistar rats were randomised into five groups (n = 8): untreated control; anaemia-only (phenylhydrazine 40 mg/kg i.p. on two days); two PHZ groups given oral vitamin D₃ (500 or 1000 IU/kg/day) for four weeks; and a PHZ group given weekly intramuscular iron dextran (10 mg/kg). Groups were compared by one-way ANOVA with Tukey's HSD; associations with 25(OH)D₃ were assessed by Pearson's correlation. Results. Phenylhydrazine reduced haemoglobin, RBC count, haematocrit, serum iron and ferritin, and prolonged PT and aPTT (all p < 0.01). Both vitamin D₃ doses improved Hb, RBC, MCV and platelets dose-dependently; the higher dose shortened PT and aPTT and raised fibrinogen. 25(OH)D₃ correlated positively with haemoglobin (r = +0.71, p < 0.001) and negatively with PT (r = −0.65, p < 0.001).
Conclusion. Oral vitamin D₃ produced dose-dependent improvement in both the blood picture and clotting profile of anaemic rats, consistent with VDR-mediated effects on erythroid maturation and vitamin K-dependent clotting factors, and supports vitamin D₃ as a plausible adjunct in anaemia management.
Article Details
Section
How to Cite
References
Aihara, K., Azuma, H., Akaike, M., Ikeda, Y., Yamashita, M., Sudo, T., Hayashi, H., Yamada, Y., Endoh, F., Fujimura, M., Yoshida, T., Yamaguchi, H., Hashizume, S., Kato, M., Yoshimura, K., Yamamoto, Y., Kato, S., & Matsumoto, T. (2004). Disruption of nuclear vitamin D receptor gene causes enhanced thrombogenicity in mice. Journal of Biological Chemistry, 279(34), 35798–35802. https://doi.org/10.1074/jbc.M404865200
Alon, D. B., Chaimovitz, C., Dvilansky, A., Lugassy, G., Douvdevani, A., Shany, S., & Nathan, I. (2002). Novel role of 1,25(OH)₂D₃ in induction of erythroid progenitor cell proliferation. Experimental Hematology, 30(5), 403–409. https://doi.org/10.1016/S0301-472X(02)00789-0
Alpakra, M., Hamed, N. F., Alfaki, A., & AlKabbani, D. M. Z. (2025). The correlation between vitamin D deficiency and anaemia: a systematic review. Cureus, 17(8), e89428. https://doi.org/10.7759/cureus.89428
Amin, F., Parvaz, N., Askari, N., Hosseini, S. M., Shariati, M., Mehrabani, M., Mahmoodi, M., & Sharififar, F. (2024). Hydrophilic extract of Pistacia vera pericarp protects against phenylhydrazine-induced hepatotoxicity and haemolytic anaemia. Avicenna Journal of Phytomedicine, 14(3), 287–299. https://doi.org/10.22038/AJP.2024.23925
Arabi, S. M., Ranjbar, G., Bahrami, L. S., Vafa, M., & Norouzy, A. (2020). The effect of vitamin D supplementation on hemoglobin concentration: a systematic review and meta-analysis. Nutrition Journal, 19(1), 11. https://doi.org/10.1186/s12937-020-0526-3
Aucella, F., Scalzulli, R. P., Gatta, G., Vigilante, M., Carella, A. M., & Stallone, C. (2003). Calcitriol increases burst-forming unit-erythroid proliferation in chronic renal failure: a synergistic effect with r-HuEpo. Nephron Clinical Practice, 95(4), c121–c127. https://doi.org/10.1159/000074837
Bacchetta, J., Zaritsky, J. J., Sea, J. L., Chun, R. F., Lisse, T. S., Zavala, K., Nayak, A., Wesseling-Perry, K., Westerman, M., Hollis, B. W., Salusky, I. B., & Hewison, M. (2014). Suppression of iron-regulatory hepcidin by vitamin D. Journal of the American Society of Nephrology, 25(3), 564–572. https://doi.org/10.1681/ASN.2013040355
Barcellini, W., & Fattizzo, B. (2015). Clinical applications of hemolytic markers in the differential diagnosis and management of hemolytic anemia. Disease Markers, 2015, Article ID 635670. https://doi.org/10.1155/2015/635670
Barminko, J., Reinholt, B. M., Emmanuelli, A., Lejeune, A. N., & Baron, M. H. (2018). Activation of the vitamin D receptor transcription factor stimulates the growth of definitive erythroid progenitors. Blood Advances, 2(11), 1207–1219. https://doi.org/10.1182/bloodadvances.2018017533
Berger, J. (2007). Phenylhydrazine haematotoxicity. Journal of Applied Biomedicine, 5(3), 125–130. https://doi.org/10.32725/jab.2007.017
Bikle, D. D. (2021). Vitamin D: production, metabolism and mechanisms of action. In K. R. Feingold, B. Anawalt, M. R. Blackman et al. (Eds.), Endotext. South Dartmouth, MA: MDText.com. Retrieved from https://www.ncbi.nlm.nih.gov/books/NBK278935/
Charoenngam, N., & Holick, M. F. (2020). Immunologic effects of vitamin D on human health and disease. Nutrients, 12(7), 2097. https://doi.org/10.3390/nu12072097
El-Shehry, M. S. E. F., Amrymi, R. A., Atia, T., Lotfy, B. M. M., Ahmed, S. H. A., Qutb, S. A., Ali, S. B., Mohamed, A. S., Mousa, M. R., Damanhory, A. A., Metawee, M. E., & Sakr, H. I. (2023). Hematopoietic effect of echinochrome on phenylhydrazine-induced hemolytic anemia in rats. PeerJ, 11, e16576. https://doi.org/10.7717/peerj.16576
Fibach, E., & Rachmilewitz, E. (2008). The role of oxidative stress in hemolytic anemia. Current Molecular Medicine, 8(7), 609–619. https://doi.org/10.2174/156652408786241384
Kautz, L., Jung, G., Valore, E. V., Rivella, S., Nemeth, E., & Ganz, T. (2014). Identification of erythroferrone as an erythroid regulator of iron metabolism. Nature Genetics, 46(7), 678–684. https://doi.org/10.1038/ng.2996
Levi, M., & Scully, M. (2018). How I treat disseminated intravascular coagulation. Blood, 131(8), 845–854. https://doi.org/10.1182/blood-2017-10-804096
Moslhy, E. A. M., Tadros, M. M. M., Thabet, R. A., Hemida, E. H. A., & Noureldeen, A. F. H. (2024). Impact of vitamin D deficiency on iron status in children with type I diabetes. Scientific Reports, 14, 12989. https://doi.org/10.1038/s41598-024-61559-5
Nie, S., Huang, P., Niu, H., Ding, F., Gong, L., Zou, C., Xiang, H., Guo, C., Xiang, Y., Cao, Y., Lu, H., & Yang, G. (2025). Vitamin D deficiency enhances platelet activation and thrombosis by regulating VDR/Akt pathway based on platelet proteomics. European Journal of Pharmacology, 985, 177112. https://doi.org/10.1016/j.ejphar.2025.177112
Patel, N. M., Gutiérrez, O. M., Andress, D. L., Coyne, D. W., Levin, A., & Wolf, M. (2010). Vitamin D deficiency and anemia in early chronic kidney disease. Kidney International, 77(8), 715–720. https://doi.org/10.1038/ki.2009.551
Pike, J. W., & Meyer, M. B. (2012). Regulation of mouse Cyp24a1 expression via promoter-proximal and downstream-distal enhancers highlights new concepts of 1,25-dihydroxyvitamin D₃ action. Archives of Biochemistry and Biophysics, 523(1), 2–8. https://doi.org/10.1016/j.abb.2011.12.003
Sikole, A. (2000). Pathogenesis of anaemia in hyperparathyroidism. Medical Hypotheses, 54(2), 236–238. https://doi.org/10.1054/mehy.1999.0027
Sim, J. J., Lac, P. T., Liu, I. L. A., Meguerditchian, S. O., Kumar, V. A., Kujubu, D. A., & Rasgon, S. A. (2010). Vitamin D deficiency and anemia: a cross-sectional study. Annals of Hematology, 89(5), 447–452. https://doi.org/10.1007/s00277-009-0850-3
Smith, E. M., & Tangpricha, V. (2015). Vitamin D and anemia: insights into an emerging association. Current Opinion in Endocrinology, Diabetes and Obesity, 22(6), 432–438. https://doi.org/10.1097/MED.0000000000000199
Targher, G., Pichiri, I., & Lippi, G. (2012). Vitamin D, thrombosis, and hemostasis: more than skin deep. Seminars in Thrombosis and Hemostasis, 38(1), 114–124. https://doi.org/10.1055/s-0031-1300957
Ullah, H., Huma, S., Ashraf, M., Tahir, N., Yasin, G., Yunus, M., Shabana, H., Muharram, M. A., Shalaby, A. H., Ali, A. A. H., Mahmoud, M. E. M., Ibrahim, A. M. A., Elbwab, A. F. A. E., Alhawy, A. M. E., Abotaha, A. A. M., Abdelraouf, M. E., Imam, M. S., Aladl, H. A. A., Shawky, T. A., et al. (2025). Relationship of vitamin D, fibrinogen and their ratio with acute coronary syndrome: a comparative analysis of unstable angina, NSTEMI, and STEMI. PLOS ONE, 20(9), e0331853. https://doi.org/10.1371/journal.pone.0331853
World Health Organization. (2015). The global prevalence of anaemia in 2011. Geneva: World Health Organization. Retrieved from https://www.who.int/publications/i/item/9789241564960
Wu-Wong, J. R., Nakane, M., & Ma, J. (2007). Vitamin D analogs modulate the expression of plasminogen activator inhibitor-1, thrombospondin-1 and thrombomodulin in human aortic smooth muscle cells. Journal of Vascular Research, 44(1), 11–18. https://doi.org/10.1159/000097812
Zughaier, S. M., Alvarez, J. A., Sloan, J. H., Konrad, R. J., & Tangpricha, V. (2014). The role of vitamin D in regulating the iron–hepcidin–ferroportin axis in monocytes. Journal of Clinical & Translational Endocrinology, 1(1), 19–25. https://doi.org/10.1016/j.jcte.2014.01.003