Significantly Elevated Vitamin B₁₂ Levels in Autoimmune Lymphoproliferative Syndrome (ALPS), a Rare Lymphoproliferative Disorder with Apoptosis Defect

Vitamin B₁₂ is an essential micronutrient that plays a fundamental role in cell division and one-carbon metabolism. Vitamin B₁₂ also plays an important role in hematopoiesis and maintaining the integrity of the nervous system. Vitamin B₁₂ in serum is bound to two proteins, haptocorrin (HC) and transcobalamin (TC). HC is a glycosylated serum protein of ~68kDa that is produced mainly by myeloid cells and binds the majority of circulating vitamin B₁₂ (70–90%). The exact function of this HC-bound vitamin B₁₂ is unknown (holo-HC), but it is believed to be biologically unavailable to most cells. The remainder of vitamin B¹² is bound to TC (holo-TC). TC is a 43 kDa non-glycosylated serum protein that is synthesized primarily in the enterocytes and carries vitamin B¹² to the tissues via a specific receptor with a high affinity for holo-TC. Only vitamin B¹² bound to TC is available for cellular uptake and use as coenzymes for L-methylmalonyl-CoA mutase and methionine synthase that synthesize succinyl-CoA and methionine, respectively.

ALPS is a rare autoimmune disease in children resulting from defective lymphocyte apoptosis and is characterized by lymphoproliferation, peripheral accumulation of DNT cells (double-negative T cells; TCR alpha/beta +, CD3+ CD4− CD8−), often leading to autoimmune multilineage cytopenias. Type Ia ALPS with Fas mutations accounting for ~75% of all reported cases is the most prevalent,

High serum vitamin B₁₂ concentrations have been observed in patients with ALPS studied at NIH Clinical Center. Levels of Vitamin B₁₂ detected in these patients range from 742 to 35365 pmol/L (median: 2489 pmol/L; mean: 4123 pmol/L; reference interval: 162–708 pmol/L) among 137 patients compiled for this study (Vitamin B₁₂: 1pmol/L=1.355 pg/mL). To date, the mechanism of the elevated vitamin B₁₂ concentrations in ALPS patients has not been determined and we sought to explore the source of elevated vitamin B₁₂.

Radio- and enzyme immunoassays, size-exclusion chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, and flow cytometry techniques were used to identify and quantitate the source of the elevated vitamin B₁₂ concentrations in peripheral blood specimens from ALPS patients and controls.

Total and holo-HC levels were 36- and 32-fold higher than the upper limit of the reference interval (Total HC: 250–840 pmol/L; Holo-HC: 240–680 pmol/L), respectively, in ALPS patients. However, total and holo-TC levels (1210 pmol/L; reference interval: 500–1500 pmol/L and 143 pmol/L; reference interval: >40 pmol/L, respectively) were not elevated in ALPS patients. The elution profile from size-exclusion chromatography of vitamin B₁₂-binding proteins did not reveal any abnormal vitamin B₁₂-binding proteins in ALPS. SDS-PAGE and western blot analysis revealed the presence of HC in lymphocyte lysates of ALPS but not apparently healthy controls. Flow cytometry studies also showed elevated levels of HC in all white blood cell types from ALPS patients compared to controls.

The results of this study show for the first time that elevated concentrations of vitamin B₁₂ found in ALPS patients were due to increased leukocyte (including lymphocytes) expression of HC. Further studies are needed to elucidate the mechanism for the increased synthesis and secretion of HC from lymphocytes of ALPS patients, but altered cytokine profiles, including increased interleukin-10 levels in ALPS may contribute.