Significantly Elevated Vitamin B12 Levels in Autoimmune Lymphoproliferative Syndrome (ALPS), a Rare Lymphoproliferative Disorder with Apoptosis Defect
Authors: Bowen Raffick AR , Dale Janet K , Brown Margaret , Drake Steven K , Moura Tarsilla , Dowdell Kennichi , Remaley Alan T , Fleisher Thomas A , Hortin Glen L , Nexo Ebba , Straus Stephen E , Rao V. Koneti 
Published: April 2020

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: &gt;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.