Elevated Vitamin B12 Levels in Autoimmune Lymphoproliferative Syndrome Attributable to Elevated Haptocorrin in Lymphocytes (2024)

Abstract

Keywords: ALPS, vitamin B12, cobalamin, germline, haptocorrin, transcobolamin

Introduction

Autoimmune lymphoproliferative syndrome (ALPS) is an immune disease characterized by lymphoproliferation, accumulation of double-negative T cells (DNT, TCRalpha/beta⁺ CD3⁺CD4⁻CD8⁻), abnormal apoptosis, and increased autoimmunity and lymphoma [1, 2]. Most patients have been genetically classified as having germline (ALPS-FAS) or somatic (ALPS-sFAS) FAS/APO-1 mutations [3]. Patients with ALPS have a number of laboratory abnormalities, including elevated vitamin B₁₂ levels most prevalent in ALPS-FAS, ALPS-sFAS and ALPS-U (genetic defect unknown) [3].

B₁₂ (Cobalamin) is an essential micronutrient that plays a fundamental role in cell division and one-carbon metabolism. Serum B₁₂ is bound to both transcobalamin (TC) and haptocorrin (HC). These proteins circulate in blood partly saturated (holo) and partly unsaturated (apo) with B₁₂ [4]. TC saturated with B₁₂ (holo-TC) constitutes 6 to 20% of endogenous circulating B₁₂. In contrast, HC binds ~80–94% of the endogenous plasma B₁₂ (holo-HC), and is largely saturated with B₁₂ or analogues [5]. We sought to determine the source of elevated B₁₂ in patients with ALPS-FAS germline mutation.

Methods

Results and Discussion

In this study, we examined B₁₂, haptocorrin and transcobalamin in patients with ALPS, first-degree relatives and healthy controls. The mean B₁₂ concentrations were significantly different among controls (274 pmol/L; N=8), relatives (500 pmol/L; N=18), and patients (3900 pmol/L; N=28) (P<0.0001). Serum B₁₂ concentrations were significantly higher in ALPS patients compared to controls and healthy relative groups (P<0.0001). Although B₁₂ levels were higher in ALPS relatives compared to controls, these differences were not statistically significant. Whereas elevated B₁₂ levels are not typically observed in lymphoproliferative disorders, there have been reports in patients with disseminated neoplasia, myeloproliferative disorders, and hypereosinophilic syndrome [7]: none of these co-morbid conditions applied to our subjects. Also, high B₁₂ concentrations are associated with renal failure [8] and hepatic disease [7]: the ALPS patients in this study showed normal renal and hepatic function. Previous studies have shown that some patients given cyano- or hydroxycobalamin have developed antibodies to TC, leading to markedly increased serum TC and B₁₂ [9]. In the ALPS patients, however, serum holo-TC concentrations were within reference intervals, with only slight increases in total TC in the presence of high B₁₂ concentrations.

We observed no significant difference in mean serum holo-TC concentrations among the three groups (P=0.93), with concentrations in controls, ALPS relatives, and ALPS patients of 81, 86, and 85 pmol/L, respectively. In contrast, we found significant differences in mean holo-HC concentrations among the three groups (P<0.0001). The mean holo-HC concentration in this part of serum B₁₂ in ALPS patients (3810 pmol/L) was significantly higher than controls (194 pmol/L) and ALPS relatives (414 pmol/L), whereas there was no significant difference in holo-HC concentrations between controls and ALPS relatives.

We evaluated whether there was an association between total B₁₂ concentrations and concentrations of B₁₂ bound to TC or HC in serum. We found no correlation between the total serum B₁₂ and holo-TC concentrations for controls (r=0.18), ALPS relatives (r=0.29), and ALPS patients (r=0.51). However, there were strong positive correlations between holo-HC concentrations and total B₁₂ concentrations in all three groups of individuals studied (r=0.93, 0.99 and 1.0 for the control, ALPS relatives and ALPS patients, respectively).

The functional B₁₂ status of a patient can be determined by the activities of the B₁₂ dependent enzymes. Therefore, the plasma concentrations of hom*ocysteine and methylmalonic acid, which are the substrates of methionine synthase and methylmalonyl CoA mutase, respectively, have a diagnostic value in tracing a functional deficiency of B₁₂ [10]. Cell damage can directly lead to reduced concentrations of intracellular B₁₂, thus significantly elevated plasma B₁₂ can be associated with a functional B₁₂ deficiency. This condition can also result from the increased binding of B₁₂ to HC, which has an inhibitory effect on B₁₂ binding to TC, the physiologic transport protein required for intracellular uptake. The paradox of high plasma B₁₂ and elevated hom*ocysteine and/or MMA has been described [10], but we found normal levels of MMA and hom*ocysteine in ALPS patients and control subjects, respectively; confirming that these individuals are not functionally deficient in B₁₂, a finding consistent with the normal transcobalamin levels in both these groups. Furthermore, the Superdex column elution profile from patients and controls did not show any unusual B₁₂ binding protein.

Two sets of experiments were performed to clarify whether the part of serum B₁₂ not accounted for by holo-TC represented B₁₂ bound to haptocorrin. First, we examined by gel filtration the occurrence of unsaturated haptocorrin, and employing labeled cobalamin we observed a major peak of unsaturated binding protein eluting similar to haptocorrin (Fig. 1). Second, we measured the amount of holo and total HC in six patients to find mean and [range] values of 15600 [854–30400] and 21700 [855–42900] pmol/L, respectively. The values are considerably higher than those observed for a reference population of 240–680 for holo-HC and 250–840 for total HC

Finally we explored the origin of the increased level of haptocorrin. Here we show the novel finding of haptocorrin in lymphocytes from patients with ALPS and high serum B₁₂ levels. Western blot analysis revealed the expected presence of haptocorrin in granulocytes from both control and ALPS-FAS subjects (data not shown); however, lymphocytes showed the presence of haptocorrin only from ALPS patients (Fig. 2). No difference was observed when lymphocyte lysates were probed for transcobalamin. Hence, lymphocytes may be synthesizing and releasing haptocorrin but not transcobalmin into serum, which may partly account for the high B₁₂ in affected ALPS subjects.

A substantial amount of knowledge with regard to the occurrence and production of HC has accumulated during the past three decades, but there are still major questions that remain. Most importantly, HC is still a protein with an unexplained function characterized by its ability to bind B₁₂ analogues, but the significance of this in relation to human pathophysiology is unclear. In patients with ALPS-FAS germline mutation, plasma levels of B₁₂ can be elevated, up to 15 times the upper limit of the reference interval, and with an even larger increase in the level of HC. The increase in HC appears to be related to an elevated production of HC by lymphocytes. When HC is released from these cells it binds B₁₂ liberated from various tissues and the expanded granulocyte pool. The possibility that the concentration of HC has prognostic value in affected ALPS subgroups warrants further investigation. Additional studies are also required to elucidate the mechanism for the increased synthesis and secretion of haptocorrin by circulating leukocytes in ALPS, particularly, double-negative T cells.

Acknowledgments

This research was supported by the intramural programs of the National Institute of Allergy and Infectious Diseases and the Warren Grant Magnuson Clinical Center.

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