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

Abstract

Objective

Identify the etiology of elevated B₁₂ in autoimmune lymphoproliferative syndrome (ALPS).

Design

Peripheral blood of ALPS patients with elevated B₁₂ and controls were evaluated.

Results

Total and holo-haptocorrin (HC) levels were 26- and 23-fold higher in ALPS patients, respectively. No abnormal B₁₂-binding proteins were found. Western blot revealed HC in lymphocyte lysates only from ALPS patients.

Conclusion

Elevated concentrations of B₁₂ found in ALPS patients were due to increased lymphocyte expression of HC.

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

Subjects

We studied 28 patients with ALPS-FAS germline mutation, 18 relatives and 8 healthy controls. Peripheral blood was obtained from subjects with signed informed consent under Institution Review Board approved protocols of the National Institute of Allergy and Infectious Diseases.

Vitamin B₁₂

Serum B₁₂ was measured on the IMMULITE 2000 analyzer (Diagnostic Products Corp., Los Angeles, CA). Total homocysteine and methymalonic acid (MMA) concentrations were measured by gas chromatography-mass spectrometry at Mayo Medical Laboratory, Rochester, MN. Holo-TC was evaluated using an RIA (Axis-Shield, Norton, MA). B₁₂-HC was calculated from the difference between total B₁₂ and holo-TC levels. Serum from ALPS and control subjects were incubated with excess ⁵⁷[Co]cyanocobalamin (10.5 Ci/mL; MP Biomedical, Solon, OH) and fractionated with a SMART system (GE Life Sciences, Pittsburgh, PA) with a Superdex 200 HR column (1.0 x 30cm) as described by Nexo et al [6]. Fractions (400μL) were collected and assayed for B₁₂ with a gamma counter and total TC and HC by ELISA.

Western Blot

Separation of granulocytes and lymphocytes was performed using dextran/ficoll separation. Protein was run on a 4%–12% gel (Expedeon, San Diego, CA) and then transferred to a nitrocellulose membrane. Haptocorrin (HC; Abcam, Cambridge, MA) in the lymphocyte lysates was detected with chicken anti-HC IgY antibodies (Abcam) and then probed for actin as a loading control.

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 homocysteine 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 homocysteine and/or MMA has been described [10], but we found normal levels of MMA and homocysteine 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

Figure 1.

Distribution of B₁₂ among the B₁₂ binding proteins. Superdex 200 HR gel filtration of serum from ALPS-FAS patient. The primary y-axis displays the ⁵⁷Co-cyanocobalamin activity in counts per minute (cpm; open circles, dashed line). The secondary y-axis displays the serum level of total TC (closed triangles, solid line). An arrow shows a major peak of unsaturated binding protein eluting similar to haptocorrin by gel filtration.

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.

Figure 2.

Western blot showing haptocorrin in ALPS-FAS lymphocytes but not in control lymphocytes. Lanes 1–3 represent three discrete ALPS-FAS patients and lanes 4–6 represent three discrete healthy controls.

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.

Highlights.

• Vitamin B12 levels were significantly increased in ALPS-FAS patients.

• Serum total and holo-haptocorrin levels were elevated in ALPS-FAS patients.

• No abnormal serum vitamin B12-binding proteins were present in ALPS-FAS patients.

• Serum transcobalamin levels were not elevated in ALPS-FAS and control subjects.

• Haptocorrin were found in lymphocyte lysates of ALPS but not control subjects.

Abbreviations

ALPS

autoimmune lymphoproliferative syndrome

DNT

double negative T cells

HC

haptocorrin

Hcy

homocysteine

TC

transcobalamin

B₁₂

vitamin B₁₂ or cobalamin

MMA

methylmalonic acid