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
]. 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
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
]. 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
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 (). 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 (more ...)
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 (). 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.
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.