Vitamin B6 challenge as a tool for detecting ALPL mutations and diagnosing hypophosphatasia

Abstract

Summary

Low serum alkaline phosphatase is the biochemical hallmark of hypophosphatasia. However, it is a non-specific finding. Here we show that a 2-day vitamin B6 challenge is useful to identify carriers of ALPL gene mutations among patients with low serum alkaline phosphatase, with specificity and sensitivity over 90%.

Purpose

Hypophosphatasia (HPP) is a disorder characterized by deficient activity of the tissue non-specific alkaline phosphatase (ALP) isoenzyme, due to pathogenic variants of the ALPL gene. The biochemical hallmark of HPP is the reduced ALP activity in serum. Pyridoxal 5'-phosphate (PLP), the major circulating form of vitamin B6, is a substrate of ALP. Thus, high PLP levels are commonly used as a diagnostic marker of HPP. This study aimed to assess the diagnostic utility of vitamin B6 supplementation for identifying patients with ALPL variants.

Patients and methods

We measured PLP in control subjects and patients with low serum ALP, with or without ALPL mutations, at baseline and after a 2-day or 6-day vitamin B6 supplementation (20 mg per day of pyridoxine hydrochloride).

Results

Although mutation carriers tended to have higher PLP values, up to 33% had baseline levels within the normal range. The vitamin B6 challenge, particularly with the 2-day protocol, improved the diagnostic performance. After 2-day supplementation, all carriers had levels above 500 nmol/l (sensitivity 100%; CI 95–100), whereas only 1 non-carrier surpassed that threshold (specificity 96%; CI 85–100).

Conclusion

A 2-day vitamin B6 supplementation test may be useful for identifying carriers of ALPL mutations among individuals with unexplained low serum ALP.

Introduction

Hypophosphatasia (HPP) is an inherited metabolic disorder characterized by deficient activity of the tissue non-specific alkaline phosphatase (ALP) isoenzyme [1]. ALP plays a critical role in the mineralization of bone and teeth, and its deficiency can result in skeletal abnormalities (e.g., pain, fractures, pseudofractures, chondrocalcinosis, calcific periarthritis), early tooth loss, and in severe cases, myopathy and life-threatening complications [2, 3]. The biochemical hallmark of HPP is the reduced ALP activity in serum. Diagnosis is typically supported by identifying a pathogenic variant in ALPL, the gene encoding tissue non-specific ALP [4, 5]. The inheritance pattern can be either recessive or dominant, with significant inter- and intrafamilial phenotypic variability [6].

Pyridoxal 5'-phosphate (PLP), a form of vitamin B6 abundant in the circulation, is a substrate of ALP, which converts PLP into pyridoxal. Thus, the impaired enzyme function in HPP elevates extracellular PLP levels, making them a useful diagnostic marker [7]. However, PLP levels are also influenced by dietary intake and supplementation of vitamin B6 [8].

Consequently, the diagnosis of HPP can be difficult, particularly when clinical manifestations are mild and genetic data are unavailable. Although serum PLP levels are frequently increased in HPP, some patients with ALPL mutations have normal PLP levels. Furthermore, approximately half of individuals with unexplained low serum ALP lack relevant genetic variants [9, 10].

Chodirker et al. studied a Mennonite community of Manitoba with a high frequency of HPP. They showed that a vitamin B6 challenge test could differentiate presumed carriers of severe infantile HPP-associated ALPL mutations from non-carriers [11]. Building on these findings, we investigated whether vitamin B6 supplementation could identify ALPL mutation carriers among individuals with unexplained low ALP levels.

Subjects and methods

We studied a convenience sample of 66 subjects, including 19 healthy volunteers (5 men and 14 women, mean age 35 ± 14 yr, range 23–62) and 47 patients (11 men and 36 women, mean age 42 ± 13 yr, range 18–78) presenting with persistently low serum ALP at our outpatient clinic. We excluded secondary causes of low ALP (e.g., drug therapy, hypothyroidism, hypoparathyroidism, celiac disease, vitamin B12 deficiency, anemia, renal failure) by extensive clinical and biochemical evaluation [10]. We also excluded individuals taking any form of vitamin B6 supplementation. ALPL gene sequencing revealed a pathogenic/likely pathogenic variant in 21 of the 47 patients. In contrast, no pathogenic/likely pathogenic or variant of unknown significance were found in 26 patients (according to ACMG criteria [12] or presence in the ALPL gene variant database[13]).

The study was approved by the Institutional Review Board (Comité de Etica de Investigación con Medicamentos de Cantabria, ref 2022.274). All participants provided written informed consent.

PLP was measured blindly in plasma samples by high-performance liquid chromatography with fluorescence detection (analysed at Reference Laboratory, Barcelona, Spain, using the kit by Immuchrom, Heppenheim, Germany). The assay's reported intra-assay CV is 2.5% and inter-assay CV is 2.9%. PLP was measured after an overnight fast, at baseline, and after 2 or 6 days of daily supplementation with 20 mg of pyridoxine hydrochloride (Becozyme C Forte, Bayer Hispania, Barcelona, Spain). The supplements were taken with breakfast. Post-supplementation blood samples were obtained 24 h (in case of 2-day supplementation) or 48 h (6-day supplementation) after the last dose. The PLP reference range is 17–177 nmol/L. Pre- and post-supplementation samples of each subject were run within the same assay. ALP was measured by an automatic colorimetric method (Atellica CH, Siemens Healthineers, USA). The assay's reported CV is < 1,5%.

Between-group differences were tested by ANOVA, using Prism software (Graphpad, Boston MA, USA). The diagnostic performance values were computed with Epidat software (www.sergas.es).

Results

As expected, all patients exhibited serum ALP levels below the reference range. No individual reported adverse effects.

Baseline serum PLP levels were 62 ± 26 nmol/l in controls (range 33–124); 75 ± 36 nmol/l (range 34–55) in the group of patients with low serum ALP not carrying an ALPL mutation; and 241 ± 119 nmol/l (range 63–506) in carriers of ALPL mutations (Fig. 1). While the PLP levels were very similar in controls and non-carriers, the difference between carriers of mutations and the other two groups was highly significant (p < 0.0001). However, only 14 of the 21 patients had PLP levels above the upper limit of the reference range, thus yielding a specificity of 100% and a sensitivity of 67% to identify subjects with an ALPL mutation.

Fig. 1.

Baseline plasma PLP values in control subjects and in patients with low serum alkaline phosphatase who were non-carriers (No Mut) or carriers (Mut) of an ALPL mutation. The horizontal dotted line at the upper normal range

Following the previously reported protocol by Chodirker et al., we explored the response to a 6-day supplementation with vitamin B6 in a subset of patients and controls. Serum PLP increased in all subjects (Fig. 2). There were no significant differences between controls (n = 14) and patients (n = 13) with low serum ALP that did not carry an ALPL mutation. The post-supplementation values remained below 500 nmol/L in all subjects of those groups. However, 10 out of 13 mutation carriers exceeded this threshold (Fig. 2), resulting in a specificity of 100% (95% confidence interval, CI 98–100), sensitivity of 77% (CI 50–100), positive predictive value (PPV) 100% (CI 95–100), and negative predictive value (NPV) 90% (CI 78–100).

Fig. 2.

Plasma PLP values at baseline and after either 6-day (upper panel) or 2-day (lower panel) supplementation with vitamin B6, in controls, non-carriers (No Mut) and carriers (“is) of an ALPL mutation

We then examined the results of a shorter two-day supplementation test conducted with a few subjects. Serum PLP levels also increased consistently in all subjects. None of the controls (n = 8) and only 1 of 15 non-carriers exceeded the 500 nmol/l threshold. In contrast, all 11 mutation carriers surpassed this threshold (Fig. 2, lower panels). Thus, the 2-day test demonstrated a specificity of 96% (CI 85–100), and sensitivity of 100% (CI 95–100) to identify individuals with ALPL mutations. The PPV was 92% (72–100) and the NPV was 100% (98–100).

Discussion

The diagnosis of HPP is usually suspected in an individual with suggestive clinical manifestations and low serum ALP levels [1, 14]. Pyrophosphate is likely the most important ALP substrate involved in the pathogenetic mechanisms of HPP. However, it is only measured in a few centers due to technical difficulties [15, 16]. Other metabolites, such as PLP and phosphoethanolamine, are more readily available as markers of ALP enzymatic activity [17].

The diagnosis of HPP is often challenging due to several factors. First, the symptoms can be non-specific, particularly in mild cases, thus requiring a low threshold for clinical suspicion. Second, in our experience, and in that of other laboratories, only approximately half of the patients with persistently low serum ALP levels harbour a relevant variant in the ALPL gene (that is, a pathogenic/likely pathogenic/unknown significance variant) [9, 18], while clinical manifestations can overlap between carriers and non-carriers of ALPL mutations [19]. Third, not all individuals with ALPL mutations exhibit elevated PLP levels, and phosphoethanolamine appears to be an even less sensitive biomarker [9].

Therefore, we aimed to explore if a vitamin B6 supplementation test could be useful to screen patients with low ALP levels, trying to identify those carrying ALPL pathogenic variants. Our results indeed confirmed that a 6-day supplementation test increased the sensitivity from 67% with the baseline determination up to 77%. These results are in line with those reported many years ago in an endogamous community presumed to harbor a single ALPL mutation [11]. Therefore, the results of the present study confirm that a similar performance can be expected in subjects carrying a variety of ALPL variants. Furthermore, our data show that good diagnostic performance is even improved when the supplementation period is shortened from 6 to 2 days, increasing the sensitivity to 100%, with only a modest loss of specificity (from 100 to 96%). These results point to the 2-day supplementation as the preferable test for feasibility, patient convenience, and diagnostic accuracy.

Overall, the vitamin B6 supplementation test is a valuable tool for clinicians evaluating patients with low serum ALP levels and suspected HPP. Interestingly, the test does not only help distinguish patients with ALPL mutations from healthy controls, but also from individuals with otherwise unexplained low ALP who do not carry a mutation. Thus, it helps identify individuals more likely to benefit from more costly and technically complex genetic testing. It is to be noted that determining the likelihood of carrying an ALPL mutation is crucial not only for symptomatic patients but also for counseling asymptomatic individuals evaluating the prospect of parenthood.

Clinically, the supplementation test can guide decisions regarding genetic testing, particularly in patients with mild or nonspecific symptoms. A normal test result strongly reduces the likelihood of carrying a pathogenic ALPL variant. However, gene sequencing may still be recommended for individuals with severe clinical manifestations, regardless of supplementation outcomes.

The study's main limitation is its relatively small sample size, which restricts precise estimations of diagnostic performance. Additionally, direct comparisons between 2-day and 6-day tests were not carried out in all patients due to practical constraints. Nevertheless, our results are in line with those of a recent study describing the response to a 6-day pyridoxine challenge in pediatric HPP [20].

In conclusion, vitamin B6 supplementation tests are valuable tools for identifying carriers of ALPL mutations among individuals with unexplained low serum ALP. The shorter 2-day protocol offers a better balance of diagnostic accuracy, cost-effectiveness, and patient convenience, making it an attractive option in clinical practice, especially in resource-limited settings or where genetic testing is not readily available.

Funding

Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

Data are available from the authors upon reasonable request.

Declarations

Conflicts of interest

Authors (JAR, SA, AO, AIV, PCC, MTG, AR, NP) do not have conflicts of interest to disclose.