rs1256335 — ALPL

ALPL rs1256335 — The Alkaline Phosphatase Gate on Vitamin B6

Your blood carries vitamin B6 mostly as pyridoxal 5'-phosphate (PLP)¹¹ pyridoxal 5'-phosphate (PLP)
The coenzyme form of vitamin B6, required for over 150 enzymatic reactions including amino acid metabolism, neurotransmitter synthesis, and one-carbon metabolism. Before PLP can enter cells from the bloodstream, it must be dephosphorylated to pyridoxal (PL) by membrane-bound tissue-nonspecific alkaline phosphatase (TNSALP)²² tissue-nonspecific alkaline phosphatase (TNSALP)
The enzyme encoded by the ALPL gene, expressed on the surface of liver, bone, kidney, and intestinal cells where it hydrolyzes phosphate groups from PLP to allow cellular uptake — then re-phosphorylated back to PLP inside the cell. ALPL is therefore the gatekeeper for vitamin B6 transport: higher ALPL activity means faster PLP dephosphorylation and quicker cellular uptake, which lowers circulating PLP while keeping intracellular B6 adequate. Lower ALPL activity does the reverse — PLP accumulates in blood while cellular delivery slows. The rs1256335 variant sits in intron 5 of ALPL and influences the gene's regulation. The G allele is associated with higher ALPL expression and enzyme activity — and therefore lower circulating PLP, because the enzyme breaks down PLP more efficiently. Carrying two G copies means measurably lower plasma B6 relative to AA individuals. This does not necessarily mean cellular B6 deficiency — faster dephosphorylation means faster PLP-to-PL conversion and potentially faster cellular uptake — but it does produce lower measured plasma PLP, which is the standard clinical marker of B6 status. Conversely, the A allele is associated with lower ALPL activity, slower PLP dephosphorylation, and higher circulating PLP.

The Mechanism

ALPL encodes tissue-nonspecific alkaline phosphatase³³ tissue-nonspecific alkaline phosphatase
TNSALP, also known as liver/bone/ kidney-type alkaline phosphatase, which is membrane-bound and expressed at highest levels in bone, liver, kidney, and intestine, a phosphomonoesterase that hydrolyzes substrates at alkaline pH. Its physiological substrates include PLP, pyridoxamine phosphate (PMP), and inorganic pyrophosphate⁴⁴ PLP, pyridoxamine phosphate (PMP), and inorganic pyrophosphate
PLP and PMP are phosphorylated vitamers of B6; inorganic pyrophosphate is a mineralization inhibitor whose hydrolysis by ALPL is essential for bone formation. For vitamin B6 metabolism specifically, ALPL cleaves the phosphate from circulating PLP to produce pyridoxal, which crosses cell membranes via facilitated transport, after which intracellular pyridoxal kinase re-phosphorylates it to PLP for enzymatic use. Functional data at this locus comes from a nearby variant, rs1256341, where homozygosity for the minor C allele is linked to reduced ALPL expression in HapMap Northern European ancestry cells⁵⁵ reduced ALPL expression in HapMap Northern European ancestry cells
Carter TC et al. Common Variants at Putative Regulatory Sites of ALPL Influence Circulating PLP. J Nutr, 2015 and correspondingly higher plasma PLP. Importantly, rs1256335 and rs1256341 are nearby but their minor alleles sit on opposite haplotypes — the minor G allele at rs1256335 is associated with lower PLP (higher ALPL activity), while the minor C allele at rs1256341 is associated with higher PLP (lower ALPL activity). These represent partially independent regulatory signals within the ALPL gene region.

The Evidence

The first genome-wide association study of B6 status was published by Tanaka et al. in 2009⁶⁶ Tanaka et al. in 2009
Tanaka T et al. Genome-wide association study of vitamin B6, vitamin B12, folate, and homocysteine blood concentrations. Am J Hum Genet, 2009, identifying the ALPL locus (rs4654748, p = 8.3 × 10⁻¹⁸) as the strongest common genetic determinant of plasma B6 levels across 3,617 participants in four cohorts. A subsequent meta-analysis by Hazra et al. 2009⁷⁷ Hazra et al. 2009
Hazra A et al. Genome-wide significant predictors of metabolites in the one-carbon metabolism pathway. Hum Mol Genet, 2009 in 4,763 subjects showed that rs1256335 specifically — the strongest ALPL signal — reaches p = 1.40 × 10⁻¹⁵ with each G allele reducing plasma PLP by approximately 0.14 standard deviation units, while the AA genotype was associated with the highest PLP levels. A candidate gene study in 2,345 Irish adults⁸⁸ candidate gene study in 2,345 Irish adults
Carter TC et al. J Nutr, 2015 found 22 significant ALPL variants, with the PLP-raising effect of the minor allele at the correlated rs1256341 locus corresponding to a median plasma PLP difference of approximately 13 nmol/L between CC (minor) and TT (major) homozygotes (92.2 vs. 78.9 nmol/L). A GWAS of B6 vitamers in cerebrospinal fluid and plasma⁹⁹ GWAS of B6 vitamers in cerebrospinal fluid and plasma
Loohuis LM et al. The Alkaline Phosphatase (ALPL) Locus Is Associated with B6 Vitamer Levels in CSF and Plasma. Genes, 2018 confirmed the ALPL locus association extends to the central nervous system: at this locus, homozygotes for the PLP-raising allele showed a 1.4-fold higher PLP-to-PL ratio in plasma and a 1.6-fold higher ratio in CSF, underscoring that ALPL controls B6 partitioning in both the systemic circulation and the brain. Why does PLP level matter clinically? PLP is an essential cofactor for over 150 enzymatic reactions. Epidemiologically, lower plasma PLP is consistently linked to higher risks: a meta-analysis of 13 prospective studies¹⁰¹⁰ meta-analysis of 13 prospective studies
Larsson SC et al. Vitamin B6 and risk of colorectal cancer: a meta-analysis. JAMA, 2010 found each 100 pmol/mL increase in plasma PLP was associated with a 49% lower risk of colorectal cancer. A large U.S. cohort study found that plasma PLP is inversely associated with CRP, IL-6, and TNF-αR2¹¹¹¹ inversely associated with CRP, IL-6, and TNF-αR2
Sakakeeny L et al. Plasma pyridoxal-5- phosphate is inversely associated with systemic markers of inflammation. J Nutr, 2012, linking B6 status to systemic inflammation independently of diet.

Practical Implications

For AA homozygotes — the majority of people (about 62%) — ALPL activity is lowest at this locus, PLP is turned over slowest, and plasma B6 levels are the highest of the three genotypes. The practical implication is reassuring: this genotype is associated with the highest circulating B6, and dietary B6 requirements may be at the lower end of normal. Standard dietary intake from B6-rich foods is likely sufficient. For GG homozygotes (about 4% of people), ALPL activity is highest, PLP is dephosphorylated most rapidly, and plasma B6 levels are on the lower end. This is not equivalent to deficiency in most cases, but it does mean that dietary B6 intake and B6 bioavailability from food sources matter more for maintaining adequate plasma PLP. Pyridoxal-5-phosphate (P5P) supplements, which bypass the need for ALPL-mediated dephosphorylation on the gut side, may be slightly more efficiently delivered at the cellular level for these individuals compared to pyridoxine supplements that require enzymatic activation.

Interactions

rs1256335 is in partial linkage disequilibrium (r² = 0.16) with rs4654748 in the nearby NBPF3 gene, which was the first GWAS top hit for plasma B6. These two loci represent partially independent signals in the same genomic region. A third variant, rs1256341, also within ALPL, has its minor allele (C) associated with reduced ALPL expression and higher PLP — the opposite direction from rs1256335's minor allele (G). These nearby variants thus capture distinct regulatory effects on ALPL despite their physical proximity. ALPL rs1256335 is also relevant in the context of methylation pathway SNPs¹²¹² methylation pathway SNPs
MTHFR C677T (rs1801133) and SHMT1 C1420T (rs1979277) both require PLP as a cofactor for their enzymatic activity: both MTHFR and SHMT1 are PLP-dependent enzymes. A GG genotype at rs1256335, by reducing circulating PLP, could modestly amplify functional B6 insufficiency when combined with high MTHFR or SHMT1 metabolic demand. However, no published study has formally quantified this combined effect, so this remains a biologically plausible but unconfirmed interaction.