Direct and Functional Biomarkers of Vitamin B6 Status

Abstract

Measures of B6 status are categorized as direct biomarkers and as functional biomarkers. Direct biomarkers measure B6 vitamers in plasma/serum, urine and erythrocytes, and among these plasma pyridoxal 5'-phosphate (PLP) is most commonly used. Functional biomarkers include erythrocyte transaminase activities and, more recently, plasma levels of metabolites involved in PLP-dependent reactions, such as the kynurenine pathway, one-carbon metabolism, transsulfuration (cystathionine), and glycine decarboxylation (serine and glycine). Vitamin B6 status is best assessed by using a combination of biomarkers because of the influence of potential confounders, such as inflammation, alkaline phosphatase activity, low serum albumin, renal function, and inorganic phosphate. Ratios between substrate-products pairs have recently been investigated as a strategy to attenuate such influence. These efforts have provided promising new markers such as the PAr index, the 3-hydroxykynurenine:xanthurenic acid ratio, and the oxoglutarate:glutamate ratio. Targeted metabolic profiling or untargeted metabolomics based on mass spectrometry allow the simultaneous quantification of a large number of metabolites, which are currently evaluated as functional biomarkers, using data reduction statistics.

Keywords: B6 vitamers; amino acids; direct biomarkers; functional biomarkers; kynurenines; metabolomics; one-carbon metabolites; targeted metabolic profiling; transaminase tests; transsulfuration metabolites.

Figure 1

The kynurenine pathway of tryptophan catabolism and enzymes and cofactors involved. The heme dioxygenases, hepatic tryptophan (2,3)-dioxygenase (TDO; EC 1.13.1.2.) and ubiquitous indoleamine (2,3)-dioxygenase (IDO; EC 1.13.11.42), catalyze the oxidation of L-tryptophan to N-formylkynurenine, which is the first and rate limiting step of tryptophan catabolism. IDO is activated by pro-inflammatory cytokines like INF-gamma and TNF-alfa (100, 106) . N-Formylkynurenine is rapidly converted by formamidase (not shown) to kynurenine (Kyn). Kyn is converted to 3-hydroxykynurenine (HK) by FAD-dependent kynurenine mono-oxygenase (KMO; EC 1.14.13.9), and then cleaved to 3-hydroxyanthranilic acid (HAA) by the PLP-dependent enzyme, kynureninase (KYNU; EC 3.7.1.3), which also catalyzes the conversion of Kyn to anthranilic acid (AA). The PLP dependent enzyme kynurenine transaminase (KAT) catalyzes the formation of two end-stage metabolites, kynurenic acid (KA, from Kyn) and xanthurenic acid (XA, from HK).

Figure 2

The kynurenine pathway of tryptophan catabolism and enzymes and cofactors involved. The heme dioxygenases, hepatic tryptophan (2,3)-dioxygenase (TDO; EC 1.13.1.2.) and ubiquitous indoleamine (2,3)-dioxygenase (IDO; EC 1.13.11.42), catalyze the oxidation of L-tryptophan to N-formylkynurenine, which is the first and rate limiting step of tryptophan catabolism. IDO is activated by pro-inflammatory cytokines like INF-gamma and TNF-alfa (101, 107). N-Formylkynurenine is rapidly converted by formamidase (not shown) to kynurenine (Kyn). Kyn is converted to 3-hydroxykynurenine (HK) by FAD-dependent kynurenine mono-oxygenase (KMO; EC 1.14.13.9), and then cleaved to 3-hydroxyanthranilic acid (HAA) by the PLP-dependent enzyme, kynureninase (KYNU; EC 3.7.1.3), which also catalyzes the conversion of Kyn to anthranilic acid (AA). The PLP dependent enzyme kynurenine transaminase (KAT) catalyzes the formation of two end-stage metabolites, kynurenic acid (KA, from Kyn) and xanthurenic acid (XA, from HK).

Figure 3

The transsulfuration pathway. Homocysteine is form by hydrolysis of S-adenosylhomocysteine, which is formed from S-adenosylmethionine during transmethylation reactions (not shown). Homocysteine is either remethylated to methionine (not shown) or converted to cysteine via the transsulfuration pathway, where homocysteine is converted to cysteine through the sequential action of two vitamin B6 (pyridoxal 5′-phosphate)-dependent enzymes, cystathionine beta-synthase (CBS; EC 4.2.1.22) and cystathionine gamma-lyase (CSE; EC 4.4.1.1).

Figure 4

Serine hydroxymethyltransferase (SHMT) and the glycine cleavage system (GCS). SHMT (EC 2.2.2.1) is a vitamin B6 (pyridoxal 5′-phosphate)-dependent enzyme that catalyses the reversible conversion of serine to glycine. In mammals there are two isoforms, a cytoplasmic (cSHMT) and mitochondrial form (mSHMT). GCS is a mitochondrial multienzyme complex that is composed of four individual proteins, three specific components (P-, T-, and H-proteins) and one house-keeping enzyme, dihydrolipoamide dehydrogenase. P-protein is a vitamin B6 (pyridoxal 5′-phosphate)-dependent glycine decarboxylase (glycine:lipoylprotein oxidoreductase, EC 1.4.4.2). This system catalyses the oxidative cleavage of glycine.

Figure 5

Aminotransferases (ATs). There are multiple ATs (transaminases). They catalyse the equilibration of amino groups among available alpha-keto acids. All ATs require B6 (pyridoxal 5′-phosphate) as prosthetic group.

Figure 6

Main characteristics of vitamin B6 biomarkers in terms of primary confounders, and linkage with metabolism and distribution of B6 vitamers and metabolites. The vertical lines to the left with a bullet at the end indicate confounders with positive (blue bullet) or negative (red bullet) effect modification. The size of the bullets indicates the effect size. Abbreviation used: 2OG, 2-oxoglutarate; 4-PA, 4-pyridoxic acid; AT, aminotransferase; Cys, cysteine; Cysta, cystathionine; Glu, glutamate; Gly, glycine; Hcy, homocysteine; HK, 3-hydroxykynurenine; Kyn, kynurenine; Met, methionine; PAr, PA/(PLP+PL) ratio; PL, pyridoxal; PLP, pyridoxal 5′-phosphate; PM, pyridoxamine; PMP, pyridoxamine 5′-phosphate; PN, pyridoxine; PNP, pyridoxine 5′-phosphate; Ser, serine; Trp, tryptophan; XA, xanthurenic acid. *Urinary excretion of kynurenines may change during inflammation.