B6 and Sulfation

A journal article, “Inhibition of phenol sulfotransferase by pyridoxal phosphate,” By R. Bartzatt and J. D. Beckmann (Biochemical Pharmacoloy, 1994) has raised some concern among parents who use vitamin B6 to help their autistic children. The study is of questionable revelance, since it involved an in vitro (test tube) experiment rather than living subjects, and used cells of bovine rather than human origin. Nevertheless, ARI decided to investigate this matter, and provided a grant to Dr. Rosemary Waring, of the university of Birmingham School of Medicine in England, a preeminent researcher on sulphation problems in autism.

Dr. Waring’s results confirm what ARI had first reported in 1973: whenever extra vitamin B6 is given, it must be accompanied by extra magnesium, or adverse effects may be seen. In our first study of vitamin B6 in autistic children, conducted in the late 1960s, a small number of the autistic children in the experiment showed increased sound sensitivity, irritability and enuresis when the B6 was started. When magnesium was added, these side effects immediately disappeared and the beneficial effects of the B6 were enhanced. Several studies by the research team led by Dr. Gilbert LeLord of Tours University Medical School, in France, confirmed our report that the combination of vitamin B6 and magnesium was markedly more effective than either vitamin B6 or magnesium alone.

—Bernard Rimland, Ph.D.

Title: The effects of pyridoxal-5-phosphate on sulfotransferase activity: actions on tyrosyl protein sulfotransferase and phenol sulfotransferase.

Authors: Rosemary H Waring, Robert M Harris and Victoria L Griffiths, School of Biosciences, University of Birmingham, Birmingham. B15 2TT. UK

Introduction

Sulfotransferase enzymes use PAPS (3’-phospho-adenosive-5’-phosphosulfate) to transfer sulfate residues onto a wide variety of substrates. TPST substrates require sulfation for efficient function while sulfation by SULT 1A1 greatly alters substrate properties, usually decreasing their activity.

a) Tyrosylprotein sulfotransferase (TPST)
Substrates - tyrosine residues on gastrin, cholecystokinin, mucin proteins

Methods

TPST activity was measured (using gastrin as substrate) with radiolabelled ³⁵S-PAPS as sulfate donor; assays were incubated at 37°C for 40 minutes. All assays were performed in quadruplicate).

Enzyme sources were a) platelet pellets prepared by centrifugation from time-expired platelet packs from the Birmingham Blood Transfusion Service and b) human colon adenocarcinoma HT-29 cells which synthesise mucin proteins and are thought to be the best model for the human g.i. tract. These were grown in McCoys 5A medium supplemented with 10% fetal bovine serum and glutamine/penicillin/streptomycin at 37°C till confluent, then harvested and centrifuged to give a cell membrane pellet. This was resuspended in phosphate buffered saline, then sonicated before assay.

Cells were also grown after confluence for 24 hours with varying concentrations of P5P. P5P was also added directly to the platelet assay (0-2.5μM) MgCl₂ was added at varying concentrations (0-5μM) before the start of the TPST assay.

Results

TPST activity was present in both human platelets and HT-29 cells. This enzyme activity was inhibited in the presence of P5P (Fig. 1). Direct effects of MgCl₂ on the assay are shown in Fig. 2. As can be seen, MgCl₂ concentration had no significant effects on the TPST activity of HT-29 cells but activated TPST activity in platelets (there are different isoforms of the enzyme).

The concentration of 0.4μM P5P was then chosen as showing clear reduction of TPST activity in initial experiments (See Fig. 1). Human platelets were treated directly with this concentration while the HT-29 cells were incubated with it for 24h. Varying amounts of MgCl₂ were then added to the assays (see Fig. 3). As can be seen, levels of MgCl₂ at 0.5μM or greater removed the inhibition caused by 0.4μM P5P. Enzyme assays and Western blotting with specific anti-TPST antibodies showed that P5P did not affect the expression of TPST.

All results were carried out in quadruplicate and are expressed as means ± SD. (SD <= 5.3%).

b) SULT1A1 (Phenolsulphotransferase)
Substrates – Phenols, catecholamines, flavonoids, steroids.

Methods

SULT1A1 activity was measured using 4-nitrophenol as a substrate (this also picks up any of the SULT1A2 isoform, although this is only present to a small extent in platelet preparations). Again radiolabelled ³⁵S-PAPS was used in the assay as a sulfate donor. The enzyme sources were cytosols from platelets and HT-29 cells, prepared as before and assayed under standard conditions.

Results

SULT1A1 activity was present in both human platelets and HT-29 cells. This activity was inhibited by P5P (Fig. 4). However, this inhibition was only significant in the platelets; the HT-29 cell SULT1A1 seemed relatively unaffected. Treatment with MgCl₂ (1.0μM) on cells exposed to 1.0 μM P5P restored the activity in platelets (Fig. 6) but had no significant effects in the HT-29 cells, which in any case were not greatly affected by the P5P (Fig. 4) SULT1A1 activity in platelets was almost unaffected by MgCl₂ although SULT1A1 activity in HT-29 cells decreased slightly with increasing Mg (Fig. 5). Our previous studies have shown that the isoenzymes in these tissues have different activities and slightly different properties. Incubation of HT-29 cells with P5P had no effect on enzyme expression, as seen by Western blotting and enzyme activity.

All results were carried out in quadruplicate and are expressed as means ±SD. (SD <= 5.4%).

Conclusions

Platelet and HT-29 cells show TPST activity which is inhibited by P5P, though only the platelet isoform is greatly affected. This inhibition is reversed by MgCl₂ in roughly equimolar amounts.
Platelet and HT-29 cells show SULT1A1 activity which is inhibited by P5P, although only the platelet isoform is greatly affected. Again this inhibition is reversed by MgCl₂ in roughly equimolar amounts.
Neither TPST nor SULT1A1 expression is altered by P5P, which only affects the enzyme activity directly.
From the literature, P5P has a pseudo-phenolic structure which is believed to interact with those phenol sulfotransferases for which phenolic rings are a substrate. However, addition of Mg²⁺ may form a complex which no longer interacts with the enzyme. From the therapeutic point of view, Mg²⁺ ions should be supplied in at least a 2:1 ratio with P5P to reverse any inhibition and activate those sulfotransferases which respond to increased magnesium levels particularly the platelet enzymes.

Fig. 1 -- Effects of varied P5P concentrations on TPST activity in platelets and HT-29 cells, expressed as a percentage of the control (0μM P5P).

Fig 2 -- Effects of different MgCl₂ concentrations on TPST activity in platelets and HT-29 cells.

Fig. 3 -- TPST activity of platelets and HT-29 cells treated with P5P (.4 μM) and varied concentrations of MgCl₂. Activity is expressed as % control (0μM P5P, 0μM, MgCl₂).

Fig. 4 -- Effects of P5P concentration on SULT1A1 activity in platelets and HT-29 cells.

Fig. 5 -- Effects of different MgCl₂ levels on SULT1A1 activity in platelets and HT-29 cells.

Fig.6 -- SULT 1A1 activity of platelets and HT-29 cells treated with P5P (1.0 μM) and varied concentration of MgCl₂ (Activity is expressed as % control (0 lμM P5P, 0μM MgCl₂).