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Liquid chromatographic studies of vitamin B6 metabolism in man
Clinica Chimicu Actu, 190 (1990) 67-80 EIsevier
67
CCA 04771
Liquid chromatographic studies of vitamin B6 metabolism in man P. Edwards, P.K.S. Liu and G.A. Rose Institute of Urology and St. Peter’s Hospitals, Lmdon (UK) (Received
3 August
Key woruk: Unidentified
1989; revision received 6 April 1990; accepted
metabolites;
Vitamin
B6; Isopyridoxal;
18 April 1990)
4Tyridoxolactone;
Vitamer
activity
The effects of increased intake of pyridoxine hydrochloride on plasma vitamin B6 metabolism within therapeutic limits (up to 800 mg/day) were investigated. Maximum plasma concentrations of pyridoxal phosphate were attained at relatively low intakes of pyridoxine hydrochloride. Two metabolism thought to be unidentified forms of vitamin B6 were present in subjects taking more than 200 mg of pyridoxine hydrochloride per day as have recently been described [l]. We investigated the possibility that these were isomer-k forms of vitamin B6. However, ‘Peak 2’ metabolite was shown to be probably 4-pyridoxolactone. The metabolism of isopyridoxal has not previously been investigated in man. We demonstrated that it is an active vitamer of the B6 complex in humans. The main fluorescent metabolite of isopyridoxal present in plasma and urine had a similar retention time to ‘Peak 1’ metabolite. Isopyridoxal was incapable of being directly phosphorylated in rat liver extract and it is therefore unlikely that peak 1 is isopyridoxal phosphate. Its nature remains unknown.
Introduction
‘Vitamin B6’ is the general name for a group of 3-hydroxy-2-methyl-pyridine derivatives exhibiting the same vitamin activity as pyridoxine. This group of compounds includes pyridoxine, pyridoxal, pyridoxamine and the respective 5’phosphate esters.
Correspondence
to: Dr. G.A. Rose, St. Paul’s Hospital,
0009-8981/90/$03.50
0 1990 EIsevier Science Publishers
EndeIl Street, London
B.V. (Biomedical
WC2, UK.
Division)
68
The recent development of an HPLC method [l] for the simultaneous measurement of all seven known components of the vitamin B6 group in human plasma showed the presence of two previously unrecognised substances thought to be forms of vitamin B6. These metabolites were shown to be present consistently in individuals taking oral supplements of more than 200 mg of pyridoxine hydrochloride per day. The discovery of these two new metabolites of pyridoxine in human plasma raises the question of their identity. We have previously suggested that since pyridoxine has methanol groups attached at two adjoining points on the heterocyclic ring, one of which is phosphorylated and the other is oxidized to the aldehyde or aminated, then the roles of these two methanol groups could be reversed, so giving rise to a series of isomers of vitamin B6. This idea was investigated.
Materials and methods Chemicals Reagent grade sodium dihydrogen orthophosphate, sodium metabisulphate, perchloric acid, triethanolamine, zinc sulphate and sodium perchlorate were obtained from BDH Chemicals Ltd, Poole, Dorset, UK. Pyridoxal phosphate, pyridoxal, pyridoxine, 4-pyridoxic acid and adenosine triphosphate were purchased from Sigma Chemical Co. Ltd., Poole, Dorset, UK. Pyridoxine phosphate was kindly donated by Dr. Stephen Coburn (Fort Wayne State Development Center, Fort Wayne, IN, USA). Pyridoxine ingestion Healthy individuals were given O-800 mg of oral pyridoxine hydrochloride per day for 1 wk, and 4 h after the final dose, blood samples were collected into potassium EDTA tubes: plasma was separated and deproteinized with perchloric acid for analysis by HPLC [l]. Isopyridoxal synthesis An isomeric form of pyridoxal, 5-hydroxy-4-hydroxymethyl-6_methylnicotinaldehyde (isopyridoxal) was synthesized from pyridoxine via isopropylidenepyridoxine hydrochloride and isopropylidene isopyridoxal by the method of Sattsangi and Argoudelis [2]. Ultraviolet spectroscopy of the product showed agreement with previously published data [3], see below. Isopyridoxal ingestion A basal blood sample was obtained from a healthy volunteer who then ingested 50 mg of isopyridoxal hydrochloride. Timed blood samples were drawn, collected into potassium EDTA tubes and analysed by HPLC. At another time, the experiment was repeated after ingestion of 100 mg of isopyridoxal hydrochloride and timed urine samples were also collected over a 24 hour period. These were analysed by HPLC after appropriate dilution (l/50) with deionized water.
69
High performance liquid chromatography (HPLC) Isocratic reverse phase HPLC using a 4.6 X 250 mm TSK ODS 120T analytical column (5 rJ.m) was used as previously described [l] with an eluent consisting of 75 mmol/l NaH,PO, buffer containing 75 mmol of NaClO,, 8.5 ml of acetonitrile and 0.5 ml of triethanolamine per litre, with the pH adjusted as required using concentrated HClO,. Post column derivatization using bisulphite ions was utilised. Synthesis of 4-pyridoxolactone 4-Pyridoxic acid was heated for 30 min in a 1 mol/l HCl in a boiling water bath, conditions known to convert 4-pyridoxic acid to its lactone [5]. Furthermore HPLC of the reaction mixture before and after showed loss of the 4-pyridoxic acid peak and the appearance of a peak of different retention time not corresponding to any other known compound. Incubation studies Purification of pyridoxal kinase enzyme source Rat liver extract was obtained by the method of McCormick [3] by homogenising one part of fresh tissue with four parts of 0.07 mol/l potassium phosphate buffer, pH 6.25, and centrifuging at 25 000 X g for 30 min. Supematant was separated and 1 mM pyridoxal was added and warmed with stirring to 55 “C and held at this temperature for 15 min. The mixture was chilled on ice and centrifuged at 18 500 x g for 15 min and supernatant was separated to yield an enzyme mixture capable of phosphorylating isopyridoxal. Incubation mixtures Incubation mixture ‘A’ containing 0.5 mmol/l isopyridoxal, 0.5 mmol/l ATP, 0.01 mmol/l Zn2’ (as the sulphate), 0.07 mol/l potassium phosphate buffer, pH 6.25 and 0.50 ml of pyridoxal kinase extract in a total volume of 2.5 ml. Incubation mixture ‘B’ contained no ATP but was otherwise the same. Mixtures were incubated at 37” C for 3 h and the reaction was stopped by deproteinising with 50 ~1 of concentrated perchloric acid. Vitamin B6 profiles were examined by HPLC [l] using 50 ~1 injections. Results Plasma vitamin B6 profiles after pyridoxine hydrochloride ingestion Table I displays the mean values of plasma-vitamin B6 compounds in healthy individuals after administration of pyridoxine hydrochloride orally in the doses shown for 1 week. Results are given as mean + SD for all B6 compounds except for pyridoxine where medians are used, since plasma pyridoxine concentrations were not normally distributed. The non-Gaussian distribution could have been due to at least two reasons. First, blood was drawn only approximately 4 h after pyridoxine administration and since the half-life of pyridoxine in plasma is about 40 min [l], small differences in timing of blood samples would be significant. Second, dietary restrictions were not imposed on subjects under study.
70 TABLE
I
Plasma vitamin subjects
B6 compounds
after 1 wk of oral pyridoxine
PNP’
Dose
PLP
PL
10 6
0 10
(34)73 595
(17; 472
_ -
9
25
(287) 631
(393) 231
-
100
(158) 518
(157) 1277
200
(130) 623
(678) 2441
8
400
(138) 732
7
800
(202) 644
(904) 4 764 (1664) 9484 (1616)
No. of subjects
PN
hydrochloride
administration
in healthy
4-PA
Peak 1
Peak 2
(1: 295
i
-
(104) 371
-
_
9 9
(182)
Results are expressed in nmol 1-l plasma for are expressed in peak height ml-’ plasma. Pyridoxal phosphate = PLP; Pyridoxal = PL; l&2. B6 compounds are expressed as mean ( f
-
(175) 1239
116
124
(60) 269
(464) 2200
(49) 208
389
(213) 120
(633) 3717
(147) 401
(318, 100
4664
(-) 245
(922) 7085 (1528)
(221) 1656
(28) 394
(506)
(196)
268
350
(-)
-
PLP, PL, PNP and 4-PA. Peak 1 and Peak 2 metabohtes Doses are in mg of pyridoxine hydrochloride per day. Pyridoxine = PN; Peak l&2 = unidentified metabolites SD) except for PN where medians are used.
It can be seen that the plasma concentrations of pyridoxal, pyridoxine and 4-pyridoxic acid rose linearly with increasing pyridoxine hydrochloride dose. Unknown peak 1 appeared at doses greater than 100 mg of pyridoxine hydrochloride per day, and unknown peak 2 appeared at doses greater than 200 mg per day. The concentration of pyridoxal phosphate in plasma showed little variation between doses of 10 mg and 800 mg of pyridoxine hydrochloride per day. Evidence for authenticity of synthesized isopyridoxai The ultraviolet spectra for the synthetic product in both 0.1 mol/l sodium hydroxide and 0.1 mol/l hydrochloric acid were similar to those reported for the natural product present in certain bacteria [3] (Fig. 1). The manufactured isopyridoxal was chromatographically homogeneous by HPLC and chromatographed between pyridoxal and pyridoxine when added to a mixed standard containing pyridoxal phosphate, pyridoxal, pyridoxine and 4-pyridoxic acid. Identity of unknown metabolite, ‘peak 2’ The unknown metabolite corresponding to peak 2 chromatographed between pyridoxal and pyridoxine (Fig. 2a). When plasma obtained from a person 4 h after a single 800 mg dose of pyridoxine hydrochloride was supplemented with a small quantity of synthetic isopyridoxal, an increase in the peak size of peak 2 was observed when chromatographed by HPLC at both pH 3.38 and pH 3.12 (Fig. 2b), without any peak splitting or separation. This suggested that the unknown metabo-
71
ISOPYRIDOXAL
ISOPYRIDOXAL
IN O.lM HCI
IN O.lM NaOH
Wavelength
Wavelength
Fig. 1. UV spectra of isopyridoxal in 0.1 mol/l HCI and 0.1 mol/l NaOH.
lite corresponding to peak 2 was isopyridoxal. However, 4-pyridoxolactone was found to co-chromatograph with isopyridoxal and peak 2 metabolite at pH (Table II). When the eluent was adjusted to pH 5.78, separation between compounds was satisfactorily achieved. It was seen that isopyridoxal did co-chromatograph with peak 2 metabolite at the pH, but Cpyridoxolactone
also 3.12 the not did
TABLE II Retention times of Cpyridoxolactone, by HPLC pH of eluent
3.12 5.78
isopyridoxal and plasma ‘peak 2’ metabolite at pH 3.12 and 5.78
Retention time @in) Isopyridoxal
CPyridoxolactone
Peak2 in plasma
Isopyridoxal +Peak2
CPyridoxolactone +peakz
11.75 10.18
11.98 12.91
11.76 12.51
11.80 ’ Isopyridoxal at 11.35 Peak 2 at 12.25
11.64 ’ 11.28 ’
’ Co-chromatography.
72
4-PA B. A.
4-PA
PN Peak
PN
2
I
PL
c P
SP 1 Fig.
2. Comparison of chromatograms pyridoxine hydrochloride
of (A) a plasma sample from an individual and (B) the same sample with added isopyridoxal.
given
800 mg
co-chromatograph with peak 2 metabolite showing that peak 2 metabolite is probably 4-pyridoxolactone. Acidification of 4-pyridoxic acid standards with perchloric acid at room temperature did not produce any lactone from 10 ng samples of treated 4-pyridoxic acid, and so does not appear to be an artifact. Metabolic Effect dose of elevation nmol 1-l elevated ingestion
studies of isopyridoxal
in man
of 50 mg ingestion of isopyridoxal hydrochloride Ingestion of a 50 mg oral the prepared isopyridoxal hydrochloride by a normal man caused an in plasma pyridoxal phosphate concentration from a basal value of 130 to a peak of 268 nmol 1-i after 3 h. Plasma pyridoxal phosphate was within 15 min (Table III). The major peak in plasma after isopyridoxal has a retention time corresponding to that of peak 1 unidentified metabo-
13 TABLE
III
The effects of ingesting Time
PLP
Basal 15 min 30 min lh 2h 3h 4h 5h 6h 24 h
130 239 190 221 239 261 239 255 202 85
50 mg of isopyridoxal
hydrochloride
Isopl metabolite (corresponding to the retention time of Peak 1)
on plasma
PL
Isopl
_
_
_
560 635 360 225 165 85 50 30 _
349 388 172 _ _
50 50 35 25 35 15
_
_
PLP, Pyridoxal phosphate; PL, pyridoxal; Isopl metabolite, isopyridoxal metabolite. height ml-‘.
vitamin
B6 metabolism PN
4-PA
78 39 49 39 39 32 15 15
60 229 328 109 60 49 38 44 44 22
Isopl, isopyridoxal; PN, pyridoxine; 4-PA, 4-pyridoxic acid; Units as nmol 1-l except for Isopl metabolite; units, peak
lsopl
metabolite
PLP Q-PA lsopl
Y
Fig. 3. HPLC chromatogram
of a plasma
sample obtained 30 min post-ingestion hydrochloride.
of 50 mg of isopyridoxal
EFFECT OF INGESTION OF ISOPYRIDOXAL ON PLASMA LEVELS OF PYRIDOXAL PHOSPHATE
420 -1” Plasma pyridoxal phosphate1 nmoles L-l
400 _;j
R
t
I\
I ‘\
380-11 It
: I
1 \
’
:
’
I
\ \
I
\
I’
\
\ \
I
\
\ I
I \
:
1
I
‘.
180 160 140 120 100 80
----100mg
60
-
0
1
0
I
1.0
I
I
2.0
I
3.0
4.0
Time after
Fig. 4. Effect of ingestion of isopyridoxal
dose 50mg dose
1
5.0
1
6.0
I
24.0
ingestion/hours
on plasma levels of pyridoxal phosphate.
lite on chromatography at pH 3.12 (Fig. 3). The effects of a 50 mg dose of isopyridoxal on plasma vitamin B6 metabolism can be seen in Table III and Figs. 4, 5 and 6. A correlation coefficient of 0.917 existed between plasma isopyridoxal and the metabolite corresponding to the retention time of unknown metabolite peak 1 ( p = 0.010). Effects of 100 mg ingestion of isopyridoxal hydrochloride plasma results Ingestion of a 100 mg oral dose of the prepared isopyridoxal hydrochloride by a healthy man
EFFECT OF INGESTION OF ISOPYRIDOXAL ON PLASMA LEVELS OF ISOPYRIDOXAL METABOLITE
1000 950 900 I350
3
Plasma Peak l/ Peak ht. mm ml-1
Time after
Fig. 5. Effect of ingestion TABLE
of isopyridoxal
ingestion/hours
on plasma
levels of its metabolite.
IV
The effects of ingesting Time
PLP
Basal 5rnin 10 min 20 min 30 min 40min lh 3h 4h Sh 24 h
522 328 364 316 215 393 267 409 215 198
100 mg of isopyridoxal
hydrochloride
on plasma
Isopl metabolite (corresponding to the retention time of Peak 1)
PL
Isopl
_
_
235 165 800 715 115 790 250 175 110 _
59 98 383 250 393 201 20 20 _ _
_ 110 145 145 140 115 20 25 20 30
154
vitamin
B6 metabolism PN
4-PA
63 54 49 54 83 34 13 112 91 29
55 55 169 251 213 207 257 68 82 71 49
PLP, Pyridoxal phosphate; Isopl metabolite, isopyridoxal metabolite; PL, pyridoxal; Isopl, isopyridoxal; PN, pyridoxine; 4-PA, Qpyridoxic acid. Units, mnol 1-l except for Isopl and Isopl metabolite; units, peak height/mm ml-‘.
EFFECT
OF
PLASMA
INGESTION LEVELS
OF OF
ISOPYRIDOXAL
4bPYRlDOXlC
ON ACID
400_ 380_ 360 340-
Plasma 4mpyridoxic acid L_
320_
’
/ n moles
1
300_ 2802602&I)_
r( 1 \
9
220-
‘I
/
’\ \
r+
200_
I
180_
:
\
160-
I I
140-
,
120,
\
I
100-1,
1OOmg
-
50mg
dose dose
\\
‘\ \ \
4
-----
\ \ \
\
I i
80
I
60
40 20 0
i
!
0
I
1.0
2.0
3.0 Time
Fig. 6. Effect of ingestion
TABLE
0
0.5 1.0 1.5 2.0 2.5 3.0 4.0 5.0 24.0
after
5.0
6.0
24.0
ingestion/hours
on plasma
levels of 4-pyridoxic
acid.
V
The excretion Time/h
of isopyridoxal
4.0
of urinary
metabolites
after ingestion
4-Pyridoxic acid excretion (nmol min-‘)
_ 10 14 18 23 33 33 13 16 14
of 100 mg of isopyridoxal
hydrochloride
Isopyridoxal metabolite excretion (peak height/mm min-‘)
_ 57 x 103 65x10’ 64x lo3 112x103 80~10~ 58 x lo3 17x103 18~10~
77
caused an elevation in plasma pyridoxal phosphate concentration from a basal value of 154 nmol 1-i to a peak value of 524 nmol 1-i after 5 min. Plasma pyridoxal phosphate levels remained elevated for at least 5 h after the ingestion of the isopyridoxal. Again the major peak observed corresponded to that of unidentified, ‘peak 1’. These effects on plasma vitamin B6 can be seen in Table IV and Figs. 4, 5 and 6. A correlation coefficient of 0.969 (p < 0.001) existed between plasma isopyridoxal and the metabolite corresponding to the retention time of unknown metabolite peak 1.
Urine results The effects of ingestion of 100 mg of isopyridoxal can be seen in Table V. The metabolite chromatographing between pyridoxal phosphate and pyridoxal (‘peak 1’) was present in the urine and decreased over 24 h after the ingestion of isopyridoxal.
TEST
BLANK (water
in place of isopyridoxal 4-PA
I-PA
PNP
PNPI
Fig. 7. HPLC chromatogram of incubation of isopyridoxal with rat liver extract showing the generation of its metabolite corresponding to the retention time of peak 1 unidentified metabolite.
78 TABLE
VI
Incubation
studies of isopyridoxal
Values as peak heights/ml
following
metabolism
in rat liver extract
50 ~1 injections
A (+ATP)
B (-ATP)
PMP = 39 PM=13 PNP = 160 PLP = 86 Isopl metabolite = 62 PL =13 PN=80 Isopl = over scale 4-PA = over scale
PMP=17 PM=22 PNP=7 PLP=8 Isopl metabolite = 90 PL=18 PN = over scale Isopl = over scale 4-PA = over scale
Difference
156% 169% 196% J 91% t 45% t 38% tt_ _
PMP, Pyridoxamine phosphate; PM, pyridoxamine; PNP, pyridoxine phosphate; PLP, pyridoxal phosphate; Isopl metabolite, isopyridoxal metabolite (not present if isopyridoxal is omitted from the reaction mixture unpublished abbreviation); PL, pyridoxal; PN, pyridoxine; Isopl, isopyridoxal.
Urinary 4-pyridoxic acid excretion rate increased up to 3 h after isopyridoxal ingestion and then decreased. Other fluorescent peaks were present in urine but were not identified. Isopyridoxal and 4-pyridoxolactone co-chromatograph at an eluent pH of 3.12. Incubation
studies
After incubation of isopyridoxal in the presence of rat liver extract, the HPLC chromatogram of the mixture showed a peak with the retention time of ‘peak 1’ unidentified metabolite in plasma and urine after isopyridoxal ingestion (Fig. 7). In a second experiment ATP was omitted from the reaction mixture to examine whether the metabolite of isopyridoxal was phosphorylated (Table VI). All phosphorylated forms of vitamin B6 decreased in the absence of added ATP, but the peak height of the isopyridoxal metabolite increased, demonstrating that this was not a phosphorylated form of isopyridoxal.
Ingestion of pyridoxine hydrochloride in doses exceeding 200 mg per day was followed by the presence in the HPLC chromatograms of two unidentified forms of vitamin B6, ‘peak 1’ and ‘peak 2’ (Fig. 2), the plasma concentrations of which increased linearly with dose of pyridoxine hydrochloride (Table I). Peak 2 was initially thought to be isopyridoxal for three reasons. First, supplementation of plasma from a healthy individual 4 hours after ingestion of 800 mg of pyridoxine hydrochloride with synthetic isopyridoxal showed an increase in peak height (Fig. 2). Second, the relative retention of isopyridoxal corresponded with that of peak 2. Third isopyridoxal and peak 2 chromatograph between pyridoxal and pyridoxine with the same retention time at pH 3.12 and 3.38. However, 4-pyridoxo-
19
lactone was found to co-chromatograph with isopyridoxal and peak 2 metabolite at pH 3.12 and after adjusting the pH of the eluent to 5.78,4-pyridoxolactone was seen to co-chromatograph with peak 2 metabolite present in plasma but isopyridoxal did not co-chromatograph with peak 2 (Table II), demonstrating that peak 2 metabolite may be 4-pyridoxolactone. Isopyridoxal has previously been identified in bacteria [5]. Prior to this finding, isopyridoxal had been synthesized and examined for biological activity [5-71. In contrast to pyridoxal, isopyridoxal was inactive in supporting growth of Streptococcus faecalis, but could support the growth of Kloeckera brevis and Saccharomyces carlsbergensis, although not as efficiently as pyridoxal [7,8]. In rats however, isopyridoxal has been shown to have approximately 4% of the growth promoting activity of pyridoxal on a molar basis [5]. In other studies, isopyridoxal was proved to be unable to replace pyridoxal as a catalyst for various biochemical reactions, such as non-enzymatic transamination [8], cysteine desulfhydration or serine dehydration [9,10] in the presence of metal ions. We initially had an interest in isopyridoxal as the possible identity of peak 2 metabolite seen in human blood after ingestion of high therapeutic doses of pyridoxine hydrochloride. This metabolite, as discussed earlier, is probably 4-pyridoxolactone. Activity of 4-pyridoxolactone has previously been determined relative to pyridoxine hydrochloride activity where it was found to be 0.24 times as active in Streptococcus Iactis, 0.02 times as active for Lactobacillus casei and 0.00025 times as active for yeast [4]. Since the metabolism of isopyridoxal, to our knowledge, has never been investigated in man, we took this opportunity to conduct studies. The synthetic isopyridoxal hydrochloride used in this experiment was shown to be the authentic product by examination of the UV spectra in 0.01 mol/l HCl, which showed maxima at 284 nm and 230 nm (shoulder peak) and 0.1 mol/l NaOH with maxima at 244 nm and 296 nm (Fig. 3), corresponding to literature values for natural isopyridoxal [5]. In the present study, it has been demonstrated that ingestion by a human volunteer of a 50-mg oral dose of isopyridoxal hydrochloride causes an increase in the plasma concentration of pyridoxal phosphate of 2 times the basal level, and when a lOO-mg oral dose of isopyridoxal hydrochloride was ingested, plasma pyridoxal phosphate rose approximately 3.5 times that of basal level (Tables III and IV; Figs. 4-6). This proves that isopyridoxal is an active form of vitamin B6 in the human subject. This was supported by the plasma and urinary increases in 4-pyridoxic acid concentration (Tables III-V). Since isopyridoxal, like pyridoxine, possesses vitamin B6 activity for some organisms but not for others (see above) it may be considered a naturally occurring member of the vitamin B6 complex. To exhibit such activity, isopyridoxal would have to be transformed in vivo to pyridoxal phosphate, which must occur by preliminary reduction to pyridoxine. This capability differs between species, for example, more efficiently in yeast than in rats [5]. The results presented here suggest that isopyridoxal is converted to pyridoxal phosphate probably via pyridoxine in man and is also converted to another
80
metabolite which chromatographs with a similar retention time to ‘peak 1’ metabolite between pyridoxal phosphate and pyridoxal and appears in both plasma and urine. Incubation studies using rat liver extract showed that this metabolite of isopyridoxal is not a phosphorylated form. This agrees with Hurwitz .[ll] who showed that analogues of pyridoxine capable of being phosphorylated by pyridoxal kinase require a hydroxymethyl group in the 5 position of the ring and should be unsubstituted in the 6 position. Isopyridoxal does not satisfy these criteria. The true nature of peak 1 therefore remains unknown. Acknowledgements We would like to thank the staff of the Department of Child Health, Institute of Child Health, great Ormond Street Hospital for laboratory facilities and St. Peter’s Research Trust for the purchase of HPLC equipment. References 1 Edwards P, Liu PKS, Rose GA. A simple liquid-chromatographic method for measuring vitamin B6 compounds in plasma. Clin Chem 1989;35:241-245. 2 Sattsangi PD, Argoudelis CJ. Synthesis of a pyridoxal analog 4,5-diformyl-3-hydroxy-2-methylpyridine. J Org Chem, 1968,33:1337-1341. 3 McCormick DB, Gregory ME, Snell EE. Pyridoxal phosphokinases. J Biol Chem 1961;236:2076-2084. 4 Huff JW, Perlzweig WA. A product of oxidative metabolism of pyridoxine, 2-methyl-3-hydroxy-4carboxy-5-hydroxy-methyl-pyridine (Cpyridoxic acid). J Biol Chem 1945;155:345-355. 5 Rodwell VW, Volcani BE, Ikawa M, Snell EE. Bacterial oxidation of vitamin B6. J Biol Chem :958;233:1548-1554. 6 Harris SA, Hey1 D, Folkers K. The structure and synthesis of pyridoxamine and pyridoxal. J Am Chem Sot 1944;66:2088-2092. 7 Snell EE, Rarmefeld AN. The vitamin activity of pyridoxal and pytidoxamine for various organisms. J Biol Chem 1945;157:475-489. 8 Metzler DE, Olivard J, Snell EE. Transamination of pyridoxamine and amino acids with glyoxylic acid. J Am Chem Sot 1954;76:644-648. 9 Metzler DE, Snell EE. Catalytic deamination of serine and cysteine by pyridoxal and metal salts. J Biol Chem 1952;198:353-361. 10 Metzler DE, Longenecker JB, Snell EE. The reversible catalytic cleavage of hydroxyamino acids by pyridoxal and metal salts. J Am Chem Sot 1954;76:639-644. 11 Hurwitz J. Enzymatic phosphorylation of vitamin B6 analogues and their effect on tyrosine decarboxylase. J Biol Chem 1955;217:513-525.
67
CCA 04771
Liquid chromatographic studies of vitamin B6 metabolism in man P. Edwards, P.K.S. Liu and G.A. Rose Institute of Urology and St. Peter’s Hospitals, Lmdon (UK) (Received
3 August
Key woruk: Unidentified
1989; revision received 6 April 1990; accepted
metabolites;
Vitamin
B6; Isopyridoxal;
18 April 1990)
4Tyridoxolactone;
Vitamer
activity
The effects of increased intake of pyridoxine hydrochloride on plasma vitamin B6 metabolism within therapeutic limits (up to 800 mg/day) were investigated. Maximum plasma concentrations of pyridoxal phosphate were attained at relatively low intakes of pyridoxine hydrochloride. Two metabolism thought to be unidentified forms of vitamin B6 were present in subjects taking more than 200 mg of pyridoxine hydrochloride per day as have recently been described [l]. We investigated the possibility that these were isomer-k forms of vitamin B6. However, ‘Peak 2’ metabolite was shown to be probably 4-pyridoxolactone. The metabolism of isopyridoxal has not previously been investigated in man. We demonstrated that it is an active vitamer of the B6 complex in humans. The main fluorescent metabolite of isopyridoxal present in plasma and urine had a similar retention time to ‘Peak 1’ metabolite. Isopyridoxal was incapable of being directly phosphorylated in rat liver extract and it is therefore unlikely that peak 1 is isopyridoxal phosphate. Its nature remains unknown.
Introduction
‘Vitamin B6’ is the general name for a group of 3-hydroxy-2-methyl-pyridine derivatives exhibiting the same vitamin activity as pyridoxine. This group of compounds includes pyridoxine, pyridoxal, pyridoxamine and the respective 5’phosphate esters.
Correspondence
to: Dr. G.A. Rose, St. Paul’s Hospital,
0009-8981/90/$03.50
0 1990 EIsevier Science Publishers
EndeIl Street, London
B.V. (Biomedical
WC2, UK.
Division)
68
The recent development of an HPLC method [l] for the simultaneous measurement of all seven known components of the vitamin B6 group in human plasma showed the presence of two previously unrecognised substances thought to be forms of vitamin B6. These metabolites were shown to be present consistently in individuals taking oral supplements of more than 200 mg of pyridoxine hydrochloride per day. The discovery of these two new metabolites of pyridoxine in human plasma raises the question of their identity. We have previously suggested that since pyridoxine has methanol groups attached at two adjoining points on the heterocyclic ring, one of which is phosphorylated and the other is oxidized to the aldehyde or aminated, then the roles of these two methanol groups could be reversed, so giving rise to a series of isomers of vitamin B6. This idea was investigated.
Materials and methods Chemicals Reagent grade sodium dihydrogen orthophosphate, sodium metabisulphate, perchloric acid, triethanolamine, zinc sulphate and sodium perchlorate were obtained from BDH Chemicals Ltd, Poole, Dorset, UK. Pyridoxal phosphate, pyridoxal, pyridoxine, 4-pyridoxic acid and adenosine triphosphate were purchased from Sigma Chemical Co. Ltd., Poole, Dorset, UK. Pyridoxine phosphate was kindly donated by Dr. Stephen Coburn (Fort Wayne State Development Center, Fort Wayne, IN, USA). Pyridoxine ingestion Healthy individuals were given O-800 mg of oral pyridoxine hydrochloride per day for 1 wk, and 4 h after the final dose, blood samples were collected into potassium EDTA tubes: plasma was separated and deproteinized with perchloric acid for analysis by HPLC [l]. Isopyridoxal synthesis An isomeric form of pyridoxal, 5-hydroxy-4-hydroxymethyl-6_methylnicotinaldehyde (isopyridoxal) was synthesized from pyridoxine via isopropylidenepyridoxine hydrochloride and isopropylidene isopyridoxal by the method of Sattsangi and Argoudelis [2]. Ultraviolet spectroscopy of the product showed agreement with previously published data [3], see below. Isopyridoxal ingestion A basal blood sample was obtained from a healthy volunteer who then ingested 50 mg of isopyridoxal hydrochloride. Timed blood samples were drawn, collected into potassium EDTA tubes and analysed by HPLC. At another time, the experiment was repeated after ingestion of 100 mg of isopyridoxal hydrochloride and timed urine samples were also collected over a 24 hour period. These were analysed by HPLC after appropriate dilution (l/50) with deionized water.
69
High performance liquid chromatography (HPLC) Isocratic reverse phase HPLC using a 4.6 X 250 mm TSK ODS 120T analytical column (5 rJ.m) was used as previously described [l] with an eluent consisting of 75 mmol/l NaH,PO, buffer containing 75 mmol of NaClO,, 8.5 ml of acetonitrile and 0.5 ml of triethanolamine per litre, with the pH adjusted as required using concentrated HClO,. Post column derivatization using bisulphite ions was utilised. Synthesis of 4-pyridoxolactone 4-Pyridoxic acid was heated for 30 min in a 1 mol/l HCl in a boiling water bath, conditions known to convert 4-pyridoxic acid to its lactone [5]. Furthermore HPLC of the reaction mixture before and after showed loss of the 4-pyridoxic acid peak and the appearance of a peak of different retention time not corresponding to any other known compound. Incubation studies Purification of pyridoxal kinase enzyme source Rat liver extract was obtained by the method of McCormick [3] by homogenising one part of fresh tissue with four parts of 0.07 mol/l potassium phosphate buffer, pH 6.25, and centrifuging at 25 000 X g for 30 min. Supematant was separated and 1 mM pyridoxal was added and warmed with stirring to 55 “C and held at this temperature for 15 min. The mixture was chilled on ice and centrifuged at 18 500 x g for 15 min and supernatant was separated to yield an enzyme mixture capable of phosphorylating isopyridoxal. Incubation mixtures Incubation mixture ‘A’ containing 0.5 mmol/l isopyridoxal, 0.5 mmol/l ATP, 0.01 mmol/l Zn2’ (as the sulphate), 0.07 mol/l potassium phosphate buffer, pH 6.25 and 0.50 ml of pyridoxal kinase extract in a total volume of 2.5 ml. Incubation mixture ‘B’ contained no ATP but was otherwise the same. Mixtures were incubated at 37” C for 3 h and the reaction was stopped by deproteinising with 50 ~1 of concentrated perchloric acid. Vitamin B6 profiles were examined by HPLC [l] using 50 ~1 injections. Results Plasma vitamin B6 profiles after pyridoxine hydrochloride ingestion Table I displays the mean values of plasma-vitamin B6 compounds in healthy individuals after administration of pyridoxine hydrochloride orally in the doses shown for 1 week. Results are given as mean + SD for all B6 compounds except for pyridoxine where medians are used, since plasma pyridoxine concentrations were not normally distributed. The non-Gaussian distribution could have been due to at least two reasons. First, blood was drawn only approximately 4 h after pyridoxine administration and since the half-life of pyridoxine in plasma is about 40 min [l], small differences in timing of blood samples would be significant. Second, dietary restrictions were not imposed on subjects under study.
70 TABLE
I
Plasma vitamin subjects
B6 compounds
after 1 wk of oral pyridoxine
PNP’
Dose
PLP
PL
10 6
0 10
(34)73 595
(17; 472
_ -
9
25
(287) 631
(393) 231
-
100
(158) 518
(157) 1277
200
(130) 623
(678) 2441
8
400
(138) 732
7
800
(202) 644
(904) 4 764 (1664) 9484 (1616)
No. of subjects
PN
hydrochloride
administration
in healthy
4-PA
Peak 1
Peak 2
(1: 295
i
-
(104) 371
-
_
9 9
(182)
Results are expressed in nmol 1-l plasma for are expressed in peak height ml-’ plasma. Pyridoxal phosphate = PLP; Pyridoxal = PL; l&2. B6 compounds are expressed as mean ( f
-
(175) 1239
116
124
(60) 269
(464) 2200
(49) 208
389
(213) 120
(633) 3717
(147) 401
(318, 100
4664
(-) 245
(922) 7085 (1528)
(221) 1656
(28) 394
(506)
(196)
268
350
(-)
-
PLP, PL, PNP and 4-PA. Peak 1 and Peak 2 metabohtes Doses are in mg of pyridoxine hydrochloride per day. Pyridoxine = PN; Peak l&2 = unidentified metabolites SD) except for PN where medians are used.
It can be seen that the plasma concentrations of pyridoxal, pyridoxine and 4-pyridoxic acid rose linearly with increasing pyridoxine hydrochloride dose. Unknown peak 1 appeared at doses greater than 100 mg of pyridoxine hydrochloride per day, and unknown peak 2 appeared at doses greater than 200 mg per day. The concentration of pyridoxal phosphate in plasma showed little variation between doses of 10 mg and 800 mg of pyridoxine hydrochloride per day. Evidence for authenticity of synthesized isopyridoxai The ultraviolet spectra for the synthetic product in both 0.1 mol/l sodium hydroxide and 0.1 mol/l hydrochloric acid were similar to those reported for the natural product present in certain bacteria [3] (Fig. 1). The manufactured isopyridoxal was chromatographically homogeneous by HPLC and chromatographed between pyridoxal and pyridoxine when added to a mixed standard containing pyridoxal phosphate, pyridoxal, pyridoxine and 4-pyridoxic acid. Identity of unknown metabolite, ‘peak 2’ The unknown metabolite corresponding to peak 2 chromatographed between pyridoxal and pyridoxine (Fig. 2a). When plasma obtained from a person 4 h after a single 800 mg dose of pyridoxine hydrochloride was supplemented with a small quantity of synthetic isopyridoxal, an increase in the peak size of peak 2 was observed when chromatographed by HPLC at both pH 3.38 and pH 3.12 (Fig. 2b), without any peak splitting or separation. This suggested that the unknown metabo-
71
ISOPYRIDOXAL
ISOPYRIDOXAL
IN O.lM HCI
IN O.lM NaOH
Wavelength
Wavelength
Fig. 1. UV spectra of isopyridoxal in 0.1 mol/l HCI and 0.1 mol/l NaOH.
lite corresponding to peak 2 was isopyridoxal. However, 4-pyridoxolactone was found to co-chromatograph with isopyridoxal and peak 2 metabolite at pH (Table II). When the eluent was adjusted to pH 5.78, separation between compounds was satisfactorily achieved. It was seen that isopyridoxal did co-chromatograph with peak 2 metabolite at the pH, but Cpyridoxolactone
also 3.12 the not did
TABLE II Retention times of Cpyridoxolactone, by HPLC pH of eluent
3.12 5.78
isopyridoxal and plasma ‘peak 2’ metabolite at pH 3.12 and 5.78
Retention time @in) Isopyridoxal
CPyridoxolactone
Peak2 in plasma
Isopyridoxal +Peak2
CPyridoxolactone +peakz
11.75 10.18
11.98 12.91
11.76 12.51
11.80 ’ Isopyridoxal at 11.35 Peak 2 at 12.25
11.64 ’ 11.28 ’
’ Co-chromatography.
72
4-PA B. A.
4-PA
PN Peak
PN
2
I
PL
c P
SP 1 Fig.
2. Comparison of chromatograms pyridoxine hydrochloride
of (A) a plasma sample from an individual and (B) the same sample with added isopyridoxal.
given
800 mg
co-chromatograph with peak 2 metabolite showing that peak 2 metabolite is probably 4-pyridoxolactone. Acidification of 4-pyridoxic acid standards with perchloric acid at room temperature did not produce any lactone from 10 ng samples of treated 4-pyridoxic acid, and so does not appear to be an artifact. Metabolic Effect dose of elevation nmol 1-l elevated ingestion
studies of isopyridoxal
in man
of 50 mg ingestion of isopyridoxal hydrochloride Ingestion of a 50 mg oral the prepared isopyridoxal hydrochloride by a normal man caused an in plasma pyridoxal phosphate concentration from a basal value of 130 to a peak of 268 nmol 1-i after 3 h. Plasma pyridoxal phosphate was within 15 min (Table III). The major peak in plasma after isopyridoxal has a retention time corresponding to that of peak 1 unidentified metabo-
13 TABLE
III
The effects of ingesting Time
PLP
Basal 15 min 30 min lh 2h 3h 4h 5h 6h 24 h
130 239 190 221 239 261 239 255 202 85
50 mg of isopyridoxal
hydrochloride
Isopl metabolite (corresponding to the retention time of Peak 1)
on plasma
PL
Isopl
_
_
_
560 635 360 225 165 85 50 30 _
349 388 172 _ _
50 50 35 25 35 15
_
_
PLP, Pyridoxal phosphate; PL, pyridoxal; Isopl metabolite, isopyridoxal metabolite. height ml-‘.
vitamin
B6 metabolism PN
4-PA
78 39 49 39 39 32 15 15
60 229 328 109 60 49 38 44 44 22
Isopl, isopyridoxal; PN, pyridoxine; 4-PA, 4-pyridoxic acid; Units as nmol 1-l except for Isopl metabolite; units, peak
lsopl
metabolite
PLP Q-PA lsopl
Y
Fig. 3. HPLC chromatogram
of a plasma
sample obtained 30 min post-ingestion hydrochloride.
of 50 mg of isopyridoxal
EFFECT OF INGESTION OF ISOPYRIDOXAL ON PLASMA LEVELS OF PYRIDOXAL PHOSPHATE
420 -1” Plasma pyridoxal phosphate1 nmoles L-l
400 _;j
R
t
I\
I ‘\
380-11 It
: I
1 \
’
:
’
I
\ \
I
\
I’
\
\ \
I
\
\ I
I \
:
1
I
‘.
180 160 140 120 100 80
----100mg
60
-
0
1
0
I
1.0
I
I
2.0
I
3.0
4.0
Time after
Fig. 4. Effect of ingestion of isopyridoxal
dose 50mg dose
1
5.0
1
6.0
I
24.0
ingestion/hours
on plasma levels of pyridoxal phosphate.
lite on chromatography at pH 3.12 (Fig. 3). The effects of a 50 mg dose of isopyridoxal on plasma vitamin B6 metabolism can be seen in Table III and Figs. 4, 5 and 6. A correlation coefficient of 0.917 existed between plasma isopyridoxal and the metabolite corresponding to the retention time of unknown metabolite peak 1 ( p = 0.010). Effects of 100 mg ingestion of isopyridoxal hydrochloride plasma results Ingestion of a 100 mg oral dose of the prepared isopyridoxal hydrochloride by a healthy man
EFFECT OF INGESTION OF ISOPYRIDOXAL ON PLASMA LEVELS OF ISOPYRIDOXAL METABOLITE
1000 950 900 I350
3
Plasma Peak l/ Peak ht. mm ml-1
Time after
Fig. 5. Effect of ingestion TABLE
of isopyridoxal
ingestion/hours
on plasma
levels of its metabolite.
IV
The effects of ingesting Time
PLP
Basal 5rnin 10 min 20 min 30 min 40min lh 3h 4h Sh 24 h
522 328 364 316 215 393 267 409 215 198
100 mg of isopyridoxal
hydrochloride
on plasma
Isopl metabolite (corresponding to the retention time of Peak 1)
PL
Isopl
_
_
235 165 800 715 115 790 250 175 110 _
59 98 383 250 393 201 20 20 _ _
_ 110 145 145 140 115 20 25 20 30
154
vitamin
B6 metabolism PN
4-PA
63 54 49 54 83 34 13 112 91 29
55 55 169 251 213 207 257 68 82 71 49
PLP, Pyridoxal phosphate; Isopl metabolite, isopyridoxal metabolite; PL, pyridoxal; Isopl, isopyridoxal; PN, pyridoxine; 4-PA, Qpyridoxic acid. Units, mnol 1-l except for Isopl and Isopl metabolite; units, peak height/mm ml-‘.
EFFECT
OF
PLASMA
INGESTION LEVELS
OF OF
ISOPYRIDOXAL
4bPYRlDOXlC
ON ACID
400_ 380_ 360 340-
Plasma 4mpyridoxic acid L_
320_
’
/ n moles
1
300_ 2802602&I)_
r( 1 \
9
220-
‘I
/
’\ \
r+
200_
I
180_
:
\
160-
I I
140-
,
120,
\
I
100-1,
1OOmg
-
50mg
dose dose
\\
‘\ \ \
4
-----
\ \ \
\
I i
80
I
60
40 20 0
i
!
0
I
1.0
2.0
3.0 Time
Fig. 6. Effect of ingestion
TABLE
0
0.5 1.0 1.5 2.0 2.5 3.0 4.0 5.0 24.0
after
5.0
6.0
24.0
ingestion/hours
on plasma
levels of 4-pyridoxic
acid.
V
The excretion Time/h
of isopyridoxal
4.0
of urinary
metabolites
after ingestion
4-Pyridoxic acid excretion (nmol min-‘)
_ 10 14 18 23 33 33 13 16 14
of 100 mg of isopyridoxal
hydrochloride
Isopyridoxal metabolite excretion (peak height/mm min-‘)
_ 57 x 103 65x10’ 64x lo3 112x103 80~10~ 58 x lo3 17x103 18~10~
77
caused an elevation in plasma pyridoxal phosphate concentration from a basal value of 154 nmol 1-i to a peak value of 524 nmol 1-i after 5 min. Plasma pyridoxal phosphate levels remained elevated for at least 5 h after the ingestion of the isopyridoxal. Again the major peak observed corresponded to that of unidentified, ‘peak 1’. These effects on plasma vitamin B6 can be seen in Table IV and Figs. 4, 5 and 6. A correlation coefficient of 0.969 (p < 0.001) existed between plasma isopyridoxal and the metabolite corresponding to the retention time of unknown metabolite peak 1.
Urine results The effects of ingestion of 100 mg of isopyridoxal can be seen in Table V. The metabolite chromatographing between pyridoxal phosphate and pyridoxal (‘peak 1’) was present in the urine and decreased over 24 h after the ingestion of isopyridoxal.
TEST
BLANK (water
in place of isopyridoxal 4-PA
I-PA
PNP
PNPI
Fig. 7. HPLC chromatogram of incubation of isopyridoxal with rat liver extract showing the generation of its metabolite corresponding to the retention time of peak 1 unidentified metabolite.
78 TABLE
VI
Incubation
studies of isopyridoxal
Values as peak heights/ml
following
metabolism
in rat liver extract
50 ~1 injections
A (+ATP)
B (-ATP)
PMP = 39 PM=13 PNP = 160 PLP = 86 Isopl metabolite = 62 PL =13 PN=80 Isopl = over scale 4-PA = over scale
PMP=17 PM=22 PNP=7 PLP=8 Isopl metabolite = 90 PL=18 PN = over scale Isopl = over scale 4-PA = over scale
Difference
156% 169% 196% J 91% t 45% t 38% tt_ _
PMP, Pyridoxamine phosphate; PM, pyridoxamine; PNP, pyridoxine phosphate; PLP, pyridoxal phosphate; Isopl metabolite, isopyridoxal metabolite (not present if isopyridoxal is omitted from the reaction mixture unpublished abbreviation); PL, pyridoxal; PN, pyridoxine; Isopl, isopyridoxal.
Urinary 4-pyridoxic acid excretion rate increased up to 3 h after isopyridoxal ingestion and then decreased. Other fluorescent peaks were present in urine but were not identified. Isopyridoxal and 4-pyridoxolactone co-chromatograph at an eluent pH of 3.12. Incubation
studies
After incubation of isopyridoxal in the presence of rat liver extract, the HPLC chromatogram of the mixture showed a peak with the retention time of ‘peak 1’ unidentified metabolite in plasma and urine after isopyridoxal ingestion (Fig. 7). In a second experiment ATP was omitted from the reaction mixture to examine whether the metabolite of isopyridoxal was phosphorylated (Table VI). All phosphorylated forms of vitamin B6 decreased in the absence of added ATP, but the peak height of the isopyridoxal metabolite increased, demonstrating that this was not a phosphorylated form of isopyridoxal.
Ingestion of pyridoxine hydrochloride in doses exceeding 200 mg per day was followed by the presence in the HPLC chromatograms of two unidentified forms of vitamin B6, ‘peak 1’ and ‘peak 2’ (Fig. 2), the plasma concentrations of which increased linearly with dose of pyridoxine hydrochloride (Table I). Peak 2 was initially thought to be isopyridoxal for three reasons. First, supplementation of plasma from a healthy individual 4 hours after ingestion of 800 mg of pyridoxine hydrochloride with synthetic isopyridoxal showed an increase in peak height (Fig. 2). Second, the relative retention of isopyridoxal corresponded with that of peak 2. Third isopyridoxal and peak 2 chromatograph between pyridoxal and pyridoxine with the same retention time at pH 3.12 and 3.38. However, 4-pyridoxo-
19
lactone was found to co-chromatograph with isopyridoxal and peak 2 metabolite at pH 3.12 and after adjusting the pH of the eluent to 5.78,4-pyridoxolactone was seen to co-chromatograph with peak 2 metabolite present in plasma but isopyridoxal did not co-chromatograph with peak 2 (Table II), demonstrating that peak 2 metabolite may be 4-pyridoxolactone. Isopyridoxal has previously been identified in bacteria [5]. Prior to this finding, isopyridoxal had been synthesized and examined for biological activity [5-71. In contrast to pyridoxal, isopyridoxal was inactive in supporting growth of Streptococcus faecalis, but could support the growth of Kloeckera brevis and Saccharomyces carlsbergensis, although not as efficiently as pyridoxal [7,8]. In rats however, isopyridoxal has been shown to have approximately 4% of the growth promoting activity of pyridoxal on a molar basis [5]. In other studies, isopyridoxal was proved to be unable to replace pyridoxal as a catalyst for various biochemical reactions, such as non-enzymatic transamination [8], cysteine desulfhydration or serine dehydration [9,10] in the presence of metal ions. We initially had an interest in isopyridoxal as the possible identity of peak 2 metabolite seen in human blood after ingestion of high therapeutic doses of pyridoxine hydrochloride. This metabolite, as discussed earlier, is probably 4-pyridoxolactone. Activity of 4-pyridoxolactone has previously been determined relative to pyridoxine hydrochloride activity where it was found to be 0.24 times as active in Streptococcus Iactis, 0.02 times as active for Lactobacillus casei and 0.00025 times as active for yeast [4]. Since the metabolism of isopyridoxal, to our knowledge, has never been investigated in man, we took this opportunity to conduct studies. The synthetic isopyridoxal hydrochloride used in this experiment was shown to be the authentic product by examination of the UV spectra in 0.01 mol/l HCl, which showed maxima at 284 nm and 230 nm (shoulder peak) and 0.1 mol/l NaOH with maxima at 244 nm and 296 nm (Fig. 3), corresponding to literature values for natural isopyridoxal [5]. In the present study, it has been demonstrated that ingestion by a human volunteer of a 50-mg oral dose of isopyridoxal hydrochloride causes an increase in the plasma concentration of pyridoxal phosphate of 2 times the basal level, and when a lOO-mg oral dose of isopyridoxal hydrochloride was ingested, plasma pyridoxal phosphate rose approximately 3.5 times that of basal level (Tables III and IV; Figs. 4-6). This proves that isopyridoxal is an active form of vitamin B6 in the human subject. This was supported by the plasma and urinary increases in 4-pyridoxic acid concentration (Tables III-V). Since isopyridoxal, like pyridoxine, possesses vitamin B6 activity for some organisms but not for others (see above) it may be considered a naturally occurring member of the vitamin B6 complex. To exhibit such activity, isopyridoxal would have to be transformed in vivo to pyridoxal phosphate, which must occur by preliminary reduction to pyridoxine. This capability differs between species, for example, more efficiently in yeast than in rats [5]. The results presented here suggest that isopyridoxal is converted to pyridoxal phosphate probably via pyridoxine in man and is also converted to another
80
metabolite which chromatographs with a similar retention time to ‘peak 1’ metabolite between pyridoxal phosphate and pyridoxal and appears in both plasma and urine. Incubation studies using rat liver extract showed that this metabolite of isopyridoxal is not a phosphorylated form. This agrees with Hurwitz .[ll] who showed that analogues of pyridoxine capable of being phosphorylated by pyridoxal kinase require a hydroxymethyl group in the 5 position of the ring and should be unsubstituted in the 6 position. Isopyridoxal does not satisfy these criteria. The true nature of peak 1 therefore remains unknown. Acknowledgements We would like to thank the staff of the Department of Child Health, Institute of Child Health, great Ormond Street Hospital for laboratory facilities and St. Peter’s Research Trust for the purchase of HPLC equipment. References 1 Edwards P, Liu PKS, Rose GA. A simple liquid-chromatographic method for measuring vitamin B6 compounds in plasma. Clin Chem 1989;35:241-245. 2 Sattsangi PD, Argoudelis CJ. Synthesis of a pyridoxal analog 4,5-diformyl-3-hydroxy-2-methylpyridine. J Org Chem, 1968,33:1337-1341. 3 McCormick DB, Gregory ME, Snell EE. Pyridoxal phosphokinases. J Biol Chem 1961;236:2076-2084. 4 Huff JW, Perlzweig WA. A product of oxidative metabolism of pyridoxine, 2-methyl-3-hydroxy-4carboxy-5-hydroxy-methyl-pyridine (Cpyridoxic acid). J Biol Chem 1945;155:345-355. 5 Rodwell VW, Volcani BE, Ikawa M, Snell EE. Bacterial oxidation of vitamin B6. J Biol Chem :958;233:1548-1554. 6 Harris SA, Hey1 D, Folkers K. The structure and synthesis of pyridoxamine and pyridoxal. J Am Chem Sot 1944;66:2088-2092. 7 Snell EE, Rarmefeld AN. The vitamin activity of pyridoxal and pytidoxamine for various organisms. J Biol Chem 1945;157:475-489. 8 Metzler DE, Olivard J, Snell EE. Transamination of pyridoxamine and amino acids with glyoxylic acid. J Am Chem Sot 1954;76:644-648. 9 Metzler DE, Snell EE. Catalytic deamination of serine and cysteine by pyridoxal and metal salts. J Biol Chem 1952;198:353-361. 10 Metzler DE, Longenecker JB, Snell EE. The reversible catalytic cleavage of hydroxyamino acids by pyridoxal and metal salts. J Am Chem Sot 1954;76:639-644. 11 Hurwitz J. Enzymatic phosphorylation of vitamin B6 analogues and their effect on tyrosine decarboxylase. J Biol Chem 1955;217:513-525.