A reassessment of tocopherol chemistry

Vol. 17, No. 5, 1964

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

ARRASSESSMRiTOFTOCOPREROLCHPlISTRY By J. F. Pennock, F. W. Hemmingand Joan D. Kerr, Department of Biochemistry, The University of Liverpool, Liverpool, 3, U.K. Received July29,

1964

This paper describes the presence in Nature of four methylated tocols (the &monomethyl, 5,8- and 7,8-dimethyl rivatives)

end the four related

McRele, Green and Marcinkiewicz, A new 'tocopherol'

tocotrienols

and 5,7,8-trimethyl

(for nomenclature see Bunyan,

1961).

from humanbone marrow

A survey of the unsaponifiable

lipids

of humanbone marrow (Kasim-

uddin, Morton, Hemmingand Pennock, 1964) revealed, compoundwhich reduced FeCl,, and showedhm, hexene, El?cm. for both maxima near 80). on silica p-

de-

gel (benzene/ Cl%.&, l/l)

.

in certain

cases, a

295 and 302 mp (cyclo-

Thin layer

chromatography (TLC)

showed the unknown had the sameRf as

and21-tocopherol but on using a solvent system capable of separating

these two tocopherols (Stove, 1963) the unknown had a lover Rf than either. Besides staining with Rumerie-Engel reagent, the unknown gave a colour with diazotised

o-dianisidine

showing the presence of a free 5-position An infrared

tocol system (Green & Marcinkiewicz,

1959).

unknown shoved that it was more like

II-tocopherol

pherol.

Hydrogenation (PtO as catalyst)

separated from X-tocopherol kiewicz

spectrum of the

than any other toco-

gave a product which could not be

by TLC. The nitroso

and Green, 1959) of /8-,

in a

X-tocopherol,

derivatives

(Marcin-

the unknown and hydroge-

nated unknown vere prepared and gave Rf values on TIC (silica

gel, CCL)

of 0.43, 0.62, 0.53 end 0.61 respectively. Since tocotrietisrelated

to p-

(Green, Memalis, Marcinkiewicz

end&-tocopherols

have been found

and McRale, 1960) it

seemedplausible

the unknown might be the tocotrienol ly 7,8-dimethyl

tocotrienol

related

to I-tocopherol.

that

According-

was synthesised (Coop and Pennock, 1964) by a 542

BIOCHEMICAL

Vol. 17, No. 5, 1964

AND BIOPHYSICAL

method used by Schudel, Mayer, Meteger, synthesis of other tocotrienols.

RESEARCH COMMUNICATIONS

aa Isler

Wegg

The product was identical

pherol from bone marrow with respect to ultraviolet

with dianisidine

and phosphomolybaic acid.

indistinguishable

by two-dimensional TIC.

spectra,

and colours produced

The two

were also

compounds

of 7 -tocopherol.

Since /3 - and X-tocopherols paper chromatography (Analytical possible that the tocotrienols together.

with the toco-

and infrared

Rf values on TIC, Rf values of nitroso-derivatives

The identity

(1963) for the

Methods Committee, 1959) it related

In fact 7 -tocopherol

tocotrienol)

are not separable by two dimensional

to these tocopherols might run

runs with f -tocopherol

and so we decided to check the identity

Green and Marcinkiewice

(1956)

seemed

identified

(5,8-dimethyl

of '3 -tocopherol.

T -tocopherol

from rice as 7-

methyl tocol but the evidence presented did not preclude our considering the possibility

of its being 7,8-aimethyl

also been described in palm oil

tocotrienol.

T -Tocopherol has

1958) ma so the nature of 3 -toco-

(Ward,

pherol from these two sources was reinvestigated. Unpolished rice with ether/light

(purchased locally)

petroleum (l/l)

was ground finely

in a Soxhlet and the oil

saponified by the recommendedmethod (Analytical Samplesof crude palm oil saponified similarly.

direction petroleum.

so obtained was

Methods Committee, 1959).

given by Merseyside Food Products) were

Aliquots of the unsaponifiable

containing 15-30 ug. total with silica

(kindly

and extracted

lipid

(0.3 - 0.5 mg.)

tocopherol were spotted on 20 x 20 cm. plates

gel'as adsorbent and the chromatogram was developed in one with CHCl, and in the second with 20% isopropyl ether/light In the CHcl, tank (lined with pager) good separations of

s-9 from/l-+?Ipropyl ether/light

and from&-tocopherols petroleum (freshly

were obtained while 2C%iso-

madeup and tank unlined) gave good

separations of p - and ~3-tocopherols (see Stowe, 1963) and also separated the methylated tocols from their 543

related

tocotrienols

(see fig&.

Vol. 17, No. 5, 1964

BIOCHEMICAL

AND BIOPHYSICAL

* ether/petrol

2c$ isopropyl

I

CHcl,

RESEARCH COMMUNICATIONS

00 QW Q

x Figure1 Two dimensional TLC of a tocopherol mixture t = = *

aC-tocopherol $ -tocopherol -tocopherol C-tocopherol I!2f-tocopherol

2' P

6 -tocopherol -

1 2 3 4

(5,7,8-trimethyl tocol) (5,7,8-trimethyl tocotrienol) (5,8-dimetbyl tocal) (5,8-dimethyl tocotrienol) (7,8-dimethyl tocol) (7,8-dimethyl tocotrielwl) (8-methyl tocol)

As can be seen in figure 1, spot 4 representsc If-Tocopherol not.

gives a stain vith

dianieidine

T?Jshov the presence of E -tocopherol

- plus x-tocopherol.

whereas E docopherol if

\I-tocopherol

present the two tocopherol.s have to be separated.

does

is also

This can be achieved

by reversed-phase chromatography or by chromatography'of

the nitmso

derivativea. Rice shoved three main spots vliich vere clearly 9 -tocopberPls. position

d-,

c-

and

So smaller spots vere present which vere identified

andbyneasw~ofnotstainingvith

dianlsidine

as /3-

and

E -tucopherols. The c)7-tocopherol ran in the sameposition as 7,8-dimethyl tocotrianol and this was confirmed by adding a sample of the latter

before chzwmatography. When the rice unsaponifiable

hydrogenated (FtO catalyst)

the spot in the (3-position

a large spot appeared in the 8 -tocopherol 544

position

lipidwas

disappeared and

which stained with

by

BIOCHEMICAL

Vol. 17, No. 5, 1964

dianisidine

and was clearly

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

&'-tocopherol

was added to rice unsaponifiable

lipid

When synthetic

and the mixture chromatographed

the 7-methyl tocol appeared as a distinct

spot being slightly

is not 7-methyl tocol but is 7,8-dimethyl similarly

appeared to be “c-, material.

In a manner identical

"3 -tocopherol toco1.

from palm oil

from rice

tocotrienol.

gave five distinct

5 -, 7 -,

more polar

Thus 7 -tocopherol

in both solvent systems than v -tocopherol.

Palm oil

7-methyl tocol

reducing spots on TLC. These

E -tocopherola

and a

8 -tocopherol-like

as for rice it was shown that the

was 7,8-dimethyl

tocotrienol

and not 7-methyl

Thus there is no evidence at present that 7-methyl tocol appears

naturally. The identity

oft

-tocopherol

Two $-tocopherols was originally -Watt (1955) c-tocophgrol McHale,

are believed to occur in Nature.

later

and

by Green and Marcinkiewicz

wae identified Mamalis

pherol in wheat and palm oil was different

es 5,7-dimethyl and Watt

8-trimethyl

tocotrienol)

of $-tocopherol

the d-tocopherol

from

tocotrienol

tocol).

rice the <-tocopherol

When synthetic

more polar thang-tocopherol of rice behaved in exactly

pherol from palm oil 5,7,8-trimethyl

ran

in the

and on hydzvgenation no epot appeared there but

from any of the rice tocopherols was observed.

The c-tocopherol

but the rice

to these two compoundsas c-(5,7,

spot had increased in size.

tocol waa a little

Green,

tocol as originally

tocol was chromatographed together with rice unsaponifiable distinct

The

More recently

and CL-tocophezP1 (5,7-dimethyl

When examining the tocopherols

and

(1959) claimed that thec-toco-

and was 5,7-dimethyl

Dunyan et -- al. (1961) refer

(19561 in rice.

tocol.

was 5,7,8-trimethyl

suggested.

position

P-Tocopherol

found in wheat, barley and rye by Green, Marcinkiewicz

Marcinkieuicz,

c-tocopherol

in rice.

545

a spot

5,7-Dimethyl

the samemanner as c-toco-

-tocopherol.

momentfor the existence of 5,7-dimethyl

lipid

of rice in both systems.

and the only <-tocopherol

tocotrienol,<

5,7-dimethyl

tocol.

80 far found in Nature is There is no evidence at the

Vol. 17, No. 5, 1964

A

BIOCHEMICAL

new 'tocopheroll It

saponifiable

When synthetic lipid

that palm oil

S -tocopherol

from palm oil

unknown was iu a position if

RESEARCH COMMUNICATIONS

from palm oil.

was mentioned earlier

compound.

AND BIOPHYSICAL

contained a 8 -tocopherol-like

uas chromatographed with the un-

it appeared as a distinct

which could be assigned to 8-methyl tocotrienol

such a compoundexisted.

The new 'tocopherol'

could be separated from

tocol by two dimensional TLC and gave a slate-coloured dine, similar

to that of s -tocopherol

given by tocol.

but different

stain with dianisifrom the brown colour

On hydrogenation the new 'tocopherol' This new 'tocopherol'

s-tocopherol.

spot and the

can be identified

was converted into as 8-methyl

tocotrienol. A revised picture We

of tocopherol chemistry.

have offered

evidence for the presence in Nature of four tocols

and four tocotrienols.

The 8-methyl group is present in all

pounds which are 8-methyl; derivatives

5,8-dimethyl;

of tocol and tocotrienol.

7,8-dimethyl

and 5,7,8-trimethyl

Table 1 shows the revised posi-

tion of tocopherol chemistry compared with the position

A

these com-

hitherto.

Table 1 Revision of Tocopherol Chemistry

Previous position

Present position

I

7-methyl tocol CT)) ) 8-methyl tocol ( 8 1 5,7-dimethyl tocol <$ )

a-methyl t0c0i (S 5,8-dimethyl toco1 7,8-dimethyl tocol

5,bdimethyl tocol

5,8-dimethyl tocotrienol (e ) 5,7,8-trimethyl tocotrienol (
5,7,8-trimethyl tocol (d ) (j-methyl tocotrienol 5,8-dimethyl tocotrienol (E ) 7,8-dimethyl tocotrienol 5,7,8-trimethyl tocotrienol&

546

1

(&3 1 ( II )

Vol.

17, No.

BIOCHEMICAL

5, 1964

The

series

authors

suggest

($,EandT

d-9

P-9

BIOPHYSICAL

the Greek

that

RESEARCH

for the

letters

COMMUNICATIONS

tocotrienol

) be dropped and these compounds be referred

2f -9 anda-tocotrienols

pherols. 43,

AND

We have found it

related convenient

to d-,

side chain of these

tocotrienols

If-

and 6 -toco-

to use the abbreviation6

to denote the presence

Y(:, and 8,

p -,

to a6

of three

dl,

double bond6 in the

(see Table 2).

Table 2 A suggested Substitution

for Tocopherols

nomenclature

Tocopherol

Tocotrienol

&methyl

%-tocopherol

(6 T)

8 -tocotrienol

(6,T)

5.8~dimethyl 7,8-dimethyl

p-tocopherol Y-tocopherol

@T) ( 8T)

B-tocotrienol Y-tocotrienol

(6,T) (\(,T)

5,7,&trimethyl

O(-tocopherol

(dT)

d-tocotrienol

&,T)

Metabolic Relations An interesting contains that

all

of the Tocopherols picture of tocopherols

four tocotrienols

the four tocotrienols

-tocopherol.

&Methyl

to form 5,8-

and&ocopherol.

tocotrienol

and 7,8-dimethyl

which

is then saturated

It

has a tocopherol

at the required is interesting

8 -tocopherols but&-tocopherol

occurred

2( -tocopherol

occurs other

of d

predominating)

to 5,7,8-trimethyl

before

arises

and is then methylated

(the latter

methylated

which

and

tocotrienol

tocol,d-tocopherol. saturation

than d-tocopherol

It

of the side chain. then saturation

step.

(1961) found that&,

that Baszyn ski during

disappeared

He found that&-tocopherol

is made first

to form .5,7,8-trimethyl

would appear that methylation

occurs

in the biosynthesis

tocotrienols

two compound6 are further

by palm oil

The possibility

are intermediates

these

If a plant

is presented

the first

days of germination

by the time a height increased

and that methionine

with

547

and

of the pea

of 3 cm.was reached.

a simultaneous

and ATP stimulated

O(-tocopherol.

v-

decrease

the synthesis

in of

Vol. 17, No. 5, 1964

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

It remains to be seen whether tocotrienol Nature or whether 8-methyl tocotrienol latter

is the firat

is the case it is possible that a related

geranyl-geranyl-1:4-benzoquinone It

itself

is hoped to publish fuller

tocotrienols

will

be found in

product.

If

the

quinone, 3-methyl-5

occura in plants. details

of the properties

of the two new

later.

We thank Dr 0. Isler Ltd) for generous gifta

(F. Roffmann-La Roche) and Dr D. &Hale (Vitamina, of chemicals. References

Analytical Methods Committee (1959) Analyst, &, 3~6 T. Baazynski (1961) Acta Sot. Botan. Polon. z, 307. J. Bunyan, D. McRsle, J. Green & S. Marcinkiewicz (19611.

Brit.

J. Nutr.

J$, 253. R. J. J. J. J.

L. Coop & J. F. Pennock (1964) Unpublished work. Green & S. Mercinkiewica (1956) Nature. 1 86. Green-& S. Marcinkiewice (1959) Analyst. -2i;4, 297. Green, S.Marcinkiewicz & P. R. Watt (1935)J. Sci. Food Agric., 6, 274. Green, P. Mamalis, S. Marcinkiewicz & D. HcIiale (1960) Chem. & Ind.

S Marcinkiewicz, P. Hamalia & P. R. Watt (1939) Sot. ;. 5362. H. Kasimuddin, R. A. Morton, F.W. Hemming& J. F. Pennock (1964) Unpublished work. S. Marcinkiewicz & J. Green (1959) Analyst, a, 304. P. Schudel, B. Mayer, J. Netzger, R. RUegg& 0. Ialer (1963) Helv. Chim.

J.

Grk73i

McHde

J. ihi.

Acta

$6, 2517.

II. D. Stowe (1963) Arch. Biochem. Biophya. B, R. J. Ward (1958) Brit. J. Niatr. Y, 231.

548

42.