Die Nahrung 26, 4.

1982, 363-367

Institute of Human Nutrition (Director: Prof. Dr. S. STAWICKI)

University of Agriculture in Poznan. Poland

Critical evaluation of the current studies

on pangamic acid - vitamin BI5

E. LAMPART-SZCZAPA
and J. SKUPIN

The research on pangamic acid (vitamin Bls) has been conducted by specialists in many fields of science
for a long time. This, however, has not put an end to controversies around the problem of its therapeutic
effect. The solution to the basic problems, i.e. defining the compound structure. finding an explicit method
of its determination and identification as well as the evidence of its biochemical and physiological functions
in the human organism appears as distant as ever. It has been found out, however, that either some prepara-
tions referred to as pangamic acid (vitamin B,s)o r their components may be detrimental to man*shealth.

Although the interest in pangamic acid has been aroused for many years, the attitude
towards the compound which is supposed to be "good for everything" has changed re-
cently. It has been also indicated that apart from not being characterised concering its
therapeutic properties it can be probably harmful. A lot of contradictory data in the de-
termination biological occurence seems to keep the problem of vitamin BI5 still open.
In the present paper some problems are discussed with respect to critical remarks. The
compound in question is classified in the vitamin group without convincing proofs. In
addition, preparations being referred to as ,,pangamic acid" are also presented.

The preparation of pangamic acid

Although almost 30 years have passed since the discovery of pangamic acid by KREBS
the details of the method of the compound isolation from plant raw material are still un-
known. I t was obtained by means of aqueous extraction: the extract produced was in
turn fractionated through precipitation and the vitamin B,, was eluted with distilled water
[I]. Thus the method of isolation of vitamin B,, does not seem to be complicated. However,
it can't be as simple as the authors considered it to be because no details concerning pan-
gamic acid production have been published so far. Hungarian scientists followed KKEBS'S
procedure uncritically. TELGDY KOVATSet al. [3] described the method of producing vit-
amin B,, from plant raw material. The following stages can be distinguished: aqueous
extraction, protein removal. purification on active carbon, separation of vitamin B,,
from vitamin B, and 9, thin-layer chromatography with the use of non-specific identi-
fication method (which will be presented later on). It is questionable whether by mean5
of this method based on aqueous extraction with a great deal of different water-soluble
compounds in plant raw material pangamic acid can be obtained. It is also worth consi-
364 LAMPART-SZCZAPA/SKUPlN

dering that KREBShimself, who described the method of chemical synthesis of vitamin B,,,
could not make a decision as far as its chemical structure was concerned. Finally, it was
estimated [4] that pangamic acid constitutes glucono-6-acetyl-N-dimethylamine. In KREBS’S
patent concerning pangamic acid synthesis, derivatives of this “vitamin” containing 4,
8 and 12 methyl groups were described [4]. Such ambiguity connected with obtaining and
structure of vitamin B15 resulted in speculations on the part of producers. CAWet al. indi-
cated [5] that on the market there are preparations characterised by vitamin B,, thera-
peutic effect, which are merely mixtures of such compounds, e.g. calcium gluconate, sly-
cine, dichloroacetate-N-diisopropylamine (Italian preparation), sodium gluconate or
calcium gluconate, dichloroacetate-N-diisopropylamine(Japanese preparation).

Determination and identification of pangamic acid

The above-mentioned difficulties in obtained and establishing the compound structure

are the result of problems connected with finding a selective, optimal and reliable method
of pangamic acid determination.
To study the chemical structure of pangamic acid in synthetic preparations spectro-
metric methods were used [2, 3, 6-81, Measurements employing ultraviolet and infrared
spectrophotometry are not considered as the basis for an explicit cvaluation of the che-
mical composition of the compound as suggested by KREBS.Hungarian scientists, [2, 3, 61
identify pangamic acid in the infrared absorption spectrum on the basis of the 1750 cm-’
band which is characteristic of ester bonds. These results were questioned by BELLAMY
who found out that the bands at the same wave length could also be found with compounds
other than esters 191. Also according to MICHEAU [7] the UV spectrum is not an adequate
structural criterion as different compounds can be characterised by similar spectra. I t
should be noted that in the case of the Russian calcium pangamian preparation maximum
absorption values determined by TELEGDY KOVATS[2] and JANICKI [lo] were different
from each other and were 250 nm and 290 nm, respectively. Descriptions of determining
vit. B,, by means of colorimetric analysis can be found in literature. The attempts to apply
GONCZAKOWA’S method of hydroxylaminolysis [I I] and MAI.AKOWA’S method [12], i.e.
the use of the reaction of dimethylglycine along with FOLIN’S reagent were suggested by
the authors for routine analysis. However, these methods proved to be unsuitable for
this purpose. The insufficient level of accuracy as well as the lack of universality resulting
from the methodological assumptions, eliminates the use of these methods for the quan-
titative determination of pangamic acid.
Another method, worked out by KRAZNER et al. [13] based on the reaction of pangamic
acid solved in dimethylformamide and methyl iodide is of no vital importance. This is
due to the fact that dimethylformamide can be decomposed into dimethylamine and the
latter can react with methyl iodine. Moreover, this method cannot be used for tlhe tleter-
mination of vitamin B,, in biological material. The above mentioned Hungarian :scientists
elaborated also methods of vitamin BI5 determination by means of thin-layer chroniato-
graphy [2. 31. These methods were questioned by Skupin et al. [8] who proved that the
compound identified as pangamic acid is really dimethylglycine. which is a product of
vitamin B,5 hydrolysis. This was also proved by extensive studies of thin-layer chromato-
graphy to determine and identify vitamin B L 5 [14]. Despite numerous experinicnts no
developing system ensured the proper separation of this compound with the use of thin-
Pangamic acid 365

layer chromatography. Thus the use of the non-specific method for the determination
of the occurrence of pangamic acid - a compound whose structure has not been finally
established - in plant raw materials in a mistake on the part of TELEGDY KOVATSet al.
12, 31.
Although gas chromatography of silyl derivatives is commonly used for studying poly-
hydroxyl compounds it is not suitable for vitamin B,, identification since it causes the
immediate breaking of ester bonds under the influence of either silyl compounds or on
the chromatographic column [7].

Evaluation of the therapeutic effect of pangamic acid

Although pangamic acid has been widely used for medical purposes, its biochemical
and physical functions in the human organism have not been specified so far. Pangamic
acid requirements of the human body with regard to biological functions of other potential
methyl group donors have not proved either. As pointed out previously, discussion on
the therapeutic effect of vitamin B,, emphasize the fact that instead of being one specific
substance, it consists of many different compounds, both synthetic preparations and mix-
ture components similar to vitamin B,, and unsynthesized components or synthesis by-
products. For example, a Russian synthetic preparation contains not less than 70 percent
of calium salt of ester of d-gluconic acid and dimethylglycine, not more than 25 per cent
of calcium gluconate and not more than 6 per cent of calcium chloride which may be toxic
1151.
KREBSand other scientists use the term pangamic acid for the product which in their
opinion may be either isolated from different plant raw materials or produced as a result
of synthetic preparation. The presence of chloride ion in the synthetic preparation of vit-
amin B,, is contrary to the assumption that this form of preparation can be obtained from
natural sources. When discussing some vitamin B,, preparations, KRAUSHAAR [ 161 points
out that some of their components, namely gluconic acid and glycine, are passive and
another component - diisopropylamine, lowering blood pressure and body temperature
-- is a toxic substance of no nutritive value. The name “pangamic acid” is connected with

its occurrence in seeds and fruit stones which may also be a source of cyanide.
Vitamin B1, is assumed to take part in various biological processes connected with
methylation. The most important question - does the human body require vitamin BI5
in the presence of other methyl group donors - has not been answered yet. Even if vit-
amin B,, were as important as it is assumed not all vitamin B,, preparations can fulfill
methylation functions. It was proved [17] that some of them contain non-labile methyl
groups : Diisopropylamine, for example (without an additional source of energy), cannot
be a donor connected with methyl group carbon. Another component, i.e. dimethylgly-
cine can be harmful to man’s health. It was proved that dimethylglycine introduced into
the diet of pregnant rats may cause embryo damages [IS]. The reaction of this compound
with “de novo” nitrates in human saliva, resulting in the carcinogenic compounds dimethyl-
nitrosamine and nitrosarcosine, may be another example of its negative effect [19, 201.
It is assumed that free dimethylglycine in some vitamin B,, tablets is more carcinogenic
than the same amount of the compound in I 0 0 g meat. Besides, preparation may not con-
tain dimethylglycine but its hydrochloride which is characterised by different properties
and reactivity.
366 LAMPART-SZCZAPA/SKUPlN

American doctors maintain that the exact composition of vitamin B15 has not been
identified yet, therefore they insist on determining which of the many mixtures known
as preparations is the proper substance. According to The Human Studies Committee
patients should be informed that pangamic acid is not a vitamin, has no nutritive value,
and when taken too long may be dangerous, and may cause cancer [17, 211. In Canada,
selling vitamin B,, is forbidden. It was also removed from the 10th impression of Merck’s
Catalogue. The editor’s decision was based on the lack of the compound identification
standard. In his opinion at present the name vitamin B15 denotes either all similar com-
pounds or none of them.
It is difficult to ascertain with precision whether the results discussed disqualify pangamic
acid completely. One thing is certain - as long as all dubious points have not been ex-
plained, the application of the preparation should be limited.

Zusammenfassung

E. LAMPART-SZCZAPA Kritische Beurteilung des Forschungsstandes zur PangaminsLure -

und J. SKUPIN:
Vitamin B,,

Langjahrige und vielseitige Forschungen zur Pangaminsaure vertieften die Kontroversen und Vorbehalte
zum Themd der therapeutischen Wirkung dieser Verbindung. Die grundsatzlichen Probleme, d. h. die Bestirn-
mung der Struktur dieser Verbindung, das Auffinden eindeutiger Methoden ihrer Kennzeichnung und
Identifizierung sowie der Nachweis der biochemischen und physiologischen Funktionen, die im Organismus
des Menschen stattfinden, bleiben weiterhin ungelost.
Es wurde festgestellt, daB einige Praparate, die Pangaminsaure (Vitamin B,s)oder deren Koniponenten
enthalten. gesundheitsschadlich sein konnen.

References

[I] KREBS,E. T.. H. H. BEARD,R. MALIN,A. F. HEWIS,and C. L. BARTLET, Int. Record Med. 164, 18-24
(1951).
[2] TELEGDY KOVATS,L., E. KRASZNER-BERNDORFER, and A. DEVAI,Z. Lebensmittel-Untersuch. u. -Forsch.
139, 61 (1969).
(31 TELEGDY KOVATS,L., E. KRASZNER-BERNDORFER, M. PETERFALVI, and T. GABOR, Z. Lebensmittel-
Untersuch. u. -Forxh. 143, 41 1 (1970).
[4] KREBS,E. T. sr., and E. T. KREBS,jr., US Patent 2710876, 1955.
151 C A W . B., A. DANSI,M. REGGIANI, and C. ZANINI,Boll. Chim. Farm. 97,3-8 (1958).
Pangamic acid 367

[6] TII-FGI~Y KOVATS,L.. E. KRASZNER-BERNDORFER, I. PAP,T. GABOR,and L. SZTARKA, Nahrung 23, 775
(1979).
[7] MICHEAU. J. C.. A. LATTE.S,M. PODESTA, G . REUX,and B. GAYREL, Bull. Chem. Terap. 2, 103 (1971).
[8] SKUPIN, J., and A. GIEC.Chemia analit. 15, 1127 (1970).
[9] B ~ L L A M YL., J., The infrared spectra of complex molecules, p. 178. Methuen, London 1964.
[lo] JAUICKI. J., J. SKUPIN. and A. GIEC. Bull. Acad. Pol. Sci. 16, 273 (1968).
[I 11 GONCZAROWA, L. A,. J. A. RUBCOW.and A. B. STARKOW, PiSE. Prom. 2. 164 (1972).
1121 MALAKOWA, U. B., Farmdcia 4, 62 (1972).
[ 131 KRASZNER-BERNDORFER. E., L. TELEFDY KOVATS,and A. ABAI,2. Lebensmittel Untersuch. u. -Forsch.
154. 257 (1974).
[I41 LAMPART-SZCZAPA, E.. Doniesienie
~ XI1 Sesja Kom. Tech. i. Chemii Zywn. 1981.
[IS] BUKIX.W. N., Pangamat Kalia, vitamin B,5, Izdatelstvo Nauka, Moskva ss. 48. 1968.
[16] KRAUSHAAR, V. A. E., W. SCHUNK.and H. F. THYM,Arzneimittel-Forsch. 13. 109 (1963).
1171 HERBERT, V., Amer. J. clin. Nutrit. 32, 1534 (1979).
[IS] WOODWARD, J. C., J. Nutrit. 100, 1215 (1970).
[ 191 FRIEDMANN. M. A.. Bull. Environm. Contamin. Toxicol. 13,226 (1975).
[20] TANNENBAUM. S. R., D. FETI,V. R. YOUNG,P. D. LAND,and W. R . BRUVE.Science 200, 1487 (1978).
[21] JUKES,T.H.. Nutrit. Notes 14. 13 (1978).

Dr. E. LAMPAR'r-SzczAPA. and Prof. Dr. habil. J. SKUPIN.University of Agriculture, Institute of Human
Nutrition, PL-60-623 Poznan, Mazowiecka 48

Eingegangen 24. 7. 1981