Correspondence concerning this article should be addressed to: Dr. Karin Björkegren Department of Public

Health and Caring Science, Family Medicine Uppsala Science Park, SE-751 85 Uppsala, Sweden. e-mail:

karin.bjorkegren@pubcare.uu.se.

2 Karin Björkegren

Keywords: Vitamin B12, deficiency, homocysteine, oral therapy.

Treatment of vitamin B12 deficiency with oral high-dose cyanocobalamin is a medical

tradition more or less unique to Sweden [1,2]. The regimen was introduced by Berlin and coworkers in 1964 [3]. By 1990, as many patients were treated with tablets as with injections.

In the period 1990-2000, the Swedish experience with oral vitamin B12 comprised about one

million patient years [1,2]. The total experience in the period 1964-2005 comprises more than

three million patient years (Mats Nilsson, personal communication).

The reason for the success of oral vitamin B12 in Sweden is thought to lie in the sevencrown reform of 1970, which made the cost of oral or parenteral B12 therapy equivalent for

physician and patient [1,2]. Thus, oral vitamin B12 for deficiency treatment steadily gained

confidence by experience in Swedish health care in the period 1964-2000.

The most comprehensive documentation of oral vitamin B12 therapy was performed by

the Berlin group (3). It should, however, be emphasized that the Berlin group worked within

an international network of about a hundred scientists, as deemed from the reference list of

the Berlins [3]. During the period 1950-1965, the basic mechanisms of vitamin B12

absorption and metabolism had been discovered. Due to the introduction of the Schilling test

for B12 malabsorption, the possibility for oral treatment of B12 deficiency had come into

focus.

In the period 1950-1965, oral treatment with vitamin B12 was distinguished by current

relapses in cases of B12 deficiency. Indeed, the clinical model of “pernicious anemia in

relapse” was a favorite tool of documentation. A few micrograms of cobalamin could trigger

a shift of iron parameters as a sign of revived erythroblast maturation, reticulocytosis,

maximal hemoglobin rise. However, body stores of vitamin B12 were still exhausted. Thus,

relapses were more rule than exception. The contribution of the Berlin group was a

comprehensive documentation of how to avoid relapses in oral B12 treatment in deficiency

states [3,4,5].

Although lucid to contemporary scientists, the concise final report of the Berlin group

has been subject to misunderstandings by modern medicine [3,4,6,7]. Their experimental

studies of vitamin B12 pharmaco-kinetics started about 1955 in Eskilstuna and Falköping.

The dose of radioactive cyanocobalamin was 0.5 mg. The results on 10 healthy probands and

64 patients with B12 malabsorption were presented at an international congress in Hamburg

in August 1961 [3].

The experimental data of the Berlin group, as well as of other scientists, were analyzed

by GBJ Glass in a major review in 1963 [5]. He concluded: “The daily oral administration of

1 mg cyanocobalamin thus not only provides safe maintenance therapy, without danger of

refractoriness or relapse for patients with pernicious anemia, but also maintains normal

concentrations of B12 in blood serum”.

The clinical studies of oral vitamin B12 appear to have started in the beginning of 1961,

when Ragnar Berlin returned to Linköping from a one-year appointment at the Swedish

education hospital in South Korea. The dose was 0.5 mg cyanocobalamin daily, in

accordance with the pharmaco-kinetic studies of the group. Furthermore, it took some time

for the message from Glass to sink in [3,5].

Commentary 3

During 1962, the first patients appear to have been switched from 0.5 mg daily to 1 mg

daily – “a dose of 1 mg daily has not caused any untoward reaction during five years´ study”

[3]. Before registration of the first commercial brand of oral cyanocobalamin in 1964, tablet

Behepan, 1 mg, all patients had been recruited (n=64). However, only 5-12 patients seem to

have been switched to 1 mg daily [cf 6,7]. From 1965 and forth, all patients were treated with

1 mg daily throughout the study [3].

Subsequent studies confirmed the documentation of the Berlin group of safe and reliable

oral vitamin B12 treatment for deficiency [8-11]. However, two teams noted a failure rate of

10-20% [9,11]; “failure” in this context is serum cobalamin concentrations below 300

pmol/L.

A recent dose-finding study verified that an efficient oral B12 dose should lie above 0.6

mg daily [12]. The calculations are consistent with clinical findings that treatment with 0.5

mg of oral cyanocobalamin daily did not improve movement and cognition in healthy elderly

citizens [13], whereas 1 mg daily improved cognition in demented patients [14].

It is reasonable to conclude that oral cyanocobalamin, 1 mg daily, is safe and reliable

deficiency treatment in most patients. Lower doses are not documented hitherto and appear

risky from a historical point of view. Doses above 1 mg daily bear a limited advantage, since

urinary excretion of vitamin B12 increases for doses above 0.5 mg [3,4,12].

An alarming token of time is that suboptimal doses of oral vitamin B12 are tried again

[13,15,16], as if the lessons from the period 1950-1965 were in vain. Such regimens lack

documentation in adequate long-term studies and could explain the poor clinical results

hitherto of homocysteine lowering [6,7,13,17]; homocysteine is a sensitive marker of vitamin

B12 and folate deficiency.

The old controversy about oral or parenteral vitamin B12 therapy for maintenance

treatment of vitamin B12 malabsorption still remains unsolved. However, there are plenty of

patients on parenteral maintenance treatment of B12 malabsorption in most post-industrial

countries. It is possible to define those patients who have a severe atrophic gastritis by serum

pepsinogen A and serum gastrin (18). Such patients constitute conclusive cases of

“pernicious anemia in maintenance therapy”.

The classical model of “pernicious anemia in relapse” (8) in its latest English version

(10) could be applied to patients with severe atrophic gastritis on parenteral maintenance with

vitamin B12. When the serum B12 approaches 300 pmol/L, the patient is randomized to

continued parenteral maintenance or oral cyanocobalamin, 1 mg daily. Thus, it would be

possible to compare efficacy, benefits, and costs of oral and parenteral maintenance with

vitamin B12 in a group of conclusive patients.

REFERENCES

[1] Nilsson M. Cobalamin communication in Sweden 1990-2000. Views, knowledge, and

practice among Swedish physicians. Dissertation, Umeå University 2005.

[2] Nilsson M, Norberg B, Hultdin J, Sandström H, Westman G, Lökk J. Medical

intelligence in Sweden. Vitamin B12: oral compared with parenteral? Postgrad Med J

2005; 81:191-93.

4 Karin Björkegren

[3] Berlin H, Berlin R, Brante G. Oral treatment of pernicious anemia with high doses of

vitamin B12 without intrinsic factor. Acta Med Scand 1968; 184:247-58

[4] Lee GR, Bitchell TC, Forster J, Athens JW, Lukens JN, eds. Wintrobe´s Clinical

Hematology, Ed 9. Philadelphia: Lea & Febiger. 1993; 777-80.

[5] Glass GBJ. Gastric intrinsic factor and its function in the metabolism of vitamin B12.

Physiol Rev 1963; 43:731,737.

[6] Norberg B. Provocative proposal – global guidelines for oral vitamin B12 therapy

[editorial]. Rondel 2006; 26. URL: http://www.rondellen.net

[7] Norberg B. Oral high-dose vitamin B12 and folate – breakthrough by broken hips

[editorial]. Rondel 2005; 24. URL: http://www.rondellen.net.

[8] Magnus EM. Cobalamin and unsaturated transcobalamin values in pernicious anaemia;

Relation to treatment. Scand J Haematol 1986; 36; 457-65.

[9] Kuzminski AM, Del Giacco EJ, Allen RH, Stabler SP, Lindenbaum J. Effective

treatment of cobalamin deficiency with oral cobalamin. Blood 1998; 92:1191-98.

[10] Nyholm E, Turpin P, Swain D, Cunningham B, Daly S, Nightingale P, Fegan C. Oral

vitamin B12 can change our practice. Postgraduate Medical Journal 2003; 79:218-20.

[11] Kwong JC, Carr D Dhalla IA, Tom-Kun D, Upshurr REG. Oral vitamin B12 therapy in

the primary care setting: a qualitative and quantitative study of patient perspectives.

BMC Family Practice 2005; 6:8, http: // www.biomedcentral.com/1471-2296/6/8.

[12] Eussen SJPM, Groot LCPG, Clarke R, Schneede J, Ueland PM, Hoefnagels WHL,

Staveren WA. Oral cyanocobalamin supplementation in older people with vitamin B12

deficiency. A dose-finding trial. Arch Intern Med 2005; 165:1167-72.

[13] Lewerin C, Matousek M, Steen G, Johansson B, Steen B, Nilsson-Ehle H. Significant

correlations of plasma homocysteine and serum methylmalonic acid with movement

and cognitive performance in elderly subjects but no improvement from short-term

vitamin therapy: a placebo-controlled randomized study. Am J Clin Nutr 2005;

81:1155-62.

[14] Nilsson K, Gustafson L, Hultberg B. Improvement of cognitive functions after

cobalamin/folate supplementation in elderly patients with dementia and elevated

plasma homocysteine. Internat J Geriatr Psychiatry 2001; 16:609-14.

[15] Bolaman Z, Kadikoylu G, Yukselen V, Yavasoglu I, Barultca S, Senturk T. Oral versus

intramuscular cobalamin treatment in megaloblastic anemia: A single-center,

prospective, randomized, open-label study. Clin Ther 2003; 25:3124-34.

[16] Andrès E, Affenberger S, Vinzio S, et al.Food-cobalamin malabsorption in elderly

patients: Clinical manifestations and treatment. Amer J Med 2005; 118:1154-59.

[17] Spence JD, Bang H, Chamblees LE, Stampfer MJ. Vitamin intervention for stroke

prevention trial. An efficacy analysis. Stroke 2005; 36:2404-09.

[18] Lindgren A, Lindstedt G, Killander AF. Advantages of serum pepsinogen A combined

with gastrin or pepsinogen C as first-line analytes in the evaluation of suspected

cobalamin deficiency: a study in patients previously not subjected to gastrointestinal

surgery. J Intern Med 1998, 244:347-49.

In: Vitamin B: New Research ISBN 978-1-60021-782-1

Editor: Charlyn M. Elliot, pp. 5-19 © 2008 Nova Science Publishers, Inc.

Chapter I

INHIBITORY EFFECT OF VITAMIN B6

COMPOUNDS ON DNA POLYMERASE, DNA

TOPOISOMERASE AND HUMAN CANCER CELL

PROLIFERATION

Yoshiyuki Mizushina1,2,∗ , Norihisa Kato3

, Hiromi Yoshida1,2 and

Kiminori Matsubara4

1

Laboratory of Food & Nutritional Sciences, Department of Nutritional Science, KobeGakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan;

2

Cooperative Research Center of Life Sciences, Kobe-Gakuin University, Nishi-ku,

Kobe, Hyogo 651-2180, Japan;

3

Graduate School of Biosphere Sciences, Hiroshima University, Kagamiyama, HigashiHiroshima, Hiroshima 739-8528, Japan;

4

Department of Nutritional Science, Faculty of Health and Welfare Science, Okayama

Prefectural University, Kuboki, Soja, Okayama 719-1197, Japan.

ABSTRACT

Vitamin B6 compounds such as pyridoxal 5'-phosphate (PLP), pyridoxal (PL),

pyridoxine (PN) and pyridoxamine (PM), which reportedly have anti-angiogenic and

anti-cancer effects, were thought to be selective inhibitors of some types of eukaryotic

DNA polymerases (pols) and human DNA topoisomerases (topos). PL moderately

inhibited only the activities of calf pol α, while PN and PM had no inhibitory effects on

any of the pols tested. On the other hand, PLP, a phosphated form of PL, was potentially

a strong inhibitor of pols α and ε from phylogenetic-wide organisms including mammals,

∗

 Correspondence concerning this article should be addressed to: Yoshiyuki Mizushina Laboratory of Food and

Nutritional Sciences, Department of Nutritional Science, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-

2180, Japan; Tel: +81-78-974-1551 (ext.3232); Fax: +81-78-974-5689; E-mail:

mizushin@nutr.kobegakuin.ac.jp.

6 Yoshiyuki Mizushina, Norihisa Kato, Hiromi Yoshida et al.

fish, insects, plants and protists. PLP also inhibited the activities of human topos I and II.

PLP did not suppress the activities of prokaryotic pols such as E. coli pol I, T4 pol and

Taq pol, or DNA metabolic enzymes such as HIV reverse transcriptase, RNA polymerase

and deoxyribonuclease I. For pols α and ε, PLP acted non-competitively with the DNA

template-primer, and competitively with the nucleotide substrate. To clarify how vitamin

B6 inhibits angiogenesis, this review was performed to examine the effect on human

umbilical vein endothelial cell (HUVEC) proliferation and HUVEC tube formation.

Consistent with the result of an ex vivo angiogenesis assay, PLP and PL markedly

suppressed the proliferation of HUVEC, while PN and PM were inactive. Suppression of

HUVEC proliferation by PLP and PL was evident in a dose-dependent manner with

LD50 values of 112 and 53.9 μM, respectively; however, HUVEC tube formation was

unaffected by PLP and PL. On the other hand, PL inhibited the growth of human

epitheloid carcinoma of the cervix (HeLa), but PLP, PN and PM had no influence. Since

PL was converted to PLP in vivo after being incorporated into human cancer cells, the

anti-angiogenic and anti-cancer effects leading to PL must have been caused by the

inhibition of pol and topo activities after conversion to PLP. These results suggest that

vitamin B6 suppresses cell proliferation and angiogenesis at least in part by inhibiting

pols α and ε, and topos I and II.

Keywords: vitamin B6, pyridoxal 5'-phosphate (PLP), pyridoxal (PL), pyridoxine (PN),

pyridoxamine (PM), DNA polymerase, DNA topoisomerase, enzyme inhibitor,

cytotoxicity, human umbilical vein endothelial cell (HUVEC), anti-cancer effect.

ABBREVIATIONS

PLP, pyridoxal 5'-phosphate; PL, pyridoxal; PN, pyridoxine; PM, pyridoxamine; pol,

DNA polymerase; topo, DNA topoisomerase; dTTP, 2'-deoxythymidine 5'-triphosphate; NP40, Nonidet P-40; IC50, 50 % inhibitory concentration; LD50, 50 % lethal dose; HUVEC,

human umbilical vein endothelial cell; VEGF, vascular endothelial growth factor.

1. INTRODUCTION

Vitamin B6 has been recognized as a cofactor for many enzymes, especially those

involved in amino acid metabolism. Apart from its role as a coenzyme, recent studies have

unveiled a new role of vitamin B6 as a chemopreventive agent. It is known that high levels of

pyridoxal (PL) or pyridoxine (PN), which are vitamin B6 compounds, suppress tumor growth

in vitro and in vivo [1-3], and that a high dietary intake of vitamin B6 suppresses herpes

simplex virus type 2-transformed (H238) cell-induced tumor growth in BALB/c mice [4].

Recent studies have also shown that vitamin B6 lowers the risk of lung and colon cancer in

epidemiological research and animal experiments [5-9]. Thus, the anti-cancer effect of

vitamin B6 has attracted considerable attention. In our study, vitamin B6 suppressed

angiogenesis in a rat aortic ring angiogenesis model, suggesting that the inhibition of

angiogenesis by vitamin B6 might partially be responsible for its anti-cancer effect [10];

Inhibition of DNA Polymerase and Topoisomerase by Vitamin B6 7

however, the mechanisms by which vitamin B6 exerts its anti-cancer effect are not fully

understood yet.

We found that some vitamin B6 compounds were inhibitors of the DNA polymerases

(pols) of various species and human DNA topoisomerases (topos) [11,12], implying that

these compounds are involved enzyme inhibition via anti-proliferation. Pol catalyzes the

addition of deoxyribonucleotides to the 3'-hydroxyl terminus of a primed double-stranded

DNA molecule [13]. In mammalian cells, at least fourteen classes of pols are reportedly

present [14]. The in vivo functions of pols α, δ and ε, act in nuclear DNA replication, and

pols β, δ, ε, ζ, η, θ, ι, κ, λ, μ, σ and Φ appear to be related to DNA repair, translation synthesis

(TLS) and/or recombination [14]. Topos catalyze the concerted breaking and rejoining of

DNA strands, and are involved in producing various necessary topological and

conformational changes in DNA [14,15]. There is no enzymatic similarity between pols and

topos, although they are critical to many cellular processes such as DNA replication, repair

and recombination, and subsequently, may act in harmony with each other. Inhibition of pols

and topos arrests the cell cycle and induces apoptosis; thus, they are molecular targets of anticancer drugs [16,17].

In this review, we described the biochemical mechanism of anti-angiogenesis and the

inhibition of human cancer cell growth by vitamin B6 compounds as inhibitors of replicative

pols and topos.

2. EFFECT OF VITAMIN B6 COMPOUNDS ON THE ACTIVITIES

OF DNA POLYMERASES, DNA TOPOISOMERASES AND OTHER

DNA METABOLIC ENZYMES

The chemical structures of vitamin B6 compounds such as pyridoxal (PL), pyridoxine

(PN), pyridoxamine (PM) and pyridoxal 5'-phosphate (PLP), which can be purchased

commercially, are shown in Figure 1. Inhibition of the activities of mammalian pols by

vitamin B6 compounds was investigated. The assay method for pol activity was described

previously [18,19]. The pol substrates were poly(dA)/oligo(dT)12-18 and 2'-deoxythymidine

5'-triphosphate (dTTP) as the DNA template-primer and nucleotide substrate (i.e., 2'-

deoxynucleotide 5'-triphosphate (dNTP)), respectively. One unit of each pol activity was

defined as the amount of enzyme that catalyzed the incorporation of 1 nmol of

deoxyribonucleoside triphosphates (i.e., dTTP) into synthetic template-primers (i.e.,

poly(dA)/oligo(dT)12-18, A/T = 2/1) in 60 min at 37 °C under the normal reaction conditions

for each enzyme [18,19].

As shown in Figure 2, 100 μM of PN and PM did not influence the activities of

mammalian pols at all. On the other hand, PL selectively inhibited calf pol α activity, but did

not suppress the activities of the other pols tested. PLP of 100 μM completely inhibited the

activities of calf pol α and human pol ε, and slightly inhibited the other pols activities.

Table 1 shows the IC50 values of vitamin B6 compounds for the activities of various

pols. Of the three non-phosphate forms of vitamin B6 compounds (i.e., PL, PN and PM), the

PL inhibitory activity of pol α from mammals (i.e., calf), fish (i.e., cherry salmon), insects

8 Yoshiyuki Mizushina, Norihisa Kato, Hiromi Yoshida et al.

(i.e., fruit fly) and plants (i.e., cauliflower) was stronger than that of PN and PM.

Interestingly, the 5'-phosphate form of PL (PLP) was a much stronger inhibitor of pol α than

PL.

N

R1

HO

H3C

R2

Vitamin B6

compound

R1 R2

PL -CHO -OH

PN -CH2OH -OH

PM -CH2NH2 -OH

PLP -CHO P OH

OH

O

O

Figure 1. Chemical structures of vitamin B6 compounds. PL: Pyridoxal, PN: Pyridoxine, PM:

Pyridoxamine, and PLP: Pyridoxal 5'-phosphate.

Calf pol α

Rat pol β

Human pol γ

Human pol δ

Human pol ε

Human pol η

Human pol ι

Human pol κ

Human pol λ

DNA polymerase activity (%)

0 20 40 60 80 100

PLP

PM

PN

PL

Figure 2. Effect of vitamin B6 compounds on the activities of mammalian DNA polymerases. Each

vitamin B6 compound (100 μM) was incubated with each pol (0.05 units). Enzymatic activity in the

absence of compound was taken as 100 %. Data are shown as the means ± SEM of three independent

experiments.

Inhibition of DNA Polymerase and Topoisomerase by Vitamin B6 9

Table 1. IC50 values of vitamin B6 compounds on the activities of various DNA

polymerases and other DNA metabolic enzymes

IC50 value of vitamin B6 compounds (μM)

PL PN PM PLP

(1) DNA polymerases

Mammalian DNA polymerases

Calf DNA polymerase α 92.0 >1000 >1000 33.8

Rat DNA polymerase β >1000 >1000 >1000 >1000

Human DNA polymerase γ >1000 >1000 >1000 86.4

Human DNA polymerase δ >1000 >1000 >1000 77.7

Human DNA polymerase ε >1000 >1000 >1000 32.6

Human DNA polymerase η >1000 >1000 >1000 >1000

Human DNA polymerase ι >1000 >1000 >1000 >1000

Human DNA polymerase κ >1000 >1000 >1000 >1000

Human DNA polymerase λ >1000 >1000 >1000 >1000

Fish DNA polymerase

Cherry salmon DNA polymerase δ >1000 >1000 >1000 74.9

Insect DNA polymerases

Fruit fly DNA polymerase α 98.5 >1000 >1000 35.5

Fruit fly DNA polymerase δ >1000 >1000 >1000 76.1

Fruit fly DNA polymerase ε >1000 >1000 >1000 33.0

Plant DNA polymerases

Cauliflower DNA polymerase α 99.7 >1000 >1000 36.6

Cauliflower DNA polymerase β >1000 >1000 >1000 >1000

Protist DNA polymerases

Yeast DNA polymerase δ >1000 >1000 >1000 75.5

Yeast DNA polymerase ε >1000 >1000 >1000 34.1

Prokaryotic DNA polymerases

E. coli DNA polymerase I >1000 >1000 >1000 >1000

T4 DNA polymerase >1000 >1000 >1000 >1000

Taq DNA polymerase >1000 >1000 >1000 >1000

(2) DNA topoisomerases

Human DNA topoisomerase I >1000 >1000 >1000 85.0

Human DNA topoisomerase II >1000 >1000 >1000 70.0

(3) Other DNA metabolic enzymes

Calf Terminal deoxynucleotidyl transferase >1000 >1000 >1000 >1000

HIV reverse transcriptase >1000 >1000 >1000 >1000

T7 RNA polymerase >1000 >1000 >1000 >1000

Bovine Deoxyribonuclease I >1000 >1000 >1000 >1000

PL: pyridoxal, PN: pyridoxine, PM: pyridoxamine, PLP: pyridoxal 5'-phosphate.

Each vitamin B6 compound (100 μM) was incubated with each enzyme (0.05 units). Enzyme activity in

the absence of compounds was taken as 100 %.

PLP inhibited the activities of mammalian pols dose-dependently (Figure 3). PLP

especially influenced pols α and ε activities, which are replicative pols, achieving 50 %

inhibition at concentrations of 33.8 and 32.6 μM, respectively. The compound moderately

10 Yoshiyuki Mizushina, Norihisa Kato, Hiromi Yoshida et al.

inhibited pols γ and δ activities, and the IC50 values were 86.4 and 77.7 μM, respectively.

PLP hardly inhibited repair-related pols such as pols β, η, ι, κ and λ (Table 1). These results

suggested that PLP could be a selective inhibitor of replicative pols rather than repair-related

pols. The effect of PLP on various species of eukaryotic pols was the same inhibitory

concentration as that of mammalian pols (Table 1).

0

20

40

60

80

100

0 20 40 60 80 100