The Effect of Vitamin C on Tetanus Toxin and Strychnine Toxicity: a Systematic Review of Animal Studies

by Harri Hemilä

Department of Public Health
University of Helsinki, Helsinki, Finland
harri.hemila@helsinki.fi

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Version May 29, 2012

Introduction
Methods
Results of the studies
Discussion
References
Table 1: Vitamin C and Tetanus Toxin
References to Table 1
Table 2: Vitamin C and Strychnine
References to Table 2
Table 3: Effect of the interval between vitamin C administration and strychnine administration (Dey 1967)
References to Table 3

Introduction

Tetanus is a disease caused by the toxin of Clostridium tetani, which may contaminate wound (see summaries eg. CDC_2008A CDC_2008B CDC 2008 Fig Todar's Textbook Wisconsin Bact ).

In a controlled trial, Jahan et al. (1984) found that 1 g/day vitamin C significantly reduced the mortality of tetanus patients in Bangladesh. Although Jahan's trial was methodologically unsatisfacory compared with current standards, the shortcomings do not give an evident explanation to the differences between the vitamin C and control groups (Hemilä & Koivula 2008). Nevertheless, there are no studies with human patients giving independent support to the proposal that vitamin C might have an effect on tetanus. Indirect support to the effects of vitamin C can be obtained from animal studies.

A large number of animal studies have found that vitamin C protects against diverse viral, bacterial and protozoal infections (see p. 7-9 and 105-118 in Hemilä (2006)). Consequently, it is possible that vitamin C affects the immune system so that it is reflected as changes in the incidence and severity of infections. Nevertheless, it is also possible that vitamin C has non-immunologic effects against infections. A number of animal studies found that vitamin C protects against the harms caused by purified bacterial toxins: diphtheria toxin (Harde 1934; Lyman & King 1936; Harris et al. 1937; Ghosh 1939a, Torrance 1940) and endotoxin (Torrance 1940; Garcia et al. 1990; Rojas et al. 1996; Benito & Bosch 1997; Yamaguchi et al. 1997; Armour et al. 2001; Victor et al. 2002). The action of vitamin C against the acute effects of bacterial toxins suggests that the effecs of vitamin C are not restricted to protection against viable bacteria. Furthermore, in guinea pigs, 0.5 MLD tetanus toxin reduced in 3 days the level of vitamin C in plasma by 44% (50/89), in adrenals by 22% (333/428), in kidney by 20% (77/97) and in the liver by 19% (568/696) (Ghosh 1939b).

In tetanus patients, blood vitamin C level is about half the level of normal controls (Chakrabarti & Bannerjee 1955, 1956). Furthermore, Chakrabarti & Bannerjee (1955) reported that among the tetanus patients, vitamin C levels were lower in those who later succumbed compared with those who survived, suggesting that the low level of vitamin C may be associated with the prognosis of the disease. Finally, the blood level of dehydroascorbte, oxidized form of vitamin C, is very low in normal controls but in tetanus patients it is higher in patients with more severe disease (Chakrabarti & Bannerjee 1955). Thus, tetanus seems to affect vitamin C metabolism in humans.

Strychnine and tetanus have similar neurophysiological effects, and because of the similarity of the symptoms, strychnine poisoning is a differential diagnosis of tetanus ().

The purpose of this systematic review is to summarize the findings of animal studies which have examined the effect of vitamin C on the effects of tetanus toxin and strychnine on animals.

Methods

Previously, extensive literature searches were carried out to find out studies that have examined the effect of vitamin C on human and animal infections (p. 20 in Hemilä 2006). The literature searches were composed of several searches using various strategies in different databases (Medline, Web of Science, Chemical Abstracts, Biological Abstracts). In some cases the search phrases were rather loose, e.g., ‘vitamin C’ alone, whereas some other searches were limited by additional phrases such as ‘infection’ or ‘immune.’ Papers citing the older seminal papers were sought in the citation data base (Web of Science). Thus, no single or simple explicit search strategy was used. For this report, additional searches, using the search terms "vitamin C"/ascorb* AND tetanus/strychnine, were carried out with modifications needed to the data bases. Old reports were scanned and are available via links of this document. Studies which reported a controlled comparison between vitamin C and control groups are presented in the tables: the effect of vitamin C on tetanus toxin (Table 1), on strychnine (Tables 2 and 3).

In studies with dichotomous data (2×2 tables), the mid-P was calculated. The rationalization of the mid-P modification of the Fisher exact test is described on p. 20-21 of Hemilä (2006) and survival data was analyzed using the logrank test (see eg BMJ Wikipedia). LD50 (lethal dose for 50% of animals) and ED50 (effective dose for 50% of animals) calculation were carried out using SAS program PROBIT.

One-tailed P-values are used in this report, because the question is whether vitamin C decreases the severity of disease or not, and this questions are unidirectional. A benefit of the 1-tailed P-value is that it shows the direction of the difference, with P>0.5 indicating the direction of harm. The 1-tailed P-value can easily be transformed to the 2-tailed P-value.

Results of the studies

Three series of studies examined the effect of vitamin C against the toxicity of tetanus toxin in rats (Table 1). Dey (1966) reported that vitamin C prevented mortality caused by tetanus toxin when vitamin C was administered either before or shortly after the toxin. Eichbaum (1977) found significant benefit of vitamin C against short term effects of tetanus toxin, but the difference in longer term mortality was nonsignificant. Frezza (1957) used two doses of vitamin C and two doses of tetanus toxin and vitamin C was significantly beneficial only in the combination of low dose toxin and high dose vitamin C. Thus, Frezza's results suggest that the ratio of vitamin C dose to the toxin dose may be crucial. Ghosh (1938) studied the effect of vitamin C against tetanus toxin toxicity in guinea pigs, and found a marginally significant benefit only in one comparison (Table 1).

Three series of experiments have examined the effect of vitamin C against strychnine toxicity in mice (Table 2). Dey (1965) found that high dose vitamin C totally prevented strychnine toxicity. In the second series, Dey (1967) used a fixed dose of strychnine and varied the dose of vitamin C over a wide range. In the absence of vitamin C, all mice died, whereas with the highest vitamin C doses, all mice survided. Based on Dey's data, the ED50 dose in mice is 76 mg/kg (95% CI: 19-149 mg/kg) for the protection against 2 mg/kg strychnine.

Eichbaum et al. (1977) varied strychnine dose in mice with and without high dose vitamin C. Based on Eichbaum's data, the LD50 value without vitamin C is 0.88 mg/kg (95% CI: 0.78-0.99 mg/kg), whereas the LD50 value was close to two times as high in animals administered high dose of vitamin C, 1.50 mg/kg (95% CI: 1.33-1.70 mg/kg). The difference between these LD50 values is statistically highly significant.

Jahan et al. (1984) studied the effect of vitamin C in chickens with two strychnine doses. In chickens administered the lower dose of strychnine, vitamin C prevented stretching of the wings. In chickens aministered the higher strychnine dose, vitamin C significantly prevented mortality.

In two studies with fish, Eichbaum et al. (1977) found that vitamin C prevented strychnine toxicity.

Dey (1967) examined the relationship between the protection against strychnine toxicity and the level of vitamin C in plasma, so that strychnine was administered 15 min to 2 hours after the vitamin (Table 3). Strychnine administered soon after high dose vitamin C did not lead to mortality, whereas strychnine administered 2 hours after vitamin C killed all mice. The effect of strychnine correlated with plasma vitamin C level, so that definite protection against strychnine toxicity occurred only when vitamin C plasma level was about 2 mg/100 ml (120 µmol/l).

Discussion

Only one of the identified experimental series failed to record an unambiguous benefit of vitamin C, namely the study by Ghosh (1938). Three series suggested dose-response by the variation of the dose of vitamin C (Dey 1967) or strychnine (Eichbaum 1977) or both vitamin C and tetanus toxin (Frezza 1957) so that a higher ratio of vitamin C to toxin led to a greater benefit in each of the three cases. Furthermore, Dey (1967) found that a delay in the administration of strychnine after a high dose vitamin C led to a parallel reduction in plasma vitamin C level and protection against strychnine (Table 3).

Although many of the studies in Tables 1 to 4 are old, it seems unlikely that administering a fixed dose of vitamin C and evaluating mortality were meaningfully different in the older days compared with modern methods. It is noteworthy that dramatic effects by vitamin C against the toxicity of tetanus toxin and strychnine have been observed in diverse animal species ranging from fish and chicken to rat and mouse. Even though caution is needed in the extrapolation of animal study results to human subjects, the consistency in the effects in such diverse species gives support to the report by Jahan et al. (1984) who found that 1 g/day vitamin C protected against tetanus in human patients.

The doses of vitamin C used in the animal studies range up to 1-2 grams per kg body weight (Tables 1 to 4). For a 70 kg person this would correspond to 70-140 grams. Only some 10 mg/day is needed to prevent scurvy, and the current recommendation of vitamin C intake in the USA is 90 mg/day. In this respect the doses in the animal studies are very high.

Lack of toxicity (Klenner, Levine)

References

Jahan K, Ahmad K, Ali MA (1984) Effect of ascorbic acid in the treatment of tetanus. Bangladesh Med Res Counc Bull 10:24-28

Armour J, Tyml K, Lidington D, Wilson JX (2001) Ascorbate prevents microvascular dysfunction in the skeletal muscle of the septic rat. J Appl Physiol 90:795-803  PubMed

Benito E, Bosch MA (1997) Impaired phosphatidylcholine biosynthesis and ascorbic acid depletion in lung during lipopolysaccharide-induced endotoxaemia in guinea pigs. Mol Cell Biochem 175:117-23 PubMed

Chakrabarti B, Banerjee S (1955) Dehydroascorbic acid level in blood of patients suffering from various infectious diseases.    Proc Soc Exp Biol Med 88:581-3 BM

Chakrabarti B, Banerjee S (1956) Studies on the functional activity of the adrenal cortes in some infectious diseases.  Indian J Med Res 44:211-215

Garcia R, Abarca S, Municio AM (1990) Adrenal gland function in reversible endotoxic shock. Circ Shock 30:365-74 PubMed

Ghosh B (1939a) Vitamins and toxins. Part III. The effect of diphtheria toxin on vitamin C metabolism. J Indian Chemical Society 16: 241-246

Ghosh B (1939b) Vitamins and toxins. Part IV. The effect of tetanus toxin on vitamin C metabolism. J Indian Chemical Society 16: 657-662

Harde E (1934) Acide ascorbique (vitamine C) et intoxications [in French]. C R Acad Sci 119:618-20

Harris LJ, Passmore R, Oxon BM, et al. (1937) Influence of infection on vitamin C content of the tissues of animals.  Lancet 230:183-6

Lyman CM, King CG (1936) The effect of diphtheria toxin on the vitamin C content of guinea pig tissues. J Pharmacol Exp Ther 56:209-15

Rojas C, Cadenas S, Herrero A, et al. (1996) Endotoxin depletes ascorbate in the guinea pig heart: protective effects of vitamins C and E against oxidative stress. Life Sci 59:649-57   PubMed

Torrance CC (1940) Diphtherial intoxication and vitamin C content of the suprarenals of guinea pigs.  J Biol Chem 132:575-84

Victor VM, Guayerbas N, de la Fuente M (2002) Changes in the antioxidant content of mononuclear leukocytes from mice with endotoxin-induced oxidative stress. Mol Cell Biochem 229:107-11   PubMed

Yamaguchi T, Hashizume T, Tanaka M, et al. (1997) Bilirubin oxidation provoked by endotoxin treatment is suppressed by feeding ascorbic acid in a rat mutant unable to synthesize ascorbic acid. Eur J Biochem 245:233-40 PubMed

Table 1: Vitamin C and Tetanus Toxin

Study Animal Exposure	Vit C	Control	Difference	Significance P(1-tail)
	No. in group	No. in group
Dey-1966 Rat Tetanus toxin inj. 2×MLD
Vit C before or simultaneusly with toxin
Animals	10	5
Mortality	0	5	-100%	.0002
Vit C after signs of tetanus appeared
Animals	15	5
Mortality	0	5	-100%	.00004

Eichbaum-1977 Rat Tetanus toxin inj. 2×MLD
Animals	5	5
Convulsions or death in 5 hours	0	5	-100%	.002
Mortality in 6 days	3	5	-40%	.1

Frezza-1957 Rats Tetanus toxin inj
Tetanus toxin 10×MLD Vit C 100 mg/kg
Animals	8	8
Survival time in hours In parenthesis: the number of animals with the survival time	30;54; 60(2); 72(2); 78(2)	54(3); 60(2); 66(3)	+10% in median survival time (66 vs. 60 h)	.065
Tetanus toxin 20×MLD Vit C 100 mg/kg
Animals	8	8
Survival time in hours In parenthesis: the number of animals with the survival time	21(3); 24; 27(2); 33;39	18; 21(2); 24(2) 27(3)	+6% in median survival time (25.5 vs. 24 h)	.2
Tetanus toxin 10×MLD Vit C 200 mg/kg
Animals	8	8
Survival time in hours In parenthesis: the number of animals with the survival time	21;54; 72;84; 102(2); 114; 138	54(3); 60(2); 66(3)	+55% in median survival time (93 vs. 60 h)	.007

Ghosh-1938 Guinea pig Tetanus toxin inj. 1×MLD
100 mg ascorbic acid inj. 1 min after tetanus toxin
Animals	9	6
Mortality	5	6	-44%	.047
100 mg neutralized ascorbic acid inj. 1 min after tetanus toxin
Animals	4	6
Mortality	4	6
100 mg ascorbic acid inj. 1 min before tetanus toxin
Animals	4	6
Mortality	2	6		0.07
50 mg/day ascorbic acid fed for 6 days before tetanus toxin
Animals	6	6
Mortality	5	6		0.3
20 mg/day ascorbic acid inj. for 6 days before tetanus toxin
Animals	6	6
Mortality	6	6

References to Table 1

Dey PK (1966) Efficacy of vitamin C in counteracting tetanus toxin toxicity. Naturwissenschaften 53:310 CH BM

Eichbaum FW, Guedes AO, Palermo Neto J, Carvalho FV (1977) Protecting effect of ascorbic acid in strychnine poisoning and in tetanus (experiments in fish, mice and rats). Int J Vitam Nutr Res Supplement 16:31-39

Frezza F (1957) Tetanus in hypervitaminosis C. Vitaminologia (Rome) 15:65-73 Abstract

Ghosh B, Guha BC (1938) Vitamins and toxins. Part II. The effect of the administration of vitamin C to guinea-pigs injected with diphtheria and tetanus toxins. J Indian Chemical Society 15:443-448

Table 2: Vitamin C and Strychnine

Study Animal Exposure	Vit C	Control	Difference	Significance P(1-tail)
	No. in group	No. in group
Dey-1965 Mice Strychnine inj.
Strychnine 2 mg/kg Vit C 1000 mg/kg
Animals	10	20
Mortality	0	20	-100%	<.000001
Strychnine 2 mg/kg Vit C 2000 mg/kg
Animals	10	20
Mortality	0	20	-100%	<.000001

Dey-1967 Mice Strychnine inj
Strychnine 2 mg/kg Vit C 50 mg/kg
Animals	10	10
Mortality	6	10	-40%	.022
Strychnine 2 mg/kg Vit C 100 mg/kg
Animals	10	10
Mortality	4	10	-60%	.003
Strychnine 2 mg/kg Vit C 500 mg/kg
Animals	10	10
Mortality	2	10	-80%	.0002
Strychnine 2 mg/kg Vit C 1000 mg/kg
Animals	10	10
Mortality	0	10	-100%	.000003
Strychnine 2 mg/kg Vit C 2000 mg/kg
Animals	10	10
Mortality	0	10	-100%	.000003
ED50 value for vitamin C	ED50=76 mg/kg (95%CI: 19-149)			P(2-tail)=.0006 Test of trend over vit C doses in PROBIT analysis

Eichbaum-1977 Mice Strychnine inj.
Strychnine 0.35 mg/kg Vit C dose 1000 mg/kg
Animals	40	40
Mortality in 2 hours	0	1
Strychnine 0.5 mg/kg Vit C dose 1000 mg/kg
Animals	25	25
Mortality in 2 hours	1	4	-75%	.11
Strychnine 0.7 mg/kg Vit C dose 1000 mg/kg
Animals	40	40
Mortality in 2 hours	3	18	-83%	.00007
Strychnine 1.0 mg/kg Vit C dose 1000 mg/kg
Animals	25	25
Mortality in 2 hours	4	13	-69%	.005
Strychnine 1.4 mg/kg Vit C dose 1000 mg/kg
Animals	40	40
Mortality in 2 hours	18	30	-40%	.004
Strychnine 2.0 mg/kg Vit C dose 1000 mg/kg
Animals	25	25
Mortality in 2 hours	20	23	-13%	.14
Strychnine 2.8 mg/kg Vit C dose 1000 mg/kg
Animals	40	40
Mortality in 2 hours	34	40	-15%	.007
LD50 values for strychnine:	LD50=1.50 mg/kg (95%CI: 1.33-1.70)	LD50=0.88 mg/kg (95%CI: 0.78-0.99)		P(2-tail)<.000001 Test of vit C and control difference in PROBIT analysis

Eichbaum-1977 Fish Strychnine in water
Strychnine 50 mg/l Vit C dose 500 mg/l
Animals	120	120
Mortality in 2 hours	32	88	-63%	<.000001
Strychnine 60 mg/l Vit C dose 500 mg/l
Animals	120	120
Mortality in 2 hours	43	99	-56%	<.000001

Jahan-1984 Chicken Strychnine inj.
Strychnine 5 µg per chick Vit C dose 30 mg/chick
Animals	15	15
Wings stretched	0	15	-100%	<.000001
Mortality	0	0
Strychnine 10 µg per chick Vit C dose 30 mg/chick
Animals	15	15
Extensor paralysis	3	15	-80%	.000003
Mortality	0	12	-100%	.000003

References to Table 2

Dey PK (1965) Protective action of lemon juice and ascorbic acid against lethality and convulsive property of strychnine. Naturwissenschaften 52:164 CH BM

Dey PK (1967) Protective action of ascorbic acid and its precursors on the convulsive and lethal actions of strychnine. Indian J Exp Biology 5(2):110-112

Eichbaum FW, Guedes AO, Palermo Neto J, Carvalho FV (1977) Protecting effect of ascorbic acid in strychnine poisoning and in tetanus (experiments in fish, mice and rats). Int J Vitam Nutr Res Supplement 16:31-39

Jahan K, Ahmad K, Ali MA (1984) Effect of ascorbic acid in the treatment of tetanus. Bangladesh Med Res Counc Bull 10:24-28

Table 3: Effect of the interval between vitamin C administration and strychnine administration (Dey 1967)

Time (min)	Vitamin C concentration in plasma		Mortality in the group	Effect on Mortality
	mg/100 ml	µmol/l
15	6.0	341	0/10	-100%
30	2.4	136	0/10	-100%
60	1.04	59	5/10	-50%
120	0.86	49	10/10	0%
Control, no vitamin C administation	0.66	38	Not tested

Vitamin C, 1000 mg/kg was administered intraperitoneally to mice, and strychnine, 2 mg/kg, was administered at the different time points and plasma vitamin C level was determined at the same time points.

References to Table 3

Dey PK (1967) Protective action of ascorbic acid and its precursors on the convulsive and lethal actions of strychnine. Indian J Exp Biology 5(2):110-112

Copyright: © 2006-2008 Harri Hemilä. This text is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.