Open Access REVIEW The cardiac and haemostatic effects of dietary hempseed
2010

- Rodriguez
Pierce Leyva
Open Access REVIEW The cardiac and haemostatic effects of dietary hempseed

Nutrition & Metabolism
7322010BioMed Central Review
Despite its use in our diet for hundreds of years, hempseed has surprisingly little research published on its physiological effects. This may have been in the past because the psychotropic properties wrongly attributed to hemp would complicate any conclusions obtained through its study. Hemp has a botanical relationship to drug/medicinal varieties of Cannabis. However, hempseed no longer contains psychotropic action and instead may provide significant health benefits. Hempseed has an excellent content of omega-3 and omega-6 fatty acids. These compounds have beneficial effects on our cardiovascular health. Recent studies, mostly in animals, have examined the effects of these fatty acids and dietary hempseed itself on platelet aggregation, ischemic heart disease and other aspects of our cardiovascular health. The purpose of this article is to review the latest developments in this rapidly emerging research field with a focus on the cardiac and vascular effects of dietary hempseed.

Introduction

Cannabis sativa L. is an annual plant in the Cannabaceae family. It has been an important source of food, fiber, medicine and psychoactive/religious drug since prehistoric times [1]. Cannabis is mentioned as a medication in ancient Egyptian medical texts: Ramesseum III Papyrus (1700 B.C.), Eber's Papyrus (1600 B.C.), the Berlin Papyrus (1300 B.C.), and the Chester Beatty VI Papyrus (1300 B.C.) [1,2].

Two main types of Cannabis Sativa L. must be distinguished, the drug and non-drug types. The first is also known as marijuana, hashish or Cannabis tincture and contains Δ 9 -Tetrahydrocannabinol (THC) in concentrations between 1-20%, high enough to exhibit psychoactivity. The second type of Cannabis Sativa L. is industrial hemp with THC concentrations < 0.3% so it has no psychoactive properties [3,4].

Canada, Australia, Austria, China, Great Britain, France and Spain are among the most important agricultural producers of hempseed. In the United States, it is not legal to cultivate hempseed. This is primarily because many believe that by legalizing hemp they may lead to a legalization of marijuana [5]. Other governments have accepted the distinction between the two types of Cannabis and, while continuing to penalize the growing of marijuana, have legalized the growing of industrial hemp [5].

Hempseed possesses excellent nutritional value. It is very rich in essential fatty acids (EFAs) and other polyunsaturated fatty acids (PUFAs). It has almost as much protein as soybean and is also rich in Vitamin E and minerals such as phosphorus, potassium, sodium, magnesium, sulfur, calcium, iron, and zinc [6,7]. The nutrient profile of hempseed is shown in Table 1. Hempseed oil contains all of the essential amino acids and also contains surprisingly high levels of the amino acid arginine, a metabolic precursor for the production of nitric oxide (NO), a molecule now recognized as a pivotal signaling messenger in the cardiovascular system that participates in the control of hemostasis, fibrinolysis, platelet and leukocyte interactions with the arterial wall, regulation of vascular tone, proliferation of vascular smooth muscle cells, and homeostasis of blood pressure [8]. In a study that included 13 401 participants, 25 years and older from the Third National Health Nutrition and Examination Survey, an independent relationship was shown between the dietary intake of L-arginine and levels of C-Reactive protein [9], a marker strongly correlated with the risk of cardiovascular disease (CVD) [10]. The results of this populated-based study suggested that individuals may be able to decrease their risk for CVD by following a diet that is high in arginine-rich foods [9]. Dietary hempseed is also particularly rich in the omega-6 fatty acid linoleic acid (LA) and also contains elevated concentrations of the omega-3 fatty acid α-linolenic acid (ALA). The LA:ALA ratio normally exists in hempseed at between 2:1 and 3:1 levels. This proportion has been proposed to be ideal for a healthy diet [11]. Other rich sources of LA [12,13] are listed in Table 2.

The long chain PUFA that is found in the body ultimately originates from the diet and through elongation and desaturation of their dietary precursors, ALA and LA. Both families of fatty acids, n-3 and n-6, share and compete for the same enzymes (Δ 6 -desaturase, Δ 5 -desaturase, and elongases) in their biosynthetic pathways. The Δ 6 -desaturase enzyme is the rate-limiting step [9]. Following its metabolism, LA can be converted into arachi-donic acid whereas ALA will be converted into the long chain fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (Figure 1). A high LA intake interferes with the desaturation and elongation of ALA [14]. Therefore, theorically, a lower ratio of omega-6/ omega-3 fatty acids is more advantageous in reducing the risk of many of the chronic diseases of high prevalence in Western societies. The ratio of ω-6 to ω-3 fatty acids ranges from 20-30:1 in Western societies instead of the traditional (historic) range of 1-2:1 on which human beings evolved [15]. This is thought to be closely associated with chronic diseases like coronary artery disease, hypertension, diabetes, arthritis, osteoporosis, inflammatory and autoimmune disorders and cancer.  Hempseed is also a rich and unusual source of the polyunsaturated fatty acid gamma linolenic acid (GLA) (18:3n6) to the body. Additionally, another important biological metabolite of ALA and LA, stearidonic acid (18:4n3; SDA) is also present in hempseed oil ( Figure 1). Both can inhibit inflammatory responses [16,17].

Recently, many studies have demonstrated that dietary interventions can play a central role in the primary and secondary prevention of several diseases. The PUFAs derived from fish, EPA and DHA, have been extensively studied. Based on the close relation between the pathways that metabolize ALA and LA, and the capacity of both to be converted into long chain fatty acids, plant sources of ALA (i.e. flaxseed, canola and others) have begun to attract more scientific attention for their potential to improve our health. However, because of legal regulations, lack of knowledge and some confusion about the differences between fiber hemp and marijuana, the growth of hempseed research has been slower than expected. In view of its long history of dietary usage, it is surprising that research on the effects of dietary hempseed in animal and humans has been limited. Furthermore, because of its expected nutritional value and the hypothetical benefits of LA and ALA against a variety of health disorders, a better understanding of the appropriate doses and presentation (oil, seed, etc) of hempseed should represent useful health-related information. It is important to point out that dietary hempseed as an energy containing food item introduces changes in the fatty acid composition of the diet and will inevitably replace other dietary components under an isocaloric condition. Previously some [18] but not all authors [19] have found differences in body weight after the administration of 30 ml/d of hempseed oil for four to eight weeks in humans. Finally, an identification of the target patient population (age, clinical condition, co-morbidities, etc) that may benefit the most from a supplementation of hempseed in the diet would also be important information.

Animal Data

The biochemical metabolism of omega-6 fatty acids like LA produces eicosanoids in the body. Eicosanoids are biologically active and contribute to the formation of thrombi and atheromas and shifts the physiological state to one that is prothrombotic and proaggregatory, with increases in blood viscosity, vasospasm, and vasocontriction and decreases in bleeding time [15]. Hempseed is rich in LA content. Therefore, hempseed has received research attention for its effects on platelet aggregation.

Richard et al [20] reported that diets supplemented with 5% and 10% hempseed (wt/wt) for 12 weeks resulted in a significant increase in total plasma PUFAs in rats. ALA and LA levels increased significantly in a concentration-dependent manner [20]. Dietary hempseed supplementation also resulted in a significant inhibition of platelet aggregation and a lower rate of aggregation. This is an important result with physiological and pathological implications. As we have become increasingly aware of the importance of blood clots to the initiation of myocar- dial infarctions and strokes, the capacity of a dietary intervention like hempseed to inhibit clot formation has obvious appeal. However, if excessive bleeding is an expected event (as would be the case during surgery), it becomes essential for the physician/surgeon to know of a prior history of dietary hempseed usage. These data on the effects of dietary hempseed on platelet aggregation have been extended into hypercholesterolemic conditions by Prociuk and colleagues [21]. They have shown that rabbits fed a high cholesterol diet for eight weeks exhibit an enhanced platelet aggregation [21]. However, when 10% hempseed was supplemented to the diet together with the high cholesterol diet, these hypercholesterolemic animals displayed normal platelet aggregation values. This normalization was not related to any correction of the elevated plasma cholesterol levels but was related in part to the increased levels of plasma gamma-linolenic acid [21]. Because most patients at high risk for coronary heart disease are hypercholesterolemic, these findings have important potential for treating or preventing cardiovascular diseases.

Two other studies have been focused on the capacity of hempseed for altering cardiac function before and after an ischemic event [22,23]. Both studies have shown no effects of a hempseed-supplemented diet on basal cardiac contractile function or electrical activity before ischemia [22,23]. However, the data on the effects of dietary hempseed on cardiac performance post-ischemia is less consistent. Al-Khalifa and colleagues [23] reported that hearts from rats fed a 5% or 10% hempseed supplemented diet for 12 weeks exhibited significantly better post-ischemic recovery of maximal contractile function and enhanced rates of tension development and relaxation during reperfusion than hearts from the control group. The authors found that these hearts were not protected from the occurrence of premature contractions, nor were the increases in resting tension altered during ischemia or reperfusion [23]. This beneficial effect of hempseed on post-ischemic cardiac performance may be species specific. The same lab found that supplementation of the diet with 10% hempseed in rabbits did not show any beneficial effects on left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP), arrhythmia incidence and arrhythmia duration during ischemia and reperfusion [22]. Some limitations of the study related to the duration of the dietary intervention (8 weeks as opposed to 12 weeks) and sample size may have influenced the capacity for the dietary hempseed to protect the heart during ischemic insult [22].

Clinical Data

The actions of dietary hempseed in humans have only been studied to a limited extent. Fatty acid bioavailability from hempseed oil was recently studied in comparison to two other dietary oils (fish and flaxseed) [24]. Hempseed and hempseed oil is enriched in LA and GLA. Eighty-six healthy subjects completed a 12 week dietary supplementation with 2 g/day of these oils. The hempseed intervention did not significantly increase the concentration of LA, GLA or any other fatty acid in the plasma of the subjects, nor did it change the level of plasma total cholesterol (TC), high density cholesterol (HDL-C), low density cholesterol (LDL-C) or triglycerides (TG) [24]. Both flaxseed and fish oils did induce significant changes in circulating fatty acid species associated with their respective oils (ALA for flaxseed; EPA and DHA for fish oil) [24]. Supplementation with hempseed oil also did not induce any change in collagen-or thrombin-stimulated platelet aggregation or in the levels of circulating inflammatory markers [24]. It was suggested that the lack of effects may be related to the dose used [24]. This hypothesis has been supported by data obtained in another dietary intervention that used higher doses of hempseed (30 ml/day) [18]. In this randomized, double-blinded, crossover design trial, hempseed and flaxseed oils were compared at the same doses. After 4 weeks of supplementation, the hempseed intervention increased the concentrations of both LA and GLA in serum cholesteryl esters (CE) and TG. The flaxseed intervention resulted in higher serum CE and TG concentrations of ALA. However, a statistically significant decrease in GLA concentrations was observed during this period of intervention. Importantly, the proportion of arachidonic acid in CE was lower after the flaxseed diet than after the hempseed supplementation but this was not statistically significant. However, the hempseed supplements resulted in a lower total cholesterol:HDL cholesterol ratio. A higher total-to-HDL cholesterol ratio has been found in association with coronary heart disease [25]. However, no significant differences were found between the effects of flaxseed and hempseed oils in terms the fasting serum total or lipoprotein lipid levels, plasma glucose levels, or insulin or hemostatic factors [18]. Callaway and colleagues [19], using 30 ml/day of hempseed oil, conducted a 20-week randomized, singleblind crossover study in 20 patients with atopic dermatitis, and found that the levels of both essential fatty acids, LA and ALA, and GLA increased in all lipid fractions after using hempseed oil, with no significant increases of arachidonic acid in any lipid fractions. Moreover, atopic dermatitis symptoms were improved after the intervention with hempseed oil [19]. These results emphasize the importance of using higher doses of hempseed oil if significant increases in fatty acid species are to be achieved. Clearly, the ingestion of two large capsules of hempseed daily (as most people in the general public may ingest), is insufficient to achieve a desired increase in LA or GLA levels in the plasma [24]. Much larger doses are required to induce beneficial physiological effects. However, this may not be possible to achieve currently in the general population. If 10-15 times the amount used by Kaul and co-workers [24] is required to achieve a significant increase in plasma fatty acid levels, it would be unpractical to expect the general public to ingest 20-30 capsules of hempseed per day. This is a significant problem that the food and supplement industry must address in the future if hempseed is to be considered a realistic dietary approach to healthy living. Supplementing the diet with tablespoons of hemp oil in addition to hemp capsules as well as ingesting foods that contain these omega-3 fatty acids may be the optimal way to obtain them.

Linoleic acid and heart disease: New research fields for hempseed

Hempseed is a rich source of LA and others nutrients. The specific pathologies or conditions in which it can be used effectively are in need of more research but the data presently available suggest that LA may have beneficial effects in certain cardiovascular circumstances.

Effects on cholesterol levels

Iacono et al [26] reported that a high LA based diet (10.8%) decreased total cholesterol by 15% and LDL-C by 22%, without producing significant changes in plasma HDL-C after 6 weeks of dietary intervention in 11 healthy middle aged, male subjects. Apolipoprotein B decreased by 37% whereas apolipoprotein A-I increased by 24% in the group of individuals supplemented with this diet [26]. In a multiple crossover design that included 56 normolipemic, healthy subjects, Zock and colleagues [27] found that those who received the LA supplemented dietary intervention for three weeks (2.0% of total energy intake as LA) obtained lower levels of serum LDL-C, and higher HDL-C levels when compared with subjects who received its hydrogenation products elaidic (trans-Cl8:ln9) and stearic acid (C18:O). Recently, Mensink et al [28] employed a meta-analysis that included 60 controlled trials to show that polyunsaturated fat (mainly LA) reduces LDL-C, triglycerides and increases HDL-C. However, others have shown that healthy individuals supplemented for 4 weeks with hempseed exhibited a lower total-to-HDL cholesterol ratio [24]. A higher total:HDL cholesterol ratio is associated with coronary heart disease and has a worse prognosis after a myocardial infarction [29,30]. Clearly, the issue is not resolved yet. The population studied (healthy vs clinically compromised), the dosages of hempseed used, the presentation administered (whole hempseed vs milled hempseed vs hemp oil vs purified LA), the duration of the dietary intervention, the composition of the diet, are all factors that may be critical in producing the effects (of lack of effects) in these studies. More research is needed in order to understand if these specific conditions influence cardiovascular efficacy and to understand which metabolic factors are most sensitive (hypertriglyceridemia, hypercholesterolemia, low HDL-C, or other hyperlipoproteinemias) to this kind of dietary intervention.

Effects on high blood pressure

Results reported by The International Study of Macro-Micronutrients and Blood Pressure, a cross-sectional epidemiological study that included 4680 individuals, suggested that dietary LA intake may contribute to prevention and control of high blood pressure [31]. Other small studies have found that supplementation with LA (4 g-23 g/day) decreased blood pressure after 4 weeks of dietary intervention [32,33]. However, these promising results are in conflict with another study that reported no association between LA intake and lower blood pressure levels [34]. Studies using hempseed as a source of LA for hypertensive patients have not been conducted. It is also important to note that the consequences of these kinds of diets on arterial stiffness and vascular perfusion characteristics are unknown. The additional effects of these diets on ventricular hypertrophy that develops secondary to high blood pressure is not known nor are the effects when hempseed is supplemented with an antihyperten-sive medication. The potential for hempseed to alter drug kinetics in the body has not been studied.

Effects on atherosclerosis

Almost three decades ago, Cornwell and Panganamala postulated that an intracellular deficiency in essential fatty acids plays a central role in the atherogenic process [35]. Recently, Das [36] showed how a defect in the activity of Δ 6 and Δ 5 desaturases may be a factor in the initiation and progression of atherosclerosis. He also provided evidence that low-grade systemic inflammatory conditions are also essential fatty acids deficient states [36]. With our current understanding of the close relationship that infectious disease and inflammation has with atherogenesis [37,38], it is not difficult to predict that foods with an optimal LA-ALA ratio will reduce inflammation under ideal dietary conditions and it may thereby attenuate atherosclerotic heart disease. Unfortunately, the effects of LA on atherosclerosis are not completely clear. Arachidonic acid can be derived from LA. This can be converted to prothrombotic and proinflammatory prostaglandins. However, changes in dietary LA within the usual dietary range do not appreciably alter arachidonic acid levels [39,40]. Consistent with this, some have suggested that LA could have anti-inflammatory effects mediated by biochemical pathways that do not involve the cyclooxygenase pathway [41]. Presently, the randomized, controlled trials that address this topic have not been able to distinguish between the effects of omega-3 and omega-6 fatty acids [42]. Both have had beneficial effects by decreasing plasma levels of soluble TNF receptor 1 and 2, indicators of TNF activity [42].

Surprisingly, studies of the effects on atherosclerotic heart disease of dietary hempseed supplementation in animals or humans have not been completed. This type of study has been successfully completed using flaxseed as a dietary intervention [43,44]. It would also be important to determine if the LA content of hempseed (and not its ALA content) is responsible for decreasing inflammatory markers and the systemic atherosclerotic process in general.

Coronary heart disease

A meta-analysis of data from 25 case-control studies strongly suggested that a lower tissue content of LA is associated with increased coronary heart disease risk [45]. More importantly, this study did not show an association between AA tissue content and the risk for coronary artery disease. The results from randomized controlled trials have not been consistent either. Some [46,47] but not all [48,49] have found reductions in coronary risk with the use of an LA diet intervention. In a recent review, Harris [45] states that reducing LA intakes to less than 5% energy would be likely to increase the risk for coronary heart disease whereas higher intakes should be beneficial even in conditions without clinical evidence of adverse effects.

What we do not know about the effects of dietary hempseed

As discussed earlier in this paper, there is a lack of knowledge regarding the usefulness of hempseed or LA in different aspects related to cardiovascular diseases. It is important to identify not only what we presently know about dietary hempseed but also what is not known. The animal data lacks systematic information about the action of hempseed on myocardial infarctions, hypertension, atherosclerosis, markers of inflammation and arrhythmias. Similarly, we need to know more about the effects of this plant on the circulating lipid profile. Primary and secondary cardiovascular prevention trials using hempseed as a source of LA have not been performed. In general, we need to understand better the bioavailability of fatty acids like LA and GLA from dietary hempseed as a function of the age or sex of the subject, or as a function of the dosage of hempseed employed. Other dietary interventions (i.e. flaxseed) are sensitive to these variables [50,51] so it is not unrealistic to hypothesize that the delivery of hempseed will be influenced by these variables as well. It will also be important to identify if the hypotensive effects attributed to LA can be reproduced by dietary hempseed. As discussed previously, the capacity of LA and/or hempseed to affect ventricular hypertrophy secondary to high blood pressure, human atherosclerosis, inflammation, as well as the co-morbidities associated with cardiovascular diseases (like metabolic syndrome, diabetes mellitus, insulin resistance, obesity, heart failure or arrhythmias) still need to be determined in carefully controlled clinical trials.

Conclusions

The data discussed above supports the hypothesis that hempseed has the potential to beneficially influence heart disease. A mix of legal issues and misunderstandings has slowed research progress in this area but enough data presently exists to argue strongly for the continued investigation into the therapeutic efficacy of dietary hempseed. There remain many questions regarding the cardiovascular effects of hempseed that demand scientific answers in order to definitively establish this food as a preventive or therapeutic dietary intervention. Cardiovascular patients may not be the only subjects who benefit from this research. Furthermore, only time will tell if other diseases that have an immunological, dermatological, neurodegenerative basis may also benefit from this new nutritional intervention.

Figure 1
1Biochemical pathway for linolenic acid and α-linolenic acid transformation. ALA = α-linolenic acid; ARA = arachidonic acid; DGLA = dihomo γ-linolenic acid; DHA = docosahexaenoic acid; DPA = docosapentaenoic acid; EPA = eicosapentaenoic acid; GLA = γ-linolenic acid; LA = linoleic acid.

Table 1 :
1Nutrient profile of hempseed*.Nutrient
Units
Value per 100
grams

Nutrient
Units
Value per 100
grams

Proximates

Energy
kcal
567
Lipids

Energy
kJ
2200
Saturated fat
g
3.3

Protein
g
24.8
16:0
g
3.44

Total lipid (fat)
g
35.5
18:0
g
1.46

Ash
g
5.6
20:0
g
0.28

Carbohydrates
g
27.6
Monounsaturated
fat

g
5 . 8

Fiber, total dietary
g
27.6
18:1n9
g
9

Digestable fiber
g
5.4
Total
polyunsaturated

g
3 6 . 2

Non-digestable
fiber

g
22.2
18:2n6
g
56

Moisture
g
6.5
18:3n6
g
4

Glucose
g
0.30
18:3n3
g
22

Fructose
g
0.45
18:4n3
g
2

Lactose
g
<0.1
Cholesterol
mg
0

Maltose
g
<0.1
Amino acids

Tryptophan
g
0.20

Minerals
Threonine
g
0.88

Calcium, Ca
mg
145
Isoleucine
g
0.98

Iron, Fe
mg
14
Leucine
g
1.72

Magnesium, Mg
mg
483
Lysine
g
1.03

Table 1 :
1Nutrient profile of hempseed*. (Continued)

Table 2 :
2Rich sources of the essential fatty acid linoleic acid*.Source of LA
LA (g/100 g)
ALA (g/100 g)
Ratio n6/n3

Safflower oil
73
0.4
>100

Corn oil
57
1
57

Hempseed Oil
56
22
2.5

Cottonseed oil
50
0.2
>100

Soybean oil
50
8
6.2

Sesame oil
40
0.3
>100

Black walnuts
37
2
18.5

English walnuts
35
6.8
5.1

Sunflower seeds
30
0.06
>100

Brazil nuts
25
0.01
>100

Margarine
22
2.1
10.4

Pumpkin and squash seeds
20
0.12
>100

Spanish peanuts
16
0.01
>100

Peanut butter
15
0.08
>100

Almonds
10
0.06
>100

*Adapted from reference [12] and [13]

Rodriguez-Leyva and Pierce Nutrition & Metabolism 2010, 7:32 http://www.nutritionandmetabolism.com/content/7/1/32
AcknowledgementsThe work was supported through a grant from the Canadian Institutes for Health Research. The indirect costs of this research were supported by the St Boniface Hospital and Research Foundation. Dr Rodriguez Leyva was a Visiting Scientist of the Heart and Stroke Foundation of Canada.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsBoth authors contributed to the creation, literature review and writing of this manuscript.
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BMC Infectious Diseases Increased incidence of traffic accidents in Toxoplasma-infected military drivers and protective effect RhD molecule revealed by a large-scale prospective cohort study

Jaroslav Flegr firstname.lastname@example.com
Department of philosophy
Faculty of Science
Charles University
Vinièná 7128 44Prague

Czech Republic

Jiří Klose
Central Medical Psychology Department
Central Military Hospital
U vojenské nemocnice 1200
169 02PragueCzech Republic

Martina Novotná martinanovotnáfirstname.lastname@example.com
Department of philosophy
Faculty of Science
Charles University
Vinièná 7128 44Prague

Czech Republic

Miroslava Berenreitterová miroslavaberenreitterováfirstname.lastname@example.com
Department of philosophy
Faculty of Science
Charles University
Vinièná 7128 44Prague

Czech Republic

Jan Havlíček
Department of Anthropology
Faculty of Humanities
Charles University
2075, 158 00Husnikova, PragueCzech Republic

BMC Infectious Diseases Increased incidence of traffic accidents in Toxoplasma-infected military drivers and protective effect RhD molecule revealed by a large-scale prospective cohort study
10.1186/1471-2334-9-72BioMed Central Page 1 of 7 (page number not for citation purposes) Research article * Corresponding author
Background: Latent toxoplasmosis, protozoan parasitosis with prevalence rates from 20 to 60% in most populations, is known to impair reaction times in infected subjects, which results, for example, in a higher risk of traffic accidents in subjects with this life-long infection. Two recent studies have reported that RhD-positive subjects, especially RhD heterozygotes, are protected against latent toxoplasmosis-induced impairment of reaction times. In the present study we searched for increased incidence of traffic accidents and for protective effect of RhD positivity in 3890 military drivers.Methods:Male draftees who attended the Central Military Hospital in Prague for regular entrance psychological examinations between 2000 and 2003 were tested for Toxoplasma infection and RhD phenotype at the beginning of their 1 to1.5-year compulsory military service. Subsequently, the data on Toxoplasma infection and RhD phenotype were matched with those on traffic accidents from military police records and the effects of RhD phenotype and Toxoplasma infection on probability of traffic accident was estimated with logistic regression.Results:We confirmed, using for the first time a prospective cohort study design, increased risk of traffic accidents in Toxoplasma-infected subjects and demonstrated a strong protective effect of RhD positivity against the risk of traffic accidents posed by latent toxoplasmosis. Our results show that RhD-negative subjects with high titers of anti-Toxoplasma antibodies had a probability of a traffic accident of about 16.7%, i.e. a more than six times higher rate than Toxoplasma-free or RhDpositive subjects.Conclusion:Our results showed that a common infection by Toxoplasma gondii could have strong impact on the probability of traffic accident in RhD negative subjects. The observed effects could provide not only a clue to the long-standing evolutionary enigma of the origin of RhD polymorphism in humans (the effect of balancing selection), but might also be the missing piece in the puzzle of the physiological function of the RhD molecule.

Background

A protozoan parasite Toxoplasma gondii infects 20-60% of the population in most countries, depending on climate, hygienic standards and cooking habits [1]. Postnatally acquired toxoplasmosis in immunocompetent subjects causes mild disease, acute toxoplasmosis, which turns spontaneously into lifelong latent toxoplasmosis. Latent toxoplasmosis is characterized by the presence of the dormant cyst stage of the parasite mainly in the neural and muscular tissues and immunity against new Toxoplasma infections [2,3]. Latent toxoplasmosis in humans is considered as clinically asymptomatic [4,5]. However, infected people have impaired reaction times [6] and about 2.6 times higher risk of traffic accidents [7,8], possibly as a result of manipulation activity of Toxoplasma aimed to increase the chance of transmission from the intermediate to the definitive host, i.e. from any bird or mammal species to any feline species, by predation.

Recently, two studies on two populations of blood donors, one of conscripts [9] and the other of university students [10], have shown that RhD-positive subjects, and RhD heterozygotes in particular, are protected against latent toxoplasmosis-induced impairment of reaction times. The RhD protein which is the RHD gene product and a major component in the Rh blood group system carries the strongest blood group immunogen, the D antigen. The structure homology data suggest that the RhD protein acts as an ion pump of uncertain specificity and unknown physiological role [11,12]. The D antigen is absent in a significant minority of the human population (RhD-negatives) due to RHD deletion or alternation. For a long time, the origin of this RhD polymorphism was an evolutionary enigma. Before the advent of modern medicine, the carriers of the rarer allele (e.g. RhD-negative mutants in the population of RhD-positives or RhD-positive mutants in the population of RhD-negatives) were at a disadvantage as some of their children (RhD-positive children born to preimmunised RhD-negative mothers) were at a higher risk of foetal or newborn death or health impairment from haemolytic disease. The higher tolerance of RhD-positive heterozygotes against Toxoplasmainduced impairment of reaction time could counterbalance the disadvantage of the rarer allele and could be responsible both for the initial spread of the RhD allele among the RhD-negative population and for a stable RhD polymorphism in most human populations. Differences in the prevalence of Toxoplasma infection between geographical regions could also explain the striking variation in the frequency of RhD-negative alleles between populations.

A real impact of the latent toxoplasmosis-associated impairment of reaction times on human life (and therefore also a possible toxoplasmosis-associated selection pressure) was estimated by comparison of the prevalence of toxoplasmosis among victims of traffic accidents and control samples of people living in the same areas in two independent studies, in Prague (Czech Republic), and Izmir and Manisa (Turkey) [7,8]. However, they were both designed as retrospective case-control studies. Therefore, it was impossible to tell whether Toxoplasma-infected subjects had a higher risk of traffic accidents or whether Toxoplasma-free subjects had a higher chance to be included into epidemiological surveys used to estimate the prevalence of toxoplasmosis in the general population (the value used for controls in case-control studies), for example because of their higher superego strength and lower suspiciousness [13]. Moreover, the RhD status of victims and controls was not monitored and included in statistical analyses of the obtained data. The aims of the present study were 1) to test whether Toxoplasma-infected subjects have a higher risk of traffic accidents or a lower probability of being enrolled in epidemiological surveys and 2) to search for possible protective effect of RhD phenotype against traffic accidents in Toxoplasma-infected subjects. For these purposes we tested for latent toxoplasmosis and RhD phenotype a large population of conscript drivers in the beginning of their 12-18-month military service and estimated the risk of traffic accidents in different subpopulations of conscripts, using military police records.

Methods

Subjects

A study population comprised of 3890 male draftees (mean age 20.1 years, s.d. 1.55) who attended the Central Military Hospital in Prague for regular entrance psychological examinations between 2000 and 2003 and consented to participate in the research project. The draftees were tested for Toxoplasma infection and RhD phenotype at the beginning of their 1 to1.5-year compulsory military service. The overall seroprevalence of Toxoplasma infection was 29.7% and 22.8% of subjects were RhD-negative. In the Czech general male population of comparable age, the seroprevalence of Toxoplasma infection would be approximately 28.7% and the rate of RhD-negative subjects would be approximately 18% [14]. It must be stressed out, however, that the seroprevalence rates of toxoplasmosis vary widely from 7 to 90% with regions of the Czech Republic [15]. We have currently no explanation for a significantly higher proportion of RhD-negative subjects in our sample of the ethnically highly homogenous Czech population. All study subjects were screened for health status prior to their enrolment in the study. In the informed consent form, the draftees were explained the general aim of the project (a study of influence of latent toxoplasmosis on human psychology and psychomotor performance) and the need for obtaining their consent to using results of their psychological and clinical examina-tions. About 80% of the conscripts consented to the use of their test results for the research project purposes and provided 5 ml of blood for serological testing. To keep the study blind, they were not informed about the Toxoplasma assay results and that the incidence rate of traffic accidents during their military service would be monitored. Subsequently, the data on Toxoplasma infection and RhD phenotype were matched with those on traffic accidents from military police records and then anonymized. The recruitment of study subjects and data handling were performed in compliance with Czech legislation in force and were approved by the Institutional Review Board of the Faculty of Science, Charles University.

Serological tests

Serological tests for toxoplasmosis were carried out in the National Reference Laboratory for Toxoplasmosis of the National Institute of Public Health, Prague. Specific IgG and IgM antibody titers for toxoplasmosis were determined by ELISA (IgG: SEVAC, Prague, IgM: TestLine, Brno), optimized for the detection of acute toxoplasmosis [16], and complement fixation test (CFT) (SEVAC, Prague). The decrease in CFT titers is more regular and therefore better reflects the length of T. gondii infection [17]. CFT titers of antibodies to Toxoplasma in sera were measured at dilutions between 1:8 and 1:1024. For the purposes of this study, the subjects with negative results in the IgM ELISA (positivity index < 0.9) and an absorbance of > 0.250, i.e. approximately 10 IU/ml, in the IgG ELISA were considered latent toxoplasmosis positive while those with an absorbance of < 0.250 in the IgG ELISA were considered latent toxoplasmosis negative. Less than ten subjects with different diagnosis obtained with CFT and IgG ELISA tests or with suspected acute toxoplasmosis were excluded from the present study. The probability of false positive results in two independent serological tests is rather low, probably less than 3% [17]; however, theoretically, the population of seronegative subjects could be contaminated with an unknown (but probably very low) number of immunocompromised subjects [18] and subjects that acquired the infection during their military service. The RhD blood group type (presence of the RhD antigen on the erythrocyte membrane) was determined in all serum samples using the human monoclonal anti-D reagents (Seraclone ® , ImmucorGamma Inc.) in the Central Military Hospital service laboratory.

Statistical analysis

The Statistica 6.1 and SPSS 16.0 programs were used for statistical testing (frequency tables, logistic regression and Generalized linear model) and to check statistical tests assumptions. All variables including the covariates entered the respective analyses are specified in the Results section.

Results

The rate of traffic accidents in 3890 male military conscripts was 2.85% (n = 111), see Table 1. In particular subgroups, the rates were as follows

Discussion

Our results show that Toxoplasma infection increased the risk of traffic accidents in military drivers. The data also indicate that RhD positivity protected infected subjects against the increase in the rate of traffic accidents. In RhDnegative subjects, the probability of traffic accidents increased with titer of anti-Toxoplasma antibodies suggesting that the risk of traffic accidents decreased with duration of Toxoplasma infection. The probability of traffic accidents increased across the years of testing. This phe- Our results are in good agreement with the results of laboratory testing of reaction times in Toxoplasma-infected and Toxoplasma-free, RhD-positive and RhD-negative subjects [6,9,10]. Latent toxoplasmosis had a strong negative effect on RhD-negative subjects and no effect on RhD-positive subjects. The strength of the negative effect of toxoplasmosis on reaction times increased with duration of infection (with decreasing anti-Toxoplasma antibody titer) [6]. In contrast, in the current study the risk of traffic accidents decreased with duration of infection. It should be noted, however, that the decrease of the risk of traffic accidents with duration of infection was already observed in a previous case-control study [7]. The most plausible explanation for such conflicting results is that, on the one hand, reaction times increase with duration of infection and, on the other hand, the infected drivers are able to gradually adapt to their impaired reaction times. Such effect has already been described in aging drivers [19].

Rates of traffic accidents in

It was previously speculated that personality differences between Toxoplasma-infected and Toxoplasma-free subjects could result in lower willingness of Toxoplasma-infected subjects to participate in serological surveys and therefore also in their underrepresentation in a control population [7]. The design of the current study, i.e. a prospective cohort study, made it possible to confirm that the increase of the risk of being involved in a traffic accident rather than a decreased chance of being enrolled in epidemiological surveys is responsible for the higher prevalence of Toxoplasma-infected subjects among victims of traffic accidents compared to the general population of the same area.

At face value, our results in some respect contradict those of the previous traffic accident studies. In the present study, the effect of latent toxoplasmosis on traffic accidents was not apparent in an unsorted population of RhD-positive and RhD-negative subjects. When infected with Toxoplasma, the RhD-negative subjects had a significantly higher and RhD-positive subjects a nonsignificantly lower incidence rate of traffic accidents than the corresponding Toxoplasma-free subjects. On the other hand, the present results are in agreement with the unpublished results of our retrospective case-control study performed in Prague between 2002-2006 using the same method as in the 1997-2000 study [7]. In that study, similarly as in the present prospective study, the effect of latent toxoplasmosis on the incidence rate of traffic accidents in the RhD unsorted population was not sig-nificant. In January 2000, a dramatic change in Czech traffic rules came into action. For example, pedestrians did not have priority at zebra crossings before the year 2000. We suppose that changes in behaviour of Czech drivers and pedestrians could be responsible for differences between results from 1997-2000 and 2001-2006.

It must also be noted that the effect of latent toxoplasmosis on reaction times varies across testing minutes [6,9,10]. The effect of toxoplasmosis on reaction time is always stronger in RhD-negative subjects than in RhDpositive subjects. However, RhD-negative Toxoplasmainfected men expressed nonsignificantly shorter reaction times (p = 0.37) than RhD-negative Toxoplasma-free subjects in the first minute of the three minute-test [10]. The authors speculated that two effects of infection might interplay in Toxoplasma-infected men. Latent toxoplasmosis is known 1) to impair reaction times in infected men [6] and animals [20] and also 2) to increase the concentration of testosterone in infected men [21][22][23]. Increased concentration of testosterone positively influences the level of personality trait competitiveness in men [24], which, in turn, could enhance performance under certain circumstances. Therefore, the negative effects of latent toxoplasmosis on psychomotor performance in men could influence the rate of traffic accidents under certain conditions only, which could explain some differences in results between the published case-control studies and present cohort study.

Our study has several limitations. Despite the fact that nearly 80% of draftees agreed to participate in our study, this population cannot be considered fully representative of the Czech general population. For example, our anecdotal observation of increased frequency of traffic accidents in 20% of drivers who did not agree to participate, suggested that this subpopulation differed in both psychological profile and probability of traffic accident from subpopulation under the study.

The subjects were screened for latent toxoplasmosis in the beginning of the military service only. Some drivers probably acquired toxoplasmosis during their 0.5-1.5 year military service. It must be stressed, however, that the presence of Toxoplasma-infected drivers in our set of Toxoplasma-free subjects (as well as possible presence of Toxoplasma-free drivers in our set of Toxoplasma-infected subjects) can only increase risk of false negative but not false positive results of our statistical tests.

According to military legislation all traffic accidents including material damage and/or injuries shall be reported to the military police. Of course, some less serious accidents might remain unreported; however, according to the experience of military traffic experts completeness of military records is much higher in comparison with the civic sphere (personal communication).

A major limitation of the present study is the absence of RHD genotype data. Previous case-control study on a large sample of blood donors has shown that RhD-positive heterozygotes are resistant to pathological effects of toxoplasmosis while RhD-positive homozygotes are only temporarily resistant: their psychomotor performance decreases with length of infection. The psychomotor performance of RhD-negative homozygotes decreases immediately after infection. Our population of RhD-positive drivers includes both RhD-positive heterozygotes and RhD-positive homozygotes (some of them with relatively low concentration of anti-Toxoplasma antibodies and therefore relatively old infection). Possible contamination of the accident protected RhD population by an unknown number of non-protected RhD-homozygotes might decrease the power of our tests and undervalue the strength of the observed effects.

The absence of data on RHD genotype of subjects makes any speculation on the mechanism of RhD positivity protection very difficult. However, the results of previous studies [6,9,10] and current knowledge about localization and probable function of RH proteins [11,12] suggest that RhD and RhCE proteins that act as ion pumps, coded for at the RH locus and localized on the erythrocyte membrane, are involved in the regulation of ion balance in some critical compartment of nerve or muscle tissue. Such regulation could be important especially in the subjects handicapped by the presence of Toxoplasma cysts in nerve and muscle tissues.

Conclusion

The influence of recent (subacute) Toxoplasma infection on the rate of traffic accidents is relatively strong. Three of seventeen RhD-negative military drivers (16.7%) with anti-Toxoplasma antibody titers of 1:64 or higher, i.e. the subjects likely to be infected with Toxoplasma for less than two years [17], had a traffic accident within the next 12-18 months while the risk rate in other groups of military drivers was about 2.6%. We suggest that RhD-negative drivers diagnosed with acute and subacute toxoplasmosis should be informed about the transiently increased risk of traffic accidents.

The observed effects could provide not only a clue to the long-standing evolutionary enigma of the origin of RhD polymorphism in humans (the effect of balancing selection), but might also be the missing piece in the puzzle of the physiological function of the RhD molecule.

: 2 .
280% (n = 69) in 2460 RhD-positive Toxoplasma-free subjects, 2.40% (n = 17) in 709 RhD-positive Toxoplasma-infected subjects, 2.59% (n = 14) in 540 RhD-negative Toxoplasma-free subjects and 6.08% (n = 11) in 181 RhD-negative Toxoplasma-infected subjects. Analyses of frequency tables showed no significant effect of Toxoplasma (Chi 2 = 0.356, P = 0.551) or RhD phenotype (Chi 2 = 1.20, P = 0.273) on probability of traffic accidents. Logistic regression with dependent variable accident (binary) and independent factors toxo, RhD and toxo-RhD interaction showed significant effects of toxo (OR 2.43, CI 95 : 1.11-5.35, t = 2.157, P = 0.027) and toxo-RhD interaction (OR 0.35, CI 95 : 0.136-0.902, t = -2.17, P = 0.028) suggesting that Toxoplasma infection increased the chance of traffic accidents while RhD positivity reduced the risk of traffic accidents in Toxoplasma-infected subjects. To eliminate the effects of possible confounding variables driver's age and year of the beginning of military

23, P = 0.026). The same analysis for RhD-positive subjects showed no significant effect of Toxoplasma infection. Similarly, analysis for Toxoplasmafree subjects showed no significant effect of RhD phenotype, while this analysis for Toxoplasma-infected subjects revealed a 0.38 times lower risk of traffic accidents in RhD-positive than in RhD-negative subjects (CI 95 : 0.177-0.84, t = -2.39, P = 0.017). To search for possible effect of anti-Toxoplasma antibody titer (indication of the length of the infection) on risk of traffic accidents, we analysed Toxoplasma-infected subjects using Generalized linear model with independent variable accident and independent binary variable RhD and ordinal variable titer of antibodies (probability distribution: binary, link function: logit). The results showed a significant effect of interaction RhD-titre of antibodies (Chi 2 = 7.85, P = 0.02). The chance of traffic accidents increased 1.42 times (CI 95 : 0.01-0.68) for each titration step in RhD-negative subjects (B = 0.35, CI 95 : 0.01-0.68, Chi 2 = 4.18, P = 0.041). However, no effect of antibody titer was observed in more numerous RhD-positive subjects (B = 0.07, CI 95 : -0.27-0.4.25, Chi 2 = 0.18, P = 0.674), Fig. 1.

RhD-negative (crosses) and RhD-positive (circles) military drivers within 12-18 months of military service Figure 1 Rates of traffic accidents in RhD-negative (crosses) and RhD-positive (circles) military drivers within 12-18 months of military service. The y-axis shows rate (probability) of traffic accidents for subjects with particular anti-Toxoplasma antibody titers. The figures indicate numbers of RhD-positive/RhD-negative subjects with particular anti-Toxoplasma antibody titers(x-axis). , probably, caused by the observed downward trend in overall quality, e.g. in intelligence, of conscripts (data not shown), related to the forthcoming abolition of compulsory military service in the Czech Republic in 2004.

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Table 1 :
1Number of subjects and age in particular categories.Toxoplasmosis
Rh
Accident No
Accident Yes
Row

No
negative
526 (13.52%)
20.23
14 (0.36%)
20.64
540 (13.88%)
20.24

No
positive
2391 (61.47%)
20.17
69 (1.77%)
20.29
2460 (63.24%)
20.17

Total
2917 (74.99%)
20.18
83 (2.13%)
20.35
3000 (77.12%)
20.18

Yes
negative
170 (4.37%)
19.98
11 (0.28%)
19.91
181 (4.65%)
19.97

Yes
positive
692 (17.79%)
20.02
17 (0.44%)
20.65
709 (18.23%)
20.04

Total
862 (22.16%)
20.01
28 (0.72%)
20.36
890 (22.88%)
20.02

Column Total
3779 (97.15%)
20.14
111 (2.85%)
20.35
3890 (100%)
20.15

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AcknowledgementsWe thank Aleš Kubĕna for his help with statistical analysis. This work was supported by grants of the Czech Ministry of Education and Sports (grant 0021620828) and Grant Agency of the Czech Republic (grant 206/07/0360).Competing interestsThe authors declare that they have no competing interests.Authors' contributionsJF conceived of the study, participated in its design and coordination, and performed all statistical analyses. JK participated in the design of the study and organized collection of data at Central Military Hospital. MN, MB and JH collected the data and participated in writing the manuscript. All authors read and approved the final manuscript.Pre-publication historyThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/72/prepub
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