Accumulation of Nitrate in Vegetables and Its Possible Implications to Human Health

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ScienceDirect Oc t o b e r 2007Agricultural Sciences in China2007, 6(10): 1246-1255Accumulationof Nitrate in Vegetables and Its Possible Implications o HumanHealthDU Shao-ting, ZHANG Yong-song and LIN Xian-yongKey Lab of Environmental Remediation and Ecosystem Health, Ministry of Educat iodCol l ege of Environmental and Resources Sciences,Zhej iang Univers i ty, Hangzhou 310029, P.R.China

AbstractIn recent times, there are two kinds of completely opposite viewpoints about the impacts of nitrate on human health. Tofurther objectively understand the effects of nitrate on hum an health, both of harm fulness and po ssible benefits of nitrateto human body, it is discussed in this review from the aspects of nitrate accumu lation in vegetables, the source of nitrateingested into human body, and the transformation of nitrate in human body , as well as the pathogenesis and physiologicalfunctions of nitrate metabolism.Key words: vegetable, nitrate, methemoglobin, carcinogenesis, human health, NO, bacteria

INTRODUCTIONNitrate is a common chemical compound in the nature,and is widely found in soils, waters, and foods.Generally, nitrate in vegetables is considered to be themain source of dietary nitrate intake (Santamaria et a l.1998). Some previous researches suggested that thevegetables with high nitrate in the diet could put a humaninto the risk of gastrointestinal cancer andmethemoglobinaemia (Bartsch et al. 1988; Slob et al.1995). Therefore, there is great concern about thenitrate content in the daily diet, especially in vegetables.In recent decades, considerable researches have beendone to minimize the nitrate accumulation invegetables, mainly in plant nutrient management,horticultural technology, and breeding. However,some epidemiological studies also proposed that thenitrate may be advantageous to human health, suchas protecting the intestine from harmful bacterial

infection (McKnight e t al. 1999; Archer 2002;Dykhuizen et a l. 1996). Besides, one of the reducednitrate products, the NO, is known to be an importantsignal molecule in regulating many physiologicalfunctions in human body, and in addition, it has alsoserved as an effective host defense against pathogens(McKnight et al. 1999; Archer 2002). Accordingly,whether the dietary nitrate is detrimental to humanhealth and environment remains a disputed topic sofar, especially after the publication of Nitrate andMan:Toxic, Harmless o r Beneficial? by LHirondel J andLHirondel J-L in France in 1996 (English edition,2001), which further activated the controversy. Thisreview summarizes the accumulation of nitrate invegetables, and the factors affecting it. Emphasis isalso placed on the metabolism mechanism of theingested nitrate and nitrite in human body and theirdeleterious and beneficial effects on human health,which can lead to a further understanding of it, and ajust evaluation.

This article is translated from its Chinese version in Scieniia Agricultura Sinica.DU Shao-ting, Ph D candidate, E-mail: [email protected]; Correspondence ZHANG Yong-song,Professor,Tel: +86-571-86971151,ax:+86-571-86971359,E-mail: yszhang@zju,edu.cn

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NITRATE ACCUMULATION IN VEGETALBESThe AD1 (acceptable daily intake) of nitrate was allocatedas 0-0.37 mg kg-' body weight, whereas, the exceedingof the AD1 limit has occurred frequently in daily humanlife (Boink and Speijers 2001). Slob et al. (1995) hadshown that in adults, 15% of the daily intake exceededthe ADI, and might rise to 45% in young children. Thedaily nitrate intake occurs from three major sources:vegetables, drinking water, and meat products.Although great differences exist in diary habits and waterquality between different countries, vegetables are stillthe major source of nitrate intake (Table 1). Theyconstitute 30-90% of the total nitrate intake. Assuminga daily consumption of 0.5 kg fresh vegetables perperson, the maximum allowed nitrate levels in vegetablesshould be 432 mg kg-I. Generally, about 45% of thetotal nitrate in the vegetables can be reduced by pickling,

Table 1 Estimation of daily nitrate intake and contribution ofvegetables in various regions (Hambridge 2003)Daily nitrate intake from Contribution of

vegetables (mg d-I) vegetables (%)CountryEastern Asia 28 45Africa 20 30Latin America 55 65Europe 155 90

and 75% reduced by cooking. Hence, the maximumallowable limit for nitrate can be increased to 785 mgkg-' for pickled vegetables and 1.440mg kg-' for cookedvegetables, respectively. In the case of a 60 kg bodyweight with the daily consumption of 100g vegetables(2500 mg kg-' of nitrate content), there would be 13%excess of the ADI. Grouping of the nitrate content inedible parts, leafy and root vegetables show higher nitratecontents, whereas, the melon and nightshade show a muchlower content (Table 2). The trends in Table 3 also showthe similar results, which are classified as vegetablevarieties. To date, no nitrate standards for vegetableshave been introduced in the USA, whereas, a suggestedmaximum level of nitrate in vegetables of 3 100 mg kg-Ihas been established in China (Zhou et al. 2000).

As shown in Tables, most vegetables can accumulatelarge amounts of nitrate. It is reported that the highestnitrate-accumulating vegetable is Diplotuxis, found inItaly, which can reach up to 9 300mg kg-' (Santamariae t al. 1999). Moreover, there i s always significantvariance of nitrate content among different genotypes,and sometimes it can even reach several folds, such asspinach. The nitrate content in the full-wrinkle genotype,half-wrinkle genotype, and smooth genotype are 446.0,315.9, and 194.0 mg kg-' FW, respectively, althoughunder the same nitrate nutrition of 18 mmol L-' (Wanget al. 2000). Besides, the nitrate content also differs in

Table 2 Nitrate and nitrite c onten t in edible part of vegetables (Wang et al. 2000)NO; (mg kgl FW)egetable types Vegetable varieties NO; (mg kg-1FW)

Root vegetables Carrot 0.02-0.23 921-1 956Datoucai leaf-mustard 0.12-0.64 709-956

Green vegetables Lettuce 0.08-2.15 123-2 678Spinach 0-0.73 239-3 872

Cabbage Chinese cabbage 0-0.65 429-1 610Pakchoi 0.09-2.42 1023-3098Cabbage 0-0.41 259-1 250Cole 3.64-5.35 766-1 365

Melon Wax gourd 0.01-0.06 358-680Cucumber 0-0.11 12-143

Nightshade Eggplant 0.07-0.49 250-424

Table 3 Classification of vegetables according to nitrate content (mg kg-'FW) (Santamaria 2006)~~ ~

Nitrate content (mg kg I FW)Very low (2500)

Vegetable varietiesArtichoke, asparagus, broad bean, eggplant, garlic, onion, green bean, mushroom, pea, pepper, potato, summersquash, sweet potato, tomato, watermelonBroccoli, carrot, cauliflower, cucumber, pumpkin, 'puntarelle' chicoryCabbage, broccoli, dill, savoy cabbage, turnipCeleriac, Chinese cabbage, endive, fennel, kohlrabi, leek, parsleyCelery, cress, chervil. lettuce, red beetroot, spinach, rocket

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various parts of the plant, such as the nitrate content instem, petiole, and leaf in mustard is 376.0, 3 10.9, and85.1 mg kg-'F W , espectively. Generally, the vegetableorgans can be listed in the decreasing order of the nitratecontent as follows: petiole > leaf > stem > root >inflorescence > tuber >bulb > fruit > seed (Santamariae t al. 2001). However, when the media nitrateconcentration is relatively low, the nitrate content willincrease in the root, but decrease in the shoot.Contrarily, it will change on the high nitrate supplycondition. The factors leading to the difference of nitratecontents in different vegetables are complex, and anumber of studies on the mechanism of nitrateaccumulation have been done, mainly on the nitrateuptake rate, nitrate reductase activity, and growth rate,which are closely related to carbon metabolism. Thecarbon metabolism in plant is determined by variousgrowth conditions, including light, temperature, CO,level, and so on (Hageman and Flesher 1960; Toselliet al . 1999; Yu et a l . 2006). Moreover, besides thegenetic factor, the growth conditions also play adecisive role in affecting the nitrate accumulation inplant. Furthermore, the nitrogen absorbed by mostvegetables from soil is nitrate which is sequentiallyreduced by nitrate reductase (NR), nitrite reductase(NiR), glutamine synthase (GS), and glutamate synthase(GOGAT). Although there are numerous researcheson the mechanisms of nitrate accumulation invegetables, it remains argumentative. However, mostof the studies suggest that the imbalance between nitrateuptake and reduction should be the fundamental factor.In addition, once the increment of nitrate accumulationis stronger than its growth, growth lag would strengthenthe nitrate accumulation through nutrient enrichment.The inconsistent distribution of nitrate and nitratereductase in plant cells is another important factorresulting in the higher nitrate contents in some parts ofvegetables. In recent years, many researches proposedthe beneficial effects of nitrate on human health, andhence, the efforts done to alleviate nitrate accumulationin vegetables are doubted. Therefore, a clearunderstanding of nitrate metabolism in human body andits relationship with the hypothesized harmful andbeneficial effects is the prerequisite for nitrate evaluation,which can also help us to set the allowable limit in foodand water.

MECHANISMOF NITRATEINHUMAN BODYNitrate widely exists in the drinking water and food,and as a result, it inevitably enters the human food chain.Therefore, understanding the metabolism mechanismof the ingested nitrate is a prerequisite for properevaluation of the hypothesized harmful and beneficialeffects on human health. Thus, the mechanism of thenitrate in human body will be demonstrated initially.

Translationof nitrate in oral cavityAs the vegetables first enter into the body by swallowing,the conversion of nitrate in the mouth is particularlyimportant. The dorsal surface of the tonguesymbiotically harbors a specialized flora of anaerobicnitrate reducing bacteria, which can rapidly reducenitrate to nitrite (Duncan et al. 1995), hence, thecontents of nitrite in saliva are generally higher, andcan reach 200 ph4 (Benjamin 2000). Some antibioticinhibition studies showed that amoxicillin (a broadspectrum antibiotic that can inhibit bacteria growth)significantly reduces the nitrite concentration n the saliva(Dougall et al. 1995), suggesting a critical role of bacteriain the conversion of nitrate to nitrite.

However, the nitrite will not be further reduced inthe mouth because of the lack of nitrite reductase.Nevertheless, t will be absorbed in the salivary glands,therefore, the nitrite concentration in saliva is activelyconcentrated by a factor of ten from the plasma, even50-100 folds of the stomach (Leifert et al. 1999).

Conversionof nitrite in stomachBecause of the absence of nitrite reductase in the oralcavity, nitrite will convert to varieties of nitrogencompounds in the stomach. The main products areNO and N-nitroso compounds.Converting to NO NO generation in human body canbe divided into the endogenous and exogenouspathways. Nitrite formed in the mouth can be acidifiedin two ways: direct acidifying in the acid environmentaround the teeth caused by microorganisriis (such asLactobacillus or Streptococcus mutans); or acidifyingafter being swallowed, in the acidic stomach, which isthought to be more important. However, acidification

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of nitrite produces nitrous acid, which is unstable,spontaneously decomposing to manifold nitrogencompounds. The most important product is NO, andthe whole process is as follows:

NO; + H -HNO,; 3HN0,- ,O + 2N0 + NO, +H+;2HN0, - H,O + N,O,; N,O, -NO + NO,Because the NO generates from the nitrate in food,this process can be defined as an exogenous pathway.It is reported that the NO concentration formed in thestomach can be increased when nitrate intake isincreased, but on the contrary, i t can also be reducedwhen stomach acidification is impaired (Lundberget a l . 1994). Recent researches indicate there is asignificant correlation between NO generation and nitritecontent in plasma (Kleinbongard et al. 2003), andthereby the nitrite is suggested to be the storage of NO(Dejamet al. 2004). Moreover, it is likely that reducingsubstances secreted in the stomach, such as ascorbicacid and reduced thiols, are responsible for the enhancedNO production (DeGroote and Fang 1995; de Belderet al . 1994). The chemistry of this process is complex,and will generate different products under varyingconditions of initial concentrations, pH, and oxido-reductive potential. Hereby, it still awaits further study.

Furthermore, a high concentration of NO could begenerated in the acidic stomach. But it was alsosuggested that the total nitrite in saliva would generateabout only one tenth of the NO that is actually measuredin the stomach (McKnight et al. 1999), indicating thatother NO generation pathways should exist. It wasconsidered that the endogenous NO was synthesizedfrom NO synthetase acting on L-arginine (Moncadaand Higgs 1993), and this NO generation pathway wasdefined as the endogenous pathway.Converting to N-nitroso compounds N-nitrosocompounds (NOCs) include nitrosamine and nitroamide.The nitosoproline formation can be detected in urineafter proline or nitrate-rich beet juice is intaked,suggesting that endogenous NOCs can be synthesizedin human body (Ohshima and Bartsch 1981). Itdemonstrates that the formation of NOCs is because ofthe acidification of salivary nitrite on entering thestomach, where it undergoes nitrosation reactions.

In the acid environment, nitrite can be converted toactive nitrosating agents, such as, nitrous anhydride(N,O,), nitrosyl isothiocyanate (GN-NCS), nitrosyl

halide (NOX), and protonic nitrite (H,NO,). Thesecompounds can easily nitrosate with secondary aminesto produce NOCs, and the reactions are as follows (Baiand Sun 2002):

2HNO,-+N,O, + H,OO; + HCl = HNO, + C1-R,N + H, - R,NH (secondary amines) + HActually, nirosation is the nucleophilic substitution

reaction between secondary amines and nitrosatingagents, converting to nitroamide probably by protonicnitrite:

2HN0,-H2N0,+ +NO; or HNO, +H+-H,NO,

R,NH + N,O, - ,N-NO + HNO,

RNH + H,NO,+- \ N - N O + H,O + H+\

RCO RCOAmide Nitroamide

The in vitru kinetics of exogenous nitrosationresearches indicated that the nitrosation reaction wasrelated to pH and the reactants concentration. The pHin the stomach of healthy humans is 1-3. In such anacidic condition, the growth of nitrate reducing bacteriawill be inhibited and almost no exogenous nitrate canbe converted to nitrite. Once achlorhydria takes place,the gastric pH will rise and then nitrate reducing bacteriawill be increased and activated, along with a largeamount of nitrate being reduced to nitrite. Thereby,there will be more nitrite converted in the chronicatrophic gastritis patients, easily leading to the formationof NOCs. Besides, gastric pH is generally increasedwith age, and hence the NOCs synthesize more easilyin older people. It is reported that more than 20% ofpeople over 50 years of age were achlorhydric becauseof the high gastric pH (Hill 1999).Nitrate recirculation between saliva andgastrointestinal ract After entering into human body,approximately 25% of dietary nitrate derived from foodand water is recirculated between the saliva and thegastrointestinal tract as follows: food or water - ralcavity- sophagus- tomach- ntestine -+ plasma- aliva- ral cavity (Duncan e t al. 1995). It wasfirst reported in 1961, that humans excrete more nitratethan they consume, which was further confirmed inthe 1970s and 1980s (Benjamin 2000), suggesting thatthere also exists a self-secrete pathway, except forexogenous nitrate intake through food and water. It isspeculated that both these methods can provide 70-75

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mg nitrate per day (Hibbs et al. 1992). Generally, theself-secrete pathway is known to exist because of theformation of nitric oxide by mammalian cells from theamino acid L-arginine (Fig.) (Moncada and Higgs 1993).Although it is still unclear whether all endogenous nitratesynthesis are derived from this route, the I5N-labelledarginine resulted that the enrichment of urinary nitratewith this heavy isotope is only about one half of thetotal lSNarginine enrichment (Li et al. 1997), indicatingthat other nitrate synthesis pathways should exist.

BENEFICIAL AND HARMFUL EFFECTS OFNITRATEGenerally, it is considered that high nitrate intakeincreased the risk of cancer and methemoglobinaemia,even hyperparathyroid, children polyuria, hypertension,and so on (McKnight et al. 1999). However, in recentyears, many researches found that the nitrate has lotsof beneficial effects on human health. Therefore, thesetwo contrary conclusions cause a controversy.

Nosogenetic effectsof nitrate and nitriteThe safety of nitrate on human health was first doubtedin 1945, when the infantile methemoglobinaemia wasassociated with dietary water. However, L'Hirondel Jand L'Hirondel J-L (2001) indicated that it should becaused by the insanitary condition and not by the nitrate

in the dietary water. Further studies also suggestedthat it was not the nitrate itself, but its metabolite, thenitrite, which could be the compound leading to infantilemethemoglobinaemia.

It is reported that the toxicity doses formethemoglobin formation, as a criterion, ranged from33 to 350mg nitrate ion kg-' body weight, especially inan nfant, and it could easily lead to death (Speijers 1996).Generally, in the early weeks of life, the infant's gut issusceptible, colonized by many bacteria (e.g.,Campylobacter, Salmonella, and E. co l i ) , and hence,nitrate in the feed is readily reduced to nitrite in thestomach and small intestine by bacterial nitratereductase, and consequently the methemoglobin caneasily accumulate through the reaction between nitriteand hemoglobin [4NO, + 4oxyHb (Fe2+)+ 4H' -4metHBb (Fe3+)+ 4NOi + 0, + 2H,O], which greatlyreduces the 0,-binding capacity and therefore the infanttissues are starved of 0, and result in cyanosis. It isgenerally accepted that 60% of the hemoglobin in theform of methemoglobin s a lethal level (Hill 1999). Laterin life the infant develops a gastric acid barrier and sothe bacteria colonized on the upper gut decrease to verylow levels, minimizing nitrite formation. Besides, anerythrocyte enzyme is formed, which destroys the nitriteand protects the hemoglobin from nitrite toxicity,reducing the methemoglobin to hemoglobin [metHBb(Fe3+)+ oxyHb (Fez+)]. Consequently, children andadults are far less susceptible to methemoglobinaemiathan young infants, unless the accidental and

Amino acid

Nitric oxide synthetase 1- ~ ~ ~ i ~ i ~ ~+ Nitric oxideIEndogenouI

Cell ___)

Lr itrate ,- SweatI L-- N i t r a t e - 1 T e a rHypochlorhydriaN-nitroso compounds 4- I I I , II Exogenous INitrate in food Mouth- Nitrite-tomach- Nitric oxideMicroorganisms I Strong acidic conditionSaliva Reducers such as VcT IrineI /aliva gland 4 ' KidneyRecycle with blood plasma

Fig. Mechanisms of translocation and transformation of nitrate in the human body.

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stomach rather than being secreted immediately,suggesting the beneficial physiological function whichis attributed to the metabolite of nitrate -NO.Signal transduction NO can be generated by the nitrateboth from exogenous nitrate intake and endogenoussynthesis. From the 1980s, many studies have alreadydemonstrated the signal transduction function of NO insignal transduction, which is an effective vasodilatoragent and platelet aggregation inhibitor (McKnightet al . 1999). Therefore, NO deficiency will cause manydiseases, such as arteriosclerosis, diabetes, low bloodpressure, and coagulation diseases (Coss et al. 2004).In addition, Stuehr and Marletta (1985) reported thatmacrophage could produce nitrate when it was sufferingfrom the toricity of bacterial compound, and theirfurther analysis showed that once macrophages wereattacked by toxic elements, they released NO to increasethe phagocytosis before the NO was converted to nitriteand nitrate. Later, DeGroote and Fang (1995) confirmedthe results above, and also found that the phagocytosiswould be impaired if NO synthesis was inhibited,demonstrating the important antibacterial role of NOduring stress induction.Cardiovascular protection Many theories have beenproffered over the last few decades in an effort to explainthe relation between cardiovascular diseases and dietswith a high content of vegetables and fruits.Undoubtedly, the antioxidant vitamins and folic acidhave an important effect on the health of thecardiovascular system, but the mechanism is still unclear(Goldin and Gorbach 1996). McKnight et al. (1997)found that large quantities of diffusible NO gas aregenerated in the stomach, which would readily lead toS-nitrosated formation with a diet that contains thiolsin the acidic environment. The platelet adhesion andaggregationarecritically related to the vascular occlusivedisease, while NO, S-nitrosothiol,and S-nitrosoglutathioneare known to be platelet aggregation inhibitors (de Belderet al. 1994). Therefore, nitrate intake can strengthenplatelet function, preventing thrombotic episodes(McKnightet al . 1999). Abrams (2002) also consideredthat the symptoms of congestive heart failure can bereduced by an appropriate amount of nitrate intake, suchas clinical nitroglycerin, but the patients who have usednitrates for over a period of years can also suffer fromweak health.Other functions McKnight et al. (1999) found that

bacterial survival was shorter after the 2 mM nitratedrink. Also, Benjamin and McKnight (1999) reportedthat a lower incidence of diarrhea happened when thevolunteer took nitrate tablets. Therefore, nitrate willhave an important physiological role in killing ingestedpathogens and reducing the normal time required.However, nitrate itself has no bactericidal effects becauseof its chemical characteristics. Recently, it was stressedthat the nitrate metabolites, including nitrite, nitrite acid,and NO, should have the antimicrobial activity forintestinal pathogen prevention (Duncan et a l. 1995;Lundberg et al . 1994). In other studies, addition ofnitrite (same concentration found in saliva) tohydrochloric acid solution with pH 2-4 increased itsantimicrobial activity up to a 100-fold, furthe rdemonstrating the significant correlation between theantimicrobial activity and acidity. Here, also the harmfuleffects of nitrite on the microorganisms that arebeneficial to human health cannot be ignored.

NO can easily diffuse across membranes from thevascular endothelium to the skin surface. Diffusion ofNO from human skin can readily be detected using asimple apparatus (Weller et al. 1996). NO release canbe increased by enhanced nitrate intake or strengthenedhumoral acidity (Lundberg et al. 2004). The healthyhuman sweat usually contains about 5 pM of nitrite,and this concentration s precisely in line with the amountof NO release from human skin (Benjamin 2000). Thisobservation maybe a host defence mechanism to protectagainst pathogenic (especially for fungi) skin infection.However, the source of these nitrites is not clear,generally from the bacterial reduction of sweat nitrateby skin organisms.

DISCUSSION AND PROSPECTAlthough nitrate itself has no toxicity, its metabolites,such as nitrite and NOCs, have a harmful effect onhuman health, especially the NOCs which are related tomany cancers. Therefore, the hazards of nitrate shouldbe mainly caused by unhygienic husbandry conditionsor improper eating habits (LHirondel J and LHirondelJ-L 2001). Contrarily, the NO from nitrate metabolismis beneficial to human health. However, the beneficialeffects of nitrate cannot be negated just because of itschemical relationship with NOCs, and both beneficial

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and harmful effects should be justly evaluated. Inconclusion, the beneficial effects of nitrate on humanhealth may occur when its dose is in a certainconcentration, but its harmful effects may happen whenits ingested amount is excessive.

However, nitrate is the precursor substance of NOCs,the inducement factors in its generation are alsounclear.Moreover, human body can secrete endogenous nitrate,accounting for 50%, which means that reducing thenitrate intake could be an acceptable way of cancerprevention (Lundberg et a l. 2004). Fresh vegetableshave low nitrite accumulation and plenty of protectiveantioxidants, which can decrease the incidence of caner.But part of the nitrate in fresh vegetables will be convertedto nitrite when it is improperly stored after harvest.Similarly, cured meat products also contain high nitrite.Once the AD1 of nitrite intake is exceeded (0-0.06mgkg-' body weight), the amine reaction easily occurs inhuman body, forming strong carcinogenic nitrosamines(Slob et al. 1995). In conclusion, the diets with morefresh vegetables, few pickled and smoked foods, lownitrate and nitrite content meat products and drinkingwater should bean mportant way to effectively preventcancer, methemoglobinaemia, and other diseases.

Besides, the legislation on human nitrate exposure tofood and water is also necessary (DeGroote and Fang1995). The AD1 of nitrate was allocated as 0-0.37 mgkg-' as a nitrite ion by the WHO, but it is always brokenbecause of daily habits (Boink and Speijers 2001).However, whether consumption exceeding the limitswould be hazardous to human health is still unknownto date. It is believed that as long as such unclear risksexist, it is very reasonable to limit the nitrate content invegetables and drinking water by legislation.Furthermore, it is important to investigate clinicalmedicine and gain a comprehensive understanding onthe mechanism of nitrate in human body and its affectfactors, including the relationship between different levelsof health status and age structure and various metabolitesfrom nitrates; different dietary habits, and variousmetabolites from nitrates, such as how to effectivelydecrease the harmful metabolites in the body, how toorient the conversion of accumulated nitrate to abeneficial direction, for example, converting it to abeneficial level of NO. Only with mastering theseproblems will it be possible to develop an effective

treatment or diet program for different patients, avoidor minimize the harm to human health, and make it auseful orientation for humans. In short, the mechanismof nitrate and its metabolites in human body is stillcritically important for future research, especially thegeneration and conversion of endogenous nitrate andthe condition of NO synthesis, which has an importantguiding role in related researches. Meanwhile, thereshould also be a continuous concern about the measuresfor reducing nitrate content in vegetables and waterbodies, and formulation of relevant standards, to ensurepublic health.

Besides diet habits and legislative attention,agricultural researches should also be conducted toreduce the accumulation of nitrate in vegetables.Generally, the nitrate content between different vegetablespecies is greatly different, even in the same variety(Wang et al. 2000). Accordingly, the selection of lownitrate accumulation varieties should be a feasible wayand the agronomic measures for coordinating the plantmetabolism between carbon and nitrogen, which isrelated to plant growth, snould be further investigated.In the previous research, it was found that carbondioxide fertilization in a greenhouse using agriculturalbio-fermentation could effectively reduce the nitratecontent in vegetables by promoting carbon metabolism(Yu et al . 2006). Moreover, some antioxidants invegetables can also help us prevent cancers, therefore,it is valuable to investigate the agricultural methods toimprove the carotenoids, vitamins C and E, dietary fiber,and plant sterols in the vegetables.

AcknowledgementsThis study was financially supported by the Programfor Changjiang Schlors and Innovative Research Teamin University, China (IRT0536), the National BasicResearch Program of China ( 973 Program,2002CB4 10806), the National Natural ScienceFoundation of China (NSFC, 30571083), and the NaturalScience Foundation of Zhejiang Province, China(2303465).

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Accumulation of Nitrate in Vegetables and Its Possible Implications to Human Health