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\���������ZZ����������������H,f��������!��"p�+�+�+�+�+�+�+$�-�d06�+Q��"���"�"�+�����,�$�$�$�"f����#&h�$�"�+�$�$�$�������
�����Y$"�$&,0H,�$�0{$�0�$�$&�0��$X�"�"�$�"�"�"�"�"�+�+�$�"�"�"H,�"�"�"�"���������������������������������������������������������������������0�"�"�"�"�"�"�"�"�"Z>�:	)Running head: Nitrate in urine and daily diet
Relationship between nitrate in urine and daily diet, Cross sectional study: citizen of Arak city, Iran
Rezaei Mohammad1, Fani Ali2*, Fani Parisa3, Mirzajani Parisa4, Malekirad Ali Akbar5
1 Dept. of Food safety and Hygiene, school of public Health, Tehran University of Medical Sciences. Tehran, Iran ( HYPERLINK "mailto:Rezaei_m@razi.tums.ac.ir" Rezaei_m@razi.tums.ac.ir)
2 Associated Prof. of Internal Medicine, Arak University of Medical Sciences, Arak, Iran ( HYPERLINK "mailto:Drfani321@yahoo.com) Tell" Drfani321@yahoo.com) Tell: +989181617447
3 MD, General Physician, Arak University of Medical Sciences, Arak, Iran ( HYPERLINK "mailto:Drparisafani@yahoo.com" Drparisafani@yahoo.com)
4 MD, General Physician, Arak University of Medical Sciences, Arak, Iran ( HYPERLINK "mailto:Mirzajani.parisa@yahoo.com" Mirzajani.parisa@yahoo.com)     
5 Postdoc in Neuropsychology and Environmental Toxicology, Payame Noor University, Tehran, Iran ( HYPERLINK "mailto:Ak_malekirad@yahoo.com" Ak_malekirad@yahoo.com)

Abstract 
The aim of this study was to determine the level of urinary nitrate in people in arak, 2012. In this study, 402 people of 10 different areas in Arak city were randomly selected and involved according to exclusion criteria.  After consent, fulfilled recall questionnaires from last 24 hrs. diet which was consumed. Simultaneously, 24 hrs. Urine (8am to 8am next day) was collected in standard jar. Firstly, the urine nitrate was reduced by diazinium salt; add xantesic acid, which changes mixture to amber-colored in the laboratory. Then, nitrate was determined with 520 nanometer spectrophotometer with DR 2500 machine made in HACH Company. In this survey, 402 urine samples were examined for total urinary nitrate. The mean of urine nitrate was 128.5�40.33 mg/L with minimum 96.5 and maximum 310.8mg/L. The fluctuations of urinary nitrate level was correlated to some daily diets (carbohydrates, cereals, vegetables and meat products) of people in arak (P<0.05). 
Key Words: Urinary, Nitrate, Daily diet, Food safety.
Introduction
High rate of nitrogen in diet and potable water is considered as source of contamination. The derivative of nitrogen is one of the combinations that constitutes a part of natural cycle of nitrogen. Nitrate ion (No3) is oxygenated and relatively stable form of nitrogen. The ion is reduced in body by microbial reactions to yields nitrite ion (No2), which is unstable. Other derivative of the ion such as oxide nitric, N-nitro, and nitrosamine�known by the WHO as carcinogen- are found in digestion system  ADDIN EN.CITE <EndNote><Cite><Author>EDITION</Author><Year>2006</Year><RecNum>77</RecNum><DisplayText>[1]</DisplayText><record><rec-number>77</rec-number><foreign-keys><key app="EN" db-id="za0dzdta5tas09epvt550tf70sfexvstvrvz">77</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>EDITION, FIRST ADDENDUM TO THIRD</author></authors></contributors><titles><title>Guidelines for Drinking-water Quality</title></titles><dates><year>2006</year></dates><urls></urls></record></Cite></EndNote>[ HYPERLINK \l "_ENREF_1" \o "EDITION, 2006 #77" 1]. Following recent surge in use of chemical fertilizers in agriculture section for higher production of crops, an increase in concentration of nitrate ions in agricultural products as well as ground water contaminations have occurred. American Environment Preservation Agency and WHO have announced the maximum acceptable level of nitrate in urban water 15mg or 44.3mg/l  ADDIN EN.CITE <EndNote><Cite><Author>Ward</Author><Year>2010</Year><RecNum>95</RecNum><DisplayText>[1, 2]</DisplayText><record><rec-number>95</rec-number><foreign-keys><key app="EN" db-id="za0dzdta5tas09epvt550tf70sfexvstvrvz">95</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Ward, Mary H</author><author>Kilfoy, Briseis A</author><author>Weyer, Peter J</author><author>Anderson, Kristin E</author><author>Folsom, Aaron R</author><author>Cerhan, James R</author></authors></contributors><titles><title>Nitrate intake and the risk of thyroid cancer and thyroid disease</title><secondary-title>Epidemiology (Cambridge, Mass.)</secondary-title></titles><periodical><full-title>Epidemiology (Cambridge, Mass.)</full-title></periodical><pages>389</pages><volume>21</volume><number>3</number><dates><year>2010</year></dates><urls></urls></record></Cite><Cite><Author>EDITION</Author><Year>2006</Year><RecNum>77</RecNum><record><rec-number>77</rec-number><foreign-keys><key app="EN" db-id="za0dzdta5tas09epvt550tf70sfexvstvrvz">77</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>EDITION, FIRST ADDENDUM TO THIRD</author></authors></contributors><titles><title>Guidelines for Drinking-water Quality</title></titles><dates><year>2006</year></dates><urls></urls></record></Cite></EndNote>[ HYPERLINK \l "_ENREF_1" \o "EDITION, 2006 #77" 1,  HYPERLINK \l "_ENREF_2" \o "Ward, 2010 #95" 2]. The addition of Nitrite/nitrate extends the shelf life of meat products as well as improves food safety ADDIN EN.CITE <EndNote><Cite><Author>Rezaei</Author><Year>2013</Year><RecNum>7</RecNum><DisplayText>[3]</DisplayText><record><rec-number>7</rec-number><foreign-keys><key app="EN" db-id="zserz2dti5xrzoevsr4pt0d80a0sztsdtzxp">7</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Rezaei, Mohammad</author><author>Shariatifar, Nabi</author><author>Jahed Khaniki, Gholamreza</author><author>Javadzadeh, Morteza</author></authors></contributors><titles><title>Nitrite in hamburgers in Arak, Iran</title><secondary-title>Food Additives &amp; Contaminants: Part B</secondary-title></titles><periodical><full-title>Food Additives &amp; Contaminants: Part B</full-title></periodical><pages>285-288</pages><volume>6</volume><number>4</number><dates><year>2013</year></dates><isbn>1939-3210</isbn><urls></urls></record></Cite></EndNote>[ HYPERLINK \l "_ENREF_3" \o "Rezaei, 2013 #7" 3]. W.G. Stillweel et al. conducted a study in Colombia regarding relationship between level of nitrate and nitrosoprolin of urine and histology of stomach. They used linear regression method to study the relationship and found a relationship between level nitrate in urine and nitrosoprolin on one hand and histology of stomach (r=0.56 & p=0.0002)  ADDIN EN.CITE <EndNote><Cite><Author>Stillwell</Author><Year>1991</Year><RecNum>79</RecNum><DisplayText>[4]</DisplayText><record><rec-number>79</rec-number><foreign-keys><key app="EN" db-id="za0dzdta5tas09epvt550tf70sfexvstvrvz">79</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Stillwell, WG</author><author>Glogowski, Joseph</author><author>Xu, HX</author><author>Wishnok, John S</author><author>Zavala, Diego</author><author>Montes, G</author><author>Correa, Pelayo</author><author>Tannenbaum, Steven R</author></authors></contributors><titles><title>Urinary excretion of nitrate, N-nitrosoproline, 3-methyladenine, and 7-methylguanine in a Colombian population at high risk for stomach cancer</title><secondary-title>Cancer research</secondary-title></titles><periodical><full-title>Cancer research</full-title></periodical><pages>190-194</pages><volume>51</volume><number>1</number><dates><year>1991</year></dates><isbn>0008-5472</isbn><urls></urls></record></Cite></EndNote>[ HYPERLINK \l "_ENREF_4" \o "Stillwell, 1991 #79" 4]. Green et al. analyzed level of nitrate and nitrite of urine ADDIN EN.CITE <EndNote><Cite><Author>Green</Author><Year>1982</Year><RecNum>96</RecNum><DisplayText>[5]</DisplayText><record><rec-number>96</rec-number><foreign-keys><key app="EN" db-id="za0dzdta5tas09epvt550tf70sfexvstvrvz">96</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Green, L. C.</author><author>Wagner, D. A.</author><author>Glogowski, J.</author><author>Skipper, P. L.</author><author>Wishnok, J. S.</author><author>Tannenbaum, S. R.</author></authors></contributors><titles><title>Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids</title><secondary-title>Anal Biochem</secondary-title><alt-title>Analytical biochemistry</alt-title></titles><periodical><full-title>Anal Biochem</full-title><abbr-1>Analytical biochemistry</abbr-1></periodical><alt-periodical><full-title>Anal Biochem</full-title><abbr-1>Analytical biochemistry</abbr-1></alt-periodical><pages>131-8</pages><volume>126</volume><number>1</number><edition>1982/10/01</edition><keywords><keyword>Autoanalysis/instrumentation</keyword><keyword>Cadmium</keyword><keyword>Gas Chromatography-Mass Spectrometry</keyword><keyword>Gastric Juice/analysis</keyword><keyword>Humans</keyword><keyword>Milk, Human/analysis</keyword><keyword>Nitrates/ analysis/blood/urine</keyword><keyword>Nitrites/ analysis/blood/urine</keyword><keyword>Nitrobenzenes/analysis</keyword><keyword>Nitrogen Isotopes</keyword><keyword>Oxidation-Reduction</keyword><keyword>Saliva/analysis</keyword></keywords><dates><year>1982</year><pub-dates><date>Oct</date></pub-dates></dates><isbn>0003-2697 (Print)&#xD;0003-2697 (Linking)</isbn><accession-num>7181105</accession-num><urls></urls><remote-database-provider>NLM</remote-database-provider><language>eng</language></record></Cite></EndNote>[ HYPERLINK \l "_ENREF_5" \o "Green, 1982 #96" 5]. They maintained that nitrate of food stuff is mainly absorbed in stomach and duodenum, which results in high concentration of nitrate and nitrite in plasma few hours after the food; in addition, concentration of nitrate and nitrite increased up to 10 times  in saliva through entro-salivary cycle. Part of the absorbed nitrate is transferred into nitrite and oxide nitric (No). Dorsal parts of tongue and pharynx are home to microbial strains that reduce nitrate into nitrite (HNO2) in short time. Nitrite has PK of 2.3 to 4. In vicinity of gastro-acid, HNO2 yields NO with the No-Synthetase enzyme as catalyzer, the same chemical reactions is continued at the frontal parts intestine and NO yields up to 10000 times of plasma�s concentration. The first conjectures regarding malignant effects of nitrate were raised in 1945; the idea was that nitrate-rich water initiate Met hemoglobin (Met Hb, (Fe3)) among children using nitrate-rich water  ADDIN EN.CITE  ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_6" \o "Lauer, 2001 #80" 6-9]. Studies regarding carcinogen effect of nitrate in food stuff and nitrite discharge in saliva have triggered debates and opponents of carcinogen effects of nitrate note vegans who use nitrate-rich diet  and receive nitrate three times more than others, while gastric cancer among vegans is much less than that of others. In addition, in spite of increase in nitrate-rich vegetable and water we observe reduction in rate of gastric cancer in the world. Also, nitric oxide protective function to prevent infection, protect of Gastric Ulcers, prevent vascular disease.  ADDIN EN.CITE  ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_10" \o "Rassaf, 2013 #97" 10-15]. Nitric oxide in gastric acid changes the bacterio-static role of gastric acid to bactericidal role; which seems positively effective in immunity system  ADDIN EN.CITE <EndNote><Cite><Author>Iijima</Author><Year>2003</Year><RecNum>85</RecNum><DisplayText>[13]</DisplayText><record><rec-number>85</rec-number><foreign-keys><key app="EN" db-id="za0dzdta5tas09epvt550tf70sfexvstvrvz">85</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Iijima, K</author><author>Fyfe, V</author><author>McColl, KEL</author></authors></contributors><titles><title>Studies of nitric oxide generation from salivary nitrite in human gastric juice</title><secondary-title>Scandinavian journal of gastroenterology</secondary-title></titles><periodical><full-title>Scandinavian journal of gastroenterology</full-title></periodical><pages>246-252</pages><volume>38</volume><number>3</number><dates><year>2003</year></dates><isbn>0036-5521</isbn><urls></urls></record></Cite></EndNote>[ HYPERLINK \l "_ENREF_13" \o "Iijima, 2003 #85" 13]. Green Lc et al. reported that endogen is the main source of excreted nitrate through urine and endogen in turn is from protein intake ADDIN EN.CITE <EndNote><Cite><Author>Green</Author><Year>1982</Year><RecNum>96</RecNum><DisplayText>[5]</DisplayText><record><rec-number>96</rec-number><foreign-keys><key app="EN" db-id="za0dzdta5tas09epvt550tf70sfexvstvrvz">96</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Green, L. C.</author><author>Wagner, D. A.</author><author>Glogowski, J.</author><author>Skipper, P. L.</author><author>Wishnok, J. S.</author><author>Tannenbaum, S. R.</author></authors></contributors><titles><title>Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids</title><secondary-title>Anal Biochem</secondary-title><alt-title>Analytical biochemistry</alt-title></titles><periodical><full-title>Anal Biochem</full-title><abbr-1>Analytical biochemistry</abbr-1></periodical><alt-periodical><full-title>Anal Biochem</full-title><abbr-1>Analytical biochemistry</abbr-1></alt-periodical><pages>131-8</pages><volume>126</volume><number>1</number><edition>1982/10/01</edition><keywords><keyword>Autoanalysis/instrumentation</keyword><keyword>Cadmium</keyword><keyword>Gas Chromatography-Mass Spectrometry</keyword><keyword>Gastric Juice/analysis</keyword><keyword>Humans</keyword><keyword>Milk, Human/analysis</keyword><keyword>Nitrates/ analysis/blood/urine</keyword><keyword>Nitrites/ analysis/blood/urine</keyword><keyword>Nitrobenzenes/analysis</keyword><keyword>Nitrogen Isotopes</keyword><keyword>Oxidation-Reduction</keyword><keyword>Saliva/analysis</keyword></keywords><dates><year>1982</year><pub-dates><date>Oct</date></pub-dates></dates><isbn>0003-2697 (Print)&#xD;0003-2697 (Linking)</isbn><accession-num>7181105</accession-num><urls></urls><remote-database-provider>NLM</remote-database-provider><language>eng</language></record></Cite></EndNote>[ HYPERLINK \l "_ENREF_5" \o "Green, 1982 #96" 5]. Wu studied 69 regions of China and concluded that concentration of nitrate in urine and N-nitroso combination is related with intake of nitrate from food and vegetable sources; he also maintained that there is a relationship between nitros and nitrite combination with esophageal cancer ADDIN EN.CITE <EndNote><Cite><Author>Wu</Author><Year>1993</Year><RecNum>94</RecNum><DisplayText>[16]</DisplayText><record><rec-number>94</rec-number><foreign-keys><key app="EN" db-id="za0dzdta5tas09epvt550tf70sfexvstvrvz">94</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Wu, Y</author><author>Chen, J</author><author>Ohshima, H</author><author>Pignatelli, B</author><author>Boreham, J</author><author>Li, J</author><author>Campbell, TC</author><author>Peto, R</author><author>Bartsch, H</author></authors></contributors><titles><title>Geographic association between urinary excretion of N nitroso compounds and oesophageal cancer mortality in china</title><secondary-title>International Journal of Cancer</secondary-title></titles><periodical><full-title>International journal of cancer</full-title></periodical><pages>713-719</pages><volume>54</volume><number>5</number><dates><year>1993</year></dates><isbn>1097-0215</isbn><urls></urls></record></Cite></EndNote>[ HYPERLINK \l "_ENREF_16" \o "Wu, 1993 #94" 16]. Owing to development of the mass media and easier access to information, an increase in public concern regarding rate of contaminations and nitrate in particular has been witnessed. It is not possible to measure concentration of nitrate intake in diet based on the ingredients. However, general intake of nitrate can be determined based on nitrate concentration in urine. In this way, the present study tries to measure level of urine discharge nitrate and its relationship with diet of citizen of Arak City. 
Material and Methods
The criteria of the participants were no record of kidney diseases (urine infection, kidney disorder) metabolic diseases, diseases featured with fever, no menstruation, and normal urine analyses results. Standard containers (2ml) of dense sulfuric acid were provided to the participants after giving them safety instructions and trainings of how to prepare 24 hours urine samples. The next day, the participants were asked to fill out a standard questionnaire of food stuff through recalling when delivering the urine samples. The samples were immediately sent to lab and level of nitrate was determined through Griess method and cadmium reduction method (method 8039). The method is able to indicate concentrations of NO3-N in water, wastewater, and sea water with accuracy of 30 mg/L. Nitrate cadmium is first reduced to nitrite and then yields diazinium salt in sulfuric acid, and its color changes into amber yellow in contact with gentisic acid, which is measurable at 520nm wavelength. The test was conducted using DR 2500 (HACH) to measure concentration of nitrate in urine. For storing the sample, if required, dense sulfuric acid 2ml was added to one liter of samples at 4�C for 24hrs storage. Before measuring nitrate, the samples were given time to reach the ambient temperature. Depending on concentration of the sample nitrate and given that the accuracy of measurement device was 30mg/l, the sample were diluted in proper volume. Finally, the obtained figure of concentration was multiplies by coefficient of dilution to reach the final figure of nitrogen (mg/l). The read figure of nitrogen concentration was obtained from discharged nitrogen in 24hrs. Having the average level of nitrate, SD of average types of food stuff, SD or relationship between types of food and nitrate in urine were analyzed through computing PV.
Statistical Analysis
Statistical analysis was performed using SPSS (Version 9) software. 
Results
About 402 adults from 10 urban districts were adopted randomly and based on household code. After introduction meeting, the participants provided 24hrs urine sample (8am to 8am). At the time of delivering the samples participants filled out the questionnaire and recorded the type and amount of food stuff taken by them in the last 24hrs using recall method. Average nitrate in urine for all the participants was 128.5�40.33mg/l with minimum of 69.8mg/l and maximum of 310mg/l. Only in case of fruits and dairy products no significant relationship was found between type of food stuff taken in the last 24hrs and level of nitrate in urine, while significant relationship was found regarding other types of food. The survey revealed that rice and bread are the main foods used by the participants (carbohydrates) (Table 1, 2).
Discussion
In this study average nitrate in urine among the participants was 128.5mg/l. Surveys of consumed foods using recall method showed that dairy products, fruits, and proteins constitute smaller portion of the diet because of economic condition. The safety of food stuff decreases with increasing concentration of toxic compounds and environmental pollutants ADDIN EN.CITE <EndNote><Cite><Author>Rezaei</Author><Year>2014</Year><RecNum>10</RecNum><DisplayText>[17]</DisplayText><record><rec-number>10</rec-number><foreign-keys><key app="EN" db-id="zserz2dti5xrzoevsr4pt0d80a0sztsdtzxp">10</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Rezaei, Mohammad</author><author>AkbariDastjerdi, Hajar</author><author>Jafari, Hassan</author><author>Farahi, Ali</author><author>Shahabi, Arman</author><author>Javdani, Hossein</author><author>Teimoory, Hossein</author><author>Yahyaei, Mohammad</author><author>Malekirad, Ali Akbar</author></authors></contributors><titles><title>Assessment of dairy products consumed on the Arakmarket as determined by heavy metal residues</title><secondary-title>Health</secondary-title></titles><periodical><full-title>Health</full-title></periodical><pages>323</pages><volume>6</volume><dates><year>2014</year></dates><urls></urls></record></Cite></EndNote>[ HYPERLINK \l "_ENREF_17" \o "Rezaei, 2014 #10" 17]. On the other hand, carbohydrates, cereals, vegetables, and fatty foods constituted the main portion of the diet. Given that the participants were selected randomly, the table above can be taken representative of the food diet of citizen of Arak city. Probably over all the specific diet of the Arak citizen (less protein and fruits and more carbohydrates and fatty foods) explains reduction of nitrate in urine samples. On the other hand, significant relationship was found between intake of protein, dairy products, vegetables, cereal and water and level of nitrate in urine. However, the role of nitrate intake from potable water is not negligible and with average nitrate of potable water of 34.4mg/l, probably the source of major portion of nitrate in urine was potable water. Morcos et al ADDIN EN.CITE <EndNote><Cite><Author>Morcos</Author><Year>2001</Year><RecNum>88</RecNum><DisplayText>[18]</DisplayText><record><rec-number>88</rec-number><foreign-keys><key app="EN" db-id="za0dzdta5tas09epvt550tf70sfexvstvrvz">88</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Morcos, Edward</author><author>Wiklund, N Peter</author></authors></contributors><titles><title>Nitrite and nitrate measurement in human urine by capillary electrophoresis</title><secondary-title>Electrophoresis</secondary-title></titles><periodical><full-title>Electrophoresis</full-title></periodical><pages>2763-2768</pages><volume>22</volume><number>13</number><dates><year>2001</year></dates><isbn>1522-2683</isbn><urls></urls></record></Cite></EndNote>[ HYPERLINK \l "_ENREF_18" \o "Morcos, 2001 #88" 18]. Also Fani et al reported that the mean of nitrate content in water in Arak (35.2�17.1), that exceed standard limit ADDIN EN.CITE <EndNote><Cite><Author>WHO</Author><Year>2008</Year><RecNum>1</RecNum><DisplayText>[19]</DisplayText><record><rec-number>1</rec-number><foreign-keys><key app="EN" db-id="d92w50xpwzrxske9zepvdvxv2wadfsv9rs0w">1</key></foreign-keys><ref-type name="Book">6</ref-type><contributors><authors><author>WHO</author></authors></contributors><titles><title>Guidelines for drinking-water quality</title></titles><edition>3</edition><dates><year>2008</year></dates><urls></urls></record></Cite></EndNote>[ HYPERLINK \l "_ENREF_19" \o "WHO, 2008 #1" 19]. Maintained that nitrate and nitrite in urine is generally accepted indicator of NO in body. They measurement of average nitrate in 24hrs showed 591�115�m and PH and density of urine did not have any effect on the measurement results. Their results are inconsistent with our results concerning amount of nitrate; which is probably due to considerable differences in food diet and level of nitrate in potable water of the participants. By measuring nitrogen (15NO3) and assigning a fixed amount of 180mm of daily nitrate, Green Lc et al ADDIN EN.CITE <EndNote><Cite><Author>Green</Author><Year>1982</Year><RecNum>96</RecNum><DisplayText>[5]</DisplayText><record><rec-number>96</rec-number><foreign-keys><key app="EN" db-id="za0dzdta5tas09epvt550tf70sfexvstvrvz">96</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Green, L. C.</author><author>Wagner, D. A.</author><author>Glogowski, J.</author><author>Skipper, P. L.</author><author>Wishnok, J. S.</author><author>Tannenbaum, S. R.</author></authors></contributors><titles><title>Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids</title><secondary-title>Anal Biochem</secondary-title><alt-title>Analytical biochemistry</alt-title></titles><periodical><full-title>Anal Biochem</full-title><abbr-1>Analytical biochemistry</abbr-1></periodical><alt-periodical><full-title>Anal Biochem</full-title><abbr-1>Analytical biochemistry</abbr-1></alt-periodical><pages>131-8</pages><volume>126</volume><number>1</number><edition>1982/10/01</edition><keywords><keyword>Autoanalysis/instrumentation</keyword><keyword>Cadmium</keyword><keyword>Gas Chromatography-Mass Spectrometry</keyword><keyword>Gastric Juice/analysis</keyword><keyword>Humans</keyword><keyword>Milk, Human/analysis</keyword><keyword>Nitrates/ analysis/blood/urine</keyword><keyword>Nitrites/ analysis/blood/urine</keyword><keyword>Nitrobenzenes/analysis</keyword><keyword>Nitrogen Isotopes</keyword><keyword>Oxidation-Reduction</keyword><keyword>Saliva/analysis</keyword></keywords><dates><year>1982</year><pub-dates><date>Oct</date></pub-dates></dates><isbn>0003-2697 (Print)&#xD;0003-2697 (Linking)</isbn><accession-num>7181105</accession-num><urls></urls><remote-database-provider>NLM</remote-database-provider><language>eng</language></record></Cite></EndNote>[ HYPERLINK \l "_ENREF_5" \o "Green, 1982 #96" 5]. Demonstrated that excretion of nitrate is not relative to amount of received nitrate. Thus, measuring nitrate in urine cannot be the indicator of received nitrate � it is four times of the received amount. They also argued that a main source of nitrate in urine is from endosion sources such as proteins. The result regarding a significant relationship between protein and dairy, drinking and cooking water although not a considerable portion of diet highlights the fact that observed nitrate is from endogen source. Studies conducted in Linzhou, China  ADDIN EN.CITE  ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_20" \o "FEI, 2006 #91" 20-22]. Also confirmed high rate of stomach and esophageal cancer in the region and direct relationship between level of nitrate and nitrite in water and total discharge of nitrate in urine. There are, currently, strict laws in China regarding minimum amount of nitrate in standard canned and bottled water (<0.002mg/l). W.G.Stillwell studied the relationship between nitrate in urine and nitroso-prolyin on one hand and histological condition of stomach mucus in Colombia and found a significant relationship (r=0.56 and P=0.002)  ADDIN EN.CITE <EndNote><Cite><Author>Stillwell</Author><Year>1991</Year><RecNum>79</RecNum><DisplayText>[4]</DisplayText><record><rec-number>79</rec-number><foreign-keys><key app="EN" db-id="za0dzdta5tas09epvt550tf70sfexvstvrvz">79</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Stillwell, WG</author><author>Glogowski, Joseph</author><author>Xu, HX</author><author>Wishnok, John S</author><author>Zavala, Diego</author><author>Montes, G</author><author>Correa, Pelayo</author><author>Tannenbaum, Steven R</author></authors></contributors><titles><title>Urinary excretion of nitrate, N-nitrosoproline, 3-methyladenine, and 7-methylguanine in a Colombian population at high risk for stomach cancer</title><secondary-title>Cancer research</secondary-title></titles><periodical><full-title>Cancer research</full-title></periodical><pages>190-194</pages><volume>51</volume><number>1</number><dates><year>1991</year></dates><isbn>0008-5472</isbn><urls></urls></record></Cite></EndNote>[ HYPERLINK \l "_ENREF_4" \o "Stillwell, 1991 #79" 4]. Although, our study showed that level of nitrate is not considerable, a wide range of change in amount of nitrate was observed (128.5 to 310mg/l). Thus, it is possible to observe condition of stomach mucus of those with high level of nitrate in urine. Furthermore, as gastric cancer is the third prevalent type of cancer in the city of Arak, it is essential to delve deeper into the relationship between nitrate and other environmental and nutritional threats. Researchers believe that micro-flora plays a role in conversion of nutrition-source nitrate into nitrite  ADDIN EN.CITE  ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_6" \o "Lauer, 2001 #80" 6-9,  HYPERLINK \l "_ENREF_12" \o "Archer, 1975 #84" 12,  HYPERLINK \l "_ENREF_13" \o "Iijima, 2003 #85" 13,  HYPERLINK \l "_ENREF_23" \o "Jones, 2013 #99" 23-25]. Also, Studies performed in the various foods in Arak city, showed that this area requires more attention to the foodstuff and regularly monitor pollutants in food  ADDIN EN.CITE  ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_3" \o "Rezaei, 2013 #7" 3,  HYPERLINK \l "_ENREF_17" \o "Rezaei, 2014 #10" 17,  HYPERLINK \l "_ENREF_26" \o "Fani, 2013 #5" 26,  HYPERLINK \l "_ENREF_27" \o "Rezaei,  #3" 27]. Taking into account the saliva-intestine cycle of nitrate, increase in nitrate intake increases probability of production nitrite and consequently the nitrite in saliva in exposure of gastric acid converts into oxide-nitrate in esophagus or stomach. This reaction finally yields derivatives of cancerogenic nitrose. Therefore, it is clear that increase in nitrate intake heightens production of nitrite and, indirectly, increases the threat of cancer indirectly. On the other hand, nitrate in vegetable and protein, if not frozen, yields nitrite in contact with bacteria. Furthermore, nitrite is used in some cases as food preservers; which is considered as a contaminator. 
Conclusion
Although average level of nitrate in urine can be considered as an indicator of intake of nitrate by the people in the region, our survey and comparison with other works showed that people of Arak city do not receive high level of nitrate. However, having in mind that many believe that role nitrite in pathogenesis of digestive system diseases is its main role, and that the region under study suffers from higher rate of gastric cancer, the present study recommends conducting further surveys of level of intake of nitrite and methods for storing food as well as level of nitrite in fast foods. 
Acknowledgement
This study was approved and supported by research and ethics committees of Arak University of Medical sciences and with regards to the Helsinki ethical principles.  
Competing interests
 The authors declare that there is no conflict of interests regarding the publication of this article.
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CJOJQJ^JaJ)jh�+"h�g
CJOJQJU^JaJ1h�+"h�5�CJOJQJ\�^JaJmH	nH	u1h�+"h�O�5�CJOJQJ\�^JaJmH	nH	u8���p�8�Z�=�،���I�͎�������t�x�����������������$d��x�xa$gd�YX���0�^��`�0�gd�YX���0��^��`�0�gd�YXa�c�����#�%���4�8���#�2�3�^���ŒČ��1�{�����΍�6�8�g��������Q���@�������J�������������R�V�t�v�x�����������������������������������������͸(h�+"h�9+CJOJQJ^JaJmH	sH	1jh�+"h�g
CJOJQJU^JaJmH	sH	Uh�YXh�YX6�h�YXh�YXh�YXh�YX5�424.	Deirmenciolu, N., et al., Evaluation of total aflatoxin, nitrate and nitrite levels in layer feed samples of companies producing their own feed in Edincik and Band1rma province of Turkey. Food Additives and Contaminants: Part B, 2012. 5(2): p. 133-139.
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