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Spinach is toxic! “But we washed the spinach ?!” Toxic Spinach? Written by Jannik Zijlstra & Lars de Bruin 5V1, Dominicus College Nijmegen April 2nd 2009 Summary It’s known for quite a while, that the body Rumor has it that, after you cook spinach converts nitrate into nitrite by itself. But we twice, it would become toxic. But how can also discovered that nitrate is converted to spinach become toxic? nitrite when vegetables are cooked! So, The substance in spinach that would be when we cook vegetables more often, toxic, is nitrite. The molecule formula for even more nitrate is being converted into nitrite is NO2-. The molecule formula for nitrite! nitrate is NO3- and as you see, these two Nitrite is a toxic substance. The molecules are very much alike. The reason why nitrite is toxic: is that it causes hypothesis is the following: nitrate is being oxygen to less easy to assimilate from the converted to nitrite when cooked twice. blood. Nitrite can, with some specific To test this hypothesis, we cooked proteins, form the really toxic nitrosamine. spinach in water after let it weak in water. These nitrosamine are carcinogenic. And One beaker is filled with uncooked that’s the main problem when cooking spinach. This beaker serves as a spinach twice: these carcinogenic reference. nitrosamines. We’ll explain these The second beaker contains spinach that nitrosamines further now. is cooked once and the third beaker contains spinach cooked twice. The idea is that we can indicate nitrite in this third beaker because there is twice cooked spinach in it. Introduction A long time ago people thought that you could warm up vegetables as long as you wanted. And even as often as you wanted. But after a period, people discovered that Nitrite there often was a huge amount of nitrate (http://www.chemistry.wustl.edu/~courses/genchem/ Tutorials/Ions/images/nitrite3.jpg) in vegetables. And then people also discovered that this gave some risks. Nitrosamines can be formed under certain circumstances. They are formed out of some nitrites and secondary amines, which are often in proteins. Nitrosamine (http://wpcontent.answers.com/wikipedia/commons/t humb/0/0b/Nitrosamine-2D.png/150px-Nitrosamine- 2D.png) Nitrate (http://www.advancedaquarist.com/2007/8/tips_albu The circumstances in which proteins are m/nitrate.jpg) being transformed to nitrosamines, are often there when spinach is cooked. But Let’s find out… how is this working exactly? Quoted from http://en.wikipedia.org/wiki/Nitrosamine “Under acidic conditions the nitrite forms nitrous acid (HNO2), which is protonated and splits into the nitrosonium cation N=O+ Experimental procedure and approach and water: H2NO2+ = H2O + NO+. The nitrosonium cation then reacts with an Well, what have we done exactly? amine to produce nitrosamine.” Right here, our work method, in items: These nitrosamines are often in nutrition. Buy some spinach at the But the government made some laws for supermarket limits of nitrosamines that may be in the nutrition. Store the spinach in the fridge There are though many cases of cancer due to this nitrosamines in trivial food. Pulverize the spinach How are nitrosamines toxic? Weigh the spinach These nitrosamines are being converted into formaldehyde and later to a carbenium ion. These are metabolites that can disturb the replication in cells. These can namely connect to our DNA. Due to changes they make in the DNA, there can occur tumours by reading mistakes. Besides, nitrosamines can easily pass a cell membrane and can than cause problems anywhere in the body. Don’t cook 50,17 grams spinach Cook 49,94 grams of spinach once Cook 50,00 grams of spinach twice. Drop all these in 400 mL of water Toxic (http://themusicteacher.files.wordpress.com/2008/01 /toxic.jpg) Well, will spinach be toxic after cooking once and even more after cooked twice? We do think so, yes. Cook the spinach for two minutes Curvet NO2- H2O Buffer Sift it after two days Nr. (mL) (mL) Sulfanil- zuur Naphtyl- amine (mL) (mL) (mL) Filtrate it after sifting 1 0,0 5,0 2,0 1,0 2,0 2 1,0 4,0 2,0 1,0 2,0 3 2,0 3,0 2,0 1,0 2,0 4 3,0 2,0 2,0 1,0 2,0 5 4,0 1,0 2,0 1,0 2,0 6 5,0 0,0 2,0 1,0 2,0 7 5,0 0,0 2,0 1,0 2,0 8 5,0 0,0 2,0 1,0 2,0 9 5,0 0,0 2,0 1,0 2,0 10 5,0 0,0 2,0 1,0 2,0 11 5,0 0,0 2,0 1,0 2,0 12 5,0 0,0 2,0 1,0 2,0 Table 1 Measuredata Take an aquarium Table 1 shows how much we need test for the samples for Nitrite testing. Make the samples for the nitrite test Nitrite test Make a calibration curve with a spectrophotometer Nitrate test Thin the cooking water Spectrophotometer (http://cripe03.rug.ac.be/Studenten/practicu mproeven/Spectrofotometer/spectrofotome ter%20dicht.JPG) Measure the amount of NO2- with this calibration curve. Results Curvet Concentration Transm. Extinction Nr. NO2- (mg/L) I (%) Log(I0/I) Figure 1 shows the concentration of 1 0 100 0 Nitrite. The diamond shows the standard 2 0,100 86 0,066 Nitrite test. The 2 squares show the test 3 0,200 69 0,161 we did. In table 2 you see the transmission 4 0,300 53 0,276 we measured with the spectrophotometer. 5 0,400 41 0,387 The results are that the spinach we 6 0,500 36 0,444 Spinach Test cooked once the same concentration has uncooked 1 7 0,367 46,5 0,333 as the spinach we cooked twice. Spinach 1x Test Cooked 1 8 0,072 86 0,066 Spinach Concentration Nitriet 2x Test Standard Nitriet Test y = 0,9053x Cooked 1 9 0,072 86 0,066 Test 1 R2 = 0,9888 Spinach 0,50 not Test 0,45 Cooked 2 10 0,119 78 0,108 0,40 Spinach 1x Test 0,35 Cooked 2 11 1,877 2 1,699 0,30 Spinach E 0,25 2x Test 0,20 Cooked 2 12 1,350 6 1,222 0,15 0,10 Table 2 Measureresults 0,05 We converted this to the extinction en then 0,00 to the concentration NO2- (mg/L). The 0,00 0,10 0,20 0,30 c (mg/L) 0,40 0,50 spinach we haven’t cooked has a bigger 0,60 concentration, but this sample is thinned Figure 1 Concentration Nitrite 50 times. The other samples, spinach cooked once and twice, are thinned 200 times. The final results, the concentration times the thin factor, are showed in table Data analysis 3. The table shows the test results. We did 2 Concen tration Thin Concentration tests, but we only used test 1. The first test NO2- makes a difference to the second test. We (mg/L) factor NO2- (mg/L) did the first test after 2 days when we Spinach Test uncooked 1 0,367 50,0 18,367 cooked the spinach. We did the second Spinach 1x Test test after a week because the first test was cooked 1 0,072 200,0 14,471 Spinach 2x Test not reliable. There was a big difference cooked 1 0,072 200,0 14,471 between the results of test 1 and test 2. We think that test 1 is the most believable, Table 3 because we think that there is more nitrate converted to nitrite in the week we did the We think that the concentration NO2- second test. (mg/L) are not reliable, because the concentration is very high. We can’t compare it with the standard nitrite test. Discussion: When we pulverized the spinach, there was lost some spinach next to the beaker. Also after weighing the spinach, there was spinach falling next to the beaker. The results are, due to that, not reliable on two significance. Cooking is off course a subjective occurrence. Sometimes you find the water cooking earlier on than the other time. So maybe the spinach is not cooked exactly two minutes. The samples we made for the calibration Bibliography curve were hard to make. We needed a lot of substances and it was not easy to http://www.steljevraag.nl/viewquestion? measure the right amount of it. That can questionID=970 also be gone wrong. The colour of the samples of our test were http://www.food-info.net/nl/qa/qa- not blank. The water was green and light fp7.htm green of the samples. We think that that’s the reason that the aquarium test didn’t http://nl.wikipedia.org/wiki/Nitriet give the right results. The problem we discovered, was that time http://en.wikipedia.org/wiki/Nitrosamine was really important in our experiment. When the spinach was laid down for a http://www.translate.google.com long time in water, especially when it was cooked, there was being formed more and more nitrite. That’s also the main reason why we did not get reliable results out of our experiment. Conclusion: Yes, there is formed nitrite after cooking spinach once and after cooking twice. There was formed the same concentration Nitrite by once cooking as by twice cooking. This means that cooking spinach twice is not worse as cooking spinach once. The best, if looking to the quite toxic nitrite, is to eat spinach uncooked. Evaluation: For further research, we have important tips: Keep eye on the time: you need to do the tests fast after each other because time is a really important factor in this research. You need to filtrate the spinach better than we did because the green colour of the spinach itself gave not reliable results when doing things as an aquarium test.
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