Spinach is toxic(1)

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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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