Order Description
Abstract
One of the essential components of the membranes of all eukaryotic organisms is plant sterols (site). Recent studies shown plant sterols has an effect on the high cholesterol levels. The first studies of effects of PS on cholestrol lowering dates back to 1950´s. PS doses of up to 50 g/day used in this studies to lower the level of cholestrol in serum (Pollak & Kritchevsky, 1981). “In the 1980’s, Japanese researchers managed to produce the saturated form of PS, known as phytostanols, which were reported to have stronger cholesterol lowering potential. Ten years later the first human trial using phytostanols (1.5 g/day) was performed and it was observed that the use of phytostanols lowered the level of cholesterol in human serum (Miettinen & Gylling, 2003).
Other experiments looked in to effects of temperature on sterol oxidation, degradation and oxysterol formation with conventional heating. However, little is known about the effects of microwave heating, this experiment looked in to those effects of temperature on sterol oxidation, degradation and oxysterol formation with microwave heating at different time parameters with and without rapid cooling. For the experiment without rapid cooling first 0.0658g sterol (5-cholesten-24ß-ethyl-3ß-OL-%54) dissolved in 25 ml of chloroform and divided in 15 test tubes each containing 1 ml mix solution, then solution evaporated using nitrogen ready to be heated in the microwave oven at five different time parameters (control 0 seconds, 30 seconds, 60 seconds, 120 seconds and 240 seconds) at 900 W and for the experiment with rapid cooling, all the procedure were same but 0.0641g sterol used and rapid cooled with ice. Experiment done in the laboratory of investigation at University of Navarra, Pamplona-Spain. Results showed degradation of sterol without any oxysterol formation. Therefor to accomplish an excitable level further scientific experiment needed.

INTRODUCTION
“Dis-moi ce que tu manges, je te dirai ce que tu es.” [Tell me what you eat and I will tell you what you are] Physiologie du Gout, ou Meditations de Gastronomie Transcendante, 1826 Anthelme Brillat-Savarin. Is it only what you eat or what you buy, how you prepare and how you cook is important. Nowadays there are different methods involved in cooking food, one of the cooking method is electromagnetic” radiation. What type of effects radiation has on Plant Sterols (PS)?

Lipids

Sterols

Cholestrol

Phytosterols

Sterol Oxidation Products (SOP’s)

Microwave

OBJECTIVE
The objective of this experiment was to find out the c. Because oxidation occurs when plant sterols exposed to air or subjected to heat at high temperature.

METHOD AND METERIALS

Experimental Procedures

For the purpose model study was develop; gas chromatography used for measuring the level of main phytosterols; campesterol, stigmasterol, and ß-sitosterol. Subsequently deviations in the phytosterols and POP´s had been measured after the experimental treatments to look for the extent of the oxidation. The intrinsic levels of the major phytosterols, campesterol, stigmasterol, and ß-sitosterol profiles were measured using gas chromatography (GC). The extent of oxidation was measured by examining changes in the phytosterols following the application of the experimental treatments.

Design of the Project

For each model system; PS dissolved in chloroform, mixed solution were transferred to 15 test tubes 15 x 100 mm with screw tops and evaporated with the help of Nitrogen (N2) following the evaporation sample tubes put in to microwave for 30, 60, 120, 240 seconds respectively and 3 tubes been the control, have not been subjected to microwave heating.
+ mixed in 25 ml
1 ml mixed solution pipetted in to 15 tubes
EVAPRATED WITH NITROGEN
PUT IN TO FOR + 3 BLANKS
Re-dissolved them with 1 ml +

Temperatures: For each experiment, temperature was measured just after the heating and during the following 10 min (without rapid cooling)

TIME(Sec)
OF
MICROWAVE HEATING
TIME (min) OF COOLING DOWN
0 0.5 1 1.5 2 2.5 3 4 5 6 7 8 9
30 Rep1 32.3 30 29.5 27.9 27.4 26.9 25 23.5 23.5 22.7 22.7 22.7 22.7
Rep2 32.6 28.9 27.5 26.5 26 25.6 24.8 24.1 23.2 22.7 22.7 22.7 22.7
Rep3 29.7 28.2 26 26.2 25.6 25.2 24.4 23.8 22.9 22.2 22.2 22.2 22.2
60 Rep1 34.5 32.5 30.9 29 28 27.8 27.2 25.4 24.5 23.8 23.8 23.8 23.8
Rep2 33 32.5 30.9 29 28 27.8 27.2 25.4 24.5 23.8 23.8 23.8 23.8
Rep3 32.5 32.5 30.9 29 28 27.8 27.2 25.4 24.5 23.8 23.8 23.8 23.8
120 Rep1 40.3 32.5 30.9 29 28 27.8 27.2 25.4 24.5 23.8 23.8 23.8 23.8
Rep2 42.9 32.5 30.9 29 28 27.8 27.2 25.4 24.5 23.8 23.8 23.8 23.8
Rep3 41.5 32.5 30.9 29 28 27.8 27.2 25.4 24.5 23.8 23.8 23.8 23.8
240 Rep1 59 32.5 30.9 29 28 27.8 27.2 25.4 24.5 23.8 23.8 23.8 23.8
Rep2 56.3 32.5 30.9 29 28 27.8 27.2 25.4 24.5 23.8 23.8 23.8 23.8
Rep3 54.1 32.5 30.9 29 28 27.8 27.2 25.4 24.5 23.8 23.8 23.8 23.8

TIME(Sec)
OF
MICROWAVE HEATING
TIME (min) OF COOLING DOWN

0 0.5 1 1.5 2 2.5 3 4 5 6 7 8 9
30 media 31.5 29.0 27.7 26.9 26.3 25.9 25.0 24.3 23.2 22.5 22.5 22.5 22.5
ds 1.6 0.9 1.8 0.9 0.9 0.9 0.7 0.6 0.3 0.3 0.3 0.3 0.29
cv 5 3 6 3 4 3 3 3 1 1 1 1 1
60 media 33.3 31.7 30.3 28.9 28.1 27.4 26.7 25.1 24.3 23.5 23.5 23.5 23.5
ds 1.0 0.7 0.5 0.1 0.1 0.3 0.4 0.3 0.2 0.3 0.3 0.3 0.3
cv 3 2 2 0 0 1 2 1 1 1 1 1 1
120 media 41.6 40.2 37.6 35.1 33.7 32.5 30.7 28.6 26.8 25.6 24.8 24.1 23.3
ds 1.3 1.1 1.0 0.8 0.6 0.7 0.6 0.4 0.3 0.4 .2 0.5 0.2
cv 3 3 3 2 2 2 2 1 1 1 1 2 0.9
240 media 56.5 51.2 48.1 45.2 41.4 39.2 37.6 35.1 31.5 29.7 28.0 26.6 25.8
ds 2.5 1.8 1.7 1.4 1.8 1.3 1.0 0.6 0.7 0.6 0.4 0.7 0.4
cv 4 3 3 3 4 3 3 2 2 2 2 3 1
Temperatures: For each experiment, temperature was measured just after the heating and the following rapid cooling with ice.
Temperatures: For each experiment, temperature was measured just after the heating and during the following
TIME (S) OF MICROWAVE HEATING
TIME (min) OF COOLING DOWN

0
30 Rep1 30.8
Rep2 31.2
Rep3 33.7
60 Rep1 31.5
Rep2 32
Rep3 34
120 Rep1 42.7
Rep2 41.2
Rep3 36.4
240 Rep1 56.1
Rep2 54.8
Rep3 53.9

Temperatures: For each experiment, temperature was measured just after the heating and during the following
TIME (S) OF MICROWAVE HEATING
TIME (min) OF COOLING DOWN

0
30 media 31.9
ds 1.6
cv 5
60 media 32.5
ds 1.3
cv 4
120 media 40.1
ds 3.3
cv 8
240 media 54.9
ds 1.1
cv 2

Analysis of sterols:
50 µl mixture of solution from each tubes pipetted in to wider test tubes and 50 µl internal standard (5a-cholestane) were added to each tube. The solvent in each tube were evaporated with the flow of N2. 400 µl of reagent Tri-sil were added to each evaporated test tubes, lids put on but not tightly closed and samples transferred in to already heated water bath to 60 °C for 45 min. The solution evaporated for the last time with flow of N2, 400 µl hexane especially for Gas Chromatography (GC) added and final solution were transferred to glass vial using syringe, needle and 0.45 µm filter ready to be analyse by GC.

Analysis of SOP’s

Three different time (0-120-240 min.) were chosen for the analysing of SOP’s. Previously prepared sample was transferred to a 3 x3 tubes of samples (an approximately 2 mg sterol), and 1 ml 19-hydroxycholesterol as internal standard (0.02 mg /1 mL) solution was added and evaporated with the help of N2. 200 µl Hex: ethyl acetate (95;5, v/v) added to evaporated sample and mixed until homogenized with the help of vortex. Homogenized solution transferred to NH2-SPE cartridge ready to be filtered.

Bickers put underneath to each cartridge used to collect the solution, because first solution is discharged. Then solution equilibrated with 3 ml hexane/ethyl acetate, each samples transferred in to a cartridge of it is concern to be filtered. Filtered samples added further 200 µl Hex: ethyl acetate shaken and transferred back to cartridges first 8 ml of Hex: ethyl acetate 95:5 added and filtered and then 10 ml of Hex: ethyl acetate 95:10 added and filtered and solution discharged. SOP’s that captured by the filter inside the cartridges washed with 10 mL acetone. Solution put in to water bath consistent temperature of 40 ºC and evaporated with under a stream of N2.
The sample solutions of SOP’s were made ready to for the chromatographic analysis as previously described for sterols.
Gas Chromatography-Mass spectrometer (Agilent 6890-5973) was used to analyse the samples;
Column: 19091S-433 HP-5ms 5% Phenyl Methyl Siloxane (30 m x 250 µm x 0.25µm)
Carrier gas: He, 1 mL/min
Oven temperatures program: 85 °C during 0.5 min
Curve 1: 50 °C/min up to 290 °C
Curve 2: 0.5 °C/min up to 298 °C
Injector temperature: 280 °C
Volume of sample injected: 1 µL, splittless mode
Transfer line to the detector: 280 °C
Source temperature:
Electron impact: 70 eV
Detector temperature: 300 °C
Mass interval: 50.00-550.00 uma
Detection mode: SCAN

Ions identification were established using Wiley library and varies different literature’s mass spectra, also retention time and mass spectrometry fragments comparison done against standard pure compounds.
Internal standard 5a-cholestane used for internal standard method, total ion chromatogram used for the quantitation of cholesterol and 5a-cholestane, although extract ion chromatograms used for PS quantification, in accordance with the quantity of specific ion for each peak for campesterol, stigmasterol and ß-sitosterol 343, 484, 357 respectively and the relative abundance of each ion had been considered (Berasategi 2012)
Retention times, ions characteristics and sterols quantitation mode in chromatographic analysis

Compound tR (min) Characteristic ions (m/z) Quantitation
5a-cholestane (is) 13.0 217, 357, 372 Total area (A)
cholesterol 17.7 329, 353, 368, 458 Total area
campesterol 20.5 343, 367, 382 343 (6.62%)*
stigmasterol 21.1 355, 394, 484 484 (3.05%)
ß-sitosterol 23.2 357, 381, 396, 486 357 (6.56%)
* ion used for integration (abundance of the ion)

Each plant sterol’s total area calculated as follows:
Calibration curves for sterols internal standard

Compound Calibration curve R2
cholesterol y = 1.1903x – 0.0288 0.9937
campesterol y = 1.1348x – 0.008 0.9923
stigmasterol y = 1.0346x – 0.0156 0.9905
ß-sitosterol y = 1.1257x – 0.0324 0.9974

;
Sterols with microwave heating at different times (times in seconds)
0 30 60 120 240
Campesterol
(µg/mg) 0,043 ± 0,00000 0,035 ± 0,00470 0,033 ± 0,00514 0,024 ± 0,00248 0,029 ± 0,00187
Stigmasterol
(µg/mg) 0,023 ± 0,00000 0,016 ± 0,00343 0,016 ± 0,00517 0,010 ± 0,00189 0,013 ± 0,00008
Sitosterol
(µg/mg) 0,082 ± 0,00000
0,067 ± 0,01118

0,063 ± 0,01087
0,046 ± 0,00290 0,053 ± 0,00374

Campesterol
% rep 1 % rep 2 average sd Cv
100 100 100 0.00000 0
73.0256703 88.4216307 80.7236505 10.88659 13
69.2740686 86.0919008 77.6829847 11.89200 15
59.767057 51.636423 55.70174 5.74923 10
64.0992971 70.2184439 67.1588705 4.32689 6
0.03948

Stigmasterol
% rep 1 % rep 2 average sd Cv

100 100 100 0.00000 0
59.3251351 80.674593 69.999864 15.09635 22
55.9204502 88.1004703 72.0104603 22.75471 32
50.8888815 39.1218279 45.0053547 8.32056 18
53.6054856 58.7338534 56.1696695 3.62630 6
0.01974
Sitosterol
% rep 1 % rep 2 average sd cv
100 100 100 0.00000 0
71.5666542 90.8098965 81.1882754 13.60703 17
67.7400095 86.4383451 77.0891773 13.22172 17
58.6773393 53.6838398 56.1805896 3.53094 6
61.1257152 67.55894 64.3423276 4.54898 7
0.0714588
Sterols with microwave heating and rapid cooling with ice at different times (times in seconds)
0 30 60 120 240
Campesterol
(µg/mg) 0,029 ± 0,00047 0,030 ± 0,00132 0,028 ± 0,00069 0,025 ± 0,00084 0,020 ± 0,00138
Stigmasterol
(µg/mg) 0,014 ± 0,00042 0,015 ± 0,00106 0,013 ± 0,00137 0,011 ± 0,00091 0,010 ± 0,00031
Sitosterol
(µg/mg) 0,053 ± 0,00096 0,053 ± 0,00186 0,051 ± 0,00210 0,044 ± 0,00005 0,037 ± 0,00267
Campesterol
mg sterol 1 mg sterol 2 % rep 1 % rep 2 average sd cv
0.02946196 0.02879328 100 100 100 0.00000 0
0.02269966 0.02575975 106.38758 102.882263 104.634922 2.47863 2
0.02737583 0.02834885 92.9192508 98.456493 95.6878719 3.91542 4
0.0241801 0.02536821 82.0722827 88.1046123 85.0884475 4.26550 5
0.02120284 0.01925472 71.966859 66.8722885 69.4195738 3.60241 5
Stigmasterol

mg sterol 1 mg sterol 2 % rep 1 % rep 2 average sd cv
0.01398498 0.01339694 100 100 100 0.00000 0
0.01216661 0.01447403 112.433323 105.769523 109.101423 4.71202 4
0.01229089 0.01422631 87.8863685 106.190741 97.038555 12.94315 13
0.01089006 0.01218078 77.8696318 90.9221131 84.3958724 9.22950 11
0.01026837 0.00983039 73.4242874 73.3778885 73.4010879 0.03281 0
Sitosterol

mg sterol 1 mg sterol 2 % rep 1 % rep 2 average sd cv
0.05207153 0.05342934 100 100 100 0.00000 0
0.04255506 0.04969144 100.673611 102.958234 101.815922 1.61547 2
0.04993595 0.0529004 95.8987574 99.0100343 97.4543958 2.20000 2
0.04453786 0.04446883 85.5320704 83.2292371 84.3806538 1.62835 2
0.0388374 0.03505448 74.5847057 65.6090442 70.0968749 6.34675 9

Retention times and characteristic ions of SOPs

Compound tR (min) Characteristic ions (m/z) Abundance (%)
19-hydroxycholesterol (is) 25.61 353 366 10.5
7a-hydroxycampesterol 25.73 470 471 472 11.0
7a-hydroxystigmasterol 26.10 482 483 484 12.0
7a-hydroxysitosterol 27.94 484 485 486 11.0
7ß-hydroxycampesterol 29.74 470 471 472 3.1
7ß-hydroxystigmasterol 29.88 482 483 484 3.5
5ß,6ß-epoxycampesterol 31.74 370 383 398 488 1.3
5a,6a-epoxycampesterol 32.28 398 380 488 11.0
7ß-hydroxysitosterol 32.54 484 485 486 3.0
5ß,6ß-epoxystigmasterol 32.59 253 382 410 500 1.5
5a,6a-epoxystigmasterol 33.13 253 392 410 500 0.8
campestanetriol 34.84 417 418 470 560 3.6
5ß,6ß-epoxysitosterol 35.14 384 394 412 502 0.14
stigmastanetriol 35.62 429 253 482 572 3.0
25-hydroxycampesterol ?? 131 470 545 560 ??
5a,6a-epoxysitosterol 35.78 394 397 412 502 0.9
25-hydroxystigmasterol ?? 131 482 487 396 ??
7-ketocampesterol 38.74 486 381 487 396 4.8
sitostanetriol 38.75 431 432 484 574 1.4
7-ketostigmasterol 39.96 357 359 498 347 4.3
25-hydroxysitosterol ?? 131 484 559 574 ??
7-ketositosterol 43.70 395 500 510 410 6.9
Ions in bold signifies the ion used for integration

SOP´s Campesterol with microwave heating at different times
(times in seconds)
0 120 240
7a-H-cam (µg/mg) 1,36 ± 0,04 1,25 ± 0,03

7ß-H-cam (µg/mg) 1,16 ± 0,03 1.06 ± 0,07
5ß,6ß-E-cam (µg/mg) 3,50 ± 0,04 3,04 ± 0,01
5a,6a-E-cam (µg/mg) 0,52 ± 0,01 0,43 ± 0,04
cam-Triol (µg/mg) 1,23 ± 0,06 1,13 ± 0,08
7-K-cam (µg/mg) 3,59 ± 0,05 3,27 ± 0,03
SOP´s Stigmasterol with microwave heating at different times
(times in seconds)
0 120 240
7a-H-stigma (µg/mg) 0,43 ± 0,01 0.41 ± 0.06
7ß-H- stigma (µg/mg) 0,69 ± 0,01 0.67 ± 0.01
5ß,6ß-E- stigma (µg/mg) 1,38 ± 0,01 1.30 ± 0.01
5a,6a-E- stigma (µg/mg)
Stigma-Triol (µg/mg) 0,46 ± 0,00 0.46 ± 0.00
7-K- stigma (µg/mg) 1,23 ± 0,03 1.14 ± 0.01

SOP´s Sitosterol with microwave heating and rapid cooling with ice at different times
(times in seconds)
0 120 240
7a-H-sito (µg/mg) 0,57 ± 0,02 0.52 ± 0.00
7ß-H- sito (µg/mg) 0,44 ± 0,01 0.40 ± 0.01
5ß,6ß-E- sito (µg/mg) 1,09 ± 0,03 0.94 ± 0.04
5a,6a-E- sito (µg/mg) 0,33 ± 0,01 0,26 ± 0.04
Sito-Triol (µg/mg) 0.79 ± 0.01 0.78 ± 0.04
7-K- sito (µg/mg) 0,87 ± 0,02 0.78 ± 0.01
SOP´s Campesterol with microwave heating and rapid cooling with ice at different times
(times in seconds)
0 120 240
7a-H-cam (µg/mg)
1,45 ± 0,12 1,21 ± 0,09 1,19 ± 0,01
7ß-H-cam (µg/mg) 1,14 ± 0,08 0,97 ± 0,16 1,03 ± 0,09
5ß,6ß-E-cam (µg/mg) 3,45 ± 0,30 3,28 ± 0,11 3,06 ± 0,33
5a,6a-E-cam (µg/mg) 0,57 ± 0,05 0,58 ± 0,13 0,37 ± 0,09
cam-Triol (µg/mg) 1,25 ± 0,06 1,08 ± 0,12 1,24 ± 0,12
7-K-cam (µg/mg) 3,47 ± 0,00 3,50 ± 0,23 3,39 ± 0,14

SOP´s Stigmasterol with microwave heating and rapid cooling with ice at different times
(times in seconds)
0 120 240
7a-H-stigma (µg/mg) 0,46 ± 0,01 0.40 ± 0.02 0.37 ± 0.02
7ß-H- stigma (µg/mg) 0,69 ± 0,02 0.68 ± 0.01 0.68 ± 0.02
5ß,6ß-E- stigma (µg/mg) 1,40 ± 0,04 1.37 ± 0.03 1.32 ± 0.12
5a,6a-E- stigma (µg/mg)
Stigma-Triol (µg/mg) 0,66 ± 0,00 0.59 ± 0.07 0.65 ± 0.02
K- stigma (µg/mg) 1,23 ± 0,03 1.18 ± 0.04 1.16 ± 0.01

SOP´s Sitosterol with microwave heating and rapid cooling with ice at different times
(times in seconds)
0 120 240
7a-H- sito (µg/mg) 0,60 ± 0,03 0.54 ± 0.01 0.53 ± 0.00
7ß-H- sito (µg/mg) 0,44 ± 0,02 0.44 ± 0.02 0.41 ± 0.02
5ß,6ß-E- sito (µg/mg) 1,06 ± 0,02 1.03 ± 0.06 0.94 ± 0.12
5a,6a-E- sito (µg/mg) 0,31 ± 0,03 0,27 ± 0.06 0.25 ± 0.05
Sito-Triol (µg/mg) 0.83 ± 0.01 0.70 ± 0.01 0.80 ± 0.19
7-K- sito (µg/mg) 0,83 ± 0,01 0.85 ± 0.02 0.84 ± 0.05

AREAS 0 120 240
1 2 1 2 1 2
19-HC 577784 1069138 708723 1186767 1109871 1190765
7a-H-cam 32431 53357 29753 55478 48522 51560
7ß-H-cam 4005 6277 2529 6915 5189 7067
5ß,6ß-E-cam 3153 5012 3268 5814 4365 5700
5a,6a-E-cam 600.77 998.62 804.43 1041 689.59 948.57
cam-Triol 3022 6183 3514 4469 6714 5778
7-K-cam 6095 11285 7107 13371 10933 12653

7a-H-stigma 10852 19178 10960 19973 16904 16793
7ß-H-stigma 1422 2291 1472 2683 2538 2346
5ß,6ß-E-stigma 942.2 1617 1104 1728 1354 1882
5a,6a-E-stigma
stigma-Triol 657.99 1233 733.24 574.18 1152 1448
7-K-stigma 1035 1766 1032 1939 1694 1770

7a-H-sito 48216 82420 50356 85755 77926 84008
7ß-H-sito 6287 10750 7596 12155 9802 11934
5ß,6ß-E-sito 3624 6543 4063 7522 5246 7087
5a,6a-E-sito 883.29 1870 889.35 1969 1313 1797
sito-Triol 4030 7265 3952 6718 8775 6207
7-K-sito 7195 13472 8987 15575 13407 16100

ION 0 120 240
ion 1 2 1 2 1 2
19-HC 0.105 5502705 10182267 6749743 11302543 10570200 11340619
7a-H-cam 0.110 294827 485064 270482 504345 441109 468727
7ß-H-cam 0.031 129194 202484 81581 223065 167387 227968
5ß,6ß-E-cam 0.013 242538 385538 251385 447231 335769 438462
5a,6a-E-cam 0.010 61935 102951 82931 107320 71092 97791
cam-Triol 0.036 83944 171750 97611 124139 186500 160500
7-K-cam 0.048 126979 235104 148063 278563 227771 263604

7a-H-stigma 0.120 90433 159817 91333 166442 140867 139942
7ß-H-stigma 0.035 40629 65457 42057 76657 72514 67029
5ß,6ß-E-stigma 0.015 62813 107800 73600 115200 90267 125467
5a,6a-E-stigma 0.008 0 0 0 0 0 0
stigma-Triol 0.030 21933 41100 24441 19139 38400 48267
7-K-stigma 0.043 24070 41070 24000 45093 39395 41163

7a-H-sito 0.110 438327 749273 457782 779591 708418 763709
7ß-H-sito 0.030 209567 358333 253200 405167 326733 397800
5ß,6ß-E-sito 0.014 258857 467357 290214 537286 374714 506214
5a,6a-E-sito 0.009 98143 207778 98817 218778 145889 199667
sito-Triol 0.014 287857 518929 282286 479857 626786 443357
7-K-sito 0.069 104275 195246 130246 225725 194304 233333

RATIO 0 120 240
1 2 1 2 1 2
7a-H-cam 0.0536 0.0476 0.0401 0.0446 0.0417 0.0413
7ß-H-cam 0.0235 0.0199 0.0121 0.0197 0.0158 0.0201
5ß,6ß-E-cam 0.0441 0.0379 0.0372 0.0396 0.0318 0.0387
5a,6a-E-cam 0.0113 0.0101 0.0123 0.0095 0.0067 0.0086
cam-Triol 0.0153 0.0169 0.0145 0.0110 0.0176 0.0142
7-K-cam 0.0231 0.0231 0.0219 0.0246 0.0215 0.0232

7a-H-stigma 0.0164 0.0157 0.0135 0.0147 0.0133 0.0123
7ß-H-stigma 0.0074 0.0064 0.0062 0.0068 0.0069 0.0059
5ß,6ß-E-stigma 0.0114 0.0106 0.0109 0.0102 0.0085 0.0111
5a,6a-E-stigma 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000
stigma-Triol 0.0040 0.0040 0.0036 0.0017 0.0036 0.0043
7-K-stigma 0.0044 0.0040 0.0036 0.0040 0.0037 0.0036

7a-H-sito 0.0797 0.0736 0.0678 0.0690 0.0670 0.0673
7ß-H-sito 0.0381 0.0352 0.0375 0.0358 0.0309 0.0351
5ß,6ß-E-sito 0.0470 0.0459 0.0430 0.0475 0.0355 0.0446
5a,6a-E-sito 0.0178 0.0204 0.0146 0.0194 0.0138 0.0176
sito-Triol 0.0523 0.0510 0.0418 0.0425 0.0593 0.0391
7-K-sito 0.0189 0.0192 0.0193 0.0200 0.0184 0.0206

ug/mg sterol
1 2 media dv cv
1.15 1.27 1.21 0.09 8
0.85 1.08 0.97 0.16 17
3.20 3.36 3.28 0.11 3
0.68 0.49 0.58 0.13 23
1.17 1.00 1.08 0.12 11
3.34 3.66 3.50 0.23 7
10.38 10.86 10.62 0.34 3

ug/mg sterol
1 2 media dv cv
1.19 1.18 1.19 0.01 1
0.96 1.09 1.03 0.09 9
2.82 3.29 3.06 0.33 11
0.30 0.43 0.37 0.09 25
1.33 1.16 1.24 0.12 10
3.29 3.49 3.39 0.14 4
9.90 10.65 10.27 0.53 5
0 7a-H-cam
120 7ß-H-cam
240 5ß,6ß-E-cam
5a,6a-E-cam
cam-Triol
7-K-cam
total cam

2 media dv cv
0.45 0.46 0.01 3
0.68 0.69 0.02 3
1.37 1.40 0.04 3

0.66 0.66 0.00 0
1.21 1.23 0.03 2
4.36 4.43 0.10 2
2 media dv cv
0.42 0.40 0.02 6
0.69 0.68 0.01 2
1.34 1.37 0.03 3

0.54 0.59 0.07 11
1.20 1.18 0.04 3
4.20 4.22 0.03 1
2 media dv cv
0.35 0.37 0.02 5
0.66 0.68 0.02 3
1.40 1.32 0.12 9

0.67 0.65 0.02 3
1.16 1.16 0.01 1
4.24 4.18 0.10 2

0 7a-H-stigma
120 7ß-H-stigma
240 5ß,6ß-E-stigma
5a,6a-E-stigma
stigma-Triol
7-K-stgima
total stigma

media dv cv
0.60 0.03 6
0.44 0.02 4
1.06 0.02 1
0.31 0.03 11
0.83 0.01 2
0.83 0.01 1
4.07 0.04 1
media dv cv
0.54 0.01 1
0.44 0.01 2
1.03 0.06 6
0.27 0.06 23
0.70 0.01 1
0.85 0.02 2
3.84 0.14 4
media dv cv
0.53 0.00 0
0.41 0.02 6
0.94 0.12 13
0.25 0.05 20
0.80 0.19 24
0.84 0.05 6
3.76 0.06 1
0 7a-H-sito
120 7ß-H-sito
240 5ß,6ß-E-sito
5a,6a-E-sito
sito-Triol
7-K-sito
total sito

Order Description
Abstract
One of the essential components of the membranes of all eukaryotic organisms is plant sterols (site). Recent studies shown plant sterols has an effect on the high cholesterol levels. The first studies of effects of PS on cholestrol lowering dates back to 1950´s. PS doses of up to 50 g/day used in this studies to lower the level of cholestrol in serum (Pollak & Kritchevsky, 1981). “In the 1980’s, Japanese researchers managed to produce the saturated form of PS, known as phytostanols, which were reported to have stronger cholesterol lowering potential. Ten years later the first human trial using phytostanols (1.5 g/day) was performed and it was observed that the use of phytostanols lowered the level of cholesterol in human serum (Miettinen & Gylling, 2003).
Other experiments looked in to effects of temperature on sterol oxidation, degradation and oxysterol formation with conventional heating. However, little is known about the effects of microwave heating, this experiment looked in to those effects of temperature on sterol oxidation, degradation and oxysterol formation with microwave heating at different time parameters with and without rapid cooling. For the experiment without rapid cooling first 0.0658g sterol (5-cholesten-24ß-ethyl-3ß-OL-%54) dissolved in 25 ml of chloroform and divided in 15 test tubes each containing 1 ml mix solution, then solution evaporated using nitrogen ready to be heated in the microwave oven at five different time parameters (control 0 seconds, 30 seconds, 60 seconds, 120 seconds and 240 seconds) at 900 W and for the experiment with rapid cooling, all the procedure were same but 0.0641g sterol used and rapid cooled with ice. Experiment done in the laboratory of investigation at University of Navarra, Pamplona-Spain. Results showed degradation of sterol without any oxysterol formation. Therefor to accomplish an excitable level further scientific experiment needed.

INTRODUCTION
“Dis-moi ce que tu manges, je te dirai ce que tu es.” [Tell me what you eat and I will tell you what you are] Physiologie du Gout, ou Meditations de Gastronomie Transcendante, 1826 Anthelme Brillat-Savarin. Is it only what you eat or what you buy, how you prepare and how you cook is important. Nowadays there are different methods involved in cooking food, one of the cooking method is electromagnetic” radiation. What type of effects radiation has on Plant Sterols (PS)?

Lipids

Sterols

Cholestrol

Phytosterols

Sterol Oxidation Products (SOP’s)

Microwave

OBJECTIVE
The objective of this experiment was to find out the c. Because oxidation occurs when plant sterols exposed to air or subjected to heat at high temperature.

METHOD AND METERIALS

Experimental Procedures

For the purpose model study was develop; gas chromatography used for measuring the level of main phytosterols; campesterol, stigmasterol, and ß-sitosterol. Subsequently deviations in the phytosterols and POP´s had been measured after the experimental treatments to look for the extent of the oxidation. The intrinsic levels of the major phytosterols, campesterol, stigmasterol, and ß-sitosterol profiles were measured using gas chromatography (GC). The extent of oxidation was measured by examining changes in the phytosterols following the application of the experimental treatments.

Design of the Project

For each model system; PS dissolved in chloroform, mixed solution were transferred to 15 test tubes 15 x 100 mm with screw tops and evaporated with the help of Nitrogen (N2) following the evaporation sample tubes put in to microwave for 30, 60, 120, 240 seconds respectively and 3 tubes been the control, have not been subjected to microwave heating.
+ mixed in 25 ml
1 ml mixed solution pipetted in to 15 tubes
EVAPRATED WITH NITROGEN
PUT IN TO FOR + 3 BLANKS
Re-dissolved them with 1 ml +

Temperatures: For each experiment, temperature was measured just after the heating and during the following 10 min (without rapid cooling)

TIME(Sec)
OF
MICROWAVE HEATING
TIME (min) OF COOLING DOWN
0 0.5 1 1.5 2 2.5 3 4 5 6 7 8 9
30 Rep1 32.3 30 29.5 27.9 27.4 26.9 25 23.5 23.5 22.7 22.7 22.7 22.7
Rep2 32.6 28.9 27.5 26.5 26 25.6 24.8 24.1 23.2 22.7 22.7 22.7 22.7
Rep3 29.7 28.2 26 26.2 25.6 25.2 24.4 23.8 22.9 22.2 22.2 22.2 22.2
60 Rep1 34.5 32.5 30.9 29 28 27.8 27.2 25.4 24.5 23.8 23.8 23.8 23.8
Rep2 33 32.5 30.9 29 28 27.8 27.2 25.4 24.5 23.8 23.8 23.8 23.8
Rep3 32.5 32.5 30.9 29 28 27.8 27.2 25.4 24.5 23.8 23.8 23.8 23.8
120 Rep1 40.3 32.5 30.9 29 28 27.8 27.2 25.4 24.5 23.8 23.8 23.8 23.8
Rep2 42.9 32.5 30.9 29 28 27.8 27.2 25.4 24.5 23.8 23.8 23.8 23.8
Rep3 41.5 32.5 30.9 29 28 27.8 27.2 25.4 24.5 23.8 23.8 23.8 23.8
240 Rep1 59 32.5 30.9 29 28 27.8 27.2 25.4 24.5 23.8 23.8 23.8 23.8
Rep2 56.3 32.5 30.9 29 28 27.8 27.2 25.4 24.5 23.8 23.8 23.8 23.8
Rep3 54.1 32.5 30.9 29 28 27.8 27.2 25.4 24.5 23.8 23.8 23.8 23.8

TIME(Sec)
OF
MICROWAVE HEATING
TIME (min) OF COOLING DOWN

0 0.5 1 1.5 2 2.5 3 4 5 6 7 8 9
30 media 31.5 29.0 27.7 26.9 26.3 25.9 25.0 24.3 23.2 22.5 22.5 22.5 22.5
ds 1.6 0.9 1.8 0.9 0.9 0.9 0.7 0.6 0.3 0.3 0.3 0.3 0.29
cv 5 3 6 3 4 3 3 3 1 1 1 1 1
60 media 33.3 31.7 30.3 28.9 28.1 27.4 26.7 25.1 24.3 23.5 23.5 23.5 23.5
ds 1.0 0.7 0.5 0.1 0.1 0.3 0.4 0.3 0.2 0.3 0.3 0.3 0.3
cv 3 2 2 0 0 1 2 1 1 1 1 1 1
120 media 41.6 40.2 37.6 35.1 33.7 32.5 30.7 28.6 26.8 25.6 24.8 24.1 23.3
ds 1.3 1.1 1.0 0.8 0.6 0.7 0.6 0.4 0.3 0.4 .2 0.5 0.2
cv 3 3 3 2 2 2 2 1 1 1 1 2 0.9
240 media 56.5 51.2 48.1 45.2 41.4 39.2 37.6 35.1 31.5 29.7 28.0 26.6 25.8
ds 2.5 1.8 1.7 1.4 1.8 1.3 1.0 0.6 0.7 0.6 0.4 0.7 0.4
cv 4 3 3 3 4 3 3 2 2 2 2 3 1
Temperatures: For each experiment, temperature was measured just after the heating and the following rapid cooling with ice.
Temperatures: For each experiment, temperature was measured just after the heating and during the following
TIME (S) OF MICROWAVE HEATING
TIME (min) OF COOLING DOWN

0
30 Rep1 30.8
Rep2 31.2
Rep3 33.7
60 Rep1 31.5
Rep2 32
Rep3 34
120 Rep1 42.7
Rep2 41.2
Rep3 36.4
240 Rep1 56.1
Rep2 54.8
Rep3 53.9

Temperatures: For each experiment, temperature was measured just after the heating and during the following
TIME (S) OF MICROWAVE HEATING
TIME (min) OF COOLING DOWN

0
30 media 31.9
ds 1.6
cv 5
60 media 32.5
ds 1.3
cv 4
120 media 40.1
ds 3.3
cv 8
240 media 54.9
ds 1.1
cv 2

Analysis of sterols:
50 µl mixture of solution from each tubes pipetted in to wider test tubes and 50 µl internal standard (5a-cholestane) were added to each tube. The solvent in each tube were evaporated with the flow of N2. 400 µl of reagent Tri-sil were added to each evaporated test tubes, lids put on but not tightly closed and samples transferred in to already heated water bath to 60 °C for 45 min. The solution evaporated for the last time with flow of N2, 400 µl hexane especially for Gas Chromatography (GC) added and final solution were transferred to glass vial using syringe, needle and 0.45 µm filter ready to be analyse by GC.

Analysis of SOP’s

Three different time (0-120-240 min.) were chosen for the analysing of SOP’s. Previously prepared sample was transferred to a 3 x3 tubes of samples (an approximately 2 mg sterol), and 1 ml 19-hydroxycholesterol as internal standard (0.02 mg /1 mL) solution was added and evaporated with the help of N2. 200 µl Hex: ethyl acetate (95;5, v/v) added to evaporated sample and mixed until homogenized with the help of vortex. Homogenized solution transferred to NH2-SPE cartridge ready to be filtered.

Bickers put underneath to each cartridge used to collect the solution, because first solution is discharged. Then solution equilibrated with 3 ml hexane/ethyl acetate, each samples transferred in to a cartridge of it is concern to be filtered. Filtered samples added further 200 µl Hex: ethyl acetate shaken and transferred back to cartridges first 8 ml of Hex: ethyl acetate 95:5 added and filtered and then 10 ml of Hex: ethyl acetate 95:10 added and filtered and solution discharged. SOP’s that captured by the filter inside the cartridges washed with 10 mL acetone. Solution put in to water bath consistent temperature of 40 ºC and evaporated with under a stream of N2.
The sample solutions of SOP’s were made ready to for the chromatographic analysis as previously described for sterols.
Gas Chromatography-Mass spectrometer (Agilent 6890-5973) was used to analyse the samples;
Column: 19091S-433 HP-5ms 5% Phenyl Methyl Siloxane (30 m x 250 µm x 0.25µm)
Carrier gas: He, 1 mL/min
Oven temperatures program: 85 °C during 0.5 min
Curve 1: 50 °C/min up to 290 °C
Curve 2: 0.5 °C/min up to 298 °C
Injector temperature: 280 °C
Volume of sample injected: 1 µL, splittless mode
Transfer line to the detector: 280 °C
Source temperature:
Electron impact: 70 eV
Detector temperature: 300 °C
Mass interval: 50.00-550.00 uma
Detection mode: SCAN

Ions identification were established using Wiley library and varies different literature’s mass spectra, also retention time and mass spectrometry fragments comparison done against standard pure compounds.
Internal standard 5a-cholestane used for internal standard method, total ion chromatogram used for the quantitation of cholesterol and 5a-cholestane, although extract ion chromatograms used for PS quantification, in accordance with the quantity of specific ion for each peak for campesterol, stigmasterol and ß-sitosterol 343, 484, 357 respectively and the relative abundance of each ion had been considered (Berasategi 2012)
Retention times, ions characteristics and sterols quantitation mode in chromatographic analysis

Compound tR (min) Characteristic ions (m/z) Quantitation
5a-cholestane (is) 13.0 217, 357, 372 Total area (A)
cholesterol 17.7 329, 353, 368, 458 Total area
campesterol 20.5 343, 367, 382 343 (6.62%)*
stigmasterol 21.1 355, 394, 484 484 (3.05%)
ß-sitosterol 23.2 357, 381, 396, 486 357 (6.56%)
* ion used for integration (abundance of the ion)

Each plant sterol’s total area calculated as follows:
Calibration curves for sterols internal standard

Compound Calibration curve R2
cholesterol y = 1.1903x – 0.0288 0.9937
campesterol y = 1.1348x – 0.008 0.9923
stigmasterol y = 1.0346x – 0.0156 0.9905
ß-sitosterol y = 1.1257x – 0.0324 0.9974

;
Sterols with microwave heating at different times (times in seconds)
0 30 60 120 240
Campesterol
(µg/mg) 0,043 ± 0,00000 0,035 ± 0,00470 0,033 ± 0,00514 0,024 ± 0,00248 0,029 ± 0,00187
Stigmasterol
(µg/mg) 0,023 ± 0,00000 0,016 ± 0,00343 0,016 ± 0,00517 0,010 ± 0,00189 0,013 ± 0,00008
Sitosterol
(µg/mg) 0,082 ± 0,00000
0,067 ± 0,01118

0,063 ± 0,01087
0,046 ± 0,00290 0,053 ± 0,00374

Campesterol
% rep 1 % rep 2 average sd Cv
100 100 100 0.00000 0
73.0256703 88.4216307 80.7236505 10.88659 13
69.2740686 86.0919008 77.6829847 11.89200 15
59.767057 51.636423 55.70174 5.74923 10
64.0992971 70.2184439 67.1588705 4.32689 6
0.03948

Stigmasterol
% rep 1 % rep 2 average sd Cv

100 100 100 0.00000 0
59.3251351 80.674593 69.999864 15.09635 22
55.9204502 88.1004703 72.0104603 22.75471 32
50.8888815 39.1218279 45.0053547 8.32056 18
53.6054856 58.7338534 56.1696695 3.62630 6
0.01974
Sitosterol
% rep 1 % rep 2 average sd cv
100 100 100 0.00000 0
71.5666542 90.8098965 81.1882754 13.60703 17
67.7400095 86.4383451 77.0891773 13.22172 17
58.6773393 53.6838398 56.1805896 3.53094 6
61.1257152 67.55894 64.3423276 4.54898 7
0.0714588
Sterols with microwave heating and rapid cooling with ice at different times (times in seconds)
0 30 60 120 240
Campesterol
(µg/mg) 0,029 ± 0,00047 0,030 ± 0,00132 0,028 ± 0,00069 0,025 ± 0,00084 0,020 ± 0,00138
Stigmasterol
(µg/mg) 0,014 ± 0,00042 0,015 ± 0,00106 0,013 ± 0,00137 0,011 ± 0,00091 0,010 ± 0,00031
Sitosterol
(µg/mg) 0,053 ± 0,00096 0,053 ± 0,00186 0,051 ± 0,00210 0,044 ± 0,00005 0,037 ± 0,00267
Campesterol
mg sterol 1 mg sterol 2 % rep 1 % rep 2 average sd cv
0.02946196 0.02879328 100 100 100 0.00000 0
0.02269966 0.02575975 106.38758 102.882263 104.634922 2.47863 2
0.02737583 0.02834885 92.9192508 98.456493 95.6878719 3.91542 4
0.0241801 0.02536821 82.0722827 88.1046123 85.0884475 4.26550 5
0.02120284 0.01925472 71.966859 66.8722885 69.4195738 3.60241 5
Stigmasterol

mg sterol 1 mg sterol 2 % rep 1 % rep 2 average sd cv
0.01398498 0.01339694 100 100 100 0.00000 0
0.01216661 0.01447403 112.433323 105.769523 109.101423 4.71202 4
0.01229089 0.01422631 87.8863685 106.190741 97.038555 12.94315 13
0.01089006 0.01218078 77.8696318 90.9221131 84.3958724 9.22950 11
0.01026837 0.00983039 73.4242874 73.3778885 73.4010879 0.03281 0
Sitosterol

mg sterol 1 mg sterol 2 % rep 1 % rep 2 average sd cv
0.05207153 0.05342934 100 100 100 0.00000 0
0.04255506 0.04969144 100.673611 102.958234 101.815922 1.61547 2
0.04993595 0.0529004 95.8987574 99.0100343 97.4543958 2.20000 2
0.04453786 0.04446883 85.5320704 83.2292371 84.3806538 1.62835 2
0.0388374 0.03505448 74.5847057 65.6090442 70.0968749 6.34675 9

Retention times and characteristic ions of SOPs

Compound tR (min) Characteristic ions (m/z) Abundance (%)
19-hydroxycholesterol (is) 25.61 353 366 10.5
7a-hydroxycampesterol 25.73 470 471 472 11.0
7a-hydroxystigmasterol 26.10 482 483 484 12.0
7a-hydroxysitosterol 27.94 484 485 486 11.0
7ß-hydroxycampesterol 29.74 470 471 472 3.1
7ß-hydroxystigmasterol 29.88 482 483 484 3.5
5ß,6ß-epoxycampesterol 31.74 370 383 398 488 1.3
5a,6a-epoxycampesterol 32.28 398 380 488 11.0
7ß-hydroxysitosterol 32.54 484 485 486 3.0
5ß,6ß-epoxystigmasterol 32.59 253 382 410 500 1.5
5a,6a-epoxystigmasterol 33.13 253 392 410 500 0.8
campestanetriol 34.84 417 418 470 560 3.6
5ß,6ß-epoxysitosterol 35.14 384 394 412 502 0.14
stigmastanetriol 35.62 429 253 482 572 3.0
25-hydroxycampesterol ?? 131 470 545 560 ??
5a,6a-epoxysitosterol 35.78 394 397 412 502 0.9
25-hydroxystigmasterol ?? 131 482 487 396 ??
7-ketocampesterol 38.74 486 381 487 396 4.8
sitostanetriol 38.75 431 432 484 574 1.4
7-ketostigmasterol 39.96 357 359 498 347 4.3
25-hydroxysitosterol ?? 131 484 559 574 ??
7-ketositosterol 43.70 395 500 510 410 6.9
Ions in bold signifies the ion used for integration

SOP´s Campesterol with microwave heating at different times
(times in seconds)
0 120 240
7a-H-cam (µg/mg) 1,36 ± 0,04 1,25 ± 0,03

7ß-H-cam (µg/mg) 1,16 ± 0,03 1.06 ± 0,07
5ß,6ß-E-cam (µg/mg) 3,50 ± 0,04 3,04 ± 0,01
5a,6a-E-cam (µg/mg) 0,52 ± 0,01 0,43 ± 0,04
cam-Triol (µg/mg) 1,23 ± 0,06 1,13 ± 0,08
7-K-cam (µg/mg) 3,59 ± 0,05 3,27 ± 0,03
SOP´s Stigmasterol with microwave heating at different times
(times in seconds)
0 120 240
7a-H-stigma (µg/mg) 0,43 ± 0,01 0.41 ± 0.06
7ß-H- stigma (µg/mg) 0,69 ± 0,01 0.67 ± 0.01
5ß,6ß-E- stigma (µg/mg) 1,38 ± 0,01 1.30 ± 0.01
5a,6a-E- stigma (µg/mg)
Stigma-Triol (µg/mg) 0,46 ± 0,00 0.46 ± 0.00
7-K- stigma (µg/mg) 1,23 ± 0,03 1.14 ± 0.01

SOP´s Sitosterol with microwave heating and rapid cooling with ice at different times
(times in seconds)
0 120 240
7a-H-sito (µg/mg) 0,57 ± 0,02 0.52 ± 0.00
7ß-H- sito (µg/mg) 0,44 ± 0,01 0.40 ± 0.01
5ß,6ß-E- sito (µg/mg) 1,09 ± 0,03 0.94 ± 0.04
5a,6a-E- sito (µg/mg) 0,33 ± 0,01 0,26 ± 0.04
Sito-Triol (µg/mg) 0.79 ± 0.01 0.78 ± 0.04
7-K- sito (µg/mg) 0,87 ± 0,02 0.78 ± 0.01
SOP´s Campesterol with microwave heating and rapid cooling with ice at different times
(times in seconds)
0 120 240
7a-H-cam (µg/mg)
1,45 ± 0,12 1,21 ± 0,09 1,19 ± 0,01
7ß-H-cam (µg/mg) 1,14 ± 0,08 0,97 ± 0,16 1,03 ± 0,09
5ß,6ß-E-cam (µg/mg) 3,45 ± 0,30 3,28 ± 0,11 3,06 ± 0,33
5a,6a-E-cam (µg/mg) 0,57 ± 0,05 0,58 ± 0,13 0,37 ± 0,09
cam-Triol (µg/mg) 1,25 ± 0,06 1,08 ± 0,12 1,24 ± 0,12
7-K-cam (µg/mg) 3,47 ± 0,00 3,50 ± 0,23 3,39 ± 0,14

SOP´s Stigmasterol with microwave heating and rapid cooling with ice at different times
(times in seconds)
0 120 240
7a-H-stigma (µg/mg) 0,46 ± 0,01 0.40 ± 0.02 0.37 ± 0.02
7ß-H- stigma (µg/mg) 0,69 ± 0,02 0.68 ± 0.01 0.68 ± 0.02
5ß,6ß-E- stigma (µg/mg) 1,40 ± 0,04 1.37 ± 0.03 1.32 ± 0.12
5a,6a-E- stigma (µg/mg)
Stigma-Triol (µg/mg) 0,66 ± 0,00 0.59 ± 0.07 0.65 ± 0.02
K- stigma (µg/mg) 1,23 ± 0,03 1.18 ± 0.04 1.16 ± 0.01

SOP´s Sitosterol with microwave heating and rapid cooling with ice at different times
(times in seconds)
0 120 240
7a-H- sito (µg/mg) 0,60 ± 0,03 0.54 ± 0.01 0.53 ± 0.00
7ß-H- sito (µg/mg) 0,44 ± 0,02 0.44 ± 0.02 0.41 ± 0.02
5ß,6ß-E- sito (µg/mg) 1,06 ± 0,02 1.03 ± 0.06 0.94 ± 0.12
5a,6a-E- sito (µg/mg) 0,31 ± 0,03 0,27 ± 0.06 0.25 ± 0.05
Sito-Triol (µg/mg) 0.83 ± 0.01 0.70 ± 0.01 0.80 ± 0.19
7-K- sito (µg/mg) 0,83 ± 0,01 0.85 ± 0.02 0.84 ± 0.05

AREAS 0 120 240
1 2 1 2 1 2
19-HC 577784 1069138 708723 1186767 1109871 1190765
7a-H-cam 32431 53357 29753 55478 48522 51560
7ß-H-cam 4005 6277 2529 6915 5189 7067
5ß,6ß-E-cam 3153 5012 3268 5814 4365 5700
5a,6a-E-cam 600.77 998.62 804.43 1041 689.59 948.57
cam-Triol 3022 6183 3514 4469 6714 5778
7-K-cam 6095 11285 7107 13371 10933 12653

7a-H-stigma 10852 19178 10960 19973 16904 16793
7ß-H-stigma 1422 2291 1472 2683 2538 2346
5ß,6ß-E-stigma 942.2 1617 1104 1728 1354 1882
5a,6a-E-stigma
stigma-Triol 657.99 1233 733.24 574.18 1152 1448
7-K-stigma 1035 1766 1032 1939 1694 1770

7a-H-sito 48216 82420 50356 85755 77926 84008
7ß-H-sito 6287 10750 7596 12155 9802 11934
5ß,6ß-E-sito 3624 6543 4063 7522 5246 7087
5a,6a-E-sito 883.29 1870 889.35 1969 1313 1797
sito-Triol 4030 7265 3952 6718 8775 6207
7-K-sito 7195 13472 8987 15575 13407 16100

ION 0 120 240
ion 1 2 1 2 1 2
19-HC 0.105 5502705 10182267 6749743 11302543 10570200 11340619
7a-H-cam 0.110 294827 485064 270482 504345 441109 468727
7ß-H-cam 0.031 129194 202484 81581 223065 167387 227968
5ß,6ß-E-cam 0.013 242538 385538 251385 447231 335769 438462
5a,6a-E-cam 0.010 61935 102951 82931 107320 71092 97791
cam-Triol 0.036 83944 171750 97611 124139 186500 160500
7-K-cam 0.048 126979 235104 148063 278563 227771 263604

7a-H-stigma 0.120 90433 159817 91333 166442 140867 139942
7ß-H-stigma 0.035 40629 65457 42057 76657 72514 67029
5ß,6ß-E-stigma 0.015 62813 107800 73600 115200 90267 125467
5a,6a-E-stigma 0.008 0 0 0 0 0 0
stigma-Triol 0.030 21933 41100 24441 19139 38400 48267
7-K-stigma 0.043 24070 41070 24000 45093 39395 41163

7a-H-sito 0.110 438327 749273 457782 779591 708418 763709
7ß-H-sito 0.030 209567 358333 253200 405167 326733 397800
5ß,6ß-E-sito 0.014 258857 467357 290214 537286 374714 506214
5a,6a-E-sito 0.009 98143 207778 98817 218778 145889 199667
sito-Triol 0.014 287857 518929 282286 479857 626786 443357
7-K-sito 0.069 104275 195246 130246 225725 194304 233333

RATIO 0 120 240
1 2 1 2 1 2
7a-H-cam 0.0536 0.0476 0.0401 0.0446 0.0417 0.0413
7ß-H-cam 0.0235 0.0199 0.0121 0.0197 0.0158 0.0201
5ß,6ß-E-cam 0.0441 0.0379 0.0372 0.0396 0.0318 0.0387
5a,6a-E-cam 0.0113 0.0101 0.0123 0.0095 0.0067 0.0086
cam-Triol 0.0153 0.0169 0.0145 0.0110 0.0176 0.0142
7-K-cam 0.0231 0.0231 0.0219 0.0246 0.0215 0.0232

7a-H-stigma 0.0164 0.0157 0.0135 0.0147 0.0133 0.0123
7ß-H-stigma 0.0074 0.0064 0.0062 0.0068 0.0069 0.0059
5ß,6ß-E-stigma 0.0114 0.0106 0.0109 0.0102 0.0085 0.0111
5a,6a-E-stigma 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000
stigma-Triol 0.0040 0.0040 0.0036 0.0017 0.0036 0.0043
7-K-stigma 0.0044 0.0040 0.0036 0.0040 0.0037 0.0036

7a-H-sito 0.0797 0.0736 0.0678 0.0690 0.0670 0.0673
7ß-H-sito 0.0381 0.0352 0.0375 0.0358 0.0309 0.0351
5ß,6ß-E-sito 0.0470 0.0459 0.0430 0.0475 0.0355 0.0446
5a,6a-E-sito 0.0178 0.0204 0.0146 0.0194 0.0138 0.0176
sito-Triol 0.0523 0.0510 0.0418 0.0425 0.0593 0.0391
7-K-sito 0.0189 0.0192 0.0193 0.0200 0.0184 0.0206

ug/mg sterol
1 2 media dv cv
1.15 1.27 1.21 0.09 8
0.85 1.08 0.97 0.16 17
3.20 3.36 3.28 0.11 3
0.68 0.49 0.58 0.13 23
1.17 1.00 1.08 0.12 11
3.34 3.66 3.50 0.23 7
10.38 10.86 10.62 0.34 3

ug/mg sterol
1 2 media dv cv
1.19 1.18 1.19 0.01 1
0.96 1.09 1.03 0.09 9
2.82 3.29 3.06 0.33 11
0.30 0.43 0.37 0.09 25
1.33 1.16 1.24 0.12 10
3.29 3.49 3.39 0.14 4
9.90 10.65 10.27 0.53 5
0 7a-H-cam
120 7ß-H-cam
240 5ß,6ß-E-cam
5a,6a-E-cam
cam-Triol
7-K-cam
total cam

2 media dv cv
0.45 0.46 0.01 3
0.68 0.69 0.02 3
1.37 1.40 0.04 3

0.66 0.66 0.00 0
1.21 1.23 0.03 2
4.36 4.43 0.10 2
2 media dv cv
0.42 0.40 0.02 6
0.69 0.68 0.01 2
1.34 1.37 0.03 3

0.54 0.59 0.07 11
1.20 1.18 0.04 3
4.20 4.22 0.03 1
2 media dv cv
0.35 0.37 0.02 5
0.66 0.68 0.02 3
1.40 1.32 0.12 9

0.67 0.65 0.02 3
1.16 1.16 0.01 1
4.24 4.18 0.10 2

0 7a-H-stigma
120 7ß-H-stigma
240 5ß,6ß-E-stigma
5a,6a-E-stigma
stigma-Triol
7-K-stgima
total stigma

media dv cv
0.60 0.03 6
0.44 0.02 4
1.06 0.02 1
0.31 0.03 11
0.83 0.01 2
0.83 0.01 1
4.07 0.04 1
media dv cv
0.54 0.01 1
0.44 0.01 2
1.03 0.06 6
0.27 0.06 23
0.70 0.01 1
0.85 0.02 2
3.84 0.14 4
media dv cv
0.53 0.00 0
0.41 0.02 6
0.94 0.12 13
0.25 0.05 20
0.80 0.19 24
0.84 0.05 6
3.76 0.06 1
0 7a-H-sito
120 7ß-H-sito
240 5ß,6ß-E-sito
5a,6a-E-sito
sito-Triol
7-K-sito
total sito