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Introduction
 Vitamin E
Vitamin E can be found in forms α-tocopherol, γ-tocopherol and δ-tocopherol. δ-tocopherol found in human milk is almost neligible.
The main biological function of vitamin E is that it acts an an antioxidant which protects the polyunsaturated fatty acids of cell membranes from free-radical damage. Vitamin E in milk is important for newborns,especially preterm babies, for their normal development.
The body needs vitamin E to help keep the immune system strong against viruses and bacteria. Vitamin E is also important in the formation of red blood cells and it helps the body use vitamin K. It also helps widen blood vessels and keep blood from clotting inside them.
Infants (adequate intake of vitamin E)
Excessive levels of vitamin E may also increase the risk of birth defects and might increase the risk for bleeding and serious bleeding in the brain.
From the experiment, we can analyse how much vitamin E is present in human milk so that we can find out whether there is a need for vitamin E supplements for newborns to prevent the lack of vitamin E in them or is it unnecessarily for it. This is crucial as both low and excessively high level of vitamin E present in body can cause problems and complications.
A LITTLE BIT ABOUT BREAST MILK...
Left hand side: Foremilk, the watery milk coming from a full breast.
Right hand side: Hindmilk, the creamy milk coming from a nearly empty breast.
Human breast milk is produced from the mammary glands from the human breast. This is to supply nutrition for the newborns before they are able to feed and digest other food. Human breast milk is known to provide health benefits to both the baby and the mother. For the baby, it has benefits such as decrease likelihood of immunological related illnesses. For the mother, it assists the uterus to reduce to pre-pregnancy size and also reduces the risk of developing breast cancer later in life. Selection of Method
Determine amount of Vitamin E present in Human Milk by High Performance Liquid Chromatography (HPLC) with UV detection
In this experiment, we will be using liquid–liquid extraction
with hexane after simple saponification for the extraction of vitamin E from human milk.
For this analysis, High Performance Liquid Chromatography (HPLC) with UV detection will be used. Chromatographic System and Conditions: 1. Waters Symmetry C18 guard column (3.9mm x 10mm) 2. Waters Symmetry C18 analytical column (3.9mm x 150mm) 3. Gradual change of mobile phase - First 10 minutes: Linear gradient of acetonitrile in water (from 95% - 100%) - Subsequent 10 minutes: 100% acetonitrile 4. Column temperature: 45 Degree Celsius 5. Flow rate: 1mL/min 6. Wavelength range used to scan: 275nm - 350nm 7. Injector volume: 25 µL * Samples were placed & stored in autosampler @ 30 degree Celsius After 30-40 milk extracts analysis, analytical column is washed with propan-2-ol under the following conditions: - Flow rate of 1mL/min for 60 minutes - Temperature of 45 degree Celsius. *This is to ensure reproducible retention on the column.
Evaluation the recovery of the δ-tocopherol spike after extraction was done using:
- 10 μL spiking solution that was diluted with 0.5 mL methanol–propan-2-ol (1:1, v/v) - 25 μL of this solution was injected on the column. - Peak areas of δ-tocopherol spikes in milk extracts were expressed as percentages of the peak areas of δ-tocopherol solutions prepared. - The amounts of native α and γ-tocopherols were calculated on the basis of their peak areas and the percentage recovery. - The latter was calculated for each sam-ple using the peak area of δ-tocopherol spike. Regression analysis was performed using Minitab software. The difference was considered significant if P < 0.05. Means and standard deviations are reported as well. Collection of Milk Samples & Obtaining a Representative Sample
Collection of Milk Samples & Obtaining a Representative Sample
- Recruitment of volunteer mothers into the study will be approved and certified by Institutional Research Board (IRB) Singapore. Informed consent will be sought from the volunteer mothers.
- Milk will be stored in sterile plastic containers using an electric pump from the neonatal unit. The milk obtained will be immediately frozen at -40 degree Celsius until analysis. Analysis will be done within 10 days from the collection to prevent any inaccuracy of results due to stale human milk.
- Stock solutions of δ-tocopherol standards (10mg/mL) were prepared in ethanol and kept for 1 week at a temperature of -20 degree Celsius.
-Spiking solutions of δ-tocopherol standards will be prepared on the day of analysis via dilutions from stock solutions. δ- tocopherol was used as internal standards as its chemical structure was similar to those of the other forms of tocopherol assayed (i.e. α and γ-tocopherol and tocopherol acetate).  Preparation of Lab Sample
Extraction of Tocopherols from Human Milk
- Milk specimens were warmed to a temperature of 38 degree Celsius after defrosting (on the actual day of analysis) and sonicated for a short while.
- The sampling of milk is then performed at 38 degree Celsius with continuous stirring. These steps are done to make sure that the sample is homogenous. - There are 2 methods, and 3 samples will be used for each method. *All milk specimens were extracted using the method and do not involve δ- tocopherol spike.
Method 1:
1. 1mL of milk will be spiked with 10µL of δ- tocopherol standards(around
7-10µg)
2. Add 1mL of methanol containing pyrogallol (3% w/v)
3. Add 1mL of aqueous potassium hydroxide (KOH) (10%, w/v)
4. Vortex sample until it is thoroughly mixed.
5. Place sample into a water bath for 30 minutes at 70 degree Celsius. This
step is called saponification.
6. After the first 15 minutes of saponification briefly vortex the tubes of
samples.
7. Cool the samples on ice.
8. Add 6M of hydrochloric acid (HCl)to acidify the sample to around pH 2. Use a pH probe to detect the pH value of the sample.
9. Add 4mL of hexane
10. Vigorously vortex the samples for 20seconds. Keep it on ice for a short
while.
11. Repeat step 10 for another 2 times.
12. Centrifuge the samples at 1300g for 10 minutes to separate the emulsion.
13. Carefully transfer the top, organic layer into a clean Pyrex container.
14. Evaporate the layer under a gentle stream of nitrogen and on a warm
plate at 40 degree Celsius.
15. The fatty residue will be reconstituted in 0.5mL methanol-propan-2-ol
(1:1 v/v) and warming up to 30 degree Celsius.
Method 2:
1. 1 mL of milk was spiked with 10 μL δ-tocopherol standard (7–10 μg)
2. 1 mL methanol containing pyrogallol (2%, w/v) was added, to disrupt milk globules.
3. Keep the tubes on ice for 10 mins
4. Add 3mL of hexane
5. Vigorously vortex the samples for 20seconds. Keep it on ice for a short
while.
6. Repeat step 10 for another 2 times.
7. Centrifuge the samples at 1300g for 10 minutes to separate the emulsion.
8. Carefully transfer the top, organic layer into a clean Pyrex container.
9. Evaporate the layer under a gentle stream of nitrogen and on a warm
plate at 40 degree Celsius.
10. The fatty residue will be reconstituted in 0.5mL methanol-propan-2-ol
(1:1 v/v) and warming up to 30 degree Celsius.
*Pyrogallol( 1%
final concentration) was used to protect vitamin E from possible
degradation during the extraction and saponification process.
Results & Discussions
The chromatograms are recorded at λ = 295 nm which shows the
elution profile of δ, γ, and α-tocopherol in spiked and unspiked human milk are shown in Fig. 1.
The native amount of δ-tocopherol in human milk is negligible in
comparison with the amount of δ-tocopherol added as the internal standard.
The major forms of
vitamin E found in milk were α and γ-tocopherol and tocopherol acetate was present in minor amounts in
some milk samples.
Fig 2A. shows the recovery of the internal standard
(δ-tocopherol spike) from human milk using the two methods of extraction.
Key: Shaded
circles: Method I,
white circles: Method II.
A. Recovery of δ-tocopherol spike from pooled milk sample using two
methods of extraction. Regression analysis (Method I, y(x) = 17311x + 255, r2 =
0.999; Method II, y(x) = 10593x, r2 = 0.998) was performed by
plotting the integrated peak areas for δ-tocopherol against the concentration
of the spike.
In Fig. 2, the ranges of the amount of δ-tocopherol spike used in both methods are
reasonably comparable. In Method I, 0.7–28 μg/mL of δ-tocopherol was detected while in Method II,
1.49–37.3 μg/mL of δ-tocopherol was detected.
Recovery of the δ-tocopherol spike from pooled milk sample was within the physiological range of concentrations for both extraction methods. The correlation coefficients (r) obtained for assay of δ-tocopherol standard in milk samples by use of both methods were indicative of very good linearity of recovery of δ-tocopherol standard. This shows that there is a directly proportional relation between the δ-tocopherol spike and δ-tocopherol peak area. The ratio of the slope coefficients of the regression lines for Methods 1 and 2 was 1.6342, which shows that the they are in good agreement with the value of the ratio for average δ-tocopherol recovery in Methods 1 and 2 (100 and 60%, respectively;). The signal to noise ratio (S/N) for more than 3 the overall limit of detection of Method I (including saponification, extraction, and HPLC analysis) was 0.65 μg/mL δ-tocopherol in milk. (The higher the signal to noise ratio, the more accurate the results will be as there are less interferences from other compounds.) The percentages for recovery of the internal standard for the two methods of extraction are shown in Fig. 2A. When extraction was performed directly (without saponification), the recovery was approximately 60% and varies from range 19%–82% depending on the milk sample. On the other hand, the recovery after saponification was nearly 100% within a more reproducible range. There are two possible reasons for this improvement for the method with saponification. First, after saponification the milk matrix does not affect the recovery. Second, the conditions of liquid–liquid extraction with hexane from saponified milk are better optimised than in direct extraction. Reduced recovery of the internal standard leads to less accurate measurement. ----------------------------------------------------------------------------------------------------------------
Fig. 3 (below) shows the correlation of
levels of α
and γ-tocopherol in
milk samples measured after direct extraction and extraction after
saponification.
Fig. 3.
Correlation of the levels of
native α and γ-tocopherol in human milk samples, measured after use of the two
methods of extraction.
A. Regression analysis (y(x) = 0.063
+ 0.952x; r2
= 0.868; P < 0.001) was performed by plotting integrated peak
areas for native α-tocopherol in twenty milk samples, measured after extraction
using Method I, against integrated peak areas for native α-tocopherol in the
same samples measured after extraction using Method II.
B. Regression analysis for γ-tocopherol (y(x) = 0.01 + 1.07x; r2 = 0.851, P < 0.01) was performed in the same way as for α-tocopherol. Average and standard deviation of three measurements for the same milk specimen with the two methods are shown.
There was a significant relationship between the two methods of
extraction for both α-tocopherol and γ-tocopherol. Levels of the major form of vitamin E, α-tocopherol, in
milk samples were variable within the range 2.8–15 μg mL−1 (Method I: 7.2 ± 4.2 μg mL−1 , Method II: 7.0 ± 4.1 μg mL−1 ).
For determination of native concentration of α-tocopherol in human milk by Methods I and II, δ-tocopherol was used as the internal standard. Although we were able to demonstrate a significant correlation between results obtained with the two methods, the method including saponification gave higher and more consistent recoveries for the internal spike and, therefore, more reliable data. The proposed saponification step was a simple addition to the method, because it was performed in the same tube as the extraction, and did not involve use of an inert gas. Other Discussion & Conclusion
Other discussion:
Vitamin
E is non-polar because when we look at the size of the molecule, and the size of the part
that is hydrocarbon only (i.e. most of it), the polarity of the -OH group will
be overwhelmed by the non-polarity of the remaining molecule. Therefore, these
molecules will be non-polar as a whole, even though they have a polar group
attached. Therefore,
reversed phase HPLC is used in this experiment. A C18 (non-polar) guard and
analytical column is used.
-For the
composition of mobile phase for the linear gradient phase, water is added to
acetonitrile to reduce the eluotropic strength of the mobile phase to the
stationary phase (because is reversed phase HPLC, water is polar). So that the
peaks will not be co-eluted due to short retention times. Short retention times will cause peaks
overlap, and give us less sharp and symmetrical peaks.
-However, in this case, the concentration for linear gradient was only from 95% to 100% acetonitrile. This was too high a concentration for elution to be be done properly and clearly to give define peaks. Therefore, we suggest that in future experiments, we can start from a lower concentration (for example 10%-100% acetonitrile) to prevent peak overlap and give define peaks for analysis.
Thereafter, an isocratic mode is used because the the analysis time is not very long, and band broadening does not occur. Thus it is better to use the isocratic mode because the initial retention time is good enough to produce a sharp and symmetrical peak. So there is not a need to use linear gradient.
-δ- tocopherol is used as internal
standard because it is present in low amounts in the human breast milk.
Moreover, δ- tocopherol has similar physiochemical properties as α and γ tocopherol. Thus, if there
are any changes in the conditions of the HPLC, then the components will be affected in the same way. This way,
the results obtained are not affected greatly.
Lastly, the UV detector is used because the detection limit for the UV range is 180-380nm, and the absorption region of Vitamin E is 275nm-350nm. Since Vitamin E is able to absorb in the UV region, then the UV detector is used.
Conclusion:
Two extraction methods for the evaluation of vitamin E in human milk were compared - With saponification & without saponification.
Direct extraction of tocopherols with hexane resulted in 60 ± 15% recovery of the internal standard, δ-tocopherol. Application of the improved method, which included a simple saponification step, resulted in 99.6 ± 4.0% recovery for the same internal standard.
There was a significant relationship between tocopherol concentrations in milk specimens measured using the two extraction procedures. This implies that both methods of extraction can be used for determination of vitamin E in human milk, although the evidence presented herein suggests that extraction after saponification may significantly improve the reliability of such data.
In
conclusion, human breast milk contains sufficient amount of Vitamin E needed
for infants because the results above showed good indication of sufficient Vitamin E needed for infants. Thus, infants rarely need external supplements of Vitamin E to aid them in their growth.
References
O. Korchazhkina, E. Jones, M. Czauderna, S. A. Spencer, and J. Kowalczyk
(2006).
HPLC WITH UV DETECTION FOR MEASUREMENT OF VITAMIN E IN HUMAN MILK,48-56, [Retrieved] July 31, 2012, http://www.us.edu.pl/uniwersytet/jednostki/wydzialy/chemia/acta/ac16/zrodla/04_AC16.pdf
A.P. De Leenheer and A.E. Claeys, Clin. Chem., 25, 425 (1979)
K.W. Miller, N.A. Lorr, and C.S. Yang, Anal. Biochem., 138, 340 (1984)
G.L. Catignani and J.G. Bieri, Clin. Chem., 29, 708 (1983)
J.E. Chappell, T. Francis, and M.T.
Clandinin, Early Human Dev., 11, 157 (1985)
Introduction:
Wikipedia, Human breast milk benefits. Available at http://en.wikipedia.org/wiki/Human_breast_milk[Accessed July 31, 2012] Medline Plus, Recommended intake for newborn Available at http://www.nlm.nih.gov/medlineplus/ency/article/002406.htm [Accessed July 31, 2012]
Pictures:
Vitamin E
structure
Human milk
Human
breast milk
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