Chapter II Experimental
II- 4. Singular value decomposition analysis
In our experiment, we measured a large number of Raman spectra (200 spectra) so we needed to shorten the exposure time (5 s for each spectrum). To improve the signal-to-noise ratio in the spectra acquired with such a short exposure time, singular value decomposition (SVD) analysis was performed [14-15, 18-20]. SVD is a mathematical technique that factorizes an arbitrary m × n matrix A into the product of three matrices as Eq. II-3 [21].
A = UWVT (Eq. II-3)
11
Here U is an m × n column-orthonormal matrix; W is an n × n diagonal matrix of positive singular values, and V is an n × n orthonormal matrix. U and V represent the spectral and positional matrices, respectively. We only retained components of U and V having significantly large singular values to reproduce matrix A, because other components with much smaller singular values contribute to the original data negligibly and can be regarded as noises. Then we reconstructed matrix A by using those components that have significantly large singular values. The number of singular values used for reconstruction depends on the data set, but it was less than 10 in all cases. The SVD was computed in Igor Pro (WaveMetrics) using LAPACK routines.
12
Figure II-1. Single colonies on the YES plate after 4–5 days of cultivation at 30 oC.
13
Figure II-2. Chemical structures of (a) (R)-(+)-lipoic acid (RLA), (b) glutathione (GSH), (c)
L-ascorbic acid (Vit C), and (d) γ-cyclodextrin (γ-CD).
Figure II-3. Schematic of the laboratory
14
Schematic of the laboratory-built confocal Raman microspectrometer.built confocal Raman microspectrometer.
15
Figure II-4. The stage-top incubator
16
Figure II-5. Estimation of the lateral (XY) resolution of the laboratory-built confocal Raman microspectrometer. Red dots, observed intensity change; blue line, the best fitting result to the model function (Eq. II-1).
Figure II-6. Estimation of the axial (Z) resolution of the laboratory-built confocal Raman microspectrometer. Red dots, observed intensity change; blue line, the best fitting result to the model function (Eq. II-1).
40x103
17
Chapter III
Results and Discussion
18
III-1. Growth curves
The growth curves of the WT and ∆ppt1 yeast strains in PMLU medium are shown in Figure III-1A. The WT strain can reach stationary phase after 30 h culture. In contrast, the
∆ppt1 strain cannot grow well in PMLU medium primarily due to the deficiency in CoQ10
production. It grows much more slowly than WT and even after 80 h culture, it seems not to reach stationary phase yet. As can be seen from Figure III-1B, however, addition of the antioxidants (RLA, GSH, Vit C, and RLA/γ-CD) was found to improve the growth of the
∆ppt1 mutant.
The cell growth was recovered in all cases, but the extent of recovery depends on antioxidants. The effect is smallest for Vit C. The cell density of the ∆ppt1 strain after 3 days of culture treated with RLA is similar to that of the WT strain, but the growth rate is still much smaller. There is no notable difference in growth curve between RLA and RLA/γ-CD;
the two growth curves look nearly identical. GSH shows the largest recovery in terms of both final cell density and growth rate. Although the ∆ppt1 strain treated with GSH takes somewhat longer time (about 50 h) to reach stationary phase than the WT strain, it indeed eventually grows to the same level as the WT strain. Based on the growth curves, we conclude that the recovery of cell growth increases in the order of Vit C, RLA (RLA/γ-CD), and GSH. This order is thought to reflect, at least to some extent, the strength of antioxidant activity. Similar restoration of the cell growth of another mutant (∆dps1) by addition of CoQ10/γ-CD complex has recently been reported by Nishida et al [22].
III-2. In vivo quantitative Raman assessment of the effects of antioxidants in fission yeast III-2-1. Raman spectra of the WT and ∆∆∆∆ppt1 strains
As shown above, the growth characteristics can clearly show the effect of the antioxidative reagents. However, the cell number does not provide any information on the molecular mechanisms for how the addition of an antioxidant affects the cell physiology of
19
the ∆ppt1 strain (e.g., metabolic activity) and results in a better growth. In this study, we used Raman microspectroscopy to understand the effect of antioxidants at the molecular level and to assess their antioxidant activity quantitatively in living cells.
Figure III-2 displays typical Raman spectra of the WT and ∆ppt1 strains. Each spectrum is the average of 200 spectra measured from 200 different cells of either WT or ∆ppt1 strain. It is important to collect data on a large number of cells in order to extract information through statistics that is not affected by cell individuality. The two spectra have been normalized to the area intensity of the 1440 cm−1 band (see Table III-1 for assignment of this band). Because the intracellular space is highly heterogeneous with many organelles and biomolecules, the observed Raman spectral pattern, in general, depends greatly on the location from which the spectrum is measured, namely, where the laser is focused. Here we focused the excitation laser on lipid droplets inside yeast cells (usually visible as tiny black spots under the microscope). Lipid droplets are composed of the core of neutral lipids such as triacylglycerols and sterol esters surrounded by a phospholipid monolayer, so they are rich in lipids.
Several representative Raman bands are observed at 1744, 1655, 1602, 1440, 1300, 1154, and 1003 cm−1 in both WT and ∆ppt1 spectra. The assignments [3] of these and other Raman bands can be found in Table 1. The averaged normalized Raman spectra of the WT and ∆ppt1 strains are quite similar to each other, except for the 1602 cm−1 band. The intensity of this band is much weaker in the ∆ppt1 strain than in WT. As already mentioned in Chapter I, this Raman band is unique in that it sharply reflects the metabolic activity of yeast cells [2, 4, 16], and it has recently been shown to arise mainly from the conjugated C=C stretching of ergosterol [7] (see Figure I-1). Because the ∆ppt1 strain is unable to synthesize CoQ10 by itself, their ability to reduce the level of intracellular reactive oxygen species (ROS) is considered weaker than WT. Note that CoQ10 also has antioxidant activity. In the presence of high levels of ROS, ergosterol can be converted to ergosterol peroxide, and the conjugated
20
C=C structure of ergosterol will be disrupted as shown in Figure I-1. Consequently, the intensity of the 1602 cm−1 band will decrease. If this is the case, the intensity of the 1602 cm−1 band should recover when intracellular oxidative stress caused by ROS is reduced by e.g., adding antioxidative reagents externally to the cell.
III-2-2. Raman intensity changes with external addition of antioxidants to the ∆∆∆∆ppt1 strain
To study the effects of exogenous antioxidants on the Raman spectrum of the ppt1 strain, we measured lipid-droplet Raman spectra from 200 living ppt1 cells grown for 10 h in the medium to which the antioxidant (RLA, GSH, Vit C, or RLA/γ-CD) was added. We obtained Raman data exclusively from lipid droplets because their Raman spectrum usually exhibits strong 1602 cm−1 band compared with the cytoplasm. Figure III-3 shows the average of 200 spectra for ∆ppt1 cells with and without the addition of antioxidants. The five averaged spectra have been normalized to the area intensity of the 1440 cm−1 band.
In all cases, the treatment with the antioxidant caused a significant increase in the intensity of the 1602 cm−1 band relative to the control (antioxidant-untreated ∆ppt1). We do not see such a drastic change in other Raman bands. To see this more quantitatively, we plot the number of spectra that give a certain intensity ratio to the 1440 cm−1 band as histograms for the bands at 1602, 1655, 1300, and 1003 cm−1 (Figure III-4–7). These figures compare the distributions of 200 spectra obtained from WT, untreated ∆ppt1 (control), and ∆ppt1 treated with RLA, GSH, Vit C, and RLA/γ-CD. The distributions do not deviate much from the normal distribution. Assuming the normal distribution, we calculated the mean of each distribution and its standard deviation (n = 200), which are also reported in Figure III-4–7.
First, let us focus on the 1602 cm−1 band (Figure III-4). As discussed above, the mean of the intensity ratio A1602/A1440, of the ∆ppt1 strain is about three times smaller than that of WT (0.06 versus 0.18). When treated with RLA, the ∆ppt1 strain shows a mean of 0.12, which is
21
double the untreated control value. The mean of the ratios A1602/A1440 is even larger for the
∆ppt1 cells treated with GSH. Upon treatment, it increases by a factor of ≈3.7 and becomes almost the same as WT. In fact, among the four antioxidative reagents tested, GSH showed the most prominent recovery of the 1602 cm−1 band. In contrast, the effect of adding Vit C was found to be little. The trend in the recovery of the band intensity at 1602 cm−1 agrees very well with our growth curve results (see the previous section). The ability of antioxidant to regain the intensity of the 1602 cm−1 band is strongest for GSH, moderate for RLA, and weakest for Vit C. An important point to note here is that using the Raman band at 1602 cm−1, we have been able to assess the activity of antioxidants in vivo and at the molecular level. It is not possible with either growth curve measurements or ordinary biochemical approaches.
We suspected the moderate antioxidant activity of RLA is due to the low solubility of RLA in water, so we also tried a complex of RLA with γ-cyclodextrin (RLA/γ-CD). In the complex, hydrophobic RLA is included in the inner cavity of γ-CD and the outer hydrophilic part makes the guest molecule solubilized in aqueous environment. Nevertheless, we observed no significant difference in the mean value of the intensity ratio A1602/A1440 between treatment with RLA alone and with RLA/γ-CD. This may be because all RLA was dissolved even without using γ-CD complex at concentration as low as 0.5 mM.
The behavior of the protein and lipid Raman bands is quite different from that of the 1602 cm−1 band. The intensity ratios of the 1655 (Figure III-5), 1300 cm−1 (Figure III-6), and 1003 cm−1 (Figure III-7) bands to the 1440 cm−1 band do not vary significantly with antioxidant treatment; the variation is within experimental uncertainties. This result clearly indicates that only the ergosterol band at 1602 cm−1 is sensitive to the treatment with antioxidants and can be used as a probe of the antioxidative effects.
To check the reproducibility of the trend, we performed the whole set of measurements (WT, untreated ∆ppt1, and antioxidant-treated ∆ppt1 strains) in triplicate. More specifically, we used the same excitation laser power and exposure time, and recorded Raman spectra of
22
200 cells in each measurement. Results are shown in Figure III-8. The overall trend in the effect of the antioxidants is fully consistent with what we have discussed on the basis of Figures III-4–7.
III-3. Time-lapse Raman measurements
We now have shown by looking into the Raman spectra measured after 10 h of culture, that the intensity of the 1602 cm−1 band of the ∆ppt1 yeast strain can be improved by external addition of the antioxidants (Figure III-3). This phenomenon should be dynamic in nature, and it is crucially important to study its temporal behavior. Therefore, we tried to do time-lapse experiments in which the change in Raman spectrum was monitored at different culture times after the antioxidant was added.
We measured 100 Raman spectra of the ∆ppt1 cells with and without addition of RLA and GSH at 0.5 mM to the medium at different culture times (0, 3, 7, 10, 15, 22, and 30 h). A time-stream of the averaged (but unnormalized) Raman spectra is shown for the control (untreated ∆ppt1), ∆ppt1 treated with RLA, and ∆ppt1 treated with GSH in Figure III- 9, 10, and 11, respectively. At 0 h, the ∆ppt1 strain showed almost no signal at 1602 cm−1 (Figure III-9). The 1602 cm−1 band emerged at 7 h and remains nearly constant for more than 20 h afterward. Note that the intensity of the 1602 cm−1 band slightly increased after long incubation even in the absence of an antioxidant. However, it increased with time more markedly when the ∆ppt1 cells were treated with RLA (Figure III-10) and GSH (Figure III-11). To better see the time evolution, we plot the averaged band intensity ratio A1602/A1440
as a function of culture time (Figure III-12). We can see a drastic increase in the ratio at around 10 h for RLA and GSH. This finding indicates that it took about 10 h for the added antioxidant to become effective. The ratio for the untreated ∆ppt1 strain decayed gradually after 10 h, whereas that for the RLA- or GSH-treated strains increased by about 20–30 times.
In stark contrast, the other Raman bands at 1655, 1300, and 1003 cm−1 did not change with
23
time as much as the 1602 cm−1 band (Figure III-13). The marked increase at 10 h can also be seen from histograms of the intensity ratio A1602/A1440 shown in Figure III-14. In both cases of RLA and GSH, the distribution shifts to a higher ratio on going from 3 h to 10 h.
III-4. Conclusion
The key findings are summarized as follows:
(1) Presumably because of the disruption of gene responsible for CoQ10 production, the
∆ppt1 strain of fission yeast may have less resistance to intracellular oxidative stress
induced by ROS. It showed much weaker 1602 cm−1 band intensity than the WT strain (Figure III-2).
(2) Adding antioxidants to the medium in which the ∆ppt1 strain was grown recovered the intensity of the 1602 cm−1 band (Figure III-4). However, similar effects of the antioxidants were not observed for other Raman bands at 1655, 1300, and 1003 cm−1 (Figures III-5-7).
(3) The antioxidant activity in living fission yeast cells evaluated by using the diminished 1602 cm−1 band of ∆ppt1 was found to be strongest for GSH (about 2.8-fold increase with respect to the untreated control), moderate for RLA and RLA/γ-CD (about 2-fold increase), and very weak for Vit C (1.2-fold increase).
(4) Time-lapse Raman measurements on the ∆ppt1 cells treated with RLA and GSH clearly revealed that the intensity of the 1602 cm−1 band begins to increase approximately 10 h after the addition and keeps growing up to 30 h.
Together, we have demonstrated that Raman microspectroscopy can be used as a novel quantitative tool for assessing the efficacy of various antioxidants in vivo. It is still unclear why only the ergosterol band recovers when cells are treated with antioxidants and other protein and lipid bands do not. More spectroscopic as well as biological experiments will be needed to clarify the molecular mechanism behind the present findings.
24
Table III-1. Assignments [3] of representative vibrational bands observed in the Raman spectrum of S. pombe
.
Peak position (cm−1) Assignment
715 Phospholipid headgroup
782 DNA/RNA
1003 Ring breathing of phenylalanine residues
1083 Antisymmetric CCC stretching
1154 C–C and C–N stretching
1266 C=C–H in-plane bend of the cis –CH=CH– linkage Amide III mode of proteins
1300 In-plane CH2 twisting mode
1340 CH2 bending of the aliphatic chain of proteins 1440 CH2 scissoring and CH3 degenerate deformation 1602 Mainly conjugated C=C stretch of ergsterol [9]
1655 cis-C=C stretching of the unsaturated lipid chains Amide I mode of proteins
1744 C=O stretch of the ester linkage of lipids
25
Figure III-1. (A) Growth curves of the WT (black) and untreated ∆ppt1 strains (red). (B) growth curves of the untreated ∆ppt1 strain (red) and that treated with RLA (green), GSH (blue), Vit C (purple), and RLA/γ-CD (orange).
6.5
log(No. of cells/ 1 mL)
80
26
Figure III-2. Averaged Raman spectra (n = 200) of the WT (black) and ∆ppt1 strain (red).
The spectra have been normalized to the area intensity of the 1440 cm−1 band.
1200 1000 800 600 400
Intensity
1800 1600 1400 1200 1000 800 600
Raman shift (cm-1)
ppt1
WT
∆
17441655 1602
1440
1300
1003
27
Figure III-3. Averaged Raman spectra (n = 200) of the untreated ∆ppt1 strain (red) and the
∆ppt1 strains treated with 0.5 mM RLA (green),0.5 mM GSH (blue), 0.5 mM Vit C (purple), and 0.5 mM RLA/γ-CD (orange).
2000 1800 1600 1400 1200 1000 800 600 400
In te ns it y
1800 1600 1400 1200 1000 800 600 Raman shift (cm-1)
ppt1 RLA GSH Vit C RLA/ CD γ
∆
28
Figure III-4. Histograms of the ratio of the area intensity of the 1602 cm−1 band to the 1440 cm−1 band, A1602/A1440. The number of bins is 20 and the bin width is 0.02. Yellow lines
29
Figure III-5. Histograms of the ratio of the area intensity of the 1655 cm−1 band to the 1440 cm−1 band, A1655/A1440. The number of bins is 20 and the bin width is 0.03. Yellow lines
30
Figure III-6. Histograms of the ratio of the area intensity of the 1300 cm−1 band to the 1440 cm−1 band, A1300/A1440. The number of bins is 20 and the bin width is 0.025. Yellow lines
31
Figure III-7. Histograms of the ratio of the area intensity of the 1003 cm−1 band to the 1440 cm−1 band, A1003/A1440. The number of bins is 20 and the bin width is 0.005. Yellow lines
32
A.
B.
C.
D.
Figure III-8. Bar graphs of the averaged intensity ratio of the 1602 (A), 1655 (B), 1300 (C), and 1003 (D) cm−1 bands to the 1440 cm−1 band.
WT Dppt1 RLA GSH Vit C RLA/gCD
Ratio to 1440 cm-1
bandarea of 1602 cm-1
***P<0.001
WT Dppt1 RLA GSH Vit C RLA/gCD
Ratio to 1440 cm-1
bandarea of 1655 cm-1
***P<0.001
WT Dppt1 RLA GSH Vit C RLA/gCD
Ratio to 1440 cm-1 bandarea of 1300 cm-1***P<0.001
0.000
WT Dppt1 RLA GSH Vit C RLA/gCD
Ratio to 1440 cm-1
bandarea of 1003 cm-1
***P<0.001
33
Figure III-9. Time-lapse Raman spectra (n =100) of the untreated ∆ppt1 strain obtained 0 h (red), 3 h (orange), 7 h (green), 10 h (blue), 15 h (purple), 22 h (mulberry), and 30 h (brown).
2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0
R am an I nt en si ty
1800 1600 1400 1200 1000 800 600 Raman shift (cm-1)
30 h 22 h 15 h 10 h 7 h 3 h 0 h without antioxidant
34
Figure III-10. Time-lapse Raman spectra (n = 100) of the ∆ppt1 strain treated with 0.5 mM RLA obtained 0 h (red), 3 h (orange), 7 h (green), 10 h (blue), 15 h (purple), 22 h (mulberry), and 30 h (brown) after addition of RLA.
2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0
R am an i nt en si ty
1800 1600 1400 1200 1000 800 600
Raman shift (cm
-1)
0 h
3 h
7 h
10 h
15 h
22 h
30 h
add 0.5 mM RLA
35
Figure III-11. Time-lapse Raman spectra (n = 100) of the ∆ppt1 strain treated with 0.5mM GSH obtained 0 h (red), 3 h (orange), 7 h (green), 10 h (blue), 15 h (purple), 22 h (mulberry), and 30 h (brown) after addition of GSH.
2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0
R am an i nt en si ty
1800 1600 1400 1200 1000 800 600
Raman shift (cm
-1)
0 h
3 h
7 h
10 h
15 h
22 h
30 h
add 0.5 mM GSH
36
Figure III-12. Time dependence of the averaged ratio of Raman band area intensity at 1602 cm−1 to that at 1440 cm−1 of the untreated ∆ppt1 (red), ∆ppt1 treated with 0.5 mM RLA (green), and ∆ppt1 treated with 0.5 mM GSH (blue).
0.25
0.20
0.15
0.10
0.05
0.00
ratio
30 25
20 15
10 5
0
Time (hr) A1602/A1440 (add GSH)
A1602/A1440 (add RLA)
A1602/A1440 (without antioxidant)
37
Figure III-13. Time dependence of the averaged ratio of Raman band area intensity at 1655 (triangle), 1300 (circle), and 1003 (square) cm−1 to that at 1440 cm−1 of the untreated ∆ppt1 (red), ∆ppt1 treated with 0.5 mM RLA (green), and ∆ppt1 treated with 0.5 mM GSH (blue).
0.5
0.4
0.3
0.2
0.1
0.0
ratio
30 25
20 15
10 5
0
Time (hr)
A1655/A1440 (non-add) A1655/A1440 (add GSH) A1655/A1440 (add RLA) A1300/A1440 (non-add) A1300/A1440 (add GSH) A1300/A1440 (add RLA) A1003/A1440 (non-add) A1003/A1440 (add GSH) A1003/A1440 (add RLA)
38
A
B
Figure III-14. Histograms of the ratio of Raman band area intensity at 1602 cm−1 to that at 1440 cm−1 3 h (A) and 10 h (B) after addition of the antioxidant (green, RLA; blue, GSH).
Yellow lines represent the mean value.
60
39
Chapter IV
Summary and Future Work
40
In this study, we used confocal Raman microspectroscopy to measure the effects of exogenous antioxidants on the CoQ-deficient (∆ppt1) strain of fission yeast. The Raman intensity of the 1602 cm−1 band, which mainly originates from the conjugated C=C stretch of ergosterol [9], is greatly suppressed in the ∆ppt1 strain. This is most likely because the ∆ppt1 strain cannot lower the level of ROS and ergosterol can potentially be oxidized by accumulated ROS to form ergosterol peroxide [12, 13], which no longer possesses the conjugated C=C moiety. Upon addition of antioxidants (RLA, GSH, Vit C, and RLA/γCD), the intensity of the 1602 cm−1 band increased about 2–3 times. However, similar recovery of the band intensity was not observed for other Raman bands at 1655, 1300, and 1003 cm−1, showing that the 1602 cm−1 band is a sensitive probe for intracellular oxidative stress.
Time-lapse Raman measurements on the ∆ppt1 cells treated with RLA and GSH clearly revealed that the intensity of the 1602 cm−1 band begins to increase approximately 10 h after the addition and keeps growing up to 30 h.
However, in these experiments, we did not discuss the effect of the concentration of exogenous antioxidants on the CoQ-deficient (∆ppt1) strain of fission yeast, so we will measure the CoQ-deficient (∆ppt1) strain of fission yeast added different concentration antioxidants. Time-lapse Raman measurements on the ∆ppt1 cells treated with RLA and GSH clearly revealed that the intensity of the 1602 cm−1 band begins to increase approximately 10 h after the addition and keeps growing up to 30 h. But the intensity of the 1602 cm-1 did not keep a balance, so we will observes the intensity of the 1602 cm-1 after 30 h.
We have thus demonstrated that Raman microspectroscopy can be used as a novel quantitative tool for assessing the efficacy of various antioxidants in vivo. Although it is still unclear why only the ergosterol band recovers when cells are treated with antioxidants and other protein and lipid bands do not, we believe this method will open new possibilities not only in fundamental cell biology but in medicine and food industry.
41
Reference
42
References
[1] H. Sies, Experimental Physiology, Vol. 82, pp. 291-295,1997.
[2] Chaudière J, Ferrari-Iliou R., Food Chem Toxicol, Vol. 37, pp. 949-962,1999.
[3] Y.-S. Huang, T. Karashima, M. Yamamoto, and H. Hamaguchi, Biochemistry, Vol. 44, pp. 10009-10019, 2005.
[4] Y.-S. Huang, T. Nakatsuka, and H. Hamaguchi, Applied Spectroscopy, Vol. 61, pp.1290-1294, 2007.
[5] Y. Naito, A. Toh-e, and H. Hamaguchi, Journal of Raman Spectroscopy, Vol. 36, pp.
[5] Y. Naito, A. Toh-e, and H. Hamaguchi, Journal of Raman Spectroscopy, Vol. 36, pp.