3.1.1.

Absorption at $355\mathrm{nm}$

The major prerequisite of singlet oxygen generation is the absorption of light or radiation, which is laser radiation at $355\mathrm{nm}$ in this study. After dissolving the fatty acid $(0.1\mathrm{mol}∕\mathrm{L})$ in ethanol, the absorption at $355\mathrm{nm}$ , was measured. The fatty acids without double bonds (palmitic acid, stearic acid) and oleic acid (one double bond) showed a rather low absorption cross section at $355\mathrm{nm}$ , and less than 0.5% of light was absorbed at a concentration of $0.1\mathrm{mol}∕\mathrm{L}$ . For fatty acids with more than two double bonds, the absorption of radiation was in the range of 3.8% to 12.4% (Table 1 ).

Table 1

Singlet oxygen produced by fatty acids.

The radiation absorption of fatty acids should fail for wavelengths longer than $220\mathrm{nm}$ . Nevertheless, auto-oxidation can occur, in particular under normal oxygen conditions. Subsequently, the absorption spectrum of fatty acids such as linoleic acid exhibit another absorption maximum at about $230\mathrm{nm}$ due to peroxidation of the fatty acid and a broad absorption at $300\mathrm{nm}$ due to decomposition products (e.g., aldehydes).²⁶ Auto-oxidation can be considered an omnipresent process, in particular in living cells that contain a sufficient amount of oxygen.

3.1.2.

Singlet oxygen luminescence

We measured the luminescence signal at $1270\mathrm{nm}$ for the seven fatty acids. In solutions with palmitic, stearic, and oleic acids, we detected a clear but weak signal, and the respective integrated luminescence signal was in the range from 1 to 4 arbitrary units (a.u.). This is well above the noise of the detector $\left(0.2\mathrm{a.u.}\right)$ . Despite the very low absorption coefficient of these fatty acids at $355\mathrm{nm}$ , the detection of singlet oxygen by its luminescence was feasible (Table 1).

The irradiation of solutions of linoleic, linolenic, arachidonic, or docosahexaenoic acids yielded a noticeable luminescence signal at $1270\mathrm{nm}$ , which is shown for arachidonic acid in Fig. 1 . The decay time of the luminescence signal was $14\pm 2\mu \mathrm{s}$ , which is equivalent to the decay time of singlet oxygen in ethanol.²⁷ These results are astonishing because fatty acids should not act as a photosensitizer due to the following reasons. First, a typical photosensitizer absorbs light and populates its triplet state. Then the energy is transferred from the triplet state to oxygen, leading to the generation of singlet oxygen (type 2 mechanism). However, the fatty acids and their oxidative derivatives are rather linear molecules, and the transition to such a triplet state is not very likely. Second, in the case of a type 2 mechanism, the luminescence signal contains the triplet state deactivation of the photosensitizer (usually the rising part of the signal) and the singlet oxygen luminescence (usually the decaying part of the signal), and both instances are on a microsecond time scale.⁹ However, Fig. 1 shows a luminescence signal with an extremely fast rise time $-$ less than a microsecond. For comparison, a luminescence signal of singlet oxygen is included that shows a typical shape for an exogenous photosensitizer. It shows a rising part of the signal and a clear maximum before decaying. In the case of fatty acids, we did not assume an energy transfer from a photosensitizer (e.g., fatty acid), but instead the involvement of a fast chemical process such as radical formation by charge transfer (type 1 mechanism). The latter was recently proven when phospholipids were investigated under UVA irradiation at $355\mathrm{nm}$ .²⁰

Fig. 1

Time-resolved singlet oxygen luminescence. The time-resolved luminescence signal of singlet oxygen at $1270\mathrm{nm}$ for arachidonic acid in ethanol showing a decay time of $14\pm 2\mu \mathrm{s}$ . For comparison, the insert shows a typical luminescence signal of singlet oxygen that has been generated by a photosensitizer via energy transfer from its triplet state (Riboflavin, ${\mathrm{H}}_2\mathrm{O}$ , excited with $355\text{-}\mathrm{nm}$ light).⁹

To double check singlet oxygen generation in all fatty acids, the luminescence signal was detected at different wavelengths in the range from $1150\text{to}1400\mathrm{nm}$ . Figure 2 shows a characteristic spectrally resolved curve of arachidonic acid that reveals a clear maximum at $1270\mathrm{nm}$ . This corresponds to the transition of singlet oxygen at the energy of $0.98\mathrm{eV}$ (Fig. 2).

Fig. 2

Spectrally resolved singlet oxygen luminescence. The luminescence signal measured at different wavelengths in the range from $1150\text{to}1400\mathrm{nm}$ . There is a clear maximum at $1270\mathrm{nm}$ that is equivalent to a singlet oxygen transition to its ground state with an energy difference of $0.98\mathrm{eV}$ .

3.2.1.

Light absorption at $355\mathrm{nm}$

In a solution of $20\mathrm{mM}$ normal egg yolk phosphatidylcholine $\left(\mathrm{L}\alpha \text{-}\mathrm{PC}\right)$ in EtOH, 14.6% of the radiation at $355\mathrm{nm}$ was absorbed, whereas 18.7% was absorbed in a solution of $20\mathrm{mM}$ sphingomyelin (SM) in EtOH (Table 2 ). Out of the three different phosphatidylcholines in solution, only 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (LI-PC) showed a clear absorption (13.4%) at $355\mathrm{nm}$ . The lipids 1,2-distearoyl-sn-glycero-3-phophocholine (ST-PC) and 1,2-dioleoyl-sn-glycero-3-phophocholine (OL-PC) absorbed less than 2% each, which is comparable to the low absorption of stearic or oleic solutions.

Table 2

Singlet oxygen produced by phospholipids.

Similar to fatty acid solutions, the fatty acids in phospholipids can undergo auto-oxidation, producing oxidative products in the solution. This has been also shown for phospholipids containing linoleic acid.²⁶

3.2.2.

Singlet oxygen luminescence

Singlet oxygen generation in aqueous or ethanol solutions of egg yolk $\mathrm{L}\alpha \text{-}\mathrm{PC}$ was recently shown.²⁰ When comparing $\mathrm{L}\alpha \text{-}\mathrm{PC}$ and sphingomyelin, $\mathrm{L}\alpha \text{-}\mathrm{PC}$ yielded twice the signal in sphingomyelin. Interestingly, the integrated luminescence signals (251 or $505\mathrm{a.u.}$ ) were significantly higher than that of the linolenic acid solution, which showed the highest luminescence signal of fatty acids (see Table 1).

3.2.3.

$\mathrm{L}\alpha \text{-}\mathrm{PC}$ and sphingomyelin

In $\mathrm{L}\alpha \text{-}\mathrm{PC}$ , there should be only a small contribution of palmitic, stearic, or oleic acid (and its oxidative products) to either the absorption of $355\text{-}\mathrm{nm}$ radiation or to singlet oxygen generation (see Table 1). However, these molecules represent 77% of the fatty acids in $\mathrm{L}\alpha \text{-}\mathrm{PC}$ used in our experiments. In addition, in the linoleic acid solution, which counted for 15% of the constituents, there was no strong signal of singlet oxygen luminescence (Table 1). Only linolenic and arachidonic acid (less than 8% constituent) effectively contributed to the generation of singlet oxygen by this phospholipid. Based upon the results of the fatty acid solutions, the small fraction of fatty acids (8%) in $\mathrm{L}\alpha \text{-}\mathrm{PC}$ should yield about $14\mathrm{a.u.}$ for $\mathrm{L}\alpha \text{-}\mathrm{PC}$ . However, we measured $505\mathrm{a.u.}$ (Table 2), which is a factor 35 higher than expected. This includes the two fatty acids that are present in one lipid molecule.

Thus, the presence of linolenic and arachidonic acids and their oxidative products might be responsible for the absorption of $355\text{-}\mathrm{nm}$ radiation (Fig. 1), but their direct contribution to singlet oxygen generation in $\mathrm{L}\alpha \text{-}\mathrm{PC}$ should be minor. To interpret the increased efficacy of singlet oxygen generation by $\mathrm{L}\alpha \text{-}\mathrm{PC}$ , we assumed the following two mechanisms. First, different oxidative products were present in $\mathrm{L}\alpha \text{-}\mathrm{PC}$ than in the respective fatty acids in solution. Second, there was an unknown synergistic effect of the polar head of the lipid molecules, leading to an enhanced generation of superoxide anions. In either case, these radicals can generate singlet oxygen by chemical reactions, as proposed by Khan, ¹⁴ and we added a scheme for illustration (Fig. 3 ).

Fig. 3

Scheme of mechanisms. The scheme shows the possible mechanisms that take place during irradiation of a lipid or fatty acids with UVA. The light could be absorbed in fatty acids, in particular in its oxidized forms. Electrons are released from the molecules that lead to the formation of superoxide anions. These oxygen radicals can generate singlet oxygen.^{14, 20} The same mechanism also may occur in pure fatty acids, but would obviously be less effective. The singlet oxygen generated causes peroxidation of fatty acids and lipids (vicious circle). In addition, singlet oxygen could be generated by the Russell mechanism—that is, two peroxyl radicals $\left({\mathrm{LOO}}^{\mathbf{\cdot }}\right)$ would form a linear tetraoxide intermediate and decompose to the corresponding products alcohol, ketone, and singlet oxygen.¹

Lipidhydroperoxidation occurs during UVA irradiation as a common process. Despite their relative stability, lipid hydroperoxides can be further decomposed by generating fatty acid-peroxyl radicals and/or alkoxyl radicals, which are responsible for the propagation of lipid peroxidation and ultimately the generation of toxic compounds.²⁸ The decomposition of lipid hydroperoxides into peroxyl radicals has been shown to be a potential source of singlet oxygen in biological systems.^{1, 29} In 1957, Russell proposed a self-reaction mechanism of such peroxyl radicals involving the formation of a cyclic mechanism from a linear tetraoxide intermediate that decomposed to give different products as well as singlet oxygen.³⁰ Mendenhall and colleagues demonstrated that such a self-reaction formation of peroxyl radicals deriving from fatty acids generated predominantly singlet oxygen.³¹ Therefore, generated fatty acid peroxyl radicals participate in reactions that lead to the formation of lipid fragmentation compounds.

Consequently, UVA radiation starts to cause lipidperoxidation, and the resulting products generate singlet oxygen, as shown by our luminescence experiments. These additional singlet oxygen molecules in turn cause the peroxidation of lipids, and cycles of singlet oxygen generation arise (see scheme in Fig. 3). This process leads to an enhancement of UVA-induced lipid peroxidation that should clearly affect cellular membranes, and therefore skin cell integrity.

These considerations are supported by the following results. When the irradiation of a lipid solution was continued for about $60\min$ , the singlet oxygen signal did not disappear, although the oxygen concentration reached the value of zero after this time span (closed cuvette). In contrast to that, in protein solutions³² and bacteria,³³ oxygen molecules and singlet oxygen luminescence disappeared very rapidly during irradiation. When oxygen was removed from the closed cuvette by adding nitrogen gas prior to UV irradiation, the luminescence signal was zero (within the experimental accuracy). This again confirms that oxygen is required right from the start to generate singlet oxygen. During the short time of singlet oxygen detection $\left(20\mathrm{s}\right)$ , the oxygen concentration was constant.

The conditions for singlet oxygen generation in sphingomyelin solution were comparable to that of $\mathrm{L}\alpha \text{-}\mathrm{PC}$ . About 76% of the fatty acids were palmitic or stearic acid, which hardly absorb at $355\mathrm{nm}$ . The luminescence energy per mol was smaller than in $\mathrm{L}\alpha \text{-}\mathrm{PC}$ , which might be partially due to a smaller percentage of unsaturated fatty acids. This effect should be even more pronounced, since the absorption of $355\text{-}\mathrm{nm}$ radiation was higher in sphingomyelin (18.7% absorption) than in phosphatidylcholine (14.6% absorption).

3.2.4.

Other phosphatidylcholines

To prove the role of fatty acids and their oxidized products in singlet oxygen generation, three different phospholipids were investigated that contained only ST-PC, OL-PC, or LI-PC. The luminescence energy per mol was weak for ST-PC and OL-PC, but again clearly higher compared to the respective fatty acid in solution (Table 1). In the case of LI-PC, the signal was high $\left(526\mathrm{a.u.}\right)$ and at a level comparable to that of $\mathrm{L}\alpha \text{-}\mathrm{PC}$ $\left(505\mathrm{a.u.}\right)$ . The signal for LI-PC was about 14-fold higher than that of the free linoleic acid $\left(37\mathrm{a.u.}\right)$ . These results support the discussions above.