2.1.

Selection of Volunteers

Thirty women volunteers were selected as research subjects; they were further equally divided into three groups: (1) 10 elderly volunteers with type II diabetes mellitus (DEW) in the age group of 65 to 80 years; (2) 10 HYW in the age group of 20 to 33 years; and, (3) 10 HEW in the age group of 65 to 80 years.

2.2.

In Vivo Confocal Raman Spectroscopy

Skin evaluations were performed by using a confocal Raman system from Rivers Diagnostics^® (Model 3510 Skin Composition Analyzer) coupled to a laser of excitation wavelength 785 nm. The laser light was focused on the skin with a microscope objective ($40\times$) located under the quartz window. The power at the sample was measured as 20 mW. The Raman spectra were measured from 70 to $130\mu \mathrm{m}$ in depth to reach the skin dermis region, with an exposure time of 45 s and two accumulations.

2.3.

Data Treatment

The subsequent preprocessing (baseline correction, four points smoothing, and vector normalization) was carried out using the Labspec software from Horiba Jobin Yvon, Lille, France and the OMNIC software from Thermo Nicolet, Madison, Wisconsin. Second derivative in conjunction with band deconvolution analysis was used to assign hidden bands in specific regions of the Raman confocal spectra of all the volunteers.

2.4.

Computational Modeling

For an interpretative model that could represent an approximate extension of the dermis collagen content in elderly women, where water is present, we elected the computational model formed by an amino acid chain: glycine-hydroxyproline-proline-alanine with six water molecules around the structure, abbreviated as $\mathrm{Gly.HP.P.Ala.}6{\mathrm{H}}_2\mathrm{O}$. By this model, the hydrogen bond among water molecules is less extensive, and by this quantity, they form less hydrogen bonds among themselves. The system $\mathrm{Gly.HP.P.Ala.}6{\mathrm{H}}_2\mathrm{O}+\text{glucose}$ represents the reactional system and cannot be considered as an interpretative model of the DEW due to its higher polarizability. (The band profile intensity of the spectra in this case is higher than the correlative intensity for the Raman spectra of skin dermis of healthy young and elderly women.) Based on the fact that the glucose solubility in water is higher than the solubility of collagen in water and the decrease of collagen content due to the higher collagen degradation on the skin dermis in elderly diabetic women by the glycation process, $\mathrm{Gly.HP.P.Ala.}6{\mathrm{H}}_2\mathrm{O}$ represents one of the reagents and glucose represents the other. Therefore, we propose a chemical reaction of water interchange between the amino acids glycine, proline, hydroxyproline, and alanine with glucose. The proposed chemical reaction can be written as follows:

Calculations were also done for proline and hydroxyproline, in which the assignments consider the variation of the equilibrium of structural parameters (bond lengths and angle between bonding atoms) in the framework structure of both amino acids. All the molecules considered in the modeling process are independent noninteracting units. For geometry optimization, the density functional theory (DFT) with the functional B3LYP was used with the 3-21G basis set in Gaussian 03 program.¹⁶ All calculations have been optimized from several initial geometries to guarantee the global minimal energy structures. After this procedure, the vibrational calculations were performed and no imaginary mode was observed. Characteristic normal modes in the amide I and in the proline-hydroxyproline region were visualized using the graphical Chemcraft program.

3.1.

Experimental and Calculated Hydroxyproline and Proline Bands

In this section, bands in the higher energy region between 2500 and $3600{\mathrm{cm}}^{-1}$ are not considered because we are not dealing with protein, lipids, and total water content in the skin dermis region. We restricted our research to collagen concentration, which expressive bands become as a result of the presence of proline and hydroxyproline amino acids and are present in the 800 to $1000{\mathrm{cm}}^{-1}$ spectral region.

In our earlier paper, we discussed the vibrational assignment of these amino acids based on assignments of other authors and in a qualitative approach, indicating the principal internal coordinates (stretching and bending), which has higher variations in the normal modes of these two amino acids.¹⁸ In the region between 1000 and $800{\mathrm{cm}}^{-1}$, DFT calculations predict the presence of six bands for P and HP, in which the wavenumbers agree with previous assignments from literature, but in several cases, the vibrational assignments are doubtful.^19–21 For the vibrational assignment of proline, we selected eight stretching coordinates and ten bending internal coordinates and for hydroxyproline, 8 stretching and 11 in-plane bending internal coordinates. In these selections, the internal coordinates that are representative of higher energy vibrations were neglected. The further assignments are based on the percentage deviation of the geometric parameter (PDPG) analysis.^22–24 All the experimental Raman bands come from the deconvoluted spectra showed in Fig. 2. Results of wavenumbers from the DFT:B3LYP/3-21G procedure were scaled by 0.965. The symbol (%) indicates the percentage variation of the bond lengths and angles, and it is given between parentheses. Our assignments in this particular spectral region are presented in Table 1.

Table 1

Selected, calculated bands of proline (P) and hydroxyproline (HP) in the spectral region of 1000 to 800  cm−1. Experimental bands determined by band deconvolution analysis. Area band values in parentheses.

The description of the different normal modes of P and HP is explained in Table 1, where the participation of different internal coordinates in the normal modes is given in percentage values (%). The normal mode of proline at 959 (DEW), 957 (HEW), and $960(\mathrm{HYW}){\mathrm{cm}}^{-1}$ is found in 37%, 41%, and 22% of the Raman confocal spectra of HYW, HEW, and DEW, respectively, and has been interpreted as follows: for the HYW volunteers, 37% of contents represent the collagen concentration; 41% of contents are expressive and indicate the participation of the $─\mathrm{C}═\mathrm{O}$ group of the carboxylate of hydroxyproline forming hydrogen bond with water with a consequence of increase in polarizability. The same trend was found for the band under amide I normalization; 22% in DEW represent collagen degradation and loss of water by the presence of glucose. The normal mode found at 939 (HYW), 938 (HEW), and $938(\mathrm{DEW}){\mathrm{cm}}^{-1}$ participates with 45%, 36%, and 20%, respectively, which indicates that the decreasing collagen concentration.

For hydroxyproline, the observed Raman band at 921 (HYW), 921 (HEW), and $922(\mathrm{DEW}){\mathrm{cm}}^{-1}$ participates with 27%, 34%, and 39%, respectively, indicating increasing water content by hydrogen bond formation, and the assignment is given in Table 1. The calculated band at $905{\mathrm{cm}}^{-1}$ for proline and at $913{\mathrm{cm}}^{-1}$ for hydroxyproline, which was observed at 914 (HYW) 33%, 904 (HEW) 46%, and $905{\mathrm{cm}}^{-1}$ (DEW) 21%, is an indication of water content among the collagen fibers, principally in the case of HEW.

It also indicates the collagen degradation in the DEW with consequent loss of water by competitive interchange with glucose.

Raman bands at 890 (HYW), 892 (HEW), and $893{\mathrm{cm}}^{-1}$ (DEW) with 69.9 and 22% area show collagen degradation in the skin of the HEW and DEW volunteers.

The value of the band centered at $875{\mathrm{cm}}^{-1}$ is the assignment for hydroxyproline with area values of 30%, 41%, and 29% for the HYW, HEW, and DEW, respectively. The 41% reflects water contents in HEW and the 29% in DEW implies collagen fiber’s degradation and water contents. The band at $856{\mathrm{cm}}^{-1}$ (HYW), $855{\mathrm{cm}}^{-1}$ (HEW), and $854{\mathrm{cm}}^{-1}$ for (DEW) for the vector normalization participates with 39%, 33%, and 28%, respectively, can be assigned as the $\nu (\mathrm{CC})+\nu (\mathrm{CO})$ mode of proline. This band shows the collagen degradation of HEW and DEW. For proline, the calculated band at $819{\mathrm{cm}}^{-1}$ was correlated to the bands found at 821 (HYW), 822 (HEW), and $823(\mathrm{DEW}){\mathrm{cm}}^{-1}$. This normal mode is considered an indication of hydrogen bond formation with water due to the increased intensity of the bands of the HEW and DEW volunteers.

Bands at 813 (DEW), 814 (HEW), and $814(\mathrm{HYW}){\mathrm{cm}}^{-1}$, assigned to hydroxyproline with area values representing the 44%, 36%, and 20% for HYW, HEW, and DEW through vector normalization, agree well with the glycation process of proteins caused by hyperglycemia conducing the thermal denaturation of collagen. The integrated band areas for the HP/P spectral region for HYW, HEW, and DEW in the spectra were 43.12 (38%), 39.74 (35%), and 31.94 (28%), respectively, in [Raman Intensity $(\text{Arbitr. Units})\times {\mathrm{cm}}^{-1}$] units. The Maillard reaction, which describes the glycation product resulting from the reaction between the aldehyde function of glucose, ribose, or fructose with the amine free function of lysine or arginine of a protein, is well known²⁶ and can be summarized as: $\mathrm{HO}─{\mathrm{CH}}_2─{(\mathrm{HCOH})}_4\mathrm{CHO}+{\mathrm{H}}_2\mathrm{N}─(\text{Lys or Arg})\iff {\mathrm{H}}_2\mathrm{O}+\mathrm{HO}─{\mathrm{CH}}_2─{(\mathrm{HCOH})}_4\mathrm{CH}═\mathrm{N}─(\text{Lys or Arg})\iff \mathrm{HO}─{\mathrm{CH}}_2─{(\mathrm{HCOH})}_4{\mathrm{CH}}_2─\mathrm{NH}─(\text{Lys or Arg})\Rightarrow \text{protein}-AGE$ or protein cross-link product. Pentosidine formation comes from oxidation and a subsequential reaction with ribose and collagen bound arginine. The other glycation product is glucosepane, which is formed by a nonoxidative reaction with collagen bound arginine.¹⁵

3.2.

Glycine-Hydroxyproline-Proline-Alanine Systems

The other systems considered in our analysis to explain the experimentally observed trends in the Raman spectra of skin dermis of young, elderly, and elderly diabetic women are the following: $\text{glycine.hydroxy.proline.proline.alanine.}6{\mathrm{H}}_2\mathrm{O}$, abbreviated as GHPPAL, $\mathrm{GHPPAL.}2{\mathrm{H}}_2\mathrm{O}$; $\mathrm{GHPPAL.}6{\mathrm{H}}_2\mathrm{O}$; GHPPAl; glucose and $\text{glucose.}4{\mathrm{H}}_2\mathrm{O}$, which also represent reagents and products of the water ligand interchange of the reaction:

These systems are shown in Fig. 3.

Fig. 3

Geometrical structure of the systems: (a) and (b) $\mathrm{GHPPAla.}6{\mathrm{H}}_2\mathrm{O}$ and glucose as reagents, and (c) and (d) $\mathrm{GHPPAl.}2{\mathrm{H}}_2\mathrm{O}$ and $\text{glucose.}4{\mathrm{H}}_2\mathrm{O}$ as products of the reaction.

3.3.

Thermodynamic Considerations

We computed the heat of formation and the Gibb free energy at 298 K of $\mathrm{GHPPAl.}2{\mathrm{H}}_2\mathrm{O}$ and $\text{glucose.}4{\mathrm{H}}_2\mathrm{O}$ via an interchange of water as ligand. Corrected values by the zero point energy are given in Table 2.

Table 2

Thermodynamics of the systems G.HP.P.Ala.4H2O, Glucose, G.HP.P.Ala.2H2O, and Glucose.4H2O.

From the values given in Table 2, we concluded that the proposed reaction of water interchange between the amino acid chain and glucose is exothermic and spontaneous. Furthermore, we evaluated the dipole moment ($\mu$) and the polarizability ($\sigma$) of these systems. Table 3 presents the calculated DFT/B3LYP:3-21G values for the low triangle of the polarizability tensor $\sigma ij$. Dipole moments are in Debye units.

Table 3

Dipolar moments and polarizability tensor components of the glycine-hydroxyproline-proline-alanine (HYW), glycine-hydroxyproline-proline-alanine. 2H2O (DEW) and glycine-hydroxyproline-proline-alanine. 6H2O (HEW), calculated by DFT/B3LYP:3-21G procedures.

The DFT/B3LYP:3-21G calculations for the above systems give the same conclusions pointed out above for the HP.P. $\mathrm{HP.P.}4{\mathrm{H}}_2\mathrm{O}$ and $\mathrm{HP.P.}2{\mathrm{H}}_2\mathrm{O}$ systems described in an earlier paper.¹⁸ According to the polarizability values, in the Raman spectra of elderly women, the bands have high intensity compared with the Raman spectra of young women and with DEW. Moreover, the intensity of the bands for the elderly healthy women must be a little higher than the intensity of the Raman spectra of young women because the attached water content inside the collagen fiber is higher in the young participants, so the polarizability became higher and, as a consequence, the intensity increased.

3.4.

Vibrational Assignments

Calculated band assignments, Raman activities, and relative Raman intensities for these systems are given in Table 4. Relative Raman intensities were calculated according to the following equation:

Table 4

Wavenumbers, Raman activities, relative Raman intensities, and assignments for the computational models of GHPPAla, GHPPAla.6H2O, and GHPPAla.2H2O in the 1000 to 800  cm−1 and in the 1500 to 1720  cm−1 spectral region.

As we pointed out, the experimental ratio $938:922{\mathrm{cm}}^{-1}$ was considered a marker of hydration.⁶ The experimental and calculated values of this ratio have the following values for HYW, HEW, and DEW: $1.48\pm 0.09$, $1.60\pm 0.13$, and $1.46\pm 0.07$ (experimental values); 0.48, 0.51, and 0.34 considering Raman activities, and 0.45, 0.47, and 0.33 considering relative Raman intensities (calculated values considering the Raman activities and the relative Raman intensities), respectively. The experimental ratio for the elderly women increases compared with the young and diabetic women, and the ratio of the diabetic women is a little under the ratio of young and elderly women. The same trends are observed in the calculated values.

3.5.

Amide I Band

Assignments of the vibrational spectrum for the band amide I were found in different publications. Movasaghi et al.²¹ reported in a review article a long list of wave numbers from 415 to $3550{\mathrm{cm}}^{-1}$ of the Raman spectra of biological tissues. Although, the Movasaghi information is valuable, their wave numbers do not express the confidence interval of spectroscopic measurement. Another interesting review is the work of Barth on the infrared absorption of amino acid side chains.²⁸ Because the amino acids are molecules of lower (or no) symmetry, Barth quoted values must be coincident in the Raman spectra of the amino acids. In one of our recent works on the confocal Raman spectra of skin dermis, we summarized vibrational assignments of different authors for amide I bands.¹⁸ According to Nguyen et al.², the amide I band assigned to collagen I is centered at $1668{\mathrm{cm}}^{-1}$ in the Raman spectra of the skin.

In Nguyen et al.’s paper, no further assignments in the skin dermis were done for the amide I spectral region. The amino acid content in collagen is variable. According to Goisses,¹⁷ the main residual components of the alpha-1 chain are glycine with a value of 345, 114 for HP, 127 residues for proline, and 127 residues for alanine; the alpha-2 chain has the values of 346, 99, 108, and 111 residues for the same amino acids described above. The band for the vibrational mode $\nu (\mathrm{C}═\mathrm{O})$ of the carbonyl in these amino acids should in principle vary in wave number position and in intensity, according to the number of residues presenting in collagen content. In the region of amide I in the Raman spectra of HEW’s skin, the observed shift to lower wave numbers is due to the hydrogen formation between the amino acid and water. Moreover, for the DEW, it is due to the hydrogen bond formation between the amino acid chains of collagen with residual water and glucose, principally through the oxydrilic groups.

In our model systems composed by the four main amino acids interacting with water molecules through hydrogen bonds, the calculated wave numbers come from the B3LYP procedure using a 3-21G basis set, and the results are listed in Table 4. Experimental wavenumbers obtained by band deconvolution analysis are given in Table 1. Figure 1 shows the Gaussian fitting of amide I profile bands for the confocal Raman spectra of HYW, HEW, and DEW. The band found at 1613 (DEW), 1609 (HEW), and $1608(\mathrm{HYW}){\mathrm{cm}}^{-1}$ has the following assignments: according to Venyaminov and Kalnin²⁹, these bands correspond to the $\delta ({\mathrm{NH}}_2)$ of asparagine or glutamine. Movasaghi et al.²¹ assigned these bands to the same $\delta ({\mathrm{NH}}_2)$ mode as cytosine. According to Huang et al.,³⁰ the amide I bands found at 1638 (DEW), 1636 (HEW), and $1635{\mathrm{cm}}^{-1}$ (HYW) can be assigned to a vibrational mode pertaining to the ${\nu }_{\mathrm{s}}({\mathrm{CN}}_3{\mathrm{H}}_5^{+})$ of arginine.²⁸

Agreeing with the wave numbers where the vibrational mode occurs, we think that the participation of the ($\mathrm{C}─\mathrm{N}$) internal coordinate must be of little extension and two conjugated $\nu (\mathrm{C}::\mathrm{N})$ internal coordinates because the protonation has higher participation in the description of the symmetrical stretching mode. The preferable nomenclature to describe this mode is $\nu s$ $(\mathrm{C}::::\mathrm{N})+\nu (\mathrm{C}─\mathrm{N})+\delta ({\mathrm{NH}}_2)+\delta (\mathrm{NH})$ because in the same region the bending $\delta ({\mathrm{NH}}_2)$ and $\delta (\mathrm{NH})$ participate. Amide I bands found at 1656 (DEW), 1657 (HEW), and $1665{\mathrm{cm}}^{-1}$ (HYW) can be assigned to the collagen content in the reticular dermis of all the 30 volunteers without age discrimination.

These assignments are coincident with the proposal of Nguyem et al.,^2,12 amide I band found at 1671 (DEW), 1673 (HEW), and $1684{\mathrm{cm}}^{-1}$ (HYW) by the deconvolution process is assigned to the $\nu (\mathrm{C}═\mathrm{O})$ of alanine or $\beta$-turns by Stuart and Movasaghi et al.,²¹ but this last assignment is general for proteins. Deconvolution process by Gaussian fitting of the amide I band between 1720 and $1580{\mathrm{cm}}^{-1}$ also gives bands at 1699 (DEW), 1697 (HEW), and $1696{\mathrm{cm}}^{-1}$ (HYW). These bands are assigned to $\delta ({\mathrm{NH}}_2)$ of glycine. Integrated area for the amide I band measured in the range of 1720 to $1580{\mathrm{cm}}^{-1}$ has a value of 77% for the HYW, 27% being expressive due to the higher collagen quantity. It is well known that one of the factors of the triple-helical structure in type I collagen is the hydrogen bonds of terminal carbonyl groups of hydroxyproline and glycine in opposite sides of the triple-helical structure or on the same side of the chain structure, which is called interchain association.

The other possibility of binding water within the triple-helix structure is called interchain and occurs between opposite chains in the triple-helix conformation. The first case forms a ring of water interacting with the carbonyl groups of hydroxyproline and with the $─\mathrm{C}═\mathrm{O}$ group of glycine; in the second case, water molecules are binding, which forms a bridge between opposite sides of the triple-helix.¹

Nguyen et al.² pointed out ratios of integrate intensities, to evaluate water/collagen interactions. In our case, the selected quotients of experimental Raman intensity bands (in arbitrary units) are: $1649/1665{\mathrm{cm}}^{-1}1\Rightarrow 0.23/0.84=0.27(\mathrm{HYW})$, $1657/1678{\mathrm{cm}}^{-1}\Rightarrow 0.30/0.59=0.51$ for HEW, and $1656/1671{\mathrm{cm}}^{-1}\Rightarrow 0.29/0.65=0.45$ for DEW, giving the values of 22%, 41%, and 37% of water/collagen interactions. We can translate this observation and state that the HYW have inside the collagen triple-helix lower water contents, with higher water contents in DEW and HEW with comparable values. Such similar ratios for the three cases are the interaction Gly/HP, $13.369/4.4102=3.03$ in the calculated spectrum of the young woman volunteers, Ala/HP, $12.6874/5.7455=3.76$ in the calculated spectrum of the HEW, and the interaction P/Ala, $13.5756/4.658=2.91$ for the diabetic women. The glycine-hydroxyproline-proline-alanine model does not contain water and was considered to represent the young woman volunteers. Wavenumber shift to low energy region was also found in the calculated spectra of the Gly.HP.P.Ala (HYW), $\mathrm{Gly.HP.P.Ala.}6{\mathrm{H}}_2\mathrm{O}$ (HEW), and the $\mathrm{Gly.HP.P.Ala.}2{\mathrm{H}}_2\mathrm{O}$, representing the DEW. Stretching $\nu (\mathrm{C}═\mathrm{O})$ for proline goes from 1645 to $1618{\mathrm{cm}}^{-1}$; for hydroxyproline, it goes from 1611 to $1605{\mathrm{cm}}^{-1}$; for alanine the shift goes down from 1739 to $1675{\mathrm{cm}}^{-1}$; and for the amino acid glycine, the wave number shift is from 1587 to $1581{\mathrm{cm}}^{-1}$ between the spectra of HYW and DEW, respectively. For the Raman spectrum of HEW, the $\nu (\mathrm{C}═\mathrm{O})$ was assigned at $1548{\mathrm{cm}}^{-1}$.

In Table 5, we add the correlative experimental wave number in the corresponding column for comparison. With the exception of $\nu (\mathrm{C}═\mathrm{O})$ of alanine calculated at $1739{\mathrm{cm}}^{-1}$, all the $\nu (\mathrm{C}═\mathrm{O})$ stretching of the amino acids that form the three models can be correlated with the experimental values obtained by band deconvolution analysis. The following wavenumbers $1620{\mathrm{cm}}^{-1}$ (HYW), $1609{\mathrm{cm}}^{-1}$ (HEW), and $1589{\mathrm{cm}}^{-1}$ (DEW) from the calculated spectra follow the experimental trends observed in the profile of the spectra of amide I band: for the young women, the amide I band is located at the right side; for the elderly women, the amide I band is in the middle; and for the diabetic women, the $\nu (\mathrm{C}═\mathrm{O})$ bands appear at lower energy. Why are there delocalized bands when we compared the amide I band of young, elderly, and elderly diabetic women? The answer is that: the strength of the $\mathrm{C}═\mathrm{O}$ bonds of amino acids of collagen becomes weaker by charge transfer to form bond interactions with the hydrogen of water (H-bond), and in the case of the DEW, glucose can also form H-bonds with the conformational amino acids of the triple-helix. Then, when the $─\mathrm{C}═\mathrm{O}$ bond becomes weaker, its wavenumber decreases. The other reason to complete this argument is that: carboxylate group of hydroxyproline in the presence of water forms a carboxylate resonance group with delocalized bonds, which are weaker than the double bond and a little stronger than the single $\mathrm{C}─\mathrm{O}$ bond. This anionic structure can form H-bonds with water in monomeric form and with the hydronium ion. In the DEW, glucose is present not only in the tail of the collagen fibers but also in the interstitial space among the fibers of collagen, and it forms H-bonds through its hydroxyl groups or with its aldehyde terminal with chemical groups of the amino acids, which form the collagen fibers.

Table 5

Lycine-hydroxyproline-proline-alanine systems with water. Carbonyl stretching assignments. Experimental wave number values obtained by band deconvolution analysis (Gaussian fitting).