Raman Spectroscopy of Advanced Glycation/Lipoxidation Endproducts


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A characteristic of human ageing is the accumulation of senescent proteins in many tissues within the human body which lead to gradual degradation in bodily function.  Such age-related modifications are linked to side-reactions that occur during the very process that sustains our activity, metabolism. Metabolism is the process of converting glucose (sugar) and fat into energy and requires a suite of highly reactive intermediate chemicals. Most of these intermediates are detoxified or immediately passed on to the next link in the chain, but some escape from the metabolic cycle. In addition to glycation, polyunsaturated fatty acids are susceptible oxidation, thereby generating reactive free radical intermediates (this is why such oils should be stored in dark, cool places and rapidly thicken ischematic of human eye showing Bruch's membranef used for frying food – non-virgin olive oil is best for balancing heating and health). These free radicals can also be stimulated by the action of UV light. Outside the cycle the intermediates can react through a chemical process called the Maillard reaction with a wide range of target molecules. In the eye a protein complex (known as Bruch's membrane, Figure 1) that filters the fluid around the photosensitive cells of the retina is particularly sensitive to Maillard chemistry.  Because within the body the vast majority of these Maillard products originate from glycation and lipid peroxidation intermediates they are referred to as Advanced Glycation/Lipoxidation endproducts (AGEs/ALEs). The proteins of Bruch's membrane gradually accumulate AGE/ALEs that alter the structure and function of the membrane, leading to compromise of the photosensitive cells of the retina and loss of vision. The chemistry underlying this process is complex (Figure 2maillard chemistry in body), with several hundred different possible modifications and a vast array of targets such as collagens and haemoglobin. 

I have studied the Maillard reaction products in the eye as they are believed to be implicated in the genesis and progression of the most common causes of irreversible vision loss in the western world. Age-related macular degeneration accounts for the majority of this vision loss in people of retirement age, while diabetic retinopathy accounts for the majority of this vision loss in people of working age. The reason the AGE/ALE chemistry is so relevant to ocular pathology is due to the inherent biochemistry of vision. Vision requires a high metabolic turnover to continuously recycle photosensitive pigment after detecting each photon of light – generating plenty of glycation intermediates. As if this was not enough, the very act of detecting light is perilous as the membranes of the photoreceptors are largely composed of dodecahexenoic acid, a fatty acid with 6 unsaturated bonds, which are liable to light-induced formation of free radicals. Thus the retina and its surrounding tissues are subjected to a very high flux of these reactive intermediates.

Bruch’s membrane integrity is essential to normal function of the retina by stabilizing the RPE and underlying choriocapillaris and regulating diffusion between these tissues. This complex extracellular matrix thickens during aging and there is profound remodelling of its constituent proteins and deposition of neutral lipids resulting in a net reduction in hydraulic conductivity (and hence increase in hydraulic resistivity) and charge selectivity (1-3).  Such alterations have implications for the RPE which relies on Bruch’s membrane for pro-survival cues and to allow effective removal of sub-cellular deposits. During aging, RPE density reduces and the surviving RPE show decreased melanin content and altered photoreceptor outer segment degradative capacity which is critical for age-related pathology (4, 5). 

Due to the complexity of the Maillard chemistry described to above, quantifying AGE/ALEs usually requires a battery of invasive extractions and immunological and/or analytical analyses. By contrast, Raman spectroscopy is an information dense technique, simultaneously yielding considerable chemical and structural information in a single measurement. Relative concentrations of molecular constituents in a sample can be directly compared and there are no concerns about sampling equivalence or extraction efficiencies that may arise using some invasive approaches. The Raman approach is ideally suited for investigating biological tissue and, in the eye, it has been employed to analyse carotenoid content of the macula (6), assess the histochemistry of retina (DHA, monounsaturated fats, 4 proteins, DNA, heme, cytochrome c and Raman spectra of advanced glycation lipoxidation endproducts AGE ALEkynurenine) (7, 8), detect ocular drugs (9) and assess lens structure (10, 11)


Figure 3 shows the Raman signals obtained from a number of AGE and ALE adducts (for more see references (12, 13)). It is clear that each adduct gives a unique signal, or ‘fingerprint’, that allows Raman spectroscopic methods to reliably identify several of these adducts within complex mixtures. Indeed several of these AGE/ALE patterns were observed in human tissue including CML, CEL, G-H1, GO, DHP-lys, pentosidine, AAA HHE and CEP, with several confirmed by chromatographic and immunostaining techniques(14).  In addition to the adducts the Raman method was able to simultaneously capture information on a considerable range of other biochemicals including several peroxidation intermediates of DHA, monounstaurated fatty acids, cholesterol, collagens 1,3 and 4, elastin, heme, cytochrome C and a number of aromatic peptides. Figure 4 shows the trends of each identified signal with age. raman spectroscopy trends  chronological age advanced glycation AGE ALE heme fat cholesterol

The proteins exhibit a mixture of age-related behaviour, with collagen 1 and alpha crystallin increasing while collagens 3 and 4 decrease as does elastin.  The heme is present a  a relatively high level in younger  donors, but this level  does continue to increase with age.  In terms of lipids no siginificant change was observed  in the levels  of monounstaurated fatty acids, while cholesterol, oxidised DHA and the endproduct of DHA oxidation (CEP) all increased significantly with age. All the identified AGE/ALE signals increased with chronological age. The most noticeable consistency is that all the changes accelerate between the age of 70 and 80, and this is the age at which incidence of age-related macular degeneration rises sharply.

Because Raman spectroscopy is a point-of-focus technique it is possible to build up a spectral image of the sample by recording spectra in a grid of points. Each signal is then processed and analysed using the multivariate models for the biochemical composition. If the predicted concentrations of each biochemical are then plotted in a grid we get a pseudo-color image of that particular biochemical’s distribution in the sample. Figure 5 show the overlay of two biochemical signal distributions, namely oxidized DHA which is red and the ALE called CEP (carboxy ethyl pyrrole) which is green. Where the red and green overlap a yellow colour is generated. There are a number of red hotspots in the images, corresponding to areas where only oxidized DHA is significantly accumulated and a number of yellow hotspots where both oxidized DHA and CEP co-accumulate. Interestingly in the younger donors the proportion of hotspots is heavily weighted towards red (oxDHA), becomes more equal in the middle donor and is predominantly yellow in the elderly donor, with one hotspot becoming green in this donor. It is readily apparent that the CEP hotspots only occur where there are DHA hotspots, which is not surprising since CEP can only be made by oxidation of DHA, so is a precursor. This also explains why the red hotspots precede the yellow and why it is only the oldest donor show any green hotspots. This gives direct visualization of the biochemical process of deposition of oxidized DHA followed by conversion to CEP over time.

Raman spectroscopy advanced glycation CEP carboxy ethyl pyrrole DHA docosenoic acid

All of the results in Figure 4 have been independantly validated by a wide range of other techniques, but using Raman spectroscopy we have for the first time been able to simulatneously examine all these biochemicals using a single measurement which allows clearer interpretation of the interrelationship of each component. There is, however, one excepetion. The trend of heme with age has never before been reported, though the Raman based detection of this protein in the Bruch's membrane has been backed up by Prof Tezel's immunostaining studies. By capturing all the information within a sample, sa Raman spectroscopy does, it is possible to detect biochemicals that were not expected and to discover upon analysis that these unexpected molecules have a potentially key role.

Such an example would be heme. Figure 6 shows the Raman distribution maps of heme and a number of different AGE/ALEs. The first noticeable feature is that the heme appears to have a uniform ‘flat-field’ distribution that is consistent between the age groups, but also displays an increase in hotspots where the heme becomes even more concentrated. This explains the high level of heme in the younger donors and also why it continues to increase with age.

In terms of the behaviour of the AGE/ALEs it is a very mixed bag. It is Raman spectroscopy heme haemoglobin advanced glycation lipoxidation endproducts CEP G-H1 CML DHP-lysclear that CML accumulates where there are gaps within the heme as there is no significant areas of yellow in the first row of Fig 6. The CML accumulates in defined spots, which grow in number and in size with age. It is known that CML disrupts the porphyrin group of the heme protein, which is the very part of it that gives the Raman signal of heme its strongest features. Hence, what appears to be happening with CML is that it reacts with the heme, disrupting its porphyrin group, releasing its iron ion and extinguishing its Raman signal. In contrast the G-H1 appears to strongly co-localise with the heme hotspots, suggesting that heme and this adduct also have some affinity, but the changes induced by G-H1 are remote from the porphyrin group and not affecting its Raman signal. The DHP-lys adduct show no consistent association with heme, with yellow red and green spots all evident. Interestingly, the adducts that showed an association with heme were those that contributed the most intensity to the Raman signals, suggesting that the association is encouraging their deposition. The Raman results suggest a critical role for heme in the deposition of the major Age/ALE adducts. This makes understanding the role heme in the human eye critical in understanding the aging of the human eye and the related dysfunction of the retina.

 

References 

1.           

1.            Starita, C., Hussain, A. A., Pagliarini, S., and Marshall, J. (1996) Hydrodynamics of ageing Bruch's membrane: implications for macular disease. Experimental eye research 62, 565-572

2.            Binder, S., Stanzel, B. V., Krebs, I., and Glittenberg, C. (2007) Transplantation of the RPE in AMD. Prog Retin Eye Res 26, 516-554

3.            Ethier, C. R., Johnson, M., and Ruberti, J. (2004) Ocular biomechanics and biotransport. Annual Review of Biomedical Engineering 6, 249-273

4.            Ambati, J., Ambati, B. K., Yoo, S. H., Ianchulev, S., and Adamis, A. P. (2003) Age-related macular degeneration: etiology, pathogenesis, and therapeutic strategies. Surv Ophthalmol 48, 257-293

5.            Boulton, M., Roanowska, M., and Wess, T. (2004) Ageing of the retinal pigment epithelium: implications for transplantation. Graefes Arch Clin Exp Ophthalmol 242, 76-84

6.            Bernstein, P. S., Zhao, D. Y., Wintch, S. W., Ermakov, I. V., McClane, R. W., and Gellermann, W. (2002) Resonance Raman measurement of macular carotenoids in normal subjects and in age-related macular degeneration patients. Ophthalmology 109, 1780-1787

7.            Beattie, J. R., Brockbank, S., McGarvey, J. J., and Curry, W. J. (2005) Effect of excitation wavelength on the Raman spectroscopy of the porcine photoreceptor layer from the area centralis. Molecular Vision 11, 825-832

8.            Beattie, J. R., Brockbank, S., McGarvey, J. J., and Curry, W. J. (2007) Raman microscopy of porcine inner retinal layers from the area centralis. Molecular Vision 13, 1106-1113

9.            Sideroudi, T. I., Pharmakakis, N. M., Papatheodorou, G. N., and Voyiatzis, G. A. (2006) Non-invasive detection of antibiotics and physiological substances in the aqueous humor by Raman spectroscopy. Lasers in Surgery and Medicine 38, 695-703

10.          Shih, S., Weng, Y. M., Chen, S. L., Huang, S. L., Huang, C. H., and Chen, W. L. (2003) FT-Raman spectroscopic investigation of lens proteins of tilapia treated with dietary vitamin E. Archives of Biochemistry and Biophysics 420, 79-86

11.          Borchman, D., Ozaki, Y., Lamba, O. P., Byrdwell, W. C., Czarnecki, M. A., and Yappert, M. C. (1995) Structural Characterization of Clear Human Lens Lipid-Membranes by near-Infrared Fourier-Transform Raman-Spectroscopy. Current Eye Research 14, 511-515

12.          Beattie, J. R., Pawlak, A. M., Boulton, M. E., Zhang, J., Monnier, V. M., Stitt, A. W., and McGarvey, J. J. (2010) Multiplex analysis of age-related protein and lipid modifications in human Bruch’s membrane. Faseb J. 24, doi:10.1096/fj.1010-166090

13.          Pawlak, A. M., Beattie, J. R., Glenn, J. V., Stitt, A. W., and McGarvey, J. J. (2008) Raman spectroscopy of advanced glycation end products (AGEs), possible markers for progressive retinal dysfunction. J. Raman Spectrosc. 39, 1635-1642

14.          Glenn, J. V., Beattie, J. R., Barrett, L., Frizzell, N., Thorpe, S. R., Boulton, M. E., McGarvey, J. J., and Stitt, A. W. (2007) Confocal Raman microscopy can quantify advanced glycation end product (AGE) modifications in Bruch's membrane leading to accurate, nondestructive prediction of ocular aging. Faseb J. 21, 3542-3552

15.          Tezel, T. H., Geng, L. J., Lato, E. B., Schaal, S., Liu, Y. Q., Dean, D., Klein, J. B., and Kaplan, H. J. (2009) Synthesis and Secretion of Hemoglobin by Retinal Pigment Epithelium. Investigative Ophthalmology & Visual Science 50, 1911-1919

   

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