Oxidative stress, fluorescent assays

Also, HPLC methods with electrochemical or fluorescent detection are used (H19, M3). In proteins, dityrosine can be estimated by immunochemical methods employing dityrosine-specific antibodies (K5). Measurements of o,o -dityrosine and o-tyrosine levels in rat urine express dityrosine contents in skeletal muscle proteins, and have been proposed as the noninvasive oxidative stress test in vivo. One should be aware, however, that A-formylkynurenine, also formed in protein oxidation, has similar fluorescence properties as dityrosine (excitation 325 nm, emission at 400-450 nm) (G29). Also, oxidation of mellitin when excited at 325 nm produces an increase in fluorescence at 400—450 nm, despite the fact that mellitin does not contain tyrosine. Oxidation of noncontaining Trp residues ribonuclease A and bovine pancreatic trypsin inhibitor with "OH produces loss of tyrosine residues with no increase in fluorescence at 410 nm (S51). There are also methods measuring the increased hydrophobicity of oxidized proteins. Assays are carried out measuring protein binding of a fluorescent probe, 8-anilino-l-naphthalene-sulfonic acid (ANS). Increase in probe binding reflects increased surface hydrophobicity (C7). 

Frolick Olson describe the conventional methods of extracting retinoids from biological samples236. They stress the delicacy of the retinoids and describe two important points to consider, (1) whether the method chosen will result in complete extraction of the retinoid of interest, and (2) whether it will contribute to the production of artifacts. Unfortunately, they do not consider whether the product itself could be degraded. They do stress the sensitivity of these labile compounds to both hydrolysis and oxidation. They also address the use of fluorescence in assays. This work points out that the use of fluorescence is a negative test for the retinoids of vision when in liquid crystalline form. They point out that mass spectrometry is one of the most specific forms of assay for the retinoids. The utility of this technique will be discussed in Chapter 6. They do not describe how the techniques mentioned are able to remove a retinoid from within the putative rhodopsin molecule, or how to do in without damage to the retinoid. 

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