Measurement of lipid oxidation and porphyrins in high oxygen modified atmosphere and vacuum-packed minced turkey and pork meat by fluorescence spectra and images.

This paper illustrates that fluorescence spectroscopy and imaging can be used to measure the extent and distribution of lipid oxidation in meat. Minced turkey thighs and pork semimembranosus muscles were stored for 7 and 12 days at 4°C in high oxygen (O(2)) modified atmosphere packages and vacuum. Turkey meat packed in high O(2) atmosphere was oxidised already after 7 days of storage. The sensory rancid odour score was 4.7 (on a scale from 1 to 9) and the TBARS value was 1.86mg MDA/kg. There was also an increase in fluorescence emission intensity in the 410-550nm region, which arises from lipid oxidation products. The combination of unsaturated fatty acids and access to O(2) resulted in lipid oxidation gradients in the turkey meat samples, and these gradients were clearly visualised by fluorescence images. In comparison, pork meat was more stable against lipid oxidation, with TBARS values <0.2mg MDA/kg and no development of fluorescent lipid oxidation products was detected. The fluorescence spectra measured in the present experiment suggest that turkey thighs and pork semimembranosus muscle in addition to protoporphyrin also have a natural content of Zn protoporphyrin. The porphyrin content was higher in pork meat than in turkey meat. It increased during storage time when the meat was packed in vacuum, and it decreased with O(2) availability. The distribution of porphyrins in the meat was visualised by fluorescence imaging.

Uptake of topically applied 5-aminolevulinic acid and production of protoporphyrin IX in normal mouse skin: dependence on skin temperature.

The temperature dependence of the uptake phase of 5-aminolevulinic acid (ALA) and the following production phase of protoporphyrin IX (PpIX) in normal mouse skin was investigated. A cream containing 20% ALA was topically applied on the skin for 10 min. The amount of ALA-induced PpIX was evaluated by measuring the fluorescence of PpIX from the treated skin. No measurable amount of PpIX was found in the skin immediately after 10 min application of ALA. The penetration of ALA into the skin was almost temperature independent while the following production of PpIX was found to be a strongly temperature-dependent process. Practically no PpIX was formed in the skin as long as skin temperature was kept low (12 degrees C).

Phototransformations of 5-aminolevulinic acid-induced protoporphyrin IX in vitro: a spectroscopic study.

Human adenocarcinoma cells of the line WiDr were incubated with 5-aminolevulinic acid to induce protoporphyrin IX (PpIX) and then exposed to laser light of wavelength 635 nm. The PpIX fluorescence decreased with increasing exposure. The decay rate was slightly dependent on the initial PpIX concentration. The PpIX fluorescence was halved by a fluence of about 40 J/cm2. Several fluorescing photoproducts were formed. The main one, supposedly the chlorine-type photoprotoporphyrin (Ppp), had a fluorescence excitation spectrum stretching out to about 680 nm with a maximum at around 668 nm. The formation kinetics of this product was dependent on the initial PpIX concentration. Moreover, it was selectively bleached by exposure to light at 670 nm. A photoproduct with an emission maximum at 652 nm, different from Ppp, remained after this exposure. Traces of a photoproduct(s) with fluorescence emission slightly blue-shifted compared with that of PpIX, supposedly water-soluble porphyrins, were also detected after light exposure.

The temperature dependence of protoporphyrin IX production in cells and tissues.

The formation of protoporphyrin IX (PpIX) in human skin during topical application of 5-aminolevulinic acid (ALA) was found to be strongly temperature dependent, with an activation energy of about 17 kcal/mol. This temperature dependence is mainly related to porphyrin production and not to ALA penetration into the skin. The penetration of ALA into mouse and human skin was almost temperature independent. The activation energy of PpIX production in mouse skin was practically identical with that in human skin. The activation energy of ALA uptake by cells in vitro was about 10 kcal/mol and that for PpIX production was about 13 kcal/mol. The latter activation energy was within the error limits similar to that for the activity of the enzyme porphobilinogen deaminase, suggesting that this enzyme might represent a rate-limiting step for PpIX production in living tissue.

The influence of UV exposure on 5-aminolevulinic acid-induced protoporphyrin IX production in skin.

The skin of nude mice was exposed to erythemogenic doses of UV radiation, which resulted in erythema with edema. An ointment containing 5-aminolevulinic acid (ALA) was topically applied on mouse and human skin. Differences in the kinetics of protoporphyrin accumulation were investigated in normal and UV-exposed skin. At 24 and 48 h after UV exposure, skin produced significantly less protoporphyrin IX (PpIX) than skin unexposed to UV. Human skin on body sites frequently exposed to solar radiation (the lower arm) also produced less PpIX than skin exposed more rarely to the sun (the upper arm). It is concluded that UV radiation introduces persisting changes in the skin, relevant to its capability of producing PpIX from ALA. The observed differences in ALA-induced PpIX fluorescence may be the result of altered penetration of ALA through the stratum corneum or altered metabolizing ability of normal and UV-exposed skin (or both).

Fluorescence spectroscopy of normal mouse skin exposed to 5-aminolaevulinic acid and red light.

Photobleaching and phototransformation of protoporphyrin IX (PpIX) was investigated in normal mouse skin. The PpIX was induced by topical application of 5-aminolaevulinic acid (ALA). Exposure to laser light (635 nm) caused photobleaching of PpIX fluorescence and formation of fluorescent products. Analysis of the fluorescence spectra revealed appearance of new fluorescent photoproducts during light exposure. The main photoproduct, supposedly chlorin-type photoprotoporphyrin (PPp), exhibited fluorescence with an emission maximum at 675 nm. The other products exhibited main fluorescence peaks at around 588 and 623 nm that can presumably be attributed to an endogenous metallo-porphyrin and water-soluble porphyrin(s), respectively. Our results indicate that light exposure causes alterations in the enzymatic pathway of PpIX synthesis from ALA and leads to accumulation of intermediate water-soluble porphyrins. ALA-induced porphyrins are transported away from the treated area and partly deposited in remote skin sites.

Topical application of 5-aminolaevulinic acid, methyl 5-aminolaevulinate and hexyl 5-aminolaevulinate on normal human skin.

BACKGROUND: 5-Aminolaevulinic acid (ALA) and its ester derivatives are used in photodynamic therapy. Despite extensive investigations, the differences in biodistribution and pharmacokinetics of protoporphyrin IX (PpIX) induced by ALA and its derivatives are still not well understood, notably for humans. OBJECTIVES: To study porphyrin accumulation after topical application of ALA and two of its ester derivatives in normal human skin. METHODS: Creams containing 0.2%, 2% and 20% (w/w) of ALA, methyl 5-aminolaevulinate (MAL) and hexyl 5-aminolaevulinate (HAL) were applied on normal human skin of six volunteers. The amount and distribution of porphyrins formed in the skin was investigated noninvasively by means of fluorescence spectroscopy. RESULTS: Fluorescence emission and excitation spectra exhibited similar spectral shapes for the all drugs, indicating that mainly PpIX was formed. Low concentrations (0.2% and 2%) of MAL induced considerably less PpIX in normal human skin than similar concentrations of ALA and HAL. A high concentration (20%) of ALA gave higher PpIX fluorescence in normal human skin than was found for MAL and HAL. CONCLUSIONS: The concentrations inducing half of the maximal PpIX fluorescence are around 2% for ALA, 8% for MAL and 1% for HAL.

Photosensitization with protoporphyrin IX inhibits attachment of cancer cells to a substratum.

Effects of photodynamic therapy (PDT) on adhesion of human adenocarcinoma cells of the line WiDr to a plastic substratum were investigated. Protoporphyrin IX induced by 5-aminolevulinic acid (ALA) was used as a photosensitizer. Light exposure inhibited attachment of suspended cells to a substratum. The adhesion was most strongly pronounced for light exposures around 200 mJ/cm(2) causing cell death. However, sub-lethal exposures (42 mJ/cm(2), 97% survival) inhibited cell adhesion as well. Sub-lethal ALA-PDT increased the intracellular space in dense colonies of WiDr cells. This was attributed to formation of lamellipodia between the cells and to increased numbers of focal contacts containing alpha(V)beta(3) integrin in some of the cells. The E-cadherin distribution was not changed by the treatment. Complex processes, including changes in cellular shape and reorganization of the cytoskeleton, are suggested to participate in the observed ALA-PDT effect on the cell adhesion.

Production of protoporphyrin IX from 5-aminolevulinic acid and two of its esters in cells in vitro and tissues in vivo.

5-Aminolevulinic acid (ALA) and two of its esters were studied in cells in vitro and in vivo on skin of healthy hairless mice. In vitro, both esters, which are more lipophilic than ALA, induced higher PpIX fluorescence at lower concentrations compared with ALA. In vivo, ALA induced PpIX fluorescence more efficiently than the esters. The difference between ALA and the esters may be related to structures in the stratum corneum or to rate of penetration through this skin layer. The stratum corneum may bind the esters temporarily, and slow down their penetration into the living cells where PpIX is formed.