In vitro simultaneous measurement of refractive index and thickness of biological tissue by the low coherence interferometry.

We proposed and demonstrated in vitro simultaneous measurement of refractive index and thickness of biological tissue. The technique is based on the low coherence interferometry combined with precise translation stages. Refractive indices were determined with the accuracy of less than 1% for tissue samples of a few hundred micron thickness, including chicken tissue, human tooth and nail. Simultaneous measurement of refractive index and thickness of multilayer tissue are also demonstrated.

Determination of thiobarbituric acid-reactive substances in oxidized lipids by high-performance liquid chromatography with a postcolumn reaction system.

A new high-performance liquid chromatography procedure with a postcolumn reaction system for determination of free malondialdehyde (MDA) and other thiobarbituric acid-reactive substances (TBA-RS) in oxidized lipids in vitro has been developed. Using this procedure, both thermally oxidized methyl linoleate and the degradation products of methyl linoleate hydroperoxides revealed many kinds of lipophilic TBA-RS, but no free MDA was detected on the high-performance liquid chromatography. Similarly, oxidized methyl arachidonate also produced certain kinds of TBA-RS in the lipophilic phase and a small amount of free MDA in the hydrophilic phase. These results indicate that lipophilic TBA-RS produced in oxidized lipids in vitro are major TBA-RS and that the production of free MDA is small, even though the degree of lipid oxidation has previously been estimated as an MDA equivalent measured by the TBA colorimetric test.

Formation of fluorescent substances from degradation products of methyl linoleate hydroperoxides with amino compound.

The degradation products formed from methyl linoleate hydroperoxides by reaction with heme were fractionated by Sephadex LH-20 column chromatography and by reverse-phase high performance liquid chromatography, and the ability of each compound to form fluorescent substances through reaction with amino compound was compared. Maximum formation of fluorescent substances was obtained from monomeric degradation products with amino compound, but low molecular weight aldehydes such as hexanal, 2-hexenal and 2,4-decadienal, formed only a small amount of fluorescent substances. However, the major monomeric degradation products described previously, the hydroxy-, keto- and epoxy-derivatives, do not significantly contribute to the formation of fluorescent substances through reaction with amino compound. It was suggested that formation of fluorescent substances from lipid peroxides with amino compound may originate from a precursor present in monomeric degradation products formed from hydroperoxide of methyl linoleate during lipid peroxidation, and that low molecular weight aliphatic aldehydes are not involved in fluorescent substance formation. Moreover, the majority of TBA-reactive substances in secondary oxidation products prepared from autoxidized methyl linoleate are also unrelated to the formation of fluorescent substances through reaction with amino compound.

Hydrolysis of a fluorescent substance formed from an oxidized phospholipid and an amino compound by phospholipase A2.

Phosphatidylcholine hydroperoxide produced a fluorescent substance (FS-III) through reaction with 1-amino-pentane after preincubation with heme methyl ester as a model system. The FS-III was retained at the 2-position of the glycerol backbone of phosphatidylcholine without breakdown into low molecular weight compounds. Phosphatidylcholine oxidized by catalysis with ferrous ion and ascorbic acid also produced the same fluorescent substance (FS-III). Phospholipase A2 specifically hydrolyzed the FS-III attached to the phospholipid, making it possible to elute the same fluorescent substance (FS-II) as that obtained from oxidized methyl linoleate. The release of FS-II by hydrolysis of FS-III attached to phospholipid increased with greater phospholipase A2 activity. It is suggested that, with aging, the accumulation of fluorescent lipofuscin pigments in biomembranes may be related to changes in the peroxidized phospholipid content and that phospholipase A2 may play a role in decreasing the formation and accumulation of fluorescent phospholipids in biomembranes.

Fluorescence formation from hydroperoxide of phosphatidylcholine with amino compound.

The hydroperoxides of methyl linoleate, 1-palmitoyl-2-linoleoyl-phosphatidylcholine and trilinolein each produced similar fluorescent substances through reaction with amino compounds after decomposition by heme methyl ester. Fluorescent substances formed from methyl linoleate with 1-aminopentane revealed characteristic fluorescence peaks on HPLC, while those obtained from 1-palmitoyl-2-linoleoyl-phosphatidylcholine and trilinolein were not eluted under the same conditions. However, when both of these fluorescent substances were transesterified to methyl ester, the same fluorescence peaks were observed. This result suggests that fluorescent substances formed from oxidized membrane lipids with amino compounds remain attached to phospholipids without being released from their glycerol backbone.

Fluorescence formation and heme degradation at different stages of lipid peroxidation.

Secondary oxidative products of autoxidized methyl linoleate were divided into three groups (SP-I, SP-II and SP-III), which were then compared as to their abilities to form fluorescent substances and to degrade heme. SP-III showed a marked ability to produce two fluorescent substances exhibiting an excitation maximum at 350-360 nm and an emission maximum at 410-430 nm, while SP-I showed a more strongly degradative effect on heme than SP-III. The heme degradation was observed in parallel with the changes of TBA value in an early stage of lipid peroxidation and the fluorescence formation markedly increased according to the decrease of TBA value in a later stage. The results suggested that there are different reactive substances which bring about fluorescence formation and heme degradation and that they are produced at different stages of lipid peroxidation.

12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) is an excellent substrate for NAD+-dependent 15-hydroxyprostaglandin dehydrogenase.

12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) was found to be an excellent substrate for NAD+ dependent 15-hydroxyprostaglandin dehydrogenase from porcine kidney. Kcat/Km value of HHT was comparable to that of prostaglandin E although HHT is not a prostanoic acid derivative. Product of enzyme catalyzed oxidation of HHT was identified as 12-keto-5,8,10-heptadecatrienoic acid by gas chromatography-mass spectrometry. The fact that HHT is an excellent substrate for 15-hydroxyprostaglandin dehydrogenase suggest that HHT may have profound unrecognized biological actions and its inactivation may be via oxidation of the hydroxyl group.