The present paper has described that the measurement of FTIR spectra of human lip surface could detect the metabolism of dietary fatty acids, especially docosahexaenoic acid (DHA), and the change of lipid hydroperoxides in situ non-invasively. Normally the metabolism of dietary fatty acids has been measured so far using human blood cells and stable-isotope labeled fatty acids , and it was shown that dietary DHA in triglyceride was incorporated into very-low-density-lipoprotein (VLDL)-triglyceride within 2 hours, and very slowly (more than 60 hours) into red blood cell phosphatidylcholine . Moreover, dietary DHA in phosphatidylcholine was incorporated maximally into triglyceride of human plasma in 6 hours after digestion and negligible incorporation into cholesterol ester was observed. These data for human body were only obtained using blood (plasma or cells), and so far no data available for human peripheral tissues and other major organs.
We found as shown in this paper that the metabolism of dietary DHA or other fatty acids/lipid hydroperoxides could be measured non-invasively through human lip. As lip surface contains thinner stratum corneum (SC) in the vermilion zone (red zone) with less barrier functions [12, 13] than normal skin SC and the red color of lip is unique to human and comes from the blood vessels in the dermis, it may be expected that the penetration or transport of biomaterials (water, electrolytes, sugars, lipids and others) from blood vessels or reuptake from the lip surface to blood vessels is more rapid than other body skin. There is a report for the permeability of solutes to human skin layers [14–16]which showed that the permeability coefficient increased with increasing the lipophilicity.
However, we have no data available so far to estimate the penetration and diffusion coefficient of chemicals in situ for human lips. Generally human lip has 4 portions anatomically, (1)appendage-bearing epidermis, (2)keratinized vermilion (red zone), (3)parakeratinized intermediate zone, and (4)labial mucosal epithelium . In the present paper, we measured the lower lip, keratinized vermilion surface (outermost stratified corneum) by FTIR-ATR method, and this portion was suitable to measure the metabolism of polyunsaturated fatty acids originated from diet. This vermilion portion normally contains several layers of partly keratinized epithelia with horny, villous appearance , and partly transparent to see the red color of lip vessels.
As shown in this paper, the dietary fatty acids, such as DHA, could be transported from blood vessels to the lip (labial vermilion) surface. However, the appearance of dietary DHA on the face skin surface could not be detected by the same FTIR-ATR method (data not shown). Normal skin surface fatty acids may be originated mainly from sebum. Because it would take time to incorporate dietary lipids from blood and synthesize lipids in sebaceous glands of skin, it may be hard to detect the dietary DHA or other polyunsaturated fatty acids of blood on the skin surface. On the other hand, the lip vermilion surface lipids may not be from sebum, because sebaceous and sweat glands in the vermilion portion were rare histologically.
The transport or movement of dietary DHA from blood vessels to the lip surface was unexpectedly rapid for younger men as shown in Fig. 5 and within 2 hours we could detect the dietary DHA on the lip surface after the intake of DHA containing diet. On the other hand, it took 4 or 5 hours for older men to detect dietary DHA on the lip surface, and this difference of transport speed of DHA between younger and older men may not be explained simply by the structural difference of lip stratum corneum, such as the intercellular matrix or lamellar membranes. As shown in Fig. 5, the time-dependent DRI changes in the control diet were similar in both, younger and older, ages groups, and the increase of polyunsaturated fatty acids of lip surface, mainly arachidonic acid (C20:4) and linoleic acid (C18:2), could be detected at 3 to 5 hours after the intake of control diets.
This difference in transport rate between DHA and other fatty acids from blood vessels to lip surface may not be simply explained by a passive penetration mechanism of DHA, but we may have to assume the presence of specific transport mechanism of DHA. The transport of DHA from blood vessels to neonate is reported to be dependent on the expression of fatty acid-transport proteins in placenta  and that lipid carriers were involved in placental transfer of DHA. It was also reported that the requirement of DHA for brain development and the coincident expression of brain lipid-binding protein (BLBP) during developmental stages indicated the involvement of BLBP in the utilization of DHA in brain .
Although the role of such fatty acid-binding protein is not known in transport of lipids from blood vessels to lip, a facilitated transport mechanism of DHA in human lip may be present as shown in this paper and this transport mechanism may be different from that of other fatty acids, such as arachidonic acid. We demonstrated also that the transport of DHA to the lip vermilion surface was carried out mainly in the form of triglyceride (or less in diglyceride form). More detailed analyses may be needed using our newly developed FTIR-ATR apparatus.
Not only polyunsaturated fatty acids were transported to lip surface but also a significant amount of LOOH was detected on the surface. These lipid hydroperoxides were non-destructively measured in situ by FTIR-ATR, and the LOOH/TG ratios were increased just before noon in our experiments. The lip lipid hydroperoxides were probably triglyceride form mainly as shown in Fig. 9. We did not determine whether the lipid hydroperoxides were dietary-origin, or metabolized and circulating lipids-origin in a body, and whether the lipid hydroperoxides were formed just on the lip surface by exposure to oxygen or UV light or other environmental pollutants (ex., cigarette smoke). In any case we could not assume the lip lipid hydroperoxides were solely dietary origin, because the increase of lipid hydroperoxides was not observed in the afternoon even at 4 hours later after lunch. In both breakfast and lunch, these diets normally contained significant amount of linoleic acid (C18:2) even in bread or rice, a base diet. The drastic increase of LOOH/TG ratio just before noon on the lip may thus indicate a physiological phenomenon. As PUFA in triglyceride of lip surface may have a rhythm to be increased just before noon or 4 to 5 hours after breakfast under the normal diet as shown in Fig. 5 in this paper, this increase of PUFA on lip surface may be a part of causal phenomena for the increase of lipid hydroperoxides. However, the precise mechanism for appearance of lipid hydroperoxides on lip vermilion surface is not clear at present.
It has been known that lipid hydroperoxides on the normal skin like face forehead is derived from mainly cholesterol  and squalene [6, 7], and the skin squalene hydroperoxide was enhanced by illumination of ultraviolet light. Normally skin squalene hydroperoxide produced could be only detected by high sensitive methods such as chemiluminescence [20, 21] and mass spectrometric  methods. However, the amount of skin squalene hydroperoxide was very little and we could not detect it by the present DMPD staining method on TLC (data not shown) under the condition where lip lipid hydroperoxide was detected with extracting lipids using PVDF membrane and EPI solvents.