**2.1** *Cis***-6-hexadecenoic acid (C16:1∆6)**

*Cis*-6-hexadecenoic acid, termed sapienic acid, is unique in the animal kingdom, being a 16 carbon mono-unsaturated fatty acid with a *cis*-double bond located at the sixth carbon from the carboxyl terminal (Fig. 1).

Fig. 1. Structure of *cis*-6-hexadecenoic acid (C16:1Δ6)

This fatty acid is an isomer of palmitoleic acid (*cis*-9-hexadecenoic acid (C16:1Δ9)), which is a common constituent of the glyceride of human adipose tissue. In addition, C16:1Δ6 is a major component of sebaceous wax esters, triacyl glycerols, and of free fatty acids found in skin and hair (Downing & Strauss, 1974; Morello & Downing 1976; Yamamoto et al., 1987). Sapienic acid represents 85% of the lipid in *Thunbergia alata* seed oil, and is obtained either by extraction of this plant or by chemical synthesis (Spencer et al., 1971).

#### **2.2 Relationship between human sebaceous fatty acid C16:1∆6 and AD**

The composition of free fatty acids in the sebum at the recovery level is shown in Figure 2 (Takigawa et al., 2005). The level of C16:1Δ6 in healthy control skin ranged from 0.43 to 5.38 μg/cm2 with a mean of 2.0 μg/cm2 (4.9 to 16.8% with a mean of 12.9% in percent of total)

Fig. 2. Amount of free fatty acid in sebum from AD patients (upper panel) and from healthy controls (lower panel). Sebum was analyzed at the recovery level.

*Cis*-6-hexadecenoic acid, termed sapienic acid, is unique in the animal kingdom, being a 16 carbon mono-unsaturated fatty acid with a *cis*-double bond located at the sixth carbon from

This fatty acid is an isomer of palmitoleic acid (*cis*-9-hexadecenoic acid (C16:1Δ9)), which is a common constituent of the glyceride of human adipose tissue. In addition, C16:1Δ6 is a major component of sebaceous wax esters, triacyl glycerols, and of free fatty acids found in skin and hair (Downing & Strauss, 1974; Morello & Downing 1976; Yamamoto et al., 1987). Sapienic acid represents 85% of the lipid in *Thunbergia alata* seed oil, and is obtained either

The composition of free fatty acids in the sebum at the recovery level is shown in Figure 2 (Takigawa et al., 2005). The level of C16:1Δ6 in healthy control skin ranged from 0.43 to 5.38 μg/cm2 with a mean of 2.0 μg/cm2 (4.9 to 16.8% with a mean of 12.9% in percent of total)

**12:0 13:0 14:0 14:1 15:0 15:1 16:0 16:1 17:0 17:1 18:0 18:1 others**

**12:0 13:0 14:0 14:1 15:0 15:1 16:0 16:1 17:0 17:1 18:0 18:1 others Chain structure**

**16:0** 

Fig. 2. Amount of free fatty acid in sebum from AD patients (upper panel) and from healthy

controls (lower panel). Sebum was analyzed at the recovery level.

by extraction of this plant or by chemical synthesis (Spencer et al., 1971).

**2.2 Relationship between human sebaceous fatty acid C16:1∆6 and AD** 

**OH**

**A AD patients**

**B Healthy controls**

**O C** =

**2.1** *Cis***-6-hexadecenoic acid (C16:1∆6)** 

Fig. 1. Structure of *cis*-6-hexadecenoic acid (C16:1Δ6)

JA02 JA03 JA05 JA06 JA09 JA10 JA11 JA12 JA13 JA14 mean

JA04 JO20 JO21 JO22 JO23 JO24 JO15 JO16 JO17 mean

**15**

**10**

**5**

**Amounts(**

**Amounts(**

**μg/cm2**

**)15**

**μg/cm2**

**)**

**0**

**10**

**5**

**0**

the carboxyl terminal (Fig. 1).

and the total level of C16:0 ranged from 0.88 to 7.64 μg/cm2 with a mean of 3.43 μg/cm2 (14.9 to 28.2% with a mean of 22.1% in percent of total). In contrast, the level of C16:1Δ6 (0.08 to 0.53 μg/cm2 with a mean of 0.26 μg/cm2) and the percent of total (1.1 to 8.7% with a mean of 3.0% in percent of total) in the skin of AD patients were lower than in the healthy controls, although the amounts of C16:0 (0.53 to 2.84 μg/cm2 with a mean of 1.44 μg/cm2) and the percent of total (6.8 to 30.7% with a mean of 16.5% in percent of total) were similar to those of the healthy controls. A significant decrease in free C16:1Δ6 content in nonlesional skin from AD patients compared with healthy controls was found.

### **2.3 Antibacterial activity of C16:1∆6: Selective antibacterial activity**

Colonization of *S. aureus* has been reported in AD patients and is regarded as an exacerbation factor (Akiyama et al., 1996). Antimicrobial agents, including C16:1Δ6, inhibited the growth of *S. aureus* at a final concentration of 100 mg/l in the following order; Benzalkonium chloride = Triclosan > C16:1Δ6 > Trichlorocarbanilide, while they inhibited the growth of *S. epidermidis* in the following order; Benzalkonium chloride = Triclosan> C16:1Δ6 = Trichlorocarbanilide (Fig. 3). Whereas C16:1Δ6 could inhibit the growth of *S. aureus* over a concentration range from 100 to 1000 ppm, it did not greatly inhibit *S. epidermidis*.

In addition, the antibacterial mechanism of C16:1Δ6 against *S. aureus* and *S. epidermidis* was investigated using protoplasts treated with lysostaphin. The results of this experiment indicated that there was no difference in the effectiveness of C16:1Δ6 against *S. aureus* and *S. epidermidis*, and suggested that the antibacterial action of C16:1Δ6 against Staphylococcal bacteria was caused by disruption of the cell membrane, not disruption of the cell wall, and that this disruption resulted in leakage of bacterial components (Data not shown).

Fig. 3. Comparison of the antimicrobial effects of C16:1Δ6 against *Staphylococcus aureus* ATCC 12600T (left panel) and *Staphylococcus epidermidis* JCM 2414T (right panel).

#### **2.4 Topical application of C16:1∆6 to AD patients**

An inverse correlation between *S. aureus* colonization and the level of C16:1Δ6 in AD patients was found (Fig. 4). In addition, the colonization of *S. aureus* was reduced by topical application of C16:1Δ6 to the skin of six to eight AD patients in a small-scale clinical study. Based on these investigations, it was considered that the decreased level of

Improvement of Atopic Dermatitis by Human Sebaceous Fatty Acids and Related Lipids 313

We found that the double mutant designated *Rhodococcus* sp. KSM-MT66 had the ability to desaturate various aliphatic compounds such as alkanes, chloro alkanes and fatty acid esters, and that this mutant strain produced unsaturated compounds extracellularly. When hexadecanoic acid esters such as methyl, propyl, isopropyl, and isobutylester were supplied as substrates to the resting cells, their corresponding *cis*-desaturated compounds were produced at a concentration of 0.5, 20, 53 and 7 g/l in 3 days. The enzyme(s) responsible for the desaturation reaction appears to recognize mainly the sixth carbon from the carbonyl carbons (Fig. 5). The bioproduction of C16:1Δ6 esters has not been previously reported (Koike et al., 1999). When alkanes were supplied to this mutant strain, the main products had a double bond at the ninth carbon from the terminal methyl group. These experiments indicated that the unsaturated position differed according to differences in the supplied

Fig. 5. The patterns of regiospecific desaturation of aliphatic substrates by *Rhodococcus* sp.

**3.2 Production of a C16:1∆6 ester by resting cells of** *Rhodococcus* **sp. KSM-MT66**  We determined the condition with resting cells of the *Rhodococcus* sp. KSM-MT66 strain to produce C16:1Δ6. The reaction mixture contained 20% (w/v) hexadecanoic acid isopropyl ester (IP-C16:0), 0.25 M phosphate buffer (pH 7.0), 1.0% (w/v) monosodium glutamate, 2

**3.1 Desaturation reaction of aliphatic substrates by a mutant strain of alkane-**

**assimilating** *Rhodococcus* **sp.** 

substrates (Koike et al., 2000a).

strain KSM-MT66 cells.

C16:1Δ6 in AD patients may be one of the factors that contribute to the colonization of *S. aureus* (Takigawa et al., 2005).

We have confirmed that C16:1Δ6 functions as a natural antibacterial component and has very unique properties including "selective antibacterial activity", in which it shows effective antibacterial activity against transient *S. aureus* but not against residential *S. epidermidis*. By topical application of this fatty acid to AD patients, the colonization of *S. aureus,* which is believed to be an exacerbation factor for this disease, was repressed, thereby indicating the effectiveness of this fatty acid. From the results of these investigations, a new approach for improvement of skin disorders, based on normalization of the microflora on human skin, was proposed using the selective antibacterial activity of C16:1Δ6.
