Rat Skin Surface Research Articles

Characteristics of the New Mast Cell-Rich Nodal Structure in the Rat Skin Surface.

: Acupuncture, practiced for millennia, lacks a clear anatomical definition for acupoints. A prevailing theory suggests that acupoints overlap with skin areas with higher mast cell density. Skin spots stained with intravenously infused Evans blue (EB), indicative of neurogenic inflammation, have recently been posited as acupoints in rats. : To demonstrate the concordance between EB-reactive skin spots and mast cell-enriched acupoints. : We employed staining and RNA-seq analysis to delineate the morphological characteristics and gene expression profiles of EB-reactive skin spots in rats. : EB infusion revealed a novel nodal structure on the rat skin surface, visible to the naked eye, with dimensions of approximately 1 mm in both diameter and height. Around 30 such nodes were identified on one side of the abdominal area, spaced roughly 3 mm apart, excluding the linea alba. RNA-seq analysis indicated that the gene expression patterns within these nodes markedly differed from both non-nodal skin areas and lymph nodes. Histological examination using toluidine blue revealed a significantly greater mast cell count in the nodes than in non-nodal skin regions. Additionally, the nodes stained positively with Alcian blue and Hemacolor, reagents known to mark primo vascular tissues. : Our findings suggest that EB-reactive nodes are indeed rich in mast cells. Further research is warranted to establish these skin nodes as surface primo nodes.

Polysulfonic acid mucopolysaccharide exerts anti scarring effect in rats through modulation of TGF β 1/Smad signaling pathway

Purpose: To determine the anti-scarring effect of polysulfonic acid mucopolysaccharide (MSP), and the implication of TGF-β1/Smad signal transduction route.Methods: Sixty (60) male Sprague Dawley (SD) rats were assigned to control, model and polysulfonic mucopolysaccharide groups, respectively, each with 20 rats. Serum inflammatory factors, scar area and scar thickness, histopathological changes and relative concentrations of TGF-β1 Smad4, collagen types I and III, and α-SMA were determined.Results: In the control group, collagen cells were closely distributed and the skin structure was intact without inflammatory infiltration. In contrast, there were numerous necrotic dermal cells on rat skin surface in model group, with obvious inflammatory infiltration and severely damaged hair follicles. In contrast, in polysulfonic mucopolysaccharide group, the thickness of skin tissue and dermis was significantly improved, with a clear layer and reduced degree of inflammatory infiltration. Types I and III collagen and α-SMA were significantly down-regulated in polysulfonic mucopolysaccharide-fed rats, relative to model rats.Conclusion: Polysulfonic acid mucopolysaccharide exerts anti-scarring effect by regulating TGFβ1/Smad signal pathway, thus has the potential for use in minimizing scarring of the skin in clinical practice.

Rat Burn Model to Study Full-Thickness Cutaneous Thermal Burn and Infection.

Burn induction methodologies are inconsistently described in rat models. A uniform burn wound model, which represents the clinical scenario, is necessary to perform reproducible burn research. The present protocol describes a simple and reproducible method to create ~20% total body surface area (TBSA) full-thickness burns in rats. Here, a 22.89 cm2 (5.4 cm diameter) copper rod heated at 97°C in a water bath was applied to the rat skin surface to induce the burn injury. A copper rod with a high thermal conductivity was able to dissipate the heat deeper in the skin tissue to create a full-thickness burn. Histology analysis shows attenuated epidermis with coagulative damage to the full-thickness extent of the dermis and the subcutaneous tissue. Additionally, this model is representative of the clinical situations observed in hospitalized burn patients following burn injury such as immune dysregulation and bacterial infections. The model can recapitulate the systemic bacterial infection by both Gram-positive and Gram-negative bacteria. In conclusion, this paper presents an easy-to-learn and robust rat burn model that mimics the clinical situations, including immune dysregulation and bacterial infections, which is of considerable utility for the development of new topical antibiotic drugs for burn wound and infections.

Establishing relation between in-vivo and in-vitro Cryospray experiments through thermal characteristics

Cryospray is a process of destructing cancerous lesions occurring on the skin. Cryogen is sprayed on the affected area and ablation is achieved through rapid freezing of the cell. Metabolic heat generation, blood perfusion and thermal properties of tissue affect the heat sink in the skin. However, these terms are neglected in in-vitro experiments because they are performed on the tissue phantom. So, the outcomes of such studies will only provide direction to dermatologist but cannot be used in clinical applications. Hence, in-vivo thermal analysis must be conducted in order to obtain precise values before proceeding for clinical application. Present study is an attempt to explore the difference between in-vivo and in-vitro experiments. An in-vivo experiment is performed on healthy male rats (Charles Foster rats) weighing about 150–200 g while the in-vitro experiment is performed on tissue phantom. Single freeze–thaw cycle (freezing 120 s and thawing 130 s) with a spraying distance of 18 mm is selected for both the experiments. Non-invasive thermal imaging technique is used to record the temperature profile on the surface. Cryoablation beneath the surface is estimated through thermocouples. A comparative study between customised multihole nozzle (MHN) and commercial single hole nozzle (SHN) is also conducted to analyze the impact of number of holes on cryoablation in in-vivo conditions. Data extracted through thermocouples advocates that biological factors have negligible impact on cryoablation. However, histopathological results suggest that in-vivo necrotic zone is larger than the in-vitro necrotic zone. Natural thawing is responsible for such behavior. The area of cryoablation on the surface of rat skin is 50 % larger when cryogen is sprayed through MHN as compared to SHN.

The Hydroalcolic Extraction of Falcaria Vulgaris used for the treatmeant of diabetic foot ulcer and its Effects

We cleaned the rat’s nape with a razor– we took a circle with 0 cm diameter from the rat skin and then we began the treatment: we used topical treatments. The day of surgery was considered as a zero-day. During different stages, compliance with ethical issues, it was tried to avoid any physical abuse and non-essential method. On the rat’s body, a part was considered as control that did not receive any herbal extract. And the other part of the rat body received the herbal extract. The results of macroscopic observations of the rat skin surface survey showed that on the third day, the wound diameter reduced and on the fourteenth day complete recovery was occurred. (The control rat‘s body)(The rat film is attached) .

Morphophysiological changes in the intact surface of rat skin under the application of silicon and gold nanoparticles

The effect of a course application of suspension of porous silicon and gold nanoparticles on the skin of female rats on the level of free radical oxidation and catalase activity in the skin homogenate was studied. MesoPSi samples were formed using the standard method of electrochemical etching of p-type c-Si wafers with a surface orientation of (100) and a specific resistance of 1-5 mOm x cm in a solution of hydrofluoric acid and ethanol (HF (50%): C2H5OH = 1: 1) at an etching current density of 60 mA / cm2 for 1 hour (45 minutes-hour). After this, mesoPSi films were separated from the silicon substrate by a short-term increase in the current density to 600 mA / cm2. Aqueous suspensions of porous silicon nanoparticles with sizes of the order of 100 nm were obtained by mechanical grinding of mesoporous silicon films in water. Aqueous suspensions of gold nanoparticles with an average size of 30-50 nm were obtained by laser ablation of gold targets in deionized water. A suspension of silicon and gold nanoparticles in physiological saline at a concentration of 0.2 mg / ml was applied once a day in the form of a thin layer on a shaved area of the skin of animals (interscapular region) and left to dry completely. Applications were carried out for 10 days. The studied silicon and gold nanoparticles did not change the initial content of malondialdehyde and catalase activity in the skin tissue but contributed to the activation of the rate of spontaneous lipid peroxidation.

Bioinspired Shear-Flow-Driven Layer-by-Layer in Situ Self-Assembly.

Layer-by-layer (LbL) assembly is widely applied as a coating technique for the nanoscale control of architecture and related properties. However, its translational applications are limited by the time-consuming and laborious nature of the process. Inspired by the blood-clotting process, herein, we develop a shear-flow-driven LbL (SF-LbL) self-assembly approach that accelerates the adsorption rate of macromolecules by mechanically configuring the polymer chain via a coil-stretch transition, which effectively simplifies and speeds the diffusion-controlled assembly process. The structural characteristics and surface homogeneity of the SF-LbL films are improved, and diverse three-dimensional structures can be achieved. Functional SF-LbL-assembled surfaces for corneal modification are successfully fabricated, and the surface of wounded rat corneas and skin can be directly decorated in situ with SF-LbL nanofilms due to the advantages of this approach. Furthermore, in situ SF-LbL self-assembly has promise as a simple approach for the wound dressing for interventional therapeutics in the clinic, as illustrated by the successful in situ fabrication of drug-free layers consisting of chitosan and heparin on the dorsal skin of diabetic mice to rescue defective wound healing. This bioinspired self-assembly approach is expected to provide a robust and versatile platform with which to explore the surface engineering of nanofilms in science, engineering, and medicine.

Effect of wintergreen oil on in vitro transdermal permeation of osthole and geniposide

The aim of this study was to investigate the transdermal penetration enhancement effect of wintergreen oil and its action mechanisms. The in vitro transdermal tests were carried out to study the transdermal penetration enhancement effect of wintergreen oil by using osthole and geniposide as the lipophilic and hydriphilic model drugs. Fourier-transform infrared spectroscopy was used to investigate the effect of wintergreen oil on the molecular structure of rat stratum corneum, and the scanning electron microscope was employed to observe the change of rat skin surface after treatment by the oil. The wintergreen oil at proper concentrations could effectively promote the transdermal permeation of osthole and geniposide, and exhibited better penetration-enhancing activity for the lipophilic osthole, close to the commonly used classical penetration enhancer azone. The infrared spectroscopy study and scanning electron microscope showed that wintergreen oil mainly acted on the stratum corneum lipids, reduced dense stratum corneum, and reduced the skin barrier function. Thus, the wintergreen oil could effectively facilitate the transdermal absorption of the lipophilic and hydrophilic drugs, resulting from the lowed skin barrier function.

Monitoring the penetration and accumulation of gold nanoparticles in rat skin ex vivo using surface-enhanced Raman scattering spectroscopy

Contamination by accidental cutaneous contact with the commercial products and the air pollutants raised a considerable health and safety issue. This study aimed to trace the dynamics of the 20 nm gold nanoparticle (GNP) penetration and accumulation in rat skin tissues using a surface-enhanced Raman scattering (SERS) technique. After the topical application of GNPs on rat skin surface, the SERS spectra were recorded for every 15 [Formula: see text]m to an overall depth of 75 [Formula: see text]m from skin surface for 150 min. The processes of GNP penetration in rat skin were accompanied by aggregation of GNPs, which affected SERS spectra. The results revealed that 20 nm GNPs can penetrate through stratum corneum layer, viable epidermis layer, and then into dermis layer. This study demonstrated for the first time the potential of SERS spectroscopy to monitor the penetration and accumulation of GNPs in rat skin.

Flux of OH and O radicals onto a surface by an atmospheric-pressure helium plasma jet measured by laser-induced fluorescence

The atmospheric-pressure helium plasma jet is of emerging interest as a cutting-edge biomedical device for cancer treatment, wound healing and sterilization. Reactive oxygen species such as OH and O radicals are considered to be major factors in the application of biological plasma. In this study, density distribution, temporal behaviour and flux of OH and O radicals on a surface are measured using laser-induced fluorescence. A helium plasma jet is generated by applying pulsed high voltage of 8 kV with 10 kHz using a quartz tube with an inner diameter of 4 mm. To evaluate the relation between the surface condition and active species production, three surfaces are used: dry, wet and rat skin. When the helium flow rate is 1.5 l min−1, radial distribution of OH density on the rat skin surface shows a maximum density of 1.2 × 1013 cm−3 at the centre of the plasma-mediated area, while O atom density shows a maximum of 1.0 × 1015 cm−3 at 2.0 mm radius from the centre of the plasma-mediated area. Their densities in the effluent of the plasma jet are almost constant during the intervals of the discharge pulses because their lifetimes are longer than the pulse interval. Their density distribution depends on the helium flow rate and the surface humidity. With these results, OH and O production mechanisms in the plasma jet and their flux onto the surface are discussed.

Enhanced dermal delivery of acyclovir using solid lipid nanoparticles.

The present investigation was enthused by the possibility to develop solid lipid nanoparticles (SLNs) of hydrophilic drug acyclovir (ACV) and evaluate their potential as the carrier for dermal delivery. ACV-loaded SLNs (ACV-SLNs) were prepared by the optimized double emulsion process using Compritol 888 ATO as solid lipid. The prepared SLNs were smooth and spherical in shape with average diameter, polydispersity index, and entrapment efficiency of 262 ± 13nm, 0.280 ± 0.01, and 40.08 ± 4.39% at 10% (w/w) theoretical drug loading with respect to Compritol 888 ATO content. Differential scanning calorimetry and powder X-ray diffraction pattern revealed that ACV was present in the amorphous state inside the SLNs. In vitro skin permeation studies on human cadaver and Sprague-Dawley rat skin revealed 17.65 and 15.17 times higher accumulation of ACV-SLNs in the dermal tissues in comparison to commercially available ACV cream after 24h. Mechanism of topical permeation and dermal distribution was studied qualitatively using confocal laser scanning microscopy. While free dye (calcein) failed to penetrate skin barrier, the same encapsulated in SLNs penetrated deeply into the dermal tissue suggesting that pilosebaceous route was followed by SLNs for skin penetration. Histological examination and transdermal epidermal water loss measurement suggested that no major morphological changes occurred on rat skin surface due to the application of SLNs. Overall, it was concluded that ACV-loaded SLNs might be beneficial in improving dermal delivery of antiviral agent(s) for the treatment of topical herpes simplex infection.

Dermal uptake and excretion of 14C-toluene diisocyante (TDI) and 14C-methylene diphenyl diisocyanate (MDI) in male rats. Clinical signs and histopathology following dermal exposure of male rats to TDI

Polyurethanes (PU) are polymers made with diisocyanates such as MDI (4,4′-methylene diphenyl diisocyanate) and TDI (2,4-toluene diisocyanate and 2,6-toluene diisocyanate). Investigations have been undertaken with MDI and TDI to assess dermal uptake and resulting systemic exposure. Absorption, distribution and excretion of MDI was studied in rats using a single dermal administration of 14C-MDI dissolved in acetone at nominal 165 mg/kg body weight and 15 mg/kg bw (4.0 and 0.4 mg/cm 2) and intradermal injection of 14C-MDI dissolved in corn oil at nominal 1.4 mg/kg bw. Dermal absorption of 14C-MDI (at both doses) was low; at or below 1% of the applied dose. Considerable amounts of the applied radioactivity were found at the application site which could not be washed off. By intradermal administration of 14C-MDI approximately 66% of applied radioactivity remained at the application site with approximately 26% recovered in excreta, cage wash, tissues and carcass. The absorption, distribution and excretion of 2,4-TDI was studied in rats following a single dermal administration of radiolabelled 14C-2,4-TDI at nominal 350 mg/kg body weight (12 mg/cm 2). Dermal absorption of 14C-2,4-TDI was at or below 1% of the applied dose. Considerable amounts of the applied radioactivity were found at the application site which could not be washed off. In summary the results show that dermal uptake of MDI and TDI is very low. Due to the chemical reactivity of isocyanates it can be expected that small amounts which might be absorbed will react with tissue constituents directly at the exposed skin area, or will be converted to adducts with biomacromolecules or to biologically inactive oligoureas. Overall it is concluded that, following dermal exposure to MDI and TDI, systemic exposures and resulting toxicity, other than the known sensitization, can be expected to be very low. In addition studies were performed with dermal application of unlabelled 2,4 and 2,6 TDI to check the availability and fate of this chemical on rat skin surface and to assess possible tissue damage. These experiments showed that unchanged test material can be detected on rat skin for up to 8 h if not washed off. Dermal treatment with 2,4 or 2,6 TDI was associated with irritation with increased severity over a 48 h period after washing with a decontaminant solution.

Sentinel Lymph Nodes in the Rat: Noninvasive Photoacoustic and US Imaging with a Clinical US System

To evaluate in vivo sentinel lymph node (SLN) mapping by using photoacoustic and ultrasonographic (US) imaging with a modified clinical US imaging system. Animal protocols were approved by the Animal Studies Committee. Methylene blue dye accumulation in axillary lymph nodes of seven healthy Sprague-Dawley rats was imaged by using a photoacoustic imaging system adapted from a clinical US imaging system. To investigate clinical translation, the imaging depth was extended up to 2.5 cm by adding chicken or turkey breast on top of the rat skin surface. Three-dimensional photoacoustic images were acquired by mechanically scanning the US transducer and light delivery fiber bundle along the elevational direction. Photoacoustic images of rat SLNs clearly help visualization of methylene blue accumulation, whereas coregistered photoacoustic/US images depict lymph node positions relative to surrounding anatomy. Twenty minutes following methylene blue injection, photoacoustic signals from SLN regions increased nearly 33-fold from baseline signals in preinjection images, and mean contrast between SLNs and background tissue was 76.0 +/- 23.7 (standard deviation). Methylene blue accumulation in SLNs was confirmed photoacoustically by using the optical absorption spectrum of the dye. Three-dimensional photoacoustic images demonstrate dynamic accumulation of methylene blue in SLNs after traveling through lymph vessels. In vivo photoacoustic and US mapping of SLNs was successfully demonstrated with a modified clinical US scanner. These results raise confidence that photoacoustic and US imaging can be used clinically for accurate, noninvasive imaging of SLNs for axillary lymph node staging in breast cancer patients.

Specific thermographic changes during Walker 256 carcinoma development: Differential infrared imaging of tumour, inflammation and haematoma

Background: Infrared imaging measures spatial variations in the skin temperature aiming to determine pathological processes; hence possible use of this non-invasive analytical method in cancer detection is emerging. Methods: Infrared thermal imaging was used to detect changes in rat skin surface temperature associated with experimental cancer development (Walker 256 carcinoma), inflammation (upon s.c. Sephadex injection) and haematoma (provoked by s.c. blood coagulate injection). Infrared camera with a geometric resolution of 76,800 pixels, spectral range of 8–14 μm and the minimal detectable temperature resolution of 0.07 °C with spatial resolution of 0.48 mm at measuring distance of 30 cm was used to obtain computerised thermal scans. Genuine ThermoWEB software developed for remote internet control as open source software was used. Results: The raise of peripheral temperature was observed after induction of local inflammation or haematoma. Opposite to that, transient decrease of the skin surface temperature was observed after tumour transplantation. Progressive growth of tumour was associated with the raise of the skin surface temperature from the 10th day after tumour inoculation, when the tumours developed supportive neoangiogenic blood supply, as verified by histology. Conclusion: While the raise of peripheral temperature in advanced tumour was caused by neoangiogenesis, the reduction in skin surface temperature in an early period after tumour cell inoculation indicated a decay of transplanted tumour cells due to the immune response and the lack of blood supply. Thus, infrared thermal imaging may have considerable value in evaluation of the tumour development and discrimination of cancer from inflammation and haematoma.

Selective use of L-valine and L-isoleucine for the biosynthesis of branched-chain fatty acids in rat skin.

Iso- and anteiso-fatty acids are detected in more than trace amounts in rat skin surface lipid. The terminal portion of even carbon number iso- and anteiso-fatty acids are synthesized respectively from valine (Val) and isoleucine (Ile) by essentially the same reaction sequences established for straight chain fatty acids. This paper describes the stereospecific biosynthesis of these branched chain fatty acids (BCFAs) and alcohols (BCFALs) in rat skin. Dependence of the concentration of these BCFAs on dietary L-Val and L-Ile was studied. Concentrations of even carbon number iso- and anteiso-fatty acid increased respectively with dietary L-Val and L-Ile. The saturation dose appeared to be 2% for L-Val and 1% for L-Ile. Supplementation of the diet with 2% D-Val, however, did not affect the concentration of even carbon number iso-fatty acid in rat skin surface lipid despite a comparable serum Val level to that of the 2% L-Val group. A similar experiment using 1% DL-Ile found that L-isomer, but not D-isomer, in the circulation was used for the biosynthesis of anteiso-fatty acids. This view was applicable to the incorporation of D-Val and DL-Ile into related BCFALs.

Precursor role of branched-chain amino acids in the biosynthesis of iso and anteiso fatty acids in rat skin

Monoester fraction of rat skin surface lipid has been shown to contain more than trace amounts of branchedlong-chain fatty acids (BCFAs) of the iso and anteiso series. These BCFAs are biosynthesized using either branched-chain amino acids (BCAAs) or branched-chain α-keto acids (BCKAs), or using both of them as precursor. This study has been carried out to address which precursor, BCAAs or BCKAs in the circulation, are mainly utilized for biosynthesis of BCFAs. Dietary supplement of [ 14C]-valine and isoleucine-induced sharp rise of serum concentration of these two amino acids and their respective α-keto acids, and elevated the levels of related BCFAs and branched-chain fatty alcohols in the monoester fraction. A larger proportion of label in the total skin surface lipid was found in the monoester fraction in which fatty acid and alcohol accounted for approx. 80% of total radioactivity. Incorporation of intravenously administered [ 14C]-BCAAs and BCKAs into the monoester fraction revealed that BCAAs were far better as precursors than BCKAs for BCFA biosynthesis in rat skin. Among three BCAAs, leucine differed from valine or isoleucine in that this amino acid was primarily utilized for production of straight-chain fatty acids rather than for production of related BCFA.

Chain Structure and Composition of Fatty Alcohol in the Monoester Fraction of Rat Skin Surface Lipid

Chain Structure and Composition of Fatty Alcohol in the Monoester Fraction of Rat Skin Surface Lipid

Percutaneous absorption of nicotinic acid, phenol, benzoic acid and triclopyr butoxyethyl ester through rat and human skin in vitro: Further validation of an in vitro model by comparison with in vivo data

The in vitro percutaneous absorption of three model compounds, nicotinic acid, phenol and benzoic acid, and the herbicide triclopyr butoxyethyl ester (triclopyr BEE) has been investigated in flow-through diffusion cells using skin from male Fischer 344 rats and humans. After the application of the four chemicals to the epidermal surface of unoccluded full-thickness rat skin, the absorption of each compound across the skin and into the receptor fluid at 72 hr reached 3.7 ± 0.3, 5.7 ± 0.6, 26.7 ± 3.7 and 48.3 ± 1.2% (mean ± SD, n = 2–7) of the applied dose for triclopyr BEE, nicotinic acid, phenol and benzoic acid, respectively. After the application of the four chemicals to the epidermal surface of unoccluded full-thickness human skin, the absorption of each compound across the skin and into the receptor fluid at 72 hr was significantly ( P < 0.05) less than through rat skin, reaching 0.7 ± 0.1, 0.7 ± 0.2, 18.8 ± 1.3 and 37.8 ± 6.9% (mean ± SD, n = 2–7) of the applied dose for triclopyr BEE, nicotinic acid, phenol and benzoic acid, respectively. Occlusion of the skin surface with teflon caps often significantly ( P < 0.05) enhanced the percutaneous absorption of the model compounds, although this effect was not uniform, varying with the compound under study and the skin (rat or human) used. When rat skin was occluded with teflon caps, the extent of absorption at 72 hr reached 8.6 ± 0.8, 36.2 ± 1.7 and 51.8 ± 3.3% (mean ± SD, n = 3–4) for nicotinic acid, phenol and benzoic acid, respectively. Corresponding values for human skin occluded with teflon caps were 3.3 ± 1.6, 47.1 ± 0.5 and 65.5 ± 7.1% (mean ± SD, n = 3–4). The experiments on the absorption of each model compound through rat and human skin were repeated and there was generally good agreement between the results from the two sets of experiments. The in vitro data reported compare favourably with data obtained by other workers using both in vitro and in vivo methodologies. The in vitro : in vivo correlation supports the use of the flow-through diffusion cell system as a model for the prediction of percutaneous absorption in vivo in the rat and in humans.

Age-related changes of the branched-chain fatty acid concentration in rat skin surface lipid

1. 1. Age-related change of the branched-chain fatty acid distribution in rat skin surface lipid was studied for 24 moths. 2. 2. The proportion of even carbon number iso-acid increased from infancy to month 5 and thereafter decreased with advancing age toward senescence. 3. 3. Concentration of odd carbon number iso-acid depicted a similar shape of time course, but with a lesser magnitude and a peak value at month 1. 4. 4. Anteiso- fatty acid reached the plateau level at month 5 month remained roughly constant through maturity to senescence.

Correlation between Serum Concentrations of the Branched Chain Amino- and .ALPHA.-Keto-Acid and the Distribution of the Branched-Chain Fatty Acids on Rat Skin Surface.

This study described the relationship between the concentration of the branched-chain fatty acid (BCFA) in rat skin surface lipid and the serum level of the branched-chain amino acid (BCAA) and branched-chain alpha-keto acid (BCKA). The concentrations of the BCFAs in the monoester fraction of the skin surface lipid, and BCAAs and BCKAs in the serum were analyzed in rats fed varying amounts of protein (0 to 40%) and different types of BCAAs (3%) for 2 weeks. The serum concentrations of BCAA were proportional to the protein level by day 10 of the feeding period. This dose response was not sustained by day 14 at the end of the feeding. Protein level dependence was not so evident in the concentration of BCKA. The concentrations of BCFA, even carbon number iso-acid in particular, increased in linear proportion to protein intake in the skin surface lipid. Supplementation of valine and isoleucine to the diet at a 3% level specifically raised the concentration of the respective BCAA and corresponding BCKA in serum, and related BCFA in the skin surface lipid. Addition of leucine, however, did not affect the related BCFA concentration in spite of elevated concentration of leucine and its alpha-keto acid. A good linear correlation between the average concentration of the substrates in the circulation and the concentrations of the product BCFA on the skin surface was thus obtained for valine, isoleucine and their respective alpha-keto acid. This relationship did not appear to hold up for leucine and its alpha-keto acid.