Introduction
The unique structure and organization of the stratum corneum, which has been pre-sented in chapter 1, gives rise to a barrier with a high diffusional resistance. The permeation of even a small molecule like water is some-what impeded, and this is of utmost impor-tance for an organism with a skin surface of
~2 m2. It has been calculated [1], that if the skin was made of only a single layer of cor-neocytes, the rate of water loss would be 2500 times greater. Knowing that a normal healthy subject releases some 0.5 L of water through the skin per day (in a rest situation), it is evi-dent that the stratum corneum plays a crucial role in water homeostasis. It is also evident that if the barrier is not fully functional due to pathological processes or it is damaged by physical or chemical agents, there will be a corresponding increase in water loss rates that directly relates to the degree of impairment [2]. The measurement of the rate of water loss from the skin is therefore a useful experimen-tal method to assess the effect of a compound
(vehicle or enhancer), of hydration (by occlu-sion, moisturizers or emollients), or of stra-tum corneum impairment (by tape stripping, solvents or detergents) on barrier function.
Methods of measurement
Three methods are currently available: the unventilated chamber, the ventilated chamber, and the open chamber. The first two methods (well reviewed in [3]) are outdated, and were used in the seventies before the emergence of the third method, which is the most widely used and the most reliable.
The open chamber method was developed by Nilsson in the late seventies [4]. It is based on Fick's first law of diffusion (see chapter 3), and on the fact that the water vapour pressure gradient close to the surface of the skin (i.e., within about 1 cm), and thereby also the water exchange, is approximately proportional to the difference between the vapour pressure measured at two separate fixed points situated on a line perpendicular to the surface and in the zone of diffusion. Thus, the following relation can be applied:
1 A
dm
dt Ddp
= − dx (1)
where
A = surface area of (m2)
m = mass of the transported water (g) t = time (h)
D = constant, 0.0877 g/mh (1/mm Hg) related to the diffusion coefficient
p = partial pressure of water vapour in the air (mm Hg)
x = distance from the surface (m)
Equation (1) indicates that the evaporation rate dm/dt is proportional to the partial pres-sure gradient, dp/dx, and thus can be deter-mined by measuring the last mentioned quan-tity.
The first device using this principle was the Evaporimeter® (Servomed, Stockholm, Sweden), which employs a sensor arrange-ment as shown in Figure 1. A small area is defined and separated from the surroundings by an open cylindrical teflon capsule. The purpose of the capsule is to protect the meas-urement area from draughts. At each of two different distances from the skin surface, 3 and 9 mm, there is a pair of transducers, one for relative humidity, the other a thermistor.
From the signals derived from these transduc-ers the instrument first computes the partial pressure of the water vapour at the two dis-tances from the surface, then the partial pres-sure gradient, and finally the evaporation rate.
The evaporimeter has been thoroughly evaluated for accuracy, reproducibility and
variability by a number of studies [5-8], and was found to provide a good method for rou-tine clinical and experimental real time TEWL measurements, even if a number of factors (albeit controllable) were likely to influence the readings. For this reason, in our studies we have adopted the evaporimeter for measuring the TEWL from the ventral fore-arm surface of human volunteers.
However, the architecture of the probe cyl-inder results in underestimation of the water flow rate at high TEWL values: at about 20 g/m2h, the evaporimeter underestimates the rate by only 10%, but at 80 g/m2h the error exceeds 50% [9]. This is due to the fact that the diffusional resistance of the static air col-umn trapped by the probe head becomes sig-nificant relative to the diffusional resistance
Figure 1: Schematic diagram of (upper) the sensor unit of the Servomed Evaporimeter®showing the relative humidity transducers on one side and the thermistor on the opposite side, and (lower) the probe applied to the skin surface. From [5].
of the skin.
Given the commercial success of the Evaporimeter®, other similar instruments have been marketed and compared to the original device: the Tewameter® (Courage-Khazaka, Cologne, Germany) [10,11], and, more recently, the DermaLab® TEWL probe (Cortex Technologies, Hadsund, Denmark) [12,13].
Practical precautions during TEWL read-ings in our studies
Various precautions must be respected when using the Evaporimeter®, since sensitiv-ity of the probe to environmental changes may perturb the readings. It is thus very im-portant to keep environmental conditions con-stant during a given series of measurements.
In all of our studies, we have followed the guidelines for TEWL measurements recom-mended by the standardization group of the European Society of Contact Dermatitis [14].
The particular experimental conditions are listed below.
The first precaution was the choice of the anatomic site of drug application: when ap-plying a formulation on the ventral forearm, it is important to avoid the wrist region, since it has been shown that TEWL readings in that
region are significantly different (p<0.002) to readings at other sites on the forearm [15].
The sweat gland density and activity, which increases towards the wrist might explain these differences.
The temperature of the room was main-tained at 22±1°C, in order to avoid sweating of the volunteers, which obviously interferes with the true transepidermal water flux [16].
However, even without sweating significant intraindividual changes in skin temperature may perturb TEWL readings, since TEWL increases exponentially as the temperature increases [17], and an increase in 7-8°C dou-bles the rate of water loss. In our studies, the mean inter-individual skin temperature of 31±2°C showed an intra-individual variation of only 1-2°C during the course of the ex-periments. This was too small a variation to compromise comparisons of TEWL meas-urements.
Relative humidity was also monitored, but not controlled, since previous studies [18]
indicate that changes below 80% relative hu-midity do not significantly affect the rate of water loss (Figure 2). The seasonal variations in the country where the studies were per-formed (eastern France) did not reach more than 60% relative humidity, even during the summer season.
Equilibration time of the volunteers prior to the first TEWL reading was also controlled in our studies. We chose an equilibrium pe-riod of 30 minutes since TEWL values tend to fluctuate during the initial 15 minutes [19].
Air convection around the probe was pre-vented in all of our studies by means of a cyl-indric open top draught shield (diameter 6 cm, height 7 cm) placed around the probe. Some authors recommend using open top boxes such as incubators when measuring the TEWL from the arm [20], but we believe that protection of the measurement area instead of the whole uninvolved arm is a more appropri-ate (and inexpensive) solution.
Another precaution (not mentioned in the guidelines), is to avoid application of organic solvents with high vapour pressures to the skin, since concentrations greater than 100 ppm may attack the polymeric sensors [21].
For this reason, vehicles such as propylene glycol, which give abnormal readings, were avoided in our studies. However, the formula-tions used in our studies did contain ethanol (vapour pressure 8 kPa at 25°C), but we re-spected a 30 minute delay before the first TEWL reading.
Physico-chemical factors affecting TEWL The water flux through the stratum corneum depends on the water gradient within the membrane [18], and the condition of its barrier function. All agents that increase the water content or alter the structure of the stra-tum corneum will therefore provoke changes in TEWL. In fact, hydration of the stratum corneum causes it to swell which again alters the characteristics of the membrane through which water is diffusing.
Hydration may be induced by means of ei-ther an occlusive dressing or emollient com-pound, or a humectant. Occlusion cuts the water flux, but once the dressing or the emol-lient compound is removed, the water desorp-tion kinetics allow the barrier's water holding capacity to be derived [22-25]. Humectants (e.g. glycerol, propylene glycol, pyrrolidone carboxylic acid) also increase the water flux but the mechanism is rather different, since they bind water rather than impeding its diffu-sion [26].
The permeability of the stratum corneum
Figure 2: Calculated in vivo water flux (J) at increas-ing relative humidities (RH), from water content data.
Modified from [18].
to a topical drug may also be affected by in-creased hydration, as studied in chapter 8 of this thesis, but the diffusion coefficient of water is not necessarily related to drug per-meability, as assessed from the concentrations in the stratum corneum.
This was anticipated in a preliminary study, where we applied four compounds (isopropyl myristate, oleic acid, urea, and sodium lauryl sulphate) on the ventral fore-arms of 7 volunteers over a period of 4 hours under a semi-occlusive polyurethane dressing.
TEWL measurements during sequential tape stripping in a given subject appeared to dis-criminate between the effects of the com-pounds (Figure 3).
However, statistical analysis of the water diffusivity and the permeability coefficients of the entire cohort revealed no differences (Figure 4).