599
Biochimica et Biophysica Acta, 437 (1976) 599--603 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 27964
A P R O T O N MAGNETIC RESONANCE STUDY OF WATER IN HUMAN STRATUM CORNEUM
M.I. FOREMAN
Scientific Development Group, Organon Laboratories Limited, Newhouse, Lanarkshire ML1 5SH (U.K.) (Received January 21st, 1976)
Summary Proton magnetic resonance spectroscopy has been used to study the nature of water in human stratum corneum. For a single planar sheet of stratum corneum m o u n t e d at a specific orientation to the applied magnetic field, three distinct absorptions m a y be seen having different chemical shifts and spin-lattice relaxation times (T,). All T1 Values for these resonances are smaller than that for normal liquid water. One absorption is unusual in that the resonance position is dependent upon the orientation of the sample within the field.
Introduction There is an increasing interest in the study of the nature of water in biological tissue, much of which has involved the use of NMR spectroscopy [1--3]. One system which has perhaps been neglected in this respect is that of skin, particularly the stratum corneum, since the physical properties of this layer are known to be very dependent u p o n the level of hydration [4]. Some studies of the proton NMR of water in keratin have been reported, but there seems to have been only one proton study of water in human stratum corneum, by Hansen and Yellin [5], which was carried o u t at 36 MHz. These authors did n o t in fact give details of the m e t h o d of observing the sample in the probe. They appear, however, to have observed a single peak. Measurement of the spin-spin relaxation time suggested that two T2 values were required to explain the relaxation behaviour, and that the observed absorption was in fact due to t w o broad, overlapping peaks. In the present work, spectra were obtained at rather higher field strengths using single sheets of stratum corneum m o u n t e d at a specific angle to the applied field. In this w a y t w o distinct resonances were observed. Measurement of the spin-lattice relaxation times indicates that the peaks in fact have slightly
600 different T1 Values. The resonance position of one of the two peaks has been f o u n d to depend very markedly on the orientation of the sample in the magnetic field. As a result of selectively relaxing these two absorptions during the measurement of the T~ Values, a third rather broader, absorption was also observed. Experimental Full thickness h u m a n abdominal skin was obtained at autopsy. Subcutaneous fat was removed and the skin stored at --4°C for not more than one week. When required, the dermis was removed, and the viable epidermis digested with trypsin. The sheets of stratum corneum, approx. 4 X 4 cm, were then thoroughly washed with distilled water and left to stand overnight (16 h) in 20 ml distilled water. A sample of the water used to soak the stratum corneum was then taken and w i t h o u t degassing, the T~ Value of the proton resonance was determined using the conventional 180°-T-90 ° pulse sequence [6]. Next, a sample of the stratum corneum sheet, approx. 3 X 2 mm, was m o u n t e d on a glass holder, allowed to stand in air for 1 h at 22°C and 30% relative humidity, and then inserted into a 5 mm diameter glass NMR tube. The sample size and its position in the tube ensured that the entire sample was inside the region of the r.f. coil. The tube was sealed with a plastic cap, placed in the probe and allowed to equilibrate for 1 h before recording commenced. The arrangement is illustrated in Fig. 1. A preliminary measurement indicated that the T1 values of the resonances were less than 1 s, a 90 ° pulse was therefore used in accumulating the free induction decay. Field locking was effected using an external water signal as the reference. T~ values for the resonances of the stratum corneum sample
Region of r.f. c o i l
I
mount
Stratum corneum
sheet
Fig. 1. M e t h o d o f m o u n t i n g o f s t r a t u m c o r n e u m s h e e t w i t h i n t h e s p e c t r o m e t e r .
601 were estimated using the "null value" m e t h o d , whereby r for a 180 °-r-90 ° pulse sequence is such that the signal has zero peak intensity. There are objections [6] to this approach b u t the precision is almost certainly adequate for the present purposes. All NMR spectra were recorded using a Jeol F.T. NMR Spectrometer operating at 100 MHz and 25°C. Results and Discussion T~ measurements of the water in which the stratum corneum had been allowed to equilibrate yielded values within the range 2.2--2.5 s. The samples were n o t degassed, which means that the relaxation behaviour will be affected by the presence of paramagnetic oxygen. However, the same may be true for water within the stratum corneum exposed to air in the normal way, although this will n o t necessarily be the case. On balance therefore, it seemed best to compare directly the relaxation behaviour of bulk water without degassing with that of the water in the stratum corneum matrix. T1 Values for the water samples were obtained by a least-squares fit of the data directly to Eqn. 1 [6]:
Ar=Ao(1-
2exp (~,))
(1)
where, for a 180°-r-90 ° pulse sequence, A0 and Ar are the peak intensities for delay times 0 and r respectively. Proton spectra were obtained for single sheets of stratum corneum from twelve different individuals. In ten cases, t w o chemically shifted absorptions were clearly appa}ent. The peak intensities were found to be dependent u p o n the hydration state of the sample; it was therefore concluded that these absorptions were due to water within the stratum corneum matrix. A typical spectrum is shown in Fig. 2 with the stratum corneum sheet lying in a plane perpendicular to the applied field direction. In two cases the peak to high frequency, marked A in the figure, was n o t apparent. Typically, for this sample orientation, the peak separation is of the order of 600 Hz. Rotation of the sample plane a b o u t an axis at right angles to the field direction causes peak A to be displaced relative to peak B; the sequence is illustrated in
605 Hz
I
I
(B)
I
i
F i g . 2. P r o t o n N M R s p e c t r u m o f s t r a t u m c o r n e u m s h e e t o r i e n t a t e d at r i g h t a n g l e s t o t h e a p p l i e d field.
602
Sample orientation with
respect
applied
--
I
~
r
I
I
r
m
to
the
field B °
n
~¢-
I
Fig. 3. E f f e c t o f s a m p l e r o t a t i o n w i t h r e s p e c t to t h e a p p l i e d field.
Fig. 3. The reason for this behaviour is n o t clear, it may be an artefact arising from the use of the glass m o u n t , or possibly be due to the anisotropic bulk magnetic susceptibility of the sample. Attempts to measure T1 Values for peaks A and B revealed a third broader peak underlying peak B. Fig. 4 shows attempts at a null signal using r = 0.25 and 0.23 s which illustrates two points. Firstly it is clear that peak A is positivegoing for r = 0.25 s whilst peak B is still negative-going. This suggests that, whatever the actual T1 Values, peak A has the shorter relaxation time. In fact,
~.
Biochimica et Biophysica Acta, 437 (1976) 599--603 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 27964
A P R O T O N MAGNETIC RESONANCE STUDY OF WATER IN HUMAN STRATUM CORNEUM
M.I. FOREMAN
Scientific Development Group, Organon Laboratories Limited, Newhouse, Lanarkshire ML1 5SH (U.K.) (Received January 21st, 1976)
Summary Proton magnetic resonance spectroscopy has been used to study the nature of water in human stratum corneum. For a single planar sheet of stratum corneum m o u n t e d at a specific orientation to the applied magnetic field, three distinct absorptions m a y be seen having different chemical shifts and spin-lattice relaxation times (T,). All T1 Values for these resonances are smaller than that for normal liquid water. One absorption is unusual in that the resonance position is dependent upon the orientation of the sample within the field.
Introduction There is an increasing interest in the study of the nature of water in biological tissue, much of which has involved the use of NMR spectroscopy [1--3]. One system which has perhaps been neglected in this respect is that of skin, particularly the stratum corneum, since the physical properties of this layer are known to be very dependent u p o n the level of hydration [4]. Some studies of the proton NMR of water in keratin have been reported, but there seems to have been only one proton study of water in human stratum corneum, by Hansen and Yellin [5], which was carried o u t at 36 MHz. These authors did n o t in fact give details of the m e t h o d of observing the sample in the probe. They appear, however, to have observed a single peak. Measurement of the spin-spin relaxation time suggested that two T2 values were required to explain the relaxation behaviour, and that the observed absorption was in fact due to t w o broad, overlapping peaks. In the present work, spectra were obtained at rather higher field strengths using single sheets of stratum corneum m o u n t e d at a specific angle to the applied field. In this w a y t w o distinct resonances were observed. Measurement of the spin-lattice relaxation times indicates that the peaks in fact have slightly
600 different T1 Values. The resonance position of one of the two peaks has been f o u n d to depend very markedly on the orientation of the sample in the magnetic field. As a result of selectively relaxing these two absorptions during the measurement of the T~ Values, a third rather broader, absorption was also observed. Experimental Full thickness h u m a n abdominal skin was obtained at autopsy. Subcutaneous fat was removed and the skin stored at --4°C for not more than one week. When required, the dermis was removed, and the viable epidermis digested with trypsin. The sheets of stratum corneum, approx. 4 X 4 cm, were then thoroughly washed with distilled water and left to stand overnight (16 h) in 20 ml distilled water. A sample of the water used to soak the stratum corneum was then taken and w i t h o u t degassing, the T~ Value of the proton resonance was determined using the conventional 180°-T-90 ° pulse sequence [6]. Next, a sample of the stratum corneum sheet, approx. 3 X 2 mm, was m o u n t e d on a glass holder, allowed to stand in air for 1 h at 22°C and 30% relative humidity, and then inserted into a 5 mm diameter glass NMR tube. The sample size and its position in the tube ensured that the entire sample was inside the region of the r.f. coil. The tube was sealed with a plastic cap, placed in the probe and allowed to equilibrate for 1 h before recording commenced. The arrangement is illustrated in Fig. 1. A preliminary measurement indicated that the T1 values of the resonances were less than 1 s, a 90 ° pulse was therefore used in accumulating the free induction decay. Field locking was effected using an external water signal as the reference. T~ values for the resonances of the stratum corneum sample
Region of r.f. c o i l
I
mount
Stratum corneum
sheet
Fig. 1. M e t h o d o f m o u n t i n g o f s t r a t u m c o r n e u m s h e e t w i t h i n t h e s p e c t r o m e t e r .
601 were estimated using the "null value" m e t h o d , whereby r for a 180 °-r-90 ° pulse sequence is such that the signal has zero peak intensity. There are objections [6] to this approach b u t the precision is almost certainly adequate for the present purposes. All NMR spectra were recorded using a Jeol F.T. NMR Spectrometer operating at 100 MHz and 25°C. Results and Discussion T~ measurements of the water in which the stratum corneum had been allowed to equilibrate yielded values within the range 2.2--2.5 s. The samples were n o t degassed, which means that the relaxation behaviour will be affected by the presence of paramagnetic oxygen. However, the same may be true for water within the stratum corneum exposed to air in the normal way, although this will n o t necessarily be the case. On balance therefore, it seemed best to compare directly the relaxation behaviour of bulk water without degassing with that of the water in the stratum corneum matrix. T1 Values for the water samples were obtained by a least-squares fit of the data directly to Eqn. 1 [6]:
Ar=Ao(1-
2exp (~,))
(1)
where, for a 180°-r-90 ° pulse sequence, A0 and Ar are the peak intensities for delay times 0 and r respectively. Proton spectra were obtained for single sheets of stratum corneum from twelve different individuals. In ten cases, t w o chemically shifted absorptions were clearly appa}ent. The peak intensities were found to be dependent u p o n the hydration state of the sample; it was therefore concluded that these absorptions were due to water within the stratum corneum matrix. A typical spectrum is shown in Fig. 2 with the stratum corneum sheet lying in a plane perpendicular to the applied field direction. In two cases the peak to high frequency, marked A in the figure, was n o t apparent. Typically, for this sample orientation, the peak separation is of the order of 600 Hz. Rotation of the sample plane a b o u t an axis at right angles to the field direction causes peak A to be displaced relative to peak B; the sequence is illustrated in
605 Hz
I
I
(B)
I
i
F i g . 2. P r o t o n N M R s p e c t r u m o f s t r a t u m c o r n e u m s h e e t o r i e n t a t e d at r i g h t a n g l e s t o t h e a p p l i e d field.
602
Sample orientation with
respect
applied
--
I
~
r
I
I
r
m
to
the
field B °
n
~¢-
I
Fig. 3. E f f e c t o f s a m p l e r o t a t i o n w i t h r e s p e c t to t h e a p p l i e d field.
Fig. 3. The reason for this behaviour is n o t clear, it may be an artefact arising from the use of the glass m o u n t , or possibly be due to the anisotropic bulk magnetic susceptibility of the sample. Attempts to measure T1 Values for peaks A and B revealed a third broader peak underlying peak B. Fig. 4 shows attempts at a null signal using r = 0.25 and 0.23 s which illustrates two points. Firstly it is clear that peak A is positivegoing for r = 0.25 s whilst peak B is still negative-going. This suggests that, whatever the actual T1 Values, peak A has the shorter relaxation time. In fact,
~.
