M e d . & Biol. Eng. & Comput,, 1978, 16, 126-134
Percutaneous electrophoresis of amino acids and urea S. Y. Shaya
C . W . Smith
J. G. Heathcote
Department of Electrical EngineeringUniversityof Salford, Salf0rd M5 4WT, England
Department of Pure & Applied Chemistry University of Salford, Salford M5 4WT, England
A b s t r a c t - - A noninvasive method for the percutaneous extraction and determination o f samples o f metabolites is described. It involves the use of a battery-operated, constant-current supply for obtaining the samples by "in-vivo electrophoresis'. These are then examined in respect of their amino a c i d and urea content by thin-layer-chromatography. The results of examinations made on the hand (thenar eminence) show an increase in the amino acids and urea collected a! the electrodes under conditions of electrophoresis, over that collected at similar electrodes without the current. Possible applications are discussed. K e y w o r d s - - C h r o m a t o g r a p h y methods, Electrodes, Electrophoresis, Skin
1 Introduction F~w analyses of metabolites in sweat are reported in the literature compared with the many analyses reported for metabolites in urine and blood. To obtain reliable values for the analysis of percutaneous metabolites, attention must be paid to the methods of stimulation and collection, skin area, age, sex, hormonal balance, temperature, humidity and previous exercise of the subject. Strictly speaking, all these factors should be carefully controlled, but it is clear that this is neither easy nor practicable. Hamilton (1965) has examined the amino acids present in thumb prints, and has carried out determinations of the quantities present. The present paper describes a method of collecting metabolites percutaneously. I t is based on the fact that all the
amino acids behave as either acids or bases (ampholytes) and may be extracted in reasonable times by electrophoresis using physiologically 'safe' values of current to obtain sufficient quantities for analysis. During these experiments, 24 individuals were examined using this method and the results were recorded (SHAYA, 1975) together with a note of the race, sex, age and state of health. Some subjects were examined on a number of different occasions. 2 Description of apparatus 2.1 Sample Collection Fig. 1 shows the form of apparatus used in the present experiments. It represents a convenient arrangement for collecting samples from the hand.
Fig. I Apparatus used for in-vivo electrophoresis experiments First received 14th March and in final form 17th March 1977 0140-0118/78/0709-0126 $1 950/0
9 IFMBE: 1978 126
Medical & Biological Engineering & Computing
March 1978
The essential features are as follows: the patientisolated, battery-operated constant-current power supply [1 ], the collection medium for the metabolite samples which in this case was two strips of chromatographic paper [2] 17 x 2 cm in size, the Perspex mount [3] containing platinum wires [4] pressing against each paper strip at its outer end and making electrical connection with the leads from the power supply, the strips of Perspex [5] which rest on each
paper strip to prevent drying out by evaporation during the sample collection, and the central block [6] to prevent an electrical short circuit and to separate the two different buffer solutions. In use, the two central ends of the paper strips [2] are folded back over the Perspex strips [5] to present known areas of contact (typically 20 x 5 mm) when the hand is placed over both of them, so completing the electrical circuit. The two outer ends provided a
~D
u~
=Ir
~L
I
vvvv
| +
Medical & Biological Engineering & Computing
March 1978
127
means of handling the paper strips; this was done with tweezers and the ends were cut off afterwards. The paper connected to the cathode was moistened with a buffer at p H 2.0 and the paper connected to the anode was moistened with a buffer at p H 11.0. These p H values were intended to avoid the isoelectric points, to ensure high values of ionic mobility for the electrophoresis. The p H 2.0 buffer was an acetic-formic acid solution and p H 11.0 buffer was an ammonium hydroxide solution (see Appendix). Unless volatile buffers are used a 'desalting' process must be employed to obtain satisfactory separations by thin-layer chromatography: details are given by HEATHCOTE et al. (1971a). The paper strips kept their respective p H values during the experiments to an accuracy determined by the absence of any visible colour change in a Universal Indicator solution. If the indicator was used during electrophoresis it was transported into the skin which then showed the same coloration as the appropriate buffer over the areas of contact with the ends of the paper strips. It took at least ten times longer to wash the indicator back out of the skin thao it took to put it there by electrophoresis. This shows that transfer of buffers and the indicator into the skin could take place by electrophoresis.
and the amino acids move electrophoretically under the influence of the applied electric field respectively into the skin and from the body into the paper strips. Preliminary experiments showed that the amino acids from the body did not reach the platinum wires at the ends of the paper strips when 30 rain of electrophoresis was used to collect the samples.
3 Experimental methods 3.1 Collection o f samples The hand was chosen as the most safe and socially acceptable location for the present experiments, and the experimental site was usually the palm of the hand (thenar eminence). This was cleaned with detergent and water, and dried before the experiment. A control sample of sweat could be taken from the other hand using a similar piece of chromatographic paper placed in the palm and covered with a disposable plastic glove. The apparatus was prepared with the paper strips (untouched by hand) and the buffer solution applied. The thenar eminence of the subject was placed so as to complete the electrical circuit between the paper strips, and the current was
2.2 Constant current power supply A constant current supply provided a stable source of current which could be set to any required value over the range 0-500/tA, which is the maximum value that should be applied percutaneously to the subject; even so, the flow of such currents in the region of the heart should be avoided. The present apparatus is of 'type BF' and the current is of the 'patient functional current' classification (BSI, 1977). Its maximum value is equal to the 'patient auxiliary current' in 'single fault condition.' It would not be safe to use these currents on subjects with pacemakers or intracutaneous electrical pathways. The circuit diagram of the supply is given in Fig. 2. It comprises a battery operated oscillator, a transformer and rectifier, followed by a constant current stabilising circuit. There is only a minimal amount of capacitance connected to the rectifier because the open-circuit, no-load output voltage can rise to 800 V. It is also desirable to arrange a pressure operated switch so that the supply cannot be energised unless the subject is connected across the output and remains so. The maximum resistance into which the supply will deliver a constant 500 p A is 500 kfL This high value permitted experiments to be made with distilled water instead of the buffer solutions. F o r routine use with lower output resistances, the noload open-circuit voltage from the rectifier might be reduced. The anions and cations of the electrolyte
128
0 Leu 0 lie 0Phe
OVel 0 Tyr c 0
Q
Glu 0 (~AIo Asp 0 0 ~Ser
Gly
0 Thr
D Second
Dimension D
Fig. 3 Chromatographic separation of e/uate with a buffer p H 11,0 at the anode
Medical & Biological Engineering & Computing
March 1978
Table I. Percentage of measurements in which the indicated amino acids were detected at the anode, cathode and in the sweat
Detection
Amino acids Ala % 100 88 77
Anode Cathode Sweat
~0
I
Asp % 100 100 66
I
Glu % 100 55 66
I
I
I
I
-5 -
Gly % 100 100 66
lie % 88 88 66
Leu % 100 100 66
I
Phe % 100 88 55
Ser % 88 100 77
Thr % 88 100 66
Tyr % 77 88 33
Val % 100 100 55
I
I
_
A
Anode
o
Cathode
x
Sweat,
U
Percutaneous electrophoresis of amino acids and urea S. Y. Shaya
C . W . Smith
J. G. Heathcote
Department of Electrical EngineeringUniversityof Salford, Salf0rd M5 4WT, England
Department of Pure & Applied Chemistry University of Salford, Salford M5 4WT, England
A b s t r a c t - - A noninvasive method for the percutaneous extraction and determination o f samples o f metabolites is described. It involves the use of a battery-operated, constant-current supply for obtaining the samples by "in-vivo electrophoresis'. These are then examined in respect of their amino a c i d and urea content by thin-layer-chromatography. The results of examinations made on the hand (thenar eminence) show an increase in the amino acids and urea collected a! the electrodes under conditions of electrophoresis, over that collected at similar electrodes without the current. Possible applications are discussed. K e y w o r d s - - C h r o m a t o g r a p h y methods, Electrodes, Electrophoresis, Skin
1 Introduction F~w analyses of metabolites in sweat are reported in the literature compared with the many analyses reported for metabolites in urine and blood. To obtain reliable values for the analysis of percutaneous metabolites, attention must be paid to the methods of stimulation and collection, skin area, age, sex, hormonal balance, temperature, humidity and previous exercise of the subject. Strictly speaking, all these factors should be carefully controlled, but it is clear that this is neither easy nor practicable. Hamilton (1965) has examined the amino acids present in thumb prints, and has carried out determinations of the quantities present. The present paper describes a method of collecting metabolites percutaneously. I t is based on the fact that all the
amino acids behave as either acids or bases (ampholytes) and may be extracted in reasonable times by electrophoresis using physiologically 'safe' values of current to obtain sufficient quantities for analysis. During these experiments, 24 individuals were examined using this method and the results were recorded (SHAYA, 1975) together with a note of the race, sex, age and state of health. Some subjects were examined on a number of different occasions. 2 Description of apparatus 2.1 Sample Collection Fig. 1 shows the form of apparatus used in the present experiments. It represents a convenient arrangement for collecting samples from the hand.
Fig. I Apparatus used for in-vivo electrophoresis experiments First received 14th March and in final form 17th March 1977 0140-0118/78/0709-0126 $1 950/0
9 IFMBE: 1978 126
Medical & Biological Engineering & Computing
March 1978
The essential features are as follows: the patientisolated, battery-operated constant-current power supply [1 ], the collection medium for the metabolite samples which in this case was two strips of chromatographic paper [2] 17 x 2 cm in size, the Perspex mount [3] containing platinum wires [4] pressing against each paper strip at its outer end and making electrical connection with the leads from the power supply, the strips of Perspex [5] which rest on each
paper strip to prevent drying out by evaporation during the sample collection, and the central block [6] to prevent an electrical short circuit and to separate the two different buffer solutions. In use, the two central ends of the paper strips [2] are folded back over the Perspex strips [5] to present known areas of contact (typically 20 x 5 mm) when the hand is placed over both of them, so completing the electrical circuit. The two outer ends provided a
~D
u~
=Ir
~L
I
vvvv
| +
Medical & Biological Engineering & Computing
March 1978
127
means of handling the paper strips; this was done with tweezers and the ends were cut off afterwards. The paper connected to the cathode was moistened with a buffer at p H 2.0 and the paper connected to the anode was moistened with a buffer at p H 11.0. These p H values were intended to avoid the isoelectric points, to ensure high values of ionic mobility for the electrophoresis. The p H 2.0 buffer was an acetic-formic acid solution and p H 11.0 buffer was an ammonium hydroxide solution (see Appendix). Unless volatile buffers are used a 'desalting' process must be employed to obtain satisfactory separations by thin-layer chromatography: details are given by HEATHCOTE et al. (1971a). The paper strips kept their respective p H values during the experiments to an accuracy determined by the absence of any visible colour change in a Universal Indicator solution. If the indicator was used during electrophoresis it was transported into the skin which then showed the same coloration as the appropriate buffer over the areas of contact with the ends of the paper strips. It took at least ten times longer to wash the indicator back out of the skin thao it took to put it there by electrophoresis. This shows that transfer of buffers and the indicator into the skin could take place by electrophoresis.
and the amino acids move electrophoretically under the influence of the applied electric field respectively into the skin and from the body into the paper strips. Preliminary experiments showed that the amino acids from the body did not reach the platinum wires at the ends of the paper strips when 30 rain of electrophoresis was used to collect the samples.
3 Experimental methods 3.1 Collection o f samples The hand was chosen as the most safe and socially acceptable location for the present experiments, and the experimental site was usually the palm of the hand (thenar eminence). This was cleaned with detergent and water, and dried before the experiment. A control sample of sweat could be taken from the other hand using a similar piece of chromatographic paper placed in the palm and covered with a disposable plastic glove. The apparatus was prepared with the paper strips (untouched by hand) and the buffer solution applied. The thenar eminence of the subject was placed so as to complete the electrical circuit between the paper strips, and the current was
2.2 Constant current power supply A constant current supply provided a stable source of current which could be set to any required value over the range 0-500/tA, which is the maximum value that should be applied percutaneously to the subject; even so, the flow of such currents in the region of the heart should be avoided. The present apparatus is of 'type BF' and the current is of the 'patient functional current' classification (BSI, 1977). Its maximum value is equal to the 'patient auxiliary current' in 'single fault condition.' It would not be safe to use these currents on subjects with pacemakers or intracutaneous electrical pathways. The circuit diagram of the supply is given in Fig. 2. It comprises a battery operated oscillator, a transformer and rectifier, followed by a constant current stabilising circuit. There is only a minimal amount of capacitance connected to the rectifier because the open-circuit, no-load output voltage can rise to 800 V. It is also desirable to arrange a pressure operated switch so that the supply cannot be energised unless the subject is connected across the output and remains so. The maximum resistance into which the supply will deliver a constant 500 p A is 500 kfL This high value permitted experiments to be made with distilled water instead of the buffer solutions. F o r routine use with lower output resistances, the noload open-circuit voltage from the rectifier might be reduced. The anions and cations of the electrolyte
128
0 Leu 0 lie 0Phe
OVel 0 Tyr c 0
Q
Glu 0 (~AIo Asp 0 0 ~Ser
Gly
0 Thr
D Second
Dimension D
Fig. 3 Chromatographic separation of e/uate with a buffer p H 11,0 at the anode
Medical & Biological Engineering & Computing
March 1978
Table I. Percentage of measurements in which the indicated amino acids were detected at the anode, cathode and in the sweat
Detection
Amino acids Ala % 100 88 77
Anode Cathode Sweat
~0
I
Asp % 100 100 66
I
Glu % 100 55 66
I
I
I
I
-5 -
Gly % 100 100 66
lie % 88 88 66
Leu % 100 100 66
I
Phe % 100 88 55
Ser % 88 100 77
Thr % 88 100 66
Tyr % 77 88 33
Val % 100 100 55
I
I
_
A
Anode
o
Cathode
x
Sweat,
U
